Deciphering the Role of the MST1/2-YAP Axis in Irisin-Treated Aplastic Anemia: Implications for Mesenchymal Stem Cell Function

Deciphering the Role of the MST1/2-YAP Axis in Irisin-Treated Aplastic Anemia: Implications for Mesenchymal Stem Cell Function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Deciphering the Role of the MST1/2-YAP Axis in Irisin-Treated Aplastic Anemia: Implications for Mesenchymal Stem Cell Function Xia Liu, Hui Li, Bingxin Guan, Dexiao Kong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4329016/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Aplastic anemia (AA) is a debilitating hematological disorder characterized by bone marrow failure. Recent advancements in mesenchymal stem cell (MSC) research have highlighted potential therapeutic avenues, particularly through the modulation of cellular pathways influenced by novel agents like Irisin. This study investigates Irisin's effects on MSCs in the context of AA using advanced techniques such as single-cell sequencing and spatial transcriptomics. Irisin administration in AA model mice significantly altered gene expression in MSCs, particularly affecting 935 genes associated with the Hippo signaling pathway, notably the MST1/2-YAP axis. These changes were linked to decreased adipogenic differentiation and enhanced mitochondrial membrane system homeostasis. In vitro experiments supported these findings, showing Irisin's capability to inhibit the MST1/2-YAP signaling pathway and suppress adipogenesis in bone marrow stem cells (BMSCs). Corresponding in vivo studies demonstrated that Irisin treatment not only downregulated Mst1 and Mst2 but also upregulated Yap expression. Importantly, these molecular alterations led to reduced bone marrow adiposity and improved hematopoietic function in AA mice, showcasing Irisin's potential as an effective treatment option. The study underscores the critical role of the MST1/2-YAP pathway in mediating Irisin's therapeutic effects, suggesting promising strategies for AA management through targeted MSC pathway modulation. Irisin Aplastic Anemia Mesenchymal Stem Cells MST1/2-YAP Pathway Gene Expression Analysis Mitochondrial Homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Aplastic anemia (AA) is a distinct blood disorder characterized by the failure of hematopoietic function [ 1 , 2 , 3 , 4 ]. Bone marrow, the primary site of hematopoiesis, often undergoes adipogenesis in AA, replacing normal hematopoietic cells with adipocytes [ 5 ]. This phenomenon also contributes to reduced hematopoietic cells, leading to clinical manifestations like anemia and bleeding in affected individuals [ 6 , 7 ]. The precise etiology of AA remains incompletely comprehended. Nonetheless, scholars posit that it potentially involves elements such as immune abnormalities, alterations in the bone marrow microenvironment, and specific genetic mutations [ 8 , 9 , 10 ]. The bone marrow microenvironment plays a critical role in the development and differentiation of hematopoietic cells, and mesenchymal stem cells (MSCs) are considered the central cells in maintaining microenvironmental homeostasis [ 11 , 12 , 13 , 14 ]. However, in AA, mesenchymal stem cells exhibit abnormal behavior by preferentially differentiating into adipocytes, which results in bone marrow adipogenesis [ 15 ]. In clinical practice, bone marrow aspirates from patients with AA demonstrate reduced precursor cells for red blood cells, white blood cells, and platelets, alongside a substantial increase in adipocytes [ 16 ]. Researchers have recently discovered a molecule called Irisin, which may be associated with bone marrow adiposity while exploring treatment methods for AA [ 17 ]. This molecule has been demonstrated to regulate cell differentiation in various diseases, particularly by inhibiting the adipogenesis process [ 18 , 19 , 20 ]. However, the mechanism of action of Irisin and its specific functions in AA remain unclear. To tackle this issue, researchers have commenced investigating Irisin on a molecular level to discover novel treatments for AA [ 21 ]. Building upon the background above, this study aims to investigate the regulatory role of Irisin in mesenchymal stem cell differentiation and its potential impact on the development of myelodysplastic syndrome. It will be accomplished by employing cutting-edge methodologies, including single-cell sequencing, spatial transcriptomics, and transcriptome sequencing. Through this research, we aim to identify new targets for treating AA, improve therapeutic outcomes for patients, and gain insights into the regulation of MSCs in other diseases. Materials and methods Clinical research ethics statement. The bone marrow tissue used in this study was collected from patients diagnosed with aplastic anemia (AA) who underwent a biopsy at our hospital from January 2020 to January 2022. The study involved 5 patients with AA and 2 healthy volunteers. Before participating in this study, all participants had to sign a written informed consent form. The age range of the patients was from 43 to 65, with an average age of 54. The gathered tissue is divided into two parts. One part is promptly preserved in liquid nitrogen, while the other is fixed in 10% formaldehyde and embedded in paraffin for sectioning [ 22 ]. This study has been approved by the Clinical Ethics Review Committee at our hospital (KYLL-2019-023) in accordance with the Helsinki Declaration. Preparation and sequencing of single-cell samples. We will select bone marrow samples from two healthy adult donors and two patients with AA from the samples collected at our hospital. Bone marrow samples were digested using 1 mg/mL of STEMxyme1 (Worthington, LS004106) and 1 mg/mL of Dispase II (ThermoFisher Scientific, 17105041). Then, they were incubated with 2% fetal bovine serum (FBS, Gibco, 10091148) in culture medium 199 (Gibco, 11150059) at 37°C for 25 minutes. After digestion, the sample was filtered through a 70 µm cell filter (Fisher Scientific, 08-771-2) and collected in a separate tube [ 23 ]. Once the samples are prepared as single-cell suspensions, the C1 Single-Cell Auto Prep System (Fluidigm, C1) is employed to capture individual cells. Once the single cells are captured, they undergo lysis within the chip to liberate mRNA. Subsequently, this mRNA is reverse-transcribed to produce complementary DNA (cDNA). The complementary DNA (cDNA) is pre-amplified in a microfluidic chip after lysis and reverse transcription, in preparation for subsequent sequencing. Library construction will be carried out on the amplified cDNA, followed by single-cell sequencing using the HiSeq 4000 Illumina platform. The sequencing parameters will include paired-end reads with a read length of 2×75 bp and approximately 20,000 reads per cell [ 24 ]. TSNE clustering analysis, cell annotation and pseudotime analysis. This article analyzes single-cell RNA sequencing (scRNA-seq) data using the Seurat package in the R software. Initially, a series of quality controls were conducted, with the corresponding filtering thresholds set as follows: nFeature_RNA > 200, nFeature_RNA < 5000, and percent.mt < 10. The canonical correlation analysis (CCA) method was employed to eliminate batch effects, followed by normalization of the data using the LogNormalize function. To reduce the dimensionality of the scRNA-Seq dataset, principal component analysis (PCA) was performed on the highly variable genes using the top 2000 genes with the highest variance. Subsequently, the top 30 principal components were selected for TSNE clustering analysis. To identify major cell subpopulations, the FindClusters function provided by Seurat should be employed with the default resolution set at 0.9 (res = 0.9). Next, the t-SNE algorithm is applied to non-linearly reduce the dimensionality of scRNA-seq sequencing data. Filtering marker genes for different cell subpopulations using the Seurat package. To further annotate the marker genes of each cell cluster, we utilized the "SingleR" package and loaded the reference dataset using the HumanPrimaryCellAtlasData function. We annotated the cells by combining well-known marker genes specific to cell lineages with the online resource CellMarker [ 25 ]. Subsequently, we conducted pseudotemporal analysis using the "monocle" package in the R software and examined cell communication using the "cellchat" package. Spatial transcriptome sequencing. Spatial transcriptomic analysis was conducted on the bone marrow of the remaining three patients diagnosed with AA, utilizing the 10x Genomics Visium platform. The 10 µm tissue sections from fresh frozen human bone marrow embedded in OCT were placed onto Visium spatial slides. It was followed by a 30-minute permeabilization step to release mRNA. The mRNA molecules attach to the barcode oligonucleotides positioned at the bottom of the slide and undergo reverse transcription following the instructions provided by the manufacturer. The cDNA libraries prepared using these samples were sequenced on the Illumina NextSeq 2000 platform, generating over 50,000 reads per position. Each library produced more than 400 million reads. We utilized the Spaceranger software (version 3.1.0, 10x Genomics) to align each Visium spatial transcriptomics slide location and acquire raw counts. Subsequently, these counts were compared to the reference data of the GRCh38 human genome. The 10x Visium spatial transcriptomics data analysis involves using the Load10X_spatial function in Seurat to integrate the raw gene expression matrix, spatial information, and tissue H&E images and create a Seurat object. Before conducting principal component analysis, the data was normalized using SCTransform. The dimensionality was subsequently reduced by selecting the top 30 principal components. To detect marker genes and perform differential gene expression analysis, the FindAllMarkers function in Seurat is used. To identify genes that exhibit spatial variation in situ, the FindSpatiallyVariableFeatures function was employed with default settings. We implemented spatial transcriptomics deconvolution and visualization to locate cells in the bone marrow tissue of three patients with AA. An anchor-based integration pipeline in Seurat was used to integrate the combined scRNA-seq dataset with 10x Visium spatial transcriptomics data. It enables the transfer of cell type annotations from single-cell RNA sequencing (scRNA-seq) to spatial transcriptomics. The cell type predictions in Seurat are imported into the R package SPOTlight, which provides annotations and visualizations of the cell types for each spatial location [ 26 , 27 ]. Construction and grouping of AA mouse models. A total of ten C57BL/6 (B6) mice aged 213 days and 86 CB6FI mice aged eight weeks were purchased from Beijing Vitonglihua Experimental Animal Technology Co., Ltd., located in Beijing, China. The mice weigh between 20–25 g and are housed in standard cages. They are subject to a 12-hour light/dark cycle that alternates regularly, and the temperature in the room is kept constant at 23 ± 1°C. The mice have unrestricted access to food and water. Prior to commencing the experiment, the subjects will undergo a one-week acclimation period during which they will be exposed to adaptive feeding practices. The Institutional Animal Ethics Committee has approved this experiment and the animal use protocol(NO. 202309001). First, we randomly divided the 86 CB6FI mice into two groups: the Control group, which comprised 16 mice, and the Model group, which included 70 mice. The construction method for the Model group AA mouse model is as described previously [ 28 ]. In brief, we retrieved the inguinal, axillary, and brachial lymph nodes from 10 C57BL/6 (B6) donor mice. The lymph nodes were then homogenized in Iscove's modified Dulbecco's medium (IMDM, ThermoFisher, 21056023) using a tissue grinder (Beyotime, E6600). Subsequently, they underwent washing, centrifugation, and filtration through a 70µm nylon mesh sieve (Labgic, 352350). The quantity of samples was determined using a Beckman Vi-Cell counter (Vi-CELL XR, USA). Subsequently, we will intravenously administer lymphocytes isolated from C57BL/6 (B6) donors, at a dosage of 5×10 6 lymphocytes per 200 µl of PBS, into CB6FI mice matched in gender. The CB6FI receptor mice underwent 5.0 Gy total body irradiation (TBI) 2–4 hours ago. A peripheral blood cell count was conducted to confirm the presence of symptoms associated with leukopenia, thereby confirming the successful construction of the model [ 29 , 30 ]. Control group: intraperitoneal injection of PBS. Model group: The AA model was constructed, and intraperitoneal injection of PBS was performed. The Control group will be randomly divided into two subgroups: The control group (8 mice, injected with PBS only) and the Control + NC-OE group (8 mice, injected with adenovirus empty vector NC-OE, which serves as the negative control for overexpression, along with PBS). Similarly, the Model group will be randomly divided into the following six subgroups: Model group (24 mice, undergoing modeling and injected with PBS), Model + Irisin group (14 mice, undergoing modeling and injected with Irisin), Model + NC-OE group (8 mice, undergoing modeling and injected with PBS and NC-OE), Model + Irisin + NC-OE group (8 mice, undergoing modeling and injected with Irisin and NC-OE), Model + Mst1/2-OE group (8 mice, undergoing modeling and injected with PBS and adenovirus vector Mst1/2 overexpression Mst1/2-OE), Model + Irisin + Mst1/2-OE group (8 mice, undergoing modeling and injected with Irisin and Mst1/2-OE). The control group received no additional treatment except for an intraperitoneal injection of PBS. Model group: Only conducting AA modeling and PBS processing. In the Model + Irisin group, Irisin (MCE, HY-P72534) was dissolved in PBS and injected intraperitoneally at a dose of 100 µg/kg per injection, once a day for a total of 14 days, starting on the 4th day after constructing the AA model [ 31 , 32 , 33 ]. Furthermore, the virus transduction was conducted in the following manner: HEK-293 cells (Procell, CL-0001) were transfected with either the adenoviral vector (Mst1/2-OE, AAV-Mst1-Mst1) carrying the mouse Mst1 and Mst2 genes, or the adenoviral empty vector (NC-OE) plasmid. The transfection was performed using the LipoFiter transfection reagent (Hanbio, HB-LF-1000). Hanbio synthesized the plasmids. After 72 hours, collect the clear liquid from the top to obtain the viral fluid. After successfully constructing the AA model, different groups of mice were injected with Mst1/2-OE or NC-OE vectors via the tail vein 4 days later. The injection was performed with a concentration of 3.5×10 12 viral genomes per mouse. Control + NC-OE group: Only tail vein injection of NC-OE and intraperitoneal injection of PBS were performed without constructing the AA model. Experimental Group: Four days after successfully constructing the AA model, NC-OE was administered through the tail vein while PBS was injected into the peritoneal cavity. Model + Irisin + NC-OE group: Accept model construction, NC-OE, and Irisin processing. Model + Mst1/2-OE group: After successfully establishing the AA model for 4 days, Mst1/2-OE was administered through a tail vein injection, followed by an intraperitoneal injection of PBS. Model: Mice treated with both Irisin and Mst1/2-OE were used to establish the AA model [ 34 , 35 ]. Except for the 24 mice in the Model group and the 14 CB6FI mice in the Model + Irisin group, eight mice were randomly assigned to each of the remaining groups. On the second day following the administration of Irisin, all the mice were euthanized. Six mice from the Model group and six from the Model + Irisin group were used for transcriptome sequencing. Ten mice from the Model group were also used to extract bone marrow mesenchymal stem cells (BMSCs) for in vitro mechanistic validation. Bone marrow smear. After decapitating the mouse, expeditiously remove the sternum and utilize hemostatic forceps to grip it, facilitating the extraction of the bone marrow. Gently place the bone marrow smear onto a microscope slide pre-treated with 0.05 mL of fetal bovine serum (FBS, Hyclone), then slide it forward. The identical procedure is employed for the bone marrow obtained from normal donors and bone marrow from patients with AA, which is collected through bonemarrow aspiration (BM aspiration). Next, allow the slides to dry at room temperature and position them on a staining rack. The cells were fixed by applying a drop of methanol for 3 minutes. Then, the working solution of Wright Giemsa (Solarbio, G1021) was added. The working solution was prepared by diluting 1 part of the stock solution with 9 parts of the buffer solution. The working solution allowed the cells to be completely covered at room temperature for 20 minutes. Thoroughly rinse the glass slides with distilled water from one end to the other. The examination and observation were conducted using an OLYMPUS BX46 upright microscope. The ImageJ software was used to quantify the percentage of nucleated cells in the bone marrow. Each group has three independent perspectives [ 31 ]. Pathological sections of femoral bone marrow. Mouse femurs were harvested, or normal donor and AA patient bone marrow were obtained through bone marrow biopsy (BM biopsy). The samples were fixed in 4% paraformaldehyde (Biosharp, BL539A) at room temperature for 48 hours. Then, rinse thrice with PBS and distilled water, each time for 20 minutes. Replace the EDTA solution (Solarbio, E1171) for decalcification every 7 days, totaling 28 days. After decalcification, rinse the femur or bone marrow using running water for 20 minutes in an embedding box. Subsequently, perform dehydration by exposing the sample to ethanol. After complete dehydration, clean with xylene. Embed the transparent mouse femur in paraffin. To obtain 4 µm thick sections, the paraffin blocks embedded in mouse femurs were sliced using a wax microtome (Leica, LECIA RM2235). Spread the sliced organisms onto a distillation water bath heated to 45°C. Then, transfer the slices onto clean glass slides, allowing them to drain and dry on a glass slide heater set at 65°C for one hour. De-waxing and dyeing procedures were carried out using an automated staining machine, with the dyeing time determined according to the manufacturer's instructions of the HE Staining Kit (Solarbio, G1120). Finally, seal the slices with neutral adhesive. Examine the bone marrow sections stained with hematoxylin and eosin under an inverted microscope (Leica, Leica DM IL LED) and photograph them at a magnification 200×. The nucleus appears blue after staining, while red blood cells and bone trabeculae are stained with eosin. Hematopoietic tissues comprise granulocytes, red blood cells, megakaryocytes, and lymphocytes, whereas non-hematopoietic tissues comprise trabecular bone, cortical bone, and adipose tissue [ 31 ]. ImageJ software was employed to identify and calculate the percentage area of hematopoietic tissue in the entire bone marrow interstitium. Three independent fields were quantified for each group. Peripheral blood cell count. Mouse tail vein blood was collected on the 5th, 10th, and 15th days after establishing the AA mouse model to determine the absolute values of white blood cells (WBC), neutrophils (NEU), platelets (PLT), red blood cells (RBC), and hemoglobin (HGB) concentration in peripheral blood. It was done using a hematology analyzer (HORIBA, ABX PENTRA XL 80) [ 33 ]. The hematopoietic colony forms experiment. Following the death of the mice, a semi-solid culture system employing colony-forming units (CFUs), including CFU-erythroid (CFU-E), CFU-granulocyte macrophage (CFU-GM), and CFU-megakaryocytic (CFU-MK), was utilized to extract nucleated cells from the bone marrow of the femur and subsequently adhere them onto the wells of the tissue culture plates. Bone marrow cells were cultured in discover-modified Dulbecco's medium (IMDM, Sigma-Aldrich, I3390). The culture medium consisted of 20% fetal bovine serum (FBS, Gibco, USA), 300 mg/L glutamine (Sigma-Aldrich, G7513), and either 10 µg/L recombinant mouse macrophage colony-stimulating factor (GM-CSF, Sigma-Aldrich, M9170) or erythropoietin (EPO, ACROBiosystems, EPO-H4214). Additionally, a viscosity carrier of 0.3% agarose was included. Cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) for the CFU-MK culture. The medium was composed of 20% fetal bovine serum, 300 mg/L of glutamine, 1% bovine serum albumin (Sigma-Aldrich, A1933), 10 − 5 mol/L of 2-mercaptoethanol (Sigma-Aldrich, M3148), and 10 µg/L of recombinant mouse thrombopoietin (Sigma-Aldrich, T4184). The experiment was repeated three times, with 10 5 nucleated cells per well, and cultured in a humid environment at 37°C with 5% CO2. Following a 5-day cultivation period, the colonies of CFU-E (≥ 8 cells) and CFU-GM (≥ 40 cells) were enumerated. As previously stated, mouse megakaryocyte cells in CFU-MK colonies were detected using acetylcholinesterase staining. The count of CFU-MK colonies with four or more cells was performed after seven days of culture [ 33 ]. Organize slice oil red o staining. Following decalcification and dehydration, the femur sections of the mouse were stained with a mixture of 0.21% Oil Red O (Sigma, 1320-06-5) and 100% isopropanol (Sigma, 67-63-0) for 10 minutes. The images were captured using the Olympus BX53 microscope, and the percentage of positive area of fat cells was quantified using ImageJ software. Three mice are randomly selected for each group, and three sections are extracted from each mouse. Data from three independent fields of view are analyzed for every section [ 28 ]. Immunofluorescence staining. Following dewaxing and dehydration, paraffin sections of the mouse femur underwent antigen retrieval at 98°C. Incubation was then carried out using 1% Triton X-100 (Sigma-Aldrich, X100) in PBS, followed by a 30-minute blocking step with goat serum (Solarbio). The BMSCs cells were fixed with 4% paraformaldehyde (Biosharp, BL539A) for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and then blocked with 1% BSA (Thermofisher, 37520) at room temperature for 1 hour. Mouse femoral bone slices and BMSCs cells were subjected to immunofluorescent staining using Perilipin antibody (1:200, Thermofisher, MA5-32597) or YAP antibody (1:500, Thermofisher, PA1-46189). The antibodies were incubated overnight at 4℃, followed by a 40-minute incubation at room temperature with Alexa FluorTM Plus 488 goat anti-rabbit IgG antibody (1:1000, Thermofisher, A32731). The samples were stained with 4',6-Diamidino-2-Phenylindole (DAPI) at 0.5µg/mL (Invitrogen, D3571). Subsequently, the stained samples were observed, and images were captured using a fluorescence microscope (Olympus FV-1000/ES). The data were analyzed using the ImageJ software. Three mice were randomly selected for each group. Three slices were taken from each mouse. The fluorescence intensity was assessed in three independent fields of view for each slice [ 36 , 37 ]. Cell culture. Bone marrow mesenchymal stem cells (BMSCs) were isolated from the bone marrow of mice by flushing the femurs and tibias of both wild-type mice and mice from different treatment groups after decapitation. The centrifugation was performed at 1000 rpm for 5 minutes. The suspension of bone marrow cells was cultured in L-DMEM (Thermo Fisher, A1443001, USA), supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin. The culture was carried out in a 10 cm² culture dish. The culture medium is changed once a day for three consecutive days to remove non-adherent cells. Afterward, change the culture medium every 3 days. Additionally, to isolate human BMSCs, three-milliliter bone marrow samples were collected from patients with AA and normal donors. Bone marrow mononuclear cells were separated from these samples using Ficoll-Paque (Cytiva, 17144003, USA) through centrifugation. Subsequently, these cells were seeded in 10 cm2 culture dishes, utilizing the previously mentioned culture medium. Remove unattached cells after 48 hours and replace the culture medium every 3 days afterward. Cultured adherent BMSCs with a cell density of 80%-90% were subjected to trypsin treatment. Isolated BMSCs were characterized using flow cytometry [ 28 , 38 ]. Flow cytometry. Surface marker analysis was conducted on BMSCs obtained from normal donors, patients with AA, and mouse bone marrow. FITC-conjugated anti-human CD45, HLA-DR, CD34, CD105, CD44, and CD29 antibodies were used for human samples, while anti-mouse CD45, HLA-DR, CD34, CD105, CD44, and CD29 antibodies were used for mouse samples. After incubating the cells at 4°C in the dark for 30 minutes, wash the BMSCs twice with PBS and then centrifuge them at 2000 rpm for 5 minutes at 4°C. We analyzed the percentage of cells expressing these surface markers using the FACSCanto II flow cytometer (BD, USA) [ 39 , 40 ]. Induction of adipogenic or osteogenic differentiation in BMSCs and cellular grouping. Bone marrow mesenchymal stem cells (BMSCs) were seeded at a density of 2.5×10 6 cells per well in a six-well plate. For differentiation induction, the cells were cultured in a medium comprising 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, I7018), 1 µM dexamethasone (Sigma-Aldrich, D4902), and 5 µg/mL insulin (Sigma-Aldrich, I3536). The medium for inducing adipogenic differentiation is changed every 3 days, and the induction process is carried out for 14 days [ 38 ]. Bone marrow-derived mesenchymal stem cells (BMSCs) were seeded at a density of 2 × 10 6 cells per well in a 6-well plate. Once the cells reached 80% confluence, they were cultured in osteogenic induction medium comprising 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.1 mM dexamethasone (Sigma-Aldrich, D4902), 10 mM β-glycerophosphate (Sigma-Aldrich, G9422), and 50 mM ascorbic acid (Sigma-Aldrich, A4403). The osteogenic induction medium is changed every 3 days [ 32 , 38 ]. The cell groups involved in the study were divided into five categories. These included the Normal group, AA group, Control group, Model group, and Model + Irisin group. The groups consisted of BMSCs isolated from clinical samples or different treatment groups of mice. BMSCs were differentiated from clinical normal donor bone marrow samples in the Normal group. BMSCs were differentiated from patient bone marrow samples with clinical AA in the AA group. In the Control group, BMSCs were differentiated from mouse bone marrow samples. In the Model group, BMSCs were differentiated from mouse bone marrow samples. In the Model + Irisin group, BMSCs were differentiated from mouse bone marrow samples with Model + Irisin treatment. Next, BMSCs cells from the Model group mice will be divided into 24 treatment groups, including the PBS group, DMSO group, PBS + DMSO group, Irisin group, Irisin + DMSO group, VP group, PBS + VP group, Irisin + VP group, Rotenone group, VP + Rotenone group, NC-OE group, Mst1/2-OE group, Yap-OE group, Mst1/2-OE + Yap-OE group, PBS + NC-OE group, PBS + DMSO + NC-OE group, Irisin + NC-OE group, Irisin + DMSO + NC-OE group, Irisin + VP + NC-OE group, PBS + Mst1/2-OE group, Irisin + Mst1/2-OE group, Irisin + DMSO + Mst1/2-OE group, PBS + Yap-OE group, and Irisin + Yap-OE group. The differentiation-inducing medium was supplemented with PBS, DMSO, Irisin, VP (verteporfin), and Rotenone. These drugs were co-treated with BMSCs, and the duration of drug treatment coincided with the induction time. In the PBS group, DMSO group, and PBS + DMSO group, the differentiation-inducing culture medium was supplemented with PBS, DMSO, or PBS + DMSO, respectively, to induce the differentiation of BMSCs. The irisin group involved dissolving irisin in PBS at 1, 5, 10, and 20 ng/ml concentrations. The dissolved irisin was then added to the differentiation-inducing culture medium to co-treat BMSCs and determine the optimal concentration. In the Irisin + DMSO group, Irisin and PBS were co-administered to treat BMSCs and induce differentiation. In the VP Group, add verteporfin (verteporfin, VP; MCE, HY-B0146) dissolved in DMSO at a concentration of 0.8 µM to the differentiation-inducing medium for simultaneous treatment with BMSCs. PBS and VP collaborate to process BMSCs and prompt differentiation. In the Irisin + VP group, both Irisin and VP were included in the differentiation-inducing medium to co-treat BMSCs. In the Rotenone group, a concentration of 100 nM of the fish poison Rotenone (Rotenone, MCE, HY-B1756) dissolved in DMSO was added to the differentiation induction medium to be co-treated with BMSCs. VP + Rotenone group incorporated VP and Rotenone into the differentiation-inducing culture medium to co-treat with BMSCs [ 41 , 42 , 43 ]. Furthermore, the AAV-Yap1 plasmid containing the mouse Yap1 gene, synthesized by Hanheng Biotechnology, will be transfected into HEK-293 cells after packaging. It will be done following the animal grouping section, along with the Mst1/2-OE vector and NC-OE vector viral solutions for transfecting BMSCs cells. BMSCs cells in the logarithmic phase (5 × 10 4 cells/well) were seeded in a 24-well plate. Transfection occurred when the cell confluence reached 50%, with a multiplicity of infection of 5 × 10 4 vp/cell. After 24 hours, the culture medium was replaced with an L-DMEM medium containing 10% FBS, and the medium was changed every three days [ 44 ]. The cell treatment after 3 days of viral transduction is as follows: In the NC-OE group, differentiation induction occurs after receiving NC-OE transfection. In the Mst1/2-OE group, differentiation induction was performed following transfection with Mst1/2 overexpression. Yap-OE group: Differentiation induction after Yap-OE transfection. In the Mst1/2-OE + Yap-OE group, differentiation induction was conducted following the co-transfection of Mst1/2-OE and Yap-OE. The PBS + NC-OE group was treated with PBS and NC-OE to induce differentiation. The PBS + DMSO + NC-OE group was treated with PBS, DMSO, and NC-OE to induce differentiation. The Irisin + NC-OE group received treatment with Irisin and NC-OE to induce differentiation. In the Irisin + DMSO + NC-OE group, the participants were treated with Irisin, DMSO, and NC-OE, which led to induced differentiation. The Irisin + VP + NC-OE group consisted of subjects who received Irisin, VP, and NC-OE treatments to induce differentiation. In the PBS + Mst1/2-OE group, cells were initially treated with PBS and then induced to differentiate by overexpressing Mst1/2 proteins. The Irisin + Mst1/2-OE group received treatment with Irisin and Mst1/2-OE to induce differentiation. Irisin + DMSO + Mst1/2-OE group: The individuals in this group received treatment with Irisin, DMSO, and Mst1/2-OE and were induced for differentiation. The PBS + Yap-OE group accepted both PBS and Yap-OE treatments to induce differentiation. The Irisin + Yap-OE group was treated with Irisin and Yap-OE to induce differentiation [ 44 , 45 ]. Oil red O staining of BMSCs. After 14 days of induction for adipogenesis, the cells were fixed in a 4% paraformaldehyde solution (Biosharp, BL539A) and stained with Oil Red O solution (Sigma-Aldrich, MAK194). The dye should be dissolved in isopropanol, and the absorbance at 510 nm should be measured [ 38 ]. Staining of alkaline phosphatase (ALP) and alizarin red was performed in BMSCs. On the seventh day of osteogenic induction, BMSCs were fixed using a 4% paraformaldehyde solution (Biosharp, BL539A) for 30 minutes. Subsequently, they were covered with a BCIP/NBT working solution (Beyotime, C3206) and incubated in the dark for 20 minutes. Observe cells under a microscope and take pictures. The protein concentration was measured using the BCA Protein Quantification Kit (ThermoFisher, 23227), and the OD values of the blank wells, standard wells, and test wells were determined at 520 nm using the alkaline phosphatase (ALP) staining kit (Jiancheng Bioengineering Research Institute, A059-2-2). Subsequently, the alkaline phosphatase activity was calculated following instructions [ 32 ]. After 21 days of osteogenic induction, the cells were fixed using 4% paraformaldehyde (Biosharp, BL539A) and stained with 2% Alizarin Red S (Sigma-Aldrich, A5533). The Xanthein Red S should be dissolved in a solution of hexadecylpyridinium chloride (Sigma-Aldrich, 1104006) and then quantitated using spectrophotometry at a wavelength of 562nm [ 32 ]. Western blot. Bone marrow stromal cells (BMSCs) were lysed using the RIPA total protein lysis buffer (AS1004, Wuhan Aspen Biotechnology Co., Ltd., China) after 7 days of osteogenic or adipogenic differentiation. The protein concentration was subsequently measured using the BCA protein quantification assay kit (23227, Thermo Fisher). Proteins were separated using SDS-PAGE and then transferred onto a PVDF membrane. The membrane was blocked with 5% BSA at room temperature for 1 hour. Afterwards, the primary antibodies were added individually, including LPL (...) Incubate the antibody at 4℃ overnight. LPL, FABP4, PPARγ, CEBPα, and PERILIPIN are markers for adipogenic differentiation, whereas Runx2, ALP, OPN, and OCN are markers for osteogenic differentiation. MST1, MST2, YAP, and p-YAP are proteins associated with the MST1/2-YAP signaling pathway. The membrane was washed three times with TBST (3×5 minutes), followed by incubation with Anti-Mouse-HRP secondary antibody (1:10000; ThermoFisher, 31430) or Goat anti-Rabbit-HRP secondary antibody (1:10000; ThermoFisher, 31460) at room temperature for 2 hours. The membrane was washed thrice with TBST (3×5 minutes). TBST should be replaced with an appropriate ECL working solution (Millipore, WBKLS0500). The transfer membrane should be incubated at room temperature for 1 minute. Excess ECL reagent should be removed, and the membrane should be sealed with cling film. Before development and fixation, the membrane should be placed in a dark box for 5–10 minutes to expose an X-ray film. The ImageJ analysis software was used to quantify the grayscale intensity of bands in Western blot images, utilizing GAPDH as the internal reference [ 39 ]. RT-qPCR. The Trizol Reagent kit (Invitrogen, 10296028CN) should be utilized for cell lysis and total RNA extraction from cells or bone marrow tissue. UV-Vis spectrophotometry (ND-1000, Nanodrop, USA) was employed to evaluate RNA quality and concentration. The PrimeScript™ RT-qPCR Kit (TaKaRa, RR037Q) was employed to measure mRNA expression levels for reverse transcription. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed using SYBR Premix Ex TaqTM (TaKaRa, RR390A) on a LightCycler 480 system (Roche Diagnostics, Pleasanton, CA, USA). GAPDH is a reference gene that functions as an internal control for mRNA. The primers utilized for amplification were designed and provided by Shanghai Universal Biotech Co., Ltd. The primer sequences can be found in Table S1 . The term 2 −ΔΔCt represents the fold difference in the target gene expression between the experimental and control groups. The formula is defined as ΔΔCT = ΔCt experimental group - ΔCt control group, where ΔCt is calculated as the difference between the Ct values of the target gene and reference gene [ 46 ]. RNA extraction, library construction, and sequencing. Total RNA was extracted from bone marrow stromal cells (BMSCs) and isolated from two groups of mice: the Model group (n = 6) and the Model + Irisin group (n = 6). The extraction was performed using a Trizol reagent (15596026, Invitrogen, USA). The concentration and purity of RNA samples were determined using a spectrophotometer instrument, specifically the Nanodrop 2000 (1011U, Nanodrop, USA). Total RNA samples meeting the following criteria are utilized for subsequent experiments: RNA Integrity Number (RIN) ≥ 7.0 and 28S:18S ratio ≥ 1.5 [ 47 ]. CapitalBio Technology, located in Beijing, China, generated and sequenced the sequencing library. Every sample utilizes a total of 5 µg of RNA. We employed the Ribo-Zero Magnetic Kit (MRZE706, Epicentre Technologies) to remove ribosomal RNA (rRNA) from total RNA. The NEB Next Ultra RNA Library Prep Kit (#E7775, New England Biolabs, USA) generates Illumina-compatible sequencing libraries. Next, the RNA fragments were fragmented into 300 base pairs (bp) using the NEB Next First Strand Synthesis Reaction Buffer (5×). The first-strand cDNA is synthesized using a reverse transcriptase primer and random primer, while the second-strand cDNA is synthesized in the reaction buffer of dUTP Mix (10×) for the second-strand synthesis. The repair of cDNA fragments involves adding polyA tails and connecting sequencing adaptors. Following the ligation of Illumina sequencing adapters, the second cDNA strand was digested using the USER enzyme (#M5508, NEB, USA) to generate strand-specific libraries. The library DNA should be amplified, purified, and enriched through PCR. Next, the libraries were evaluated using the Agilent 2100 system and quantified using the KAPA Library Quantification Kit (KK4844, KAPA Biosystems). Lastly, we conducted paired-end sequencing using the Illumina NextSeq CN500 sequencer [ 48 , 49 ]. Quality control of sequencing data and its alignment to a reference genome. The quality of the paired-end reads in the raw sequencing data was assessed using FastQC software version 0.11.8. The raw data underwent preprocessing using Cutadapt software version 1.18, which involved removing Illumina sequencing adapters and poly(A) tail sequences. Filter out reads with an N content exceeding 5% using a perl script. Using the FASTX Toolkit software version 0.0.13, we extracted reads with a base quality of 20 or higher, which accounted for 70% of the total. Repair the paired-end sequences using BBMap software. Finally, the filtered fragments of high-quality reads were aligned to the mouse reference genome using hisat2 software (version 0.7.12) [ 50 , 51 ]. Differential expression gene bioinformatics analysis. The limma package in R was used to identify differentially expressed genes (DEGs) in the raw count matrix. DEGs were selected based on a threshold of |log fold change (FC)| > 1 and a P value < 0.05. The ggplot2 package in R was used to plot the volcano plot. The clustering heat map of differentially expressed genes (DEGs) was generated using the "pheatmap" package in the R programming language. We conducted enrichment analysis using the R language and several packages, including the "clusterProfiler", "org.Hs.eg.db", "org.Mm.eg.db", "enrichplot", "ggplot2", and "pathview" packages. The analysis focused on Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) [ 52 ]. Bioinformatics analysis. We extracted 26 mouse genes related to the Hippo signaling pathway (HRGs) from the Reactome database ( https://reactome.org/ ). Conduct a protein-protein interaction (PPI) analysis of HRGs using the STRING database ( https://string-db.org/ ) and optimize the results using Cytoscape 3.10.0 software. The Jvenn website generates Venn diagrams to identify the intersection of differentially expressed genes (HR-DEGs) associated with the Hippo pathway. The clustering heatmap, volcano plot, correlation analysis heatmap, and scatter plot for highly-regulated differentially expressed genes (HR-DEGs) were generated using the R packages "ggplot2" and "pheatmap" [ 53 ]. GO and KEGG are utilized to perform functional enrichment analysis of HR-DEGs. GO and KEGG analyses were conducted using the 'clusterProfiler,' 'org.Hs.eg.db,' 'enrichplot,' 'DOSE,' and 'ggplot2' packages in the R programming language [ 54 ]. We applied machine learning algorithms such as lasso regression, SVM-RFE, and random forest using the "glmnet", "e1071", and "randomForest" packages in the R programming language. The Venn diagram was plotted using the "venn" package in R [ 55 ]. CCK-8. Cell viability was assessed following the guidelines provided in the CCK-8 assay kit (ab228554, Abcam, USA). Cells from each group were seeded into individual wells of 96-well plates (2500 cells per well) and cultured for 1, 3, and 5 days, respectively. At the designated time point, add 10µl of CCK-8 to each well. After incubating for 2 hours, the absorbance at 450 nm should be measured using an enzyme-linked immunosorbent assay reader (M1000 PRO, Tecan) [ 41 ]. Live-death staining. The LIVE/DEAD Cell Viability/Cytotoxicity Assay Kit (Invitrogen, L3224) was employed to assess cell death objectively. The assay kit offers two types of molecular probes: one that labels live cells as green based on intracellular esterase activity and another that labels dead cells red due to compromised membrane integrity. Conduct the testing in accordance with the plan provided by the manufacturer. In brief, cells were seeded in a 24-well plate and incubated overnight. Afterward, they were treated with Irisin for 3 days. Subsequently, the cells were incubated with a fluorescent dye (2.0 µM) for 15 minutes, and a fluorescence microscope (FV-1000/ES, Olympus) was used to capture microscopic images. The software ImageJ was employed to identify and calculate the percentage of viable and nonviable cells [ 56 ]. MitoTracker staining. After inducing adipogenic differentiation of BMSCs for 14 days, the mitochondria staining reagent, mitoTracker Green FM (Invitrogen, M7514), was applied at a concentration of 100 nM. The cells were then incubated in a cell culture incubator for 30 minutes. Next, the Hoechst 33342 Live Cell Stain (Beyotime, C1029) should be applied in a dark environment at 37°C for 10 minutes. Subsequently, the cells were washed twice with preheated PBS, and a fresh culture medium was added. Imaging was then promptly conducted using a Zeiss LSM 510 META confocal microscope (Zeiss). Merge and scale the original image using ImageJ software [ 43 , 57 ]. Quantitative analysis of mitochondrial DNA. Total genomic and mitochondrial DNA could be extracted using the QIAamp DNA Mini kit (Qiagen, 51304), following the manufacturer's instructions. Adjust the DNA template concentration to 10 ng/µl. Refer to the report by Malik et al. [ 58 ]. Evaluating the amount of mitochondrial DNA through RT-qPCR for absolute quantification. We utilized mouse mitochondrial DNA primers (Mito) and mouse nuclear DNA primers (β2-microglobulin, mB2M) to amplify the corresponding products in mouse genomic DNA. The primer sequences are available in Table S2 [ 59 ]. Measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The Seahorse XF24 extracellular flux analyzer (Agilent, Seahorse XF24, 24-well plate) was employed, following the described method, to assess the oxygen consumption rate (OCR) of adipogenic-induced BMSCs after 7 days [ 60 , 61 ]. The OCR determination was conducted using the Seahorse XF Cell Mito Stress Test Kit (Agilent, 103672-100). The assay medium consisted of Seahorse XF DMEM culture medium (Agilent, 103680-100) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose, adjusted to pH 7.4. Sixty thousand cells were inoculated per well (0.32 cm2 growth area) into XF24 24-well culture microplates containing 500 µL of assay medium. The plates were incubated overnight in a humid environment at 37℃, with 95% air and 5% carbon dioxide. The culture medium should be removed before the experiment, and 500 µL of fresh assay medium should be added. The cells are pre-incubated in ambient air at 37°C for one hour. We used oligomycin at a concentration of 4 µg/mL to assess ATP synthesis driven by oxidative phosphorylation and respiration driven by proton leak. Following three measurement cycles, a decoupling agent, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), was added at a concentration of 5 µM to assess the maximum respiratory capacity. After three additional measurement cycles, 1 µM of Rotenone should be added to inhibit complex I and 1 µM of antimycin A should be used to inhibit complex III, thereby suppressing mitochondrial respiration. Furthermore, the Seahorse XF Glycolysis Stress Test Kit (Agilent, 103020-100) was used to accurately measure the glycolytic rate. The cells should be replaced with XF DMEM medium (Agilent, 103575-100) supplemented with 5 mM HEPES at pH 7.4. Additionally, 1 mM pyruvate (Agilent, 103578-100), 2 mM glutamine (Agilent, 103579-100), and 10 mM glucose (Agilent, 103577-100) should be added. The cells should then be incubated in a CO2-free incubator at 37℃ for 1 hour. Before commencing the test, the culture medium should be replaced again, following the guidelines provided by the manufacturer. After establishing the baseline, 1 µm fisetin and antifungal A were added sequentially, along with 50 mM 2-deoxyglucose, and the response was measured. The OCR, ECAR, glycolytic proton efflux rate, and ATP production rate were determined using the Seahorse XFe96 software, version 2.6 [ 62 ]. ROS detection. The levels of reactive oxygen species (ROS) inside the cells were measured in mouse bone marrow-derived mesenchymal stem cells (BMSCs) or BMSCs induced for adipogenic differentiation for 7 days from different groups, following the guidelines provided by the manufacturer. The measurement was performed using a ROS assay kit (Beyotime, S0033S). The fluorescent probe DCFH-DA (10 mM) should be diluted 1000-fold in serum-free L-DMEM before adding it to the cells. The cells should be incubated in the dark at 37℃ for 20 minutes. After rinsing with PBS three times, we examined cell morphology using a fluorescence microscope (FV-1000/ES, Olympus). Subsequently, the cells were harvested, and the fluorescence intensity was quantified using a TriStar3 multimode reader (Berthold Technologies) with an excitation wavelength set at 488 nm and an emission wavelength of 525 nm. Normalize intracellular ROS levels to total cell number [ 63 ]. Mitochondrial membrane potential (MMP) detection. After isolating bone marrow-derived mesenchymal stem cells (BMSCs) or differentiating them into adipocytes for 7 days, we assessed the mitochondrial membrane potential (MMP) using the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, C2006) [ 64 ]. In summary, the cells are incubated in a culture medium containing JC-1 for 30 minutes. Subsequently, they are loaded into the BD FACSMelody flow cytometer (BD) with an excitation wavelength of 480 nm and emission wavelengths of 525 nm and 590 nm [ 65 ]. We analyzed 10,000 cells using FlowJo X10 software and repeated the experiment three times. ATP level determination. The total ATP production of BMSCs in each group was quantified using the ATP assay kit (Beyotime, S0026B). Initially, the cells are inoculated into a 96-well plate. Subsequently, PBS washing, lysis, and centrifugation are performed. Subsequently, the luminescence in the supernatant was measured with the BioTek Synergy 2 microplate reader (BioTek Instruments Inc.). The MicroBCA Protein Assay Kit (ThermoFisher, 23235) was utilized to determine the protein concentration and subsequently performed normalization. Finally, the ATP content should be determined using the ATP standard curve, and the experiment should be repeated three times [ 66 ]. Statistical analysis. Each experiment was repeated independently at least three times, and the data are presented as the mean ± standard deviation (SD). To compare the differences between groups, we utilize either an independent samples t-test or a one-way analysis of variance. If the variance analysis results reveal differences, we will conduct Tukey's HSD post-hoc test to examine the disparities between each group. When dealing with data that is not normally distributed or exhibits heteroscedasticity, we will employ either the Mann-Whitney U or Kruskal-Wallis H test. Statistical analyses were conducted using GraphPad Prism 8.0 software [ 28 ]. A p-value less than 0.05 is considered statistically significant. Results Comprehensive single-cell rna sequencing analysis of bone marrow microenvironment in aplastic anemia: unraveling cellular diversity and pathological alterations. Aplastic anemia (AA) is a serious hematological disorder characterized by insufficient development of the bone marrow, accompanied by fatty degeneration. There are numerous unresolved issues concerning the pathogenesis and treatment options [ 67 ]. Single-cell RNA sequencing (scRNA-seq) could uncover alterations in the transcriptome of distinct cellular subpopulations, thus facilitating the investigation of crucial pathways that might impact the development of AA [ 68 ]. To examine the developmental process of AA and the alterations in the bone marrow microenvironment, single-cell RNA sequencing (scRNA-seq) was conducted on bone marrow samples from two healthy donors and two patients with AA. We conducted quality control and normalization of single-cell RNA sequencing (scRNA-seq) data using the "Seurat" package in the R software. After processing, the distribution of cellular RNA is presented in Fig. S1 A. The correlation coefficient between nCount and percent.mt is -0.13 (r = -0.13), and the correlation coefficient between nCount and nFeature is 0.74 (r = 0.74). These findings suggest that the filtered cell data exhibits good quality and is suitable for subsequent analysis (Fig. S1 B). Subsequently, we further analyzed the filtered cells. After filtering, we chose the top 2000 genes with high variance in gene expression for further analysis (Fig. S1 C). The cell cycle was calculated using the CellCycleScoring function (Fig. S1 D), and the data were subsequently normalized. Subsequently, we applied linear dimensionality reduction to the data using principal component analysis (PCA), utilizing the highly variable genes previously selected. We obtained a total of 50 principal components (PCs). A smaller p-value and a larger standard deviation suggest a greater importance of the PCs (Fig. S1 E-F). The results suggest that principal components PC_1-PC_30 effectively capture the information in the selected highly variable genes and hold analytical value. In this study, we provide the characteristic genes of PC_1 and PC_2, as shown in Fig. S1 G. Additionally, we present the expression heatmaps of the major correlated genes of PC_1 - PC_6, depicted in Fig. S1 H. Furthermore, we also illustrated the distribution of various sample cells across PC_1 and PC_2 (Fig. S1 I). The results indicate the presence of a distinct batch effect across various samples. To mitigate batch effects among samples and enhance the accuracy of cell clustering, we employed the harmony package to correct batch variation in the sample data (Fig. 1 A). The corrected results indicate that the batch effect of the samples has been successfully eliminated (Fig. 1 B). Next, we utilized the TSNE algorithm to reduce non-linear dimensionality on the initial 30 principal components (PCs). Subsequently, we conducted cluster analysis with a resolution of 0.9 (Fig. S2 ). We utilized clustering to generate 29 clusters and determine each respective cluster's marker gene expression patterns (Fig. 1 C-D). Next, we annotated the cells by conducting a literature search and utilizing the online resource CellMarker, which provided us with identified marker genes specific to cell lineages (Fig. 1 E). Overall, we obtained a total of 15 cell types: Hematopoietic stem and progenitor cells (HSPCs), Mesenchymal stem cells (MSCs), Adipocytes, T cells, Neutrophils, Myeloid cells, Plasmacytoid dendritic cells (pDCs), Red blood cells, Monocytes, Macrophages, B cells, Common lymphoid progenitor cells (CLPs), Plasma cells, Natural killer cells (NK cells), and Megakaryocytes (Fig. 1 F). The results above indicate that bone marrow samples from normal donors and patients with AA could be categorized into 28 clusters, comprising 15 cellular subgroups. A decrease in hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) could be observed in the bone marrow tissue of patients with AA, while the number of adipocytes increases significantly. Elucidating the role of mesenchymal stem cells and adipocytes in aplastic anemia: a deep dive into cellular dynamics and intercellular communication. Mesenchymal stem cells (MSCs) and adipocytes play a role in the excessive differentiation of bone marrow stromal cells, thereby contributing to the occurrence of AA [ 28 ]. Therefore, we initially confirmed the annotation of MSCs and Adipocytes (Fig. 2 A). Following this, Seurat analysis was used to calculate the proportions of different cell types in both the AA and Normal groups (Fig. 2 B). The results demonstrated a decrease in MSC quantity and an increase in Adipocyte quantity in the AA group compared to the Normal group. To further validate the essential role played by MSCs in differentiating into adipocytes in AA, we conducted a pseudo-temporal analysis of MSC subpopulations using the 'monocle' package. We generated plots representing the trajectories of cells changing over time and examined the disparities in their involvement in the pathogenesis of AA compared to normal conditions. The results demonstrated that, by considering the highly variable genes, MSCs could be classified into eight distinct expression patterns or branches (Fig. 2 C). The visualization of pseudotime results demonstrates that the trajectory of MSCs undergoes a transition from State 1 to State 8 (Fig. 2 D). Subsequently, the cells were sorted into different groups, and the results indicated that states 3, 4, 5, 6, and 7 represented AA-specific differentiation or developmental stages during the differentiation or development process of MSCs (Fig. 2 E). Subsequently, we plotted the pseudo-temporal gene expression changes of marker genes specific to adipocytes. The results revealed an increase in marker gene expression in states 3, 4, 5, 6, and 7 over time (Fig. 2 F), suggesting that adipogenesis of MSCs may be a crucial factor in the development of AA. Furthermore, we employed the "CellChat" package to examine the intercellular communication between MSCs and Adipocyte cells. The findings revealed that the connection between MSCs and Adipocytes was stronger in the AA group compared to the Normal group, with a heightened interaction observed in the bone marrow tissue (Fig. 2 G-H). In conclusion, the excessive differentiation of mesenchymal stem cells (MSCs) into adipocytes may play a critical role in the development of AA. Integrating spatial transcriptomics and single-cell RNA sequencing to uncover the cellular landscape and intercellular interactions in aplastic anemia bone marrow. Before tissue dissociation, single-cell RNA sequencing (scRNA-seq) results in the loss of spatial information. Integrating spatial transcriptomics technology with scRNA-seq could compensate for the loss of spatial information caused by scRNA-seq and has wide-ranging applications in biology [ 69 ]. To accurately describe the distribution of various cell types in the bone marrow tissues of AA patients, we utilized the spatial transcriptomics (ST) method to analyze frozen sections of bone marrow tissues from the remaining three AA patients. This analysis aimed to provide an unbiased mapping of tissue transcript expression. The Seurat package is utilized for integrating ST sequencing data. Initially, we analyzed the count of genes (nFeature_Spatial), mRNA molecules (nCount_Spatial), and the proportion of mitochondrial genes (percent.mt) in all cells of the spatial transcriptomics (ST) dataset. The results indicate that most cells exhibit nFeature_Spatial values less than 10000, nCount_Spatial values less than 50000, and percent.mt values less than 20% (Fig. S3 A). The correlation analysis of sequencing depth showed that, after filtering, the correlation coefficient between nCount_Spatial and percent.mt was not applicable (r = NA). Meanwhile, the correlation coefficient between nCount_Spatial and nFeature_Spatial was 0.96, and the correlation coefficient between nCount_Spatial and percent.HB was − 0.04 (Fig. S3 B). This result suggests that the ST data is of good quality and could be utilized for further analysis. Fig. S3 C depicts the distribution of nCount_Spatial on tissue sections obtained from various organizations. The CellCycleScoring function was utilized to calculate the cellular cycle of the samples (Fig. S3 D), and subsequently, the data were normalized (Fig. S3 E). Subsequently, genes exhibiting high variance in gene expression were selected and the top 3000 genes with the highest variance were chosen for subsequent analysis (Fig. 3 A). Subsequently, we conducted principal component analysis (PCA) on the selected genes with high variance to reduce the dimensionality linearly, producing a principal component analysis plot (Fig. 3 B). Meanwhile, we used ElbowPlot to standardize the principal components and sorted them by standard deviation (Fig. 3 C). The expression heatmap of the correlated genes from principal component 1 to principal component 6 is shown here (Fig. S3 F). The results show that principal components 1 to 30 effectively capture the information from the selected high-variance genes and have analytical value. Following this, we used the TSNE algorithm to reduce non-linear dimensionality on the initial 30 principal components. A resolution of 0.4 was then chosen for cluster analysis. We inferred the enrichment of specific cell types in a given tissue region and annotated cells in the spatial transcriptomics (ST) data (Fig. 3 D) by quantifying the overlap between genes identified by scRNA-seq data and genes specific to a particular cell type and region. The distribution of mesenchymal stem cells (MSCs) and mature adipocytes can be observed in Fig. 3 E-F. We utilized the "SPOTlight" package in R to extract data concerning cell spatial interactions. Subsequently, we generated a circular graph (Fig. 3 G) to illustrate the magnitude of intercellular interactions. A heat map (Fig. 3 H) was also created to portray the correlations between cells. The results demonstrate an interaction between MSCs and Adipocytes, with a negative correlation observed between MSCs and Adipocytes. This finding further confirms the previous scRNA-seq results. In summary, the results above indicate a strong coexistence of MSCs and adipocytes in the bone marrow samples of AA patients, implying their contribution to the microenvironment of AA bone marrow. Enhanced adipogenic differentiation of bone marrow mesenchymal stem cells in aplastic anemia: insights from histological analysis and molecular characterization. To further investigate the adipogenic potential of bone marrow-derived multipotent mesenchymal stem cells (BMSCs) in AA patients and their influence on the pathogenesis of AA, Wright-Giemsa and H&E staining was performed on bone marrow tissues obtained from both AA patients and healthy individuals. The results revealed that patients with AA exhibited bone marrow hypoplasia and an elevated presence of fat globules compared to the control group (Fig. 4 A). Subsequently, we characterized the bone marrow mesenchymal stem cells (BMSCs) isolated from both healthy donors and patients diagnosed with aplastic anemia. The flow cytometry results revealed no differences in the expression of surface markers CD45, HLA-DR, CD34, CD29, CD105, and CD44 between the two groups of bone marrow stromal stem cells. Among them, bone marrow stromal cells (BMSCs) express positive surface antigens CD29, CD105, and CD44, while negative expression is observed for CD45, HLA-DR, and CD34 (Fig. 4 B). However, compared to the normal group, the number of lipid droplets in BMSCs from AA patients increased during adipocyte differentiation, indicating an enhanced ability of BMSCs to differentiate into adipocytes in AA patients (Fig. 4 C). In accordance with this finding, the mRNA and protein levels of adipogenic markers, including LPL, FABP4, PPARγ, CEBPα, and PERILIPIN, were notably increased in BMSCs from the AA group (Fig. 4 D-E). The above results provide additional confirmation of the findings obtained from ScRNA-seq and ST techniques, suggesting that there is an increase in adiposity within the bone marrow of AA patients. Moreover, the results indicate an improved capacity of BMSCs to undergo adipocyte differentiation. Irisin as a therapeutic agent: reversing aplastic anemia-induced bone marrow failure and marrow adiposity by regulating mesenchymal stem cell differentiation. Previous studies have demonstrated that Irisin has an inhibitory effect on adipocyte generation and a promoting effect on osteoblast generation during lineage-specific differentiation [ 41 , 70 ]. The buildup of fatty cells in the bone marrow can potentially hinder the processes of hematopoiesis and osteogenesis [ 71 ]. Therefore, we propose a scientific hypothesis: Irisin regulates the differentiation of BMSCs into osteoblasts and inhibits their differentiation into adipocytes, thereby improving adipogenesis. We initially established a mouse model of acquired aplastic anemia (AA) to test this hypothesis. The blood routine results of peripheral blood from mice in the Model group showed a decrease in cell concentration compared to the Control group. However, the administration of Irisin in the Model + Irisin group reversed this change, as indicated in Table 1 . The results of the hematopoietic colony formation experiment revealed a reduction in the number of hematopoietic colonies in the mice of the Model group compared to those in the Control group. However, upon Irisin treatment, the colony formation in the Model + Irisin group increased compared to the Model group (Table 2 ). It suggests that Irisin greatly improves hematopoietic disorders resulting from AA and enhances the proliferation of hematopoietic cells. Table 1 The peripheral blood counts and hemoglobin concentration of WBC, NEU, PLT, and RBC in each group of mice (mean ± standard deviation, n = 8) Group Time WBC (10 9 /L) NEU absolute value (10 9 /L) PLT (10 9 /L) RBC (10 12 /L) HGB (g/L) Control Day 5 5.74 ± 0.41 0.32 ± 0.01 449.4 ± 45.4 12.0 ± 1.0 154.2 ± 10.6 Day 10 5.57 ± 0.39 0.31 ± 0.02 431.1 ± 33 11.7 ± 1.0 156.2 ± 10.6 Day 15 5.83 ± 0.45 0.32 ± 0.02 479.3 ± 36.5 11.7 ± 1.0 156.2 ± 13.6 Model Day 5 0.87 ± 0.17* 0.02 ± 0.01* 95.5 ± 8.0* 7.1 ± 0.8* 90.2 ± 7.5* Day 10 0.81 ± 0.13* 0.03 ± 0.01* 89.6 ± 6.48* 7.2 ± 0.8* 91.2 ± 8.5* Day 15 0.80 ± 0.19* 0.03 ± 0.01* 90.1 ± 9.42* 7.2 ± 0.9* 94.3 ± 7.9* Model +Irisin Day 5 2.59 ± 0.11 △ 0.17 ± 0.01 △ 263.0 ± 27.2 △ 8.8 ± 0.7 △ 107.0 ± 8.7 △ Day 10 3.25 ± 0.35 △ 0.21 ± 0.01 △ 333.5 ± 29.4 △ 10.1 ± 0.7 △ 120.0 ± 8.7 △ Day 15 4.01 ± 0.17 △ 0.27 ± 0.02 △ 414.0 ± 31.3 △ 10.9 ± 1.1 △ 147.4 ± 9.7 △ Note: WBC, white blood cell; NEU, neutrophil; PLT, platelets; RBC, red blood cell; HGB, hemoglobin; * indicates a significant difference compared to the Control group with P < 0.05, △ indicates a significant difference compared to the Model group with P < 0.05 Table 2 The number of colony-forming units of erythroid lineage (CFU-E), granulomonocytic lineage (CFU-GM), and megakaryocytic lineage (CFU-MK) in each group of mice (mean ± standard deviation, n = 8) Group CFU-E (10 5 cells) CFU-GM (10 5 cells) CFU-MK (10 5 cells) Control 133.0 ± 14.4 92.1 ± 11.9 50.2 ± 5.9 Model 21.8 ± 2.5* 18.2 ± 1.4* 10.1 ± 2.1* Model + Irisin 92.5 ± 3.5△ 73.0 ± 5.0△ 40.0 ± 5.6△ Note: CFU, colony forming unit; E, erythroid; GM, granulocyte macrophage; MK, megakaryocytic; * indicates a significant difference compared to the Control group, P < 0.05, △ indicates a significant difference compared to the Model group, P < 0.05. The results of Wright-Giemsa staining showed a decrease in the number of nucleated cells in the sternum bone marrow of the Model group compared to the Control group. However, the administration of Irisin, in addition to the Model group, restored the number of nucleated cells (Fig. 5 A). The pathological results demonstrate a substantial disparity in developmental abnormalities between the Model and Control groups. The Control group exhibited bone marrow hyperplasia, with an average area of hematopoietic tissue of 82.33% and identifiable megakaryocytes. On the other hand, the Model group displayed bone marrow underdevelopment, reduced nuclear cells, and incomplete maturation of megakaryocytes, with an average area of hematopoietic tissue of only 38.12%. Additionally, there was an increase in fatty particles in the Model group (Fig. 5 B). In the Model + Irisin group, treatment with Irisin reverses this phenomenon (Fig. 5 B). This result indicates the successful establishment of an AA mouse model, in which Irisin improved bone marrow failure and marrow adiposity induced by AA. In order to further validate the inhibitory effect of Irisin on bone marrow adipogenesis, we conducted experiments on murine femurs using Oil Red O and Perilipin immunofluorescence staining. This result allowed us to investigate the changes in adipocytes within the murine bone marrow. The Oil Red O staining results revealed an increase in bone marrow adipocytes in the Model group of mice compared to the Control group. However, treatment of Model mice with Irisin resulted in a decrease in adipocytes, thereby confirming the inhibitory effect of Irisin on marrow adipogenesis (Fig. 5 C). The immunofluorescent staining results are consistent with it (Fig. 5 D). Subsequently, we extracted bone marrow mesenchymal stem cells (BMSCs) from the femur and tibia of mice in the Control, Model, and Model + Irisin groups to investigate alterations in their adipogenic and osteogenic differentiation capacity. Bone marrow mesenchymal stem cells (BMSCs) were identified using flow cytometry, where they showed positive expression of surface markers CD29, CD105, and CD44, and negative expression of CD45, HLA-DR, and CD34 (Fig. S4 ). Oil Red O staining revealed increased differentiation of BMSCs into adipocytes in the Model group compared to the Control group. However, in the Model + Irisin group, the differentiation of BMSCs into adipocytes was inhibited (Fig. 5 E). In contrast, the differentiation of BMSCs into osteoblasts was reduced in the Model group mice. However, treatment with Irisin effectively reversed this phenomenon, as shown in Fig. 5 F-G. Additionally, the expression of adipogenic-related genes, including LPL, FABP4, PPARγ, CEBPα, and PERILIPIN, was up-regulated in mouse BMSCs from the Model group compared to the Control group. Conversely, the expression of osteogenic-related genes, such as Runx2, ALP, OPN, and OCN, was down-regulated. However, Irisin treatment inhibited this phenomenon, as shown in Fig. 5 H-K. The results suggest that Irisin could alleviate AA mice's bone marrow failure and marrow adiposity. It is attributed to Irisin's ability to enhance the differentiation of AA mouse BMSCs into osteoblasts and inhibit their differentiation into adipocytes. Irisin's role in redirecting mesenchymal stem cell fate: a transcriptomic analysis unveiling the involvement of the hippo signaling pathway. We collected bone marrow-derived mesenchymal stem cells (BMSCs) from mice in both the Model and Model + Irisin groups to investigate how Irisin enhances adipogenesis. Through transcriptome sequencing, 935 differentially expressed genes (DEGs) were identified in the Model group of mouse BMSCs treated with Irisin. Four hundred sixty-one genes were upregulated, while 474 were downregulated (Fig. 6 A-B). Functional enrichment analysis on differentially expressed genes (DEGs) was conducted using the GO and KEGG databases. The results of the GO enrichment analysis revealed that the differentially expressed genes (DEGs) were primarily enriched in biological processes related to tissue remodeling and bone remodeling (Biological process, BP). Additionally, they were also enriched in cellular components such as secretory vesicles and transport vesicles (Cellular component, CC), as well as in molecular functions such as receptor-ligand activity and growth factor activity (Molecular function, MF) (Fig. 6 C). The KEGG enrichment analysis revealed that the differentially expressed genes (DEGs) were primarily enriched in the Hippo signaling pathway (Fig. 6 D), as illustrated in Fig. 6 E. The validation was performed using GSEA enrichment analysis (Fig. 6 F). Previous studies have demonstrated the involvement of the Hippo signaling pathway in regulating the differentiation fate of BMSCs toward osteogenic and adipogenic lineages [ 72 ]. In conclusion, it is believed that Irisin has the potential to rectify the biased tendency of BMSCs towards adipocyte differentiation through the regulation of the Hippo signaling pathway. Irisin's modulatory effect on the hippo signaling pathway: unraveling the mechanism behind its role in reducing adipogenesis and improving aplastic anemia. To further investigate the mechanism through which Irisin regulates the Hippo pathway, we initially retrieved 26 genes related to the Hippo signaling pathway (HRGs) from the Reactome database. The interconnections among these genes are shown in Fig. 7 A-B. The HRGs should intersect with the differentially expressed genes (DEGs) identified in the Model group and Model + Irisin group of mouse BMSCs. This process will ultimately allow the identification of 7 differentially expressed genes (HR-DEGs) associated with the Hippo pathway. These genes include Tead1, Wwtr1 (also known as Taz), Stk3 (also known as Mst2), Yap1 (also known as Yap), Mst1 (also known as Stk4), Lats2, and Lats1 (Fig. 7 C). Among them, Tead1, Yap1, and Wwtr1 exhibited high expression in the samples of mouse BMSCs from the Model + Irisin group, whereas Stk3, Mst1, Lats2, and Lats1 showed low expression (Fig. 7 D-E). We further conducted correlation analysis on HR-DEGs. Correlations were observed among HR-DEGs in the heat map of correlation analysis (Fig. 7 F), which aligns with the findings of the correlation scatter plot (Fig. S5 ). This result suggests that HR-DEGs play a role in regulating the Hippo signaling pathway. Subsequently, we performed functional enrichment analysis on the 7 differentially expressed genes related to human resources. The Gene Ontology (GO) analysis revealed an enrichment of differentially expressed genes (DEGs) involved in various biological processes (BP), including Hippo signaling and regulation of the canonical Wnt signaling pathway (Fig. 7 G). Furthermore, these DEGs were found to be associated with specific cellular components (CC), such as the spindle pole and RNA polymerase II transcription regulator (Fig. 7 H), as well as molecular functions (MF), such as protein serine/threonine kinase activity and magnesium ion binding (Fig. 7 I). The KEGG analysis revealed that the HR-DEGs were enriched in pathways, including the Hippo signaling pathway, as depicted in Fig. 7 J-K. The results suggest that Irisin can potentially mitigate adipogenesis in BMSCs and enhance AA by regulating the Hippo pathway through HR-DEGs. Deciphering the irisin-mediated modulation of lipotoxicity in BMSCs through the MST1/2-YAP axis: a comprehensive analysis in aplastic anemia. To further investigate the role of Irisin in improving lipotoxicity of BMSCs in AA, we utilized three different algorithms to identify potential key HR-DEGs that have the potential to mitigate BMSCs lipotoxicity. Initially, we conducted LASSO regression analysis on the 7 HR-DEGs (Fig. 8 A). Subsequently, the SVM-RFE analysis method was applied to extract feature genes (Fig. 8 B). Finally, we assessed the significance of genes using the random forest algorithm (Fig. 8 C). Finally, we identified three overlapping genes: Mst1, Stk3 (Mst2), and Yap1 (Yap) (Fig. 8 D). MST1/2 is the central kinase in the Hippo pathway, with YAP acting as the downstream effector. Upon activation of MST1/2, it facilitates the phosphorylation of YAP, causing its sequestration in the cytoplasm. Consequently, YAP gets inhibited, fostering adipocyte differentiation [ 72 , 73 ]. Thus, Irisin has the potential to enhance the impact of excessive adipogenesis in BMSCs on AA by activating the MST1/2-YAP pathway within the Hippo signaling pathway. Validation results from RT-qPCR showed downregulation of Yap1 expression in the BMSCs of mice in the Model group compared to the Control group. Conversely, the expression of Mst1 and Mst2 was upregulated in the Model group. However, the use of Irisin in the Model group effectively blocked the upregulation of Mst1 and Mst2 expression and restored the downregulated expression of Yap1 (Fig. 8 E). In line with this, BMSCs isolated from AA patients' bone marrow showed upregulated expression of Mst1 and Mst2, while the expression of Yap1 was downregulated, as compared to the Normal group (Fig. 8 F). In conclusion, the MST1/2-YAP pathway is involved in correcting the bias of differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) towards adipocytes and influencing the core pathway of adipogenesis in Irisin. It is accomplished by inhibiting the activation of MST1/2, promoting YAP's expression and nuclear translocation. Irisin's modulatory effect on BMSC differentiation: inhibiting adipogenesis and enhancing osteogenesis through the MST1/2-YAP axis. Based on the previous bioinformatics analysis results, we continued to validate the subsequent mechanistic study of BMSCs extracted from mice with AA. CCK-8 experiments revealed that Irisin concentrations ranging from 1 to 20 ng/mL had no impact on the proliferation of BMSCs. It is illustrated in Fig. 9 A. The results of the Live-Death staining also corroborated similar findings (Fig. 9 B-C). Hence, a concentration of 20 ng/mL of Irisin was selected for the subsequent experiments. The results of staining with Oil Red O, Alizarin Red S, and ALP confirmed that Irisin inhibited the differentiation of BMSCs into adipocytes and promoted their differentiation into osteoblasts, compared to the PBS group (Fig. 9 D-F). Western blot analysis revealed that Irisin treatment inhibited MST1 and MST2 expression in BMSCs during osteogenic or adipogenic differentiation induction, compared to the PBS group. Additionally, Irisin treatment led to an increase in the expression level of YAP (Fig. 9 G-H). Furthermore, Irisin decreases the expression of adipogenic genes such as LPL, FABP4, PPARγ, CEBPα, and PERILIPIN while increasing the expression levels of osteogenic genes like Runx2, ALP, OPN, and OCN (Fig. 9 I-L). Immunofluorescence staining revealed that treatment with Irisin promoted the co-localization of YAP with the nucleus in BMSCs during the induction process of osteogenic or adipogenic differentiation (Fig. 9 M-N). In conclusion, the ytg6results above demonstrate that Irisin inhibits the expression of MST1/2, increases YAP's expression, and facilitates YAP's nuclear localization. As a result, it effectively hinders the differentiation of BMSCs into adipocytes. Irisin's regulatory mechanism on BMSC adipogenesis: a comprehensive study unveiling the inhibitory role of the MST1/2-YAP signaling pathway. To further investigate whether Irisin regulates adipogenesis of BMSCs through the MST1/2-YAP pathway, we initially constructed the MST1/2 overexpression model (Mst1/2-OE). It was achieved by overexpressing the Mst1/2 genes using adenoviral vectors (Fig. 10 A). In the Irisin + Mst1/2-OE group, overexpression of Mst1/2 reversed the inhibitory effect of Irisin on the differentiation of BMSCs into adipocytes. Additionally, overexpression of Mst1/2 also blocked the upregulation of YAP expression by Irisin during the adipogenic differentiation of BMSCs compared to the Irisin + NC-OE group (Fig. 10 B-D). This finding suggests that Irisin suppresses the adipogenic differentiation of BMSCs through the MST1/2 pathway. The YAP inhibitor verteporfin (VP) consistently reversed the corrective effect of Irisin on the differentiation of BMSCs into adipocytes (Fig. 10 E-G). In contrast, the overexpression of YAP amplified the inhibitory impact of Irisin on the differentiation process of BMSCs into adipocytes (Fig. 10 H-K). Moreover, the overexpression of VP and YAP did not result in changes in the expression levels of Mst1 and Mst2 (Fig. 10 F and Fig. 10 J). This finding suggests that Irisin may potentially suppress the adipogenesis of BMSCs by interfering with the MST1/2-YAP pathway. To further validate this conclusion, we co-transfected Mst1/2-OE and Yap-OE plasmids (Fig. 10 L). The results of Oil Red O staining showed that in the Mst1/2-OE + Yap-OE group, YAP was overexpressed and inhibited the adipocyte differentiation that was promoted by Mst1/2-OE (Fig. 10 M). The western blot results indicate that the overexpression of YAP counteracted the inhibitory effect of MST1/2 overexpression on YAP expression (Fig. 10 N). Furthermore, real-time quantitative PCR (RT-qPCR) analysis demonstrated that overexpression of YAP reversed the upregulation of lipogenesis-related genes, including LPL, FABP4, PPARγ, CEBPα, and PERILIPIN induced by MST1/2 overexpression, as depicted in Fig. 10 O. In summary, the results suggest that Irisin inhibits the differentiation of bone marrow mesenchymal stem cells (BMSCs) into adipocytes by suppressing the MST1/2-YAP signaling pathway. Irisin mediates mitochondrial homeostasis and suppresses adipogenic differentiation in BMSCs through MST1/2-YAP signaling. Mitochondria play a vital role in the lineage differentiation of MSCs [ 74 , 75 ]. The inner mitochondrial membrane (IMM) plays a pivotal role in numerous functions, including the electron transport chain (ETC), oxidative phosphorylation (OXPHOS), energy transfer, and ion transport. Maintaining the homeostasis of the IMM is crucial for preserving normal mitochondrial function [ 76 , 77 , 78 ]. Studies have demonstrated that the MST1/2-YAP signaling pathway could enhance the regulation of mitochondrial function, leading to improvements in various disease states [ 79 , 80 , 81 ]. Hence, we propose that Irisin has the potential to enhance the homeostasis of the mitochondrial inner membrane system and suppress adipocyte differentiation of BMSCs via the MST1/2-YAP signaling pathway. To confirm this hypothesis, we initially examined the impact of Irisin on mitochondrial biogenesis during the process of adipogenic differentiation in BMSCs. The results from MitoTracker staining and quantification demonstrated that the Irisin group decreased the fluorescence intensity of MitoTracker compared to the PBS group (Fig. 11 A). This result suggests that Irisin plays a role in suppressing mitochondrial biogenesis during adipocyte differentiation. The Western blot results consistently demonstrated that the Irisin group suppressed the expression of PGC-1α compared to the PBS group (Fig. 11 B). Moreover, the analysis of mitochondrial DNA quantification revealed that treatment with Irisin reduced the mitochondrial DNA/nuclear DNA ratio compared to the PBS group (Fig. 11 C). This finding further confirms that Irisin hinders the process of mitochondrial biogenesis and enhances the quantity of mitochondria during adipogenesis. To investigate whether Irisin affects mitochondrial biogenesis and its impact on mitochondrial respiration, we observed the oxidative phosphorylation and glycolysis in BMSCs cells treated with Irisin under adipogenic differentiation conditions. The results of the mitochondrial oxygen consumption rate (OCR) detection during the adipogenic differentiation process of BMSCs demonstrated that Irisin treatment suppressed OCR, including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity, in comparison to the PBS group (Fig. 11 D-E). The Irisin group showed a higher extracellular acidification rate (ECAR) than the PBS group, as shown in Fig. 11 F. These findings suggest that Irisin inhibits the mitochondrial oxidative phosphorylation activity and enhances the glycolytic pathway during adipogenic differentiation of BMSCs. Furthermore, compared to PBS, the administration of Irisin increased the production of glycolytic ATP and total ATP during adipogenesis (Fig. 11 G). Next, we conducted additional investigations to examine the effects of Irisin on reactive oxygen species (ROS) and membrane potential. DCFH-DA staining and the obtained quantitative results demonstrated that treatment with Irisin inhibited the levels of reactive oxygen species (ROS) in cells during the adipogenic differentiation process (Fig. 11 H). Additionally, it was observed that Irisin treatment increased the mitochondrial membrane potential (MMP) (Fig. 11 I). These findings suggest that Irisin treatment improves mitochondrial function impairment during the adipogenic differentiation process of BMSCs. Furthermore, the mRNA expression of uncoupling proteins UCP1, UCP2, and UCP3 was observed to be downregulated in the Irisin group (Fig. 11 J), thus suggesting a decrease in the activity of proton leakage channels and a subsequent reduction in proton leakage. It resulted in the accumulation of protons on the mitochondrial inner membrane, increasing the mitochondrial membrane potential. This finding shows that while Irisin decreases mitochondrial respiration levels, it increases the potential across the inner membrane. Additionally, RT-qPCR analysis demonstrated a decrease in Irisin expression at the mRNA level, specifically in the mitochondrial inner membrane fusion protein OPA1 (Fig. 11 K). This result suggests that Irisin facilitates the fusion of mitochondrial inner membranes, thereby inhibiting the adipogenic differentiation of BMSCs. The results above indicate that Irisin inhibits mitochondrial biogenesis, the mitochondrial respiratory chain (ETC), and reactive oxygen species (ROS) levels. Additionally, it inhibits mitochondrial inner membrane fusion. Furthermore, it increases the mitochondrial inner membrane potential to enhance the homeostasis of the mitochondrial inner membrane system. As a result, it inhibits the differentiation of BMSCs into adipocytes. Irisin enhances mitochondrial inner membrane homeostasis and inhibits adipogenic differentiation in BMSCs via the MST1/2-YAP signaling pathway. To further investigate whether Irisin regulates the homeostasis of the mitochondrial inner membrane system and inhibits the differentiation of BMSCs into adipocytes through the MST1/2-YAP signaling pathway, we detected the mitochondrial OCR during BMSCs' adipogenic differentiation. It was observed that Mst1/2-OE and the YAP inhibitor VP both blocked the Irisin-mediated inhibition of OCR during the induction of adipogenesis (Fig. 12 A) and reversed the upregulation of ECAR, glycolytic ATP, and total ATP production induced by Irisin (Fig. 12 B-C) in comparison to the Irisin + DMSO + NC-OE group. Furthermore, the overexpression of Mst1/2 and VP antagonized the inhibitory effect of Irisin on intracellular ROS levels and the beneficial effect on mitochondrial membrane potential during adipogenesis (Fig. 12 D-E). Additionally, Irisin treatment restored the suppressed proton leak channel activity and fusion of the mitochondrial inner membrane (Fig. 12 F). It suggests that Irisin may enhance the homeostasis of the mitochondrial inner membrane system via the involvement of MST1/2 and YAP. We then proceeded to validate whether Irisin regulates the homeostasis of the mitochondrial inner membrane through the MST1/2-YAP pathway. The results of the OCR analysis demonstrated that Yap overexpression in the Mst1/2-OE + Yap-OE group effectively inhibited the increase in OCR caused by Mst1/2 overexpression. Yap overexpression also led to a decrease in ECAR, glycolytic ATP, and total ATP production (Fig. 12 G-I). Simultaneously, Yap overexpression counteracted the increase in intracellular ROS levels and the decrease in mitochondrial membrane potential observed during adipogenesis (Fig. 12 J-K). The alteration in membrane potential is linked to the recovery of increased proton leak channel activity resulting from Yap overexpression in Mst1/2-OE treated cells (Fig. 12 L). Furthermore, the overexpression of Yap in the Mst1/2-OE + Yap-OE group blocked the promoting effect of Mst1/2 overexpression on mitochondrial inner membrane fusion during adipogenic induction, as observed in the Mst1/2-OE group (Fig. 12 L). Furthermore, this finding provides additional evidence of the impact of Yap overexpression on mitochondrial homeostasis disruption. It supports the hypothesis that Irisin enhances the stability of the mitochondrial inner membrane system via the MST1/2-YAP pathway. Furthermore, we investigated whether Irisin regulates the homeostasis of the mitochondrial inner membrane system to inhibit the differentiation of BMSCs into adipocytes via the MST1/2-YAP signaling pathway. We treated BMSCs with the mitochondrial respiratory chain inhibitor Rotenone to achieve this. Compared to the DMSO group, treatment with the YAP inhibitor VP enhanced mitochondrial oxidative phosphorylation activity during adipogenesis, inhibition of glycolysis, and reduction in both glycolytic and total ATP production. However, the effects of VP were attenuated by the addition of fisetin, as demonstrated in Fig. S6 A-C. In contrast, YuTeng ketone mitigated the heightened VP levels during lipogenesis through the reduction of cellular ROS levels and mitochondrial membrane potential (Fig. S6 D-E). The results obtained from RT-qPCR showed an upregulation of mRNA expression levels of coupling proteins UCP1, UCP2, UCP3, and mitochondrial inner membrane fusion protein OPA1 in the group treated with VP compared to the DMSO group. However, the group treated with VP + Rotenone showed a restoration of this change when Rotenone was administered (Fig. S6 F). Furthermore, using fish triterpenes has counteracted the promoting effect of VP on the differentiation of BMSCs into adipocytes (Fig. S6 G-H). The results above suggest that Irisin enhances the homeostasis of the mitochondrial inner membrane system by inhibiting the MST1/2-YAP signaling pathway, consequently inhibiting the differentiation of BMSCs into adipocytes. Irisin ameliorates adipogenic differentiation and mitochondrial dysfunction in BMSCs through MST1/2-YAP pathway in an AA mouse model. Our previous research has confirmed that Irisin plays a role in inhibiting the differentiation of BMSCs into adipocytes, leading to improvements in adiposity. To further investigate the inhibitory effect of Irisin on the differentiation of mice BMSCs into adipocytes, we intravenously injected Mst1/2-OE, which was constructed in vitro , into mice to explore the progression of adipogenesis. The blood cell counts in the peripheral blood of mice were analyzed. The Model + NC-OE group exhibited a decrease in blood cell counts compared to the Control + NC-OE group. Conversely, the Model + Irisin + NC-OE group showed a notable increase in blood cell counts compared to the Model + NC-OE group. However, the overexpression of Mst1/2 diminished the beneficial effect of Irisin on blood cell counts according to Table 3 . The Model + Irisin group exhibited a notable increase in hematopoietic colony formation compared to the Model group, as supported by the experimental findings. Nevertheless, the overexpression of Mst1/2 counteracted the hematopoietic cell proliferation enhancement induced by Irisin (Table 4 ). This result suggests that the overexpression of Mst1/2 undermines the hematopoietic improvement facilitated by Irisin in AA. Table 3 Peripheral blood WBC, NEU, PLT, RBC counts, and HGB concentration (mean ± standard deviation, n = 8) in each group of mice Group Time WBC (10 9 /L) NEU absolute value (10 9 /L) PLT (10 9 /L) RBC (10 12 /L) HGB (g/L) Control + NC-OE Day 5 5.74 ± 0.07 0.81 ± 0.01 449.4 ± 45.4 12.0 ± 0.3 154.2 ± 1.6 Day 10 5.57 ± 0.09 0.81 ± 0.02 431.1 ± 33 11.7 ± 0.3 156.2 ± 1.6 Day 15 5.83 ± 0.05 0.83 ± 0.02 479.3 ± 36.5 11.7 ± 0.3 156.2 ± 3.6 Model + NC-OE Day 5 0.87 ± 0.07* 0.22 ± 0.01* 95.5 ± 3.0* 7.1 ± 0.1* 90.2 ± 0.5* Day 10 0.81 ± 0.03* 0.23 ± 0.01* 89.6 ± 2.48* 7.2 ± 0.1* 91.2 ± 0.5* Day 15 0.80 ± 0.09* 0.23 ± 0.01* 90.1 ± 3.42* 7.2 ± 0.1* 94.3 ± 0.9* Model +Irisin + NC-OE Day 5 2.59 ± 0.11 △ 0.65 ± 0.01 △ 263.0 ± 27.2 △ 8.8 ± 0.2 △ 107.0 ± 0.7 △ Day 10 3.25 ± 0.15 △ 0.71 ± 0.01 △ 333.5 ± 29.4 △ 10.1 ± 0.2 △ 120.0 ± 0.8 △ Day 15 4.01 ± 0.17 △ 0.77 ± 0.02 △ 414.0 ± 31.3 △ 10.9 ± 0.1 △ 147.4 ± 0.7 △ Model + Mst1/2-OE Day 5 0.61 ± 0.02 △ 0.11 ± 0.01 △ 49.5 ± 1.2 △ 6.2 ± 0.1 △ 80.1 ± 0.7 △ Day 10 0.41 ± 0.03 △ 0.08 ± 0.01 △ 45.4 ± 1.4 △ 6.0 ± 0.1 △ 78.5 ± 0.7 △ Day 15 0.32 ± 0.02 △ 0.06 ± 0.02 △ 40.6 ± 1.3 △ 5.7 ± 0.1 △ 77.9 ± 0.7 △ Model +Irisin + Mst1/2-OE Day 5 1.17 ± 0.02 ▲ 0.30 ± 0.02 ▲ 116.9 ± 1.2 ▲ 7.3 ± 0.1 ▲ 92.2 ± 0.6 ▲ Day 10 1.25 ± 0.02 ▲ 0.33 ± 0.02 ▲ 123.8 ± 1.2 ▲ 7.5 ± 0.1 ▲ 94.3 ± 0.6 ▲ Day 15 1.36 ± 0.02 ▲ 0.35 ± 0.02 ▲ 130.3 ± 1.2 ▲ 7.7 ± 0.1 ▲ 98.1 ± 0.5 ▲ Note: WBC, white blood cell; NEU, neutrophil; PLT, platelets; RBC, red blood cell; HGB, hemoglobin; * indicates a significant difference compared to the Control + NC-OE group with a P value < 0.05, △ indicates a significant difference compared to the Model + NC-OE group with a P value < 0.05, ▲ indicates a significant difference compared to the Model + Irisin + NC-OE group with a P value < 0.05. Table 4 The number of CFU-E, CFU-GM, and CFU-MK colonies in each group of mice (mean ± standard deviation, n = 8). Group CFU-E (10 5 cells) CFU-GM (10 5 cells) CFU-MK (10 5 cells) Control + NC-OE 133.0 ± 1.4 92.1 ± 1.9 50.2 ± 1.9 Model + NC-OE 21.8 ± 1.0* 18.2 ± 1.4* 10.1 ± 2.1* Model + Irisin + NC-OE 92.5 ± 1.5 △ 73.0 ± 1.5 △ 40.0 ± 1.6 △ Model + Mst1/2-OE 6.5 ± 0.5 △ 5.5 ± 0.5 △ 2.1 ± 0.5 △ Model + Irisin + NC-OE 27.3 ± 1.2 ▲ 25.4 ± 1.2 ▲ 14.9 ± 1.2 ▲ Note: CFU stands for colony forming unit; E represents erythroid; GM represents granulocyte macrophage; MK represents megakaryocytic; * indicates a significant difference compared to the Control + NC-OE group with a p-value < 0.05, △ indicates a significant difference compared to the Model + NC-OE group with a p-value < 0.05, ▲ indicates a significant difference compared to the Model + Irisin + NC-OE group with a p-value < 0.05. Additional HE staining showed that the overexpression of Mst1/2 in the Model + Irisin + Mst1/2-OE group blocked the rescuing effect of Irisin on bone marrow hypoplasia compared to the Model + Irisin + NC-OE group. This result also reduced nucleated cell count, impaired megakaryocyte development, increased lipid droplets, and decreased hematopoietic tissue area in Model mice (Fig. 13 A). These findings suggest that the overexpression of Mst1/2 undermines the positive effects of Irisin on bone marrow failure and excessive marrow adiposity induced by AA. To further confirm the inhibitory effect of Irisin on adipogenesis in the bone marrow of the mouse model through overexpression of Mst1/2, we performed experiments on the femurs of mice. We utilized Oil Red O and Perilipin immunofluorescence staining to examine alterations in adipocytes within the bone marrow. The Oil red O staining results indicated an increase in the number of adipocytes in the mouse bone marrow of the Model + Irisin + Mst1/2-OE group compared to the Model + Irisin + NC-OE group (Fig. 13 B). Immunofluorescence staining results supported these findings, suggesting that the overexpression of Mst1/2 in the Model + Irisin + Mst1/2-OE group inhibited the suppressive effect of Irisin on adipocytes in the mouse bone marrow (Fig. 13 C). The expression of lipogenic-related genes in mouse bone marrow was determined using RT-qPCR. The findings indicated that Mst1/2 overexpression mitigated the downregulation of LPL, FABP4, PPARγ, CEBPα, and PERILIPIN gene expression by Irisin (Fig. 13 D). These results suggest that the overexpression of Mst1/2 counteracted Irisin's inhibitory effect on bone marrow failure and marrow adipogenesis in the mouse model. Additionally, Western blot analysis of bone marrow samples showed that the Model + Irisin + NC-OE group exhibited a reversal in the upregulation of MST1 and MST2, as well as YAP downregulation, which was induced by AA treatment, compared to the Model + NC-OE group (Fig. 13 E). Nevertheless, the upregulation of YAP expression induced by Irisin was reversed upon overexpression of Mst1/2 (Fig. 13 F). This finding suggests that Irisin activation occurs through the inhibition of the MST1/2-YAP pathway, suppressing the differentiation of mouse BMSCs into adipocytes and improving adipose tissue function. To further validate the improvement of mitochondrial inner membrane homeostasis by Irisin through the MST1/2-YAP pathway, we inhibited the differentiation of BMSCs into adipocytes and evaluated its impact on acidosis (AA) in a mouse model. Subsequently, BMSCs were isolated from the bone marrow of mice in each group. The detection of total ATP and ROS levels exhibited the following results: compared to the Control + NC-OE group, the Model + NC-OE group showed a decrease in ATP levels and an increase in ROS levels. These findings suggest that AA induces damage to the respiratory chain and energy conversion function of BMSCs' mitochondria and impairs the homeostasis of the mitochondrial inner membrane. Compared to the Model + NC-OE group, the Model + Irisin + NC-OE group showed a rescue in the decrease of ATP levels and an increase in ROS levels caused by AA in BMSCs after Irisin treatment. It improved the damage to the mitochondrial respiratory chain and energy conversion function in the BMSCs of model mice. Nevertheless, the rescuing effect of Irisin on mitochondrial inner membrane homeostasis was hindered by the overexpression of Mst1/2, as shown in Fig. 14 A-B. Furthermore, treatment with Irisin ameliorated the reduced mitochondrial membrane potential in BMSCs induced by AA. However, when comparing the Model + Irisin + NC-OE group to the Model + Irisin + Mst1/2-OE group, overexpression of Mst1/2 in the latter group counteracted the favorable effects of Irisin on the mitochondrial membrane potential in the experimental mice (Fig. 14 C). Irisin treatment restores the upregulation of mRNA expressions of uncoupling proteins UCP1, UCP2, and UCP3, which AA induces. This restoration inhibits the increased activity of mitochondrial inner membrane proton leak channels caused by AA. On the other hand, the beneficial effect was hindered by the overexpression of Mst1/2 in the Model + Irisin + Mst1/2-OE group (Fig. 14 D). Furthermore, Irisin treatment in BMSCs decreased the mRNA expression of OPA1, a protein involved in mitochondrial inner membrane fusion, compared to the Model + NC-OE group. This result suggests that Irisin can potentially enhance AA-induced mitochondrial inner membrane fusion. However, based on this premise, the expression of Mst1/2 reversed this alteration (Fig. 14 D). The results mentioned above confirm that Irisin improves damage to the homeostasis of the mitochondrial membrane system in model mice BMSCs by inhibiting the activation of the MST1/2-YAP pathway. In summary, Irisin enhances the homeostasis of the mitochondrial inner membrane system by inhibiting the activation of the MST1/2-YAP pathway. It also prevents the differentiation of BMSCs into adipocytes and relieves AA-induced bone marrow failure and marrow adiposity, ultimately mitigating acidosis. Discussion Aplastic anemia (AA) is a condition marked by the dysfunction of hematopoiesis, resulting in the abnormal production of red blood cells, white blood cells, and platelets in the bone marrow [ 2 , 82 , 83 ]. Previous studies have shown that patients with AA undergo adipogenesis in their bone marrow, where hematopoietic cells are replaced by adipocytes [ 84 ]. This study further elucidated that lipidization results from the excessive differentiation of mesenchymal stem cells (MSCs) into adipocytes. Previous studies have identified irisin as a hormone linked to fat and metabolism [ 85 ]. This study revealed that Irisin is involved in fat metabolism and influences the differentiation direction of MSCs. Irisin could delay the differentiation of MSCs into adipocytes by inhibiting the activation of the MST1/2-YAP signaling pathway. This slowdown or mitigation of the progression of AA occurs as a result. The MST1/2-YAP signaling pathway regulates cell proliferation, apoptosis, and differentiation [ 86 , 87 , 88 ]. This study confirms that the signaling pathway also has a regulatory role in differentiating MSCs into adipocytes. In comparison to previous studies, we have provided additional clarification regarding the involvement of Irisin in this pathway, subsequently influencing the differentiation of MSCs. Mitochondria play a vital role in cellular energy production and regulation. This study indicates, for the first time, that the differentiation of MSCs could be influenced by modulating the homeostasis of the mitochondrial inner membrane system. Compared to previous studies focused on mitochondrial function and cell fate decisions, this study offers a fresh and distinct understanding of the mitochondrial function of MSCs. This study employed two advanced techniques, single-cell sequencing and spatial transcriptomics, enabling us to examine the differentiation characteristics of MSCs at the single-cell level and accurately determine their specific location within the bone marrow tissue. Compared to previous research methods that rely on group averages, these two techniques offer more extensive and precise information [ 89 ]. Considering the role of Irisin in inhibiting MSC differentiation into adipocytes and improving mitochondrial inner membrane system homeostasis, it can potentially become a promising therapeutic target for AA. However, additional clinical research is required to ascertain Irisin's precise mechanisms, dosage, treatment timing, and potential side effects. Additionally, future studies could investigate additional potential factors that regulate MSC differentiation. Based on the results above, the following preliminary conclusions could be drawn: Irisin can increase the expression of Yap and facilitate its nuclear translocation by suppressing the expression of Mst1/2. This action restores the stability of the mitochondrial inner membrane system in adriamycin-damaged mesenchymal stem cells, preventing their differentiation into adipocytes and subsequently ameliorating adriamycin-induced nephropathy (Fig. 15). This study revealed the therapeutic mechanism of Irisin in the treatment of AA, offering novel insights into the pathogenesis of this condition. Unfortunately, because of time and budget limitations, we could not validate the mechanism using clinical samples. It undermined the reliability and scientific validity of our conclusions. Henceforth, we will persist in exploring clinical mechanisms. Bone marrow fatty infiltration is a prominent characteristic of acquired aplastic anemia (AA) [ 90 ]. This study uncovered how the excessive differentiation of mesenchymal stem cells (MSCs) into adipocytes contributes to AA, offering novel insights into the mechanistic understanding of the disease. Previous research has demonstrated that Irisin can enhance the homeostasis of the mitochondrial inner membrane system by inhibiting the activation of the Mst1/2-YAP signaling pathway. Consequently, this inhibition leads to the suppression of mesenchymal stem cell differentiation into adipocytes, offering a promising new approach for treating AA. Advanced techniques, such as single-cell sequencing, spatial transcriptomics, and transcriptome sequencing, have been utilized to provide methodological references for future studies. The intervention of Irisin could reverse adipogenesis in AA bone marrow, thereby restoring hematopoietic function and providing practical clinical benefits to patients. This study primarily focused on AA model mice. However, it is important to consider that the physiological differences between humans and mice may impact the translation of research findings to clinical settings. While this study primarily investigates the MST1/2-YAP signaling pathway, it is important to note that other intricate signaling pathways may also regulate the differentiation of mesenchymal stem cells. The study utilized Irisin as a treatment; however, the potential side effects and long-term safety in the human body have yet to be definitively established. Additional clinical trials are required to assess the effectiveness of Irisin in the treatment of AA and to evaluate its safety and efficacy in humans. This study could offer guidance in finding potential drugs capable of regulating the MST1/2-YAP signaling pathway. Given the strong correlation between the mechanisms addressed in this study and the hematopoietic function of the bone marrow, additional research should be undertaken to examine their potential therapeutic applications in other hematopoietic disorders. This study aims to investigate the precise regulation of the MST1/2-YAP signaling pathway by Irisin and contribute to a more solid theoretical foundation for its clinical application. This research has profound implications as it introduces new directions and methods for thalassemia treatment. However, further clinical studies are necessary to validate its effectiveness and safety in humans. Declarations Data availability The data that supports the findings of this study are available on request from the corresponding author. Acknowledgment Not applicable. Ethical Statement This study was approved by our Clinical Ethics Committee and complied with the Declaration of Helsinki (KYLL-2019-023). This experimental program and animal use protocol were approved by our institutional animal ethics committee (NO. 202309001). Authors contributions XL and HL designed the study. BG collated the data, designed and developed the database, carried out data analyses and produced the initial draft of the manuscript. DK contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript. Competing interests The authors declare that they have no competing interests. Funding This study was supported by SDU-KI Collaborative Research Project of Qilu Medical College of Shandong University (Project No. SDU-KI-2019-15) and Natural Science Foundation of Shandong Province (Project No. ZR2023MH341). Consent for publication Consent for publication was obtained from the participants. References Moore CA, Krishnan K. Aplastic Anemia. In: StatPearls. Treasure Island (FL): StatPearls Publishing; July 17, 2023. DeZern AE, Churpek JE. Approach to the diagnosis of aplastic anemia. Blood Adv. 2021;5(12):2660–71. 10.1182/bloodadvances.2021004345 . Wang L, Liu H. Pathogenesis of aplastic anemia. Hematology. 2019;24(1):559–66. 10.1080/16078454.2019.1642548 . Javan MR, Saki N, Moghimian-Boroujeni B. Aplastic anemia, cellular and molecular aspects. Cell Biol Int. 2021;45(12):2395–402. 10.1002/cbin.11689 . Li CJ, Xiao Y, Sun YC et al. Senescent immune cells release grancalcin to promote skeletal aging [published correction appears in Cell Metab. 2022;34(1):184–185]. 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Supplementary Files FigureS1.pdf Fig. S1 Quality Control, Filtering, and Principal Component Analysis of the scRNA-seq Data. Note: (A) Violin plots showing the number of genes (nFeature_RNA), mRNA molecules (nCount_RNA), and the percentage of mitochondrial genes (percent.mt) in each cell of the scRNA-seq data from normal donors (n=2) and AA patients (n=2). (B) Scatter plots showing the correlation between filtered data nCount_RNA and percent.mt, as well as nCount_RNA and nFeature_RNA. (C) Analysis of variance to select highly variable genes in the samples (red dots represent highly variable genes, black dots represent invariant genes). (D) Cell cycle status of each cell in the scRNA-seq data, with S.Score representing the S phase and G2M.Score representing the G2M phase. (E) p-values of the top 50 PCs obtained from PCA analysis. (F) Distribution of standard deviations of PCs, where important PCs have larger standard deviations. (G) Heatmap showing PCA's top 15 major correlated gene expressions for PC_1 - PC_6. (H) Heatmap showing the expression levels of the top 15 major correlated genes for PC_1 - PC_6 in PCA, where yellow represents upregulation and purple represents downregulation. (I) Distribution of cell distributions in PC_1 and PC_2 before batch correction, with each point representing a cell and different colors representing different samples. AA: aplastic anemia. FigureS2.pdf Fig. S2 T-SNE Clustering Tree of scRNA-seq data. Note: Branches represent the clustering relationship between cells, while dots represent cell clusters, with the size of the dots typically indicating cell abundance. FigureS3.pdf Fig. S3 Quality Control and PCA Dimensionality Reduction of ST data. Note: (A) Violin plots depicting the number of genes (nFeature_Spatial), mRNA molecule count (nCount_Spatial), and percentage of mitochondrial genes (percent.mt) in each cell of the scRNA-seq data (n=3); (B) Scatter plots showing the correlation between nCount_Spatial and percent.mt, nCount_Spatial and nFeature_Spatial, and nCount_Spatial and percent.HB in the ST data (n=3); (C) Distribution of nCount_Spatial in tissue sections of the ST data (n=3), with redder color indicating higher expression levels of nCount_Spatial in that spot; (D) Cell cycle status of each cell in the ST data, where S.Score represents the S phase and G2M.Score represents the G2M phase (n=3); (E) Visualization of the SCTransform and LogNormalize normalized results in the ST data (n=3); (F) Heatmap of the top 15 correlated genes in PC_1 - PC_6 in the PCA, where yellow represents upregulation and purple represents downregulation (n=3). FigureS4.pdf Fig. S4 Identification results of surface markers on BMSCs isolated from different mouse groups using flow cytometry. FigureS5.pdf Fig. S5 Scatter plot depicting the correlation analysis of HR-DEGs. FigureS6.pdf Fig. S6 Irisin affects the homeostatic regulation of the mitochondrial inner membrane in BMSCs via the MST1/2-YAP signaling pathway. Note: (A) Oxygen consumption rates of various groups on the 7th day of adipogenic differentiation induction; (B) Extracellular acidification rate (ECAR) of various groups; (C) Rates of ATP production from mitochondrial respiration (mito-ATP) and glycolytic respiration (glyco-ATP), solid line represents the ratio of total ATP production rates between the two groups, dashed line represents the ratio of glycolytic ATP production rates between the two groups; (D) DCFH-DA staining images (scale bar = 100 μm) of BMSCs from each group and quantification of fluorescent intensity representing ROS generation levels; (E) JC-1 staining and MMP detection results using flow cytometry, along with quantitative assessment of MMP; (F) Expression levels of uncoupling proteins UCP1, UCP2, UCP3, and mitochondrial inner membrane fusion protein genes detected via RT-qPCR; (G) Oil Red O staining images (scale bar = 100 μm) and quantification of absorbance at 510 nm as a representative result; (H) Expression levels of adipogenic differentiation-related genes in each group detected via RT-qPCR; VP: verteporfin, PLIN: PERILIPIN; * indicates a difference between the two groups with a p-value < 0.05, and all experiments were repeated at least three times. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4329016","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296026666,"identity":"760d20be-5e60-496b-8e24-b2f9cb8e33a0","order_by":0,"name":"Xia Liu","email":"","orcid":"","institution":"Department of Respiratory Intervention, Children’s Hospital Affiliated to Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Liu","suffix":""},{"id":296026667,"identity":"7ab592c7-22da-4df1-b876-209a605e1042","order_by":1,"name":"Hui Li","email":"","orcid":"","institution":"Department of Hematology, The Second Hospital, Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Li","suffix":""},{"id":296026670,"identity":"98f2e868-7ef0-4e6b-945e-a7170c9b1704","order_by":2,"name":"Bingxin Guan","email":"data:image/png;base64,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","orcid":"","institution":"Department of Pathology, the Second Hospital, Cheeloo College of Medicine, Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Bingxin","middleName":"","lastName":"Guan","suffix":""},{"id":296026673,"identity":"76fe4059-fa16-4e40-ae83-204018d7d3d4","order_by":3,"name":"Dexiao Kong","email":"","orcid":"","institution":"Department of Hematology, The Second Hospital, Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Dexiao","middleName":"","lastName":"Kong","suffix":""}],"badges":[],"createdAt":"2024-04-26 10:46:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4329016/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4329016/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55701729,"identity":"5a906da2-fd4d-4292-a417-7df7ec6b87d5","added_by":"auto","created_at":"2024-05-02 03:32:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":401019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell clustering and annotation of scRNA-seq data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Batch correction process of Harmony, with the number of iterations on the x-axis; (B) Distribution of cells after batch correction in PC_1 and PC_2, where each point represents a cell and different colors represent different samples; (C) tSNE visualization of the clustering results, showing the aggregation and distribution of normal donors (n=2) and AA patients (n=2) bone marrow cells, with red representing normal donor bone marrow samples and blue representing AA patient bone marrow samples; (D) tSNE visualization of the clustering results, showing the aggregation and distribution of cells from different sources (n=2), with each color representing a cluster; (E) Expression of known cell lineage-specific marker genes in different clusters, with deeper red indicating higher average expression levels and larger circles indicating a higher number of cells expressing the gene; (F) Annotation results of normal group (n=2) and AA group (n=2) cells based on tSNE clustering, with each color representing a cell subset; AA: aplastic anemia.\u003c/p\u003e","description":"","filename":"Figure117.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/6450f54a8b036ef899802f62.png"},{"id":55701731,"identity":"35e3e949-ebef-43ec-a5b9-06a34146a6bb","added_by":"auto","created_at":"2024-05-02 03:32:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":493737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePseudotime analysis and cell communication analysis of scRNA-seq data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Expression of MSCs and Adipocytes marker genes in different cell subsets of normal donor bone marrow tissue (n=2) and AA patient bone marrow tissue (n=2), with deeper purple indicating higher average expression levels; (B) Proportions of different cell subsets in different sample groups (n=2), with different colors representing different subsets; (C) Cell trajectory differentiation plot of MSCs, with different colors representing different states; (D) Cell trajectory plot visualized in pseudotime, with darker colors indicating earlier time points; (E) Cell trajectory plot visualized for different sample groups (n=2), with different colors representing different groups; (F) Expression-temporal curve of the Adipocytes marker gene ADIPOR1 in pseudotime, with time on the x-axis and gene expression levels on the y-axis; (G) Interactions among cells in the Normal group samples (n=2), with line thickness representing the strength of interaction; (H) Interactions among cells in the AA group samples (n=2), with line thickness representing the strength of interaction.\u003c/p\u003e","description":"","filename":"Figure214.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/057132da7000430865d4036d.png"},{"id":55700885,"identity":"7fb7f692-984b-4574-8366-61041b7dd883","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2444449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis results combining scRNA-seq and ST.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Variance analysis of highly variable genes, with red representing the top 3000 highly variable genes and black representing low variable genes, with the top 10 gene names (n=3); (B) PCA analysis showing the distribution of cells in PC_1 and PC_2, with each point representing a cell (n=3); (C) Distribution of standard deviations of principal components (PCs), with important PCs having larger standard deviations (n=3); (D) Distribution of different cell types in AA patient bone marrow tissue (n=3) on ST data, with pie charts representing the proportions of different cell distributions in each spot; (E) Distribution of MSCs in AA patient bone marrow tissue (n=3) on ST data, with darker red indicating a higher percentage of MSCs in each spot; (F) Distribution of Adipocytes in AA patient bone marrow tissue (n=3) on ST data, with darker red indicating a higher percentage of Adipocytes in each spot; (G) Circle plot showing the strength of interactions between different cells based on ST data, with thicker lines representing stronger interactions (n=3); (H) Heatmap showing the cell correlation on the ST data, with the values representing correlation coefficients between cells (n=3).\u003c/p\u003e","description":"","filename":"Figure39.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/a3ce55d5c4fcc2922fcd2dfd.png"},{"id":55701730,"identity":"a83602b1-5f79-40f5-bc47-50a0d852bfec","added_by":"auto","created_at":"2024-05-02 03:32:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2541853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdipogenic differentiation of BMSCs in AA patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Representative images of Wright-Giemsa staining (left) and H\u0026amp;E staining (right) of the normal donor (n=3) and AA patient (n=3) bone marrow tissue, scale bar=100 μm; (B) Flow cytometry analysis of the percentages of BMSCs expressing surface markers (CD45, HLA-DR, CD34, CD29, CD105, and CD44) in different groups; (C) Oil red staining for adipogenic differentiation of BMSCs in different groups (n=8), with scale bar=200 μm, and quantification of Oil Red O staining absorbance at 510nm; (D) RT-qPCR analysis of the expression of adipogenic differentiation-related mRNAs in BMSCs in different groups (n=8); (E) Western blot analysis of the expression of adipogenic differentiation-related proteins in BMSCs in different groups (n=8), and quantification of the results; PLIN: PERILIPIN; * indicates differences between groups with p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure42.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/7f421892bbffa523d0f906fd.jpg"},{"id":55700896,"identity":"674b2d2a-84ba-48c4-8d14-785abb78f599","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5035178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Irisin on bone marrow phenotype and osteogenic and adipogenic differentiation capacity of BMSCs in AA mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Wright-Giemsa staining of bone marrow tissue from different groups of mice (n=8), scale bar=100 μm; (B) H\u0026amp;E staining of bone marrow tissue from different groups of mice (n=8), representative images with scale bar=100 μm, and quantification of the area percentage occupied by hematopoietic tissue in the total marrow cavity; (C) Oil red O staining of femur from different groups of mice (n=8), representative images with scale bar=100 μm, and quantification of the area percentage occupied by adipocytes; (D) Immunofluorescence staining of Perilipin in femur from different groups of mice (n=8), representative images with scale bar=200 μm, and quantification of fluorescence intensity; (E) Oil red O staining for adipogenic differentiation of BMSCs in different groups of mice (n=8), representative images with scale bar=100 μm, and quantification of Oil Red O staining absorbance at 510 nm; (F) Alizarin Red S staining of osteogenic differentiation of BMSCs in different groups of mice (n=8), representative images with scale bar=100 µm, and quantification of Alizarin Red S staining absorbance at 562 nm; (G) Alkaline phosphatase (ALP) staining of BMSCs in different groups of mice (n=8), representative images with scale bar=200 µm, and quantification of ALP activity; (H) Western blot analysis of the expression of adipogenic differentiation-related proteins in BMSCs of different groups of mice (n=8), and quantification of the results; (I) RT-qPCR analysis of the expression of adipogenic differentiation-related mRNAs in BMSCs of different groups of mice (n=8); (J) Western blot analysis of the expression of osteogenic differentiation-related proteins in BMSCs of different groups of mice (n=8), and quantification of the results; (K) RT-qPCR analysis of the expression of osteogenic differentiation-related mRNAs in BMSCs of different groups of mice (n=8); PLIN: PERILIPIN; * indicates differences between groups with p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure52.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/4106b263c0437ac5ea8ecb8b.jpg"},{"id":55700898,"identity":"1fab5498-3aac-42bc-a32a-1b8311bf2740","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":232445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiological information analysis of DEGs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Volcano plot of the transcriptome sequencing results of BMSCs samples from the Model group (n=6) and the Model+Irisin group (n=6); (B) Heatmap of DEGs between BMSCs samples from the Model group (n=6) and the Model+Irisin group (n=6); (C) Bubble plot of GO enrichment analysis of DEGs; (D) Bubble plot of KEGG enrichment analysis of DEGs; (E) Hippo signaling pathway diagram from KEGG enrichment analysis; (F) GSEA enrichment analysis validates the enrichment of the Hippo signaling pathway.\u003c/p\u003e","description":"","filename":"Figure64.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/322f7544a4c7a71f3e317f85.png"},{"id":55700899,"identity":"dc881f22-05f0-40f2-9d58-54f27478eec0","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":579151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelection and enrichment analysis of HR-DEGs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Protein-protein interaction network of 26 HRGs in mice; (B) Optimized Cytoscape diagram of protein-protein interactions of HRGs, where node size and color are proportional to degree values; (C) Venn diagram showing the intersection of HR-DEGs obtained from the DEGs and HRGs; (D) Volcano plot of expression fold changes of HR-DEGs in BMSCs samples from the Model group (n=6) and the Model+Irisin group (n=6); (E) Heatmap of expression of HR-DEGs in BMSCs samples from the Model group (n=6) and the Model+Irisin group (n=6); (F) Heatmap of correlation analysis of HR-DEGs, with Pearson's method used for evaluation; (G) Bubble chart of GO enrichment analysis of HR-DEGs in Biological process (BP); (H) Bubble chart of GO enrichment analysis of HR-DEGs in Cellular component (CC); (I) Bubble chart of GO enrichment analysis of HR-DEGs in Molecular function (MF); (J) Bar plot of KEGG enrichment analysis of HR-DEGs; (K) Chord diagram of KEGG enrichment analysis of HR-DEGs and gene participation; DEGs: differentially expressed genes, HRGs: Hippo signaling pathway related genes; * indicates difference between two groups (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure74.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/63e1f2b52e41ada809d0bd5c.png"},{"id":55700892,"identity":"2b503148-e704-4435-b121-d4b0c65b8966","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":152698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelection and expression verification of core HR-DEGs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Lasso coefficient selection plot of HR-DEGs; (B) SVM-RFE analysis plot of HR-DEGs; (C) Random forest analysis results plot of HR-DEGs; (D) Venn diagram showing the intersection of HR-DEGs selected by lasso regression, SVM-RFE, and random forest algorithms to improve the adipogenic features of BMSCs; (E) RT-qPCR validation of the expression of Mst1, Mst2, and Yap1 in BMSCs from the Control group, Model group, and Model+Irisin group (n=3); (F) RT-qPCR detection of the expression of Mst1, Mst2, and Yap1 in BMSCs isolated from normal donors and AA patients (n=3); * indicates difference between two groups (P\u0026lt;0.05), RT-qPCR experiments repeated 3 times.\u003c/p\u003e","description":"","filename":"Figure83.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/573ed145a8ad0f744c7a52d4.png"},{"id":55700897,"identity":"df31d80f-e49e-4feb-a199-4ef6d343122a","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1660532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Irisin on osteogenic and adipogenic fate differentiation of BMSCs through MST1, MST2, and YAP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Cell viability detected by CCK-8 assay after treatment with different concentrations of Irisin for 1, 3, and 5 days; (B) Live-Dead staining results after treatment with different concentrations of Irisin for 3 days, scale bar = 200 μm; (C) Quantification of dead cells/live cells ratio by Live-Dead staining; (D) Representative images of Oil Red O staining (scale bar = 100 μm) and quantification of absorbance at 510 nm; (E) Representative images of Sudan Red staining (scale bar = 100 μm) and quantification of absorbance at 562 nm; (F) Representative images of ALP staining (scale bar = 200 μm) and quantification of ALP activity; (G) Western blot analysis and quantification of MST1, MST2, and YAP expression in BMSCs during adipogenic differentiation; (H) Western blot analysis and quantification of MST1, MST2, and YAP expression in BMSCs during osteogenic differentiation; (I) Western blot analysis and quantification of adipogenic differentiation-related gene expression in BMSCs during adipogenic differentiation; (J) RT-qPCR analysis of gene expression related to adipogenic differentiation in different groups; (K) Western blot analysis and quantification of osteogenic differentiation-related gene expression in BMSCs during osteogenic differentiation; (L) RT-qPCR analysis of gene expression related to osteogenic differentiation in different groups; (M) Immunofluorescence staining of BMSCs undergoing adipogenic differentiation (scale bar = 25 μm), fluorescence intensity of YAP co-localized with the nucleus in each cell, YAP (green), DAPI (nucleus, blue); (N) Immunofluorescence staining of BMSCs undergoing osteogenic differentiation (scale bar = 25 μm), fluorescence intensity of YAP co-localized with the nucleus in each cell, YAP (green), DAPI (nucleus, blue); PLIN: PERILIPIN; * indicates difference between two groups (P\u0026lt;0.05), all experiments repeated for a minimum of 3 times.\u003c/p\u003e","description":"","filename":"Figure92.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/5fab7d3eef634557bddbf8e2.png"},{"id":55700905,"identity":"cd22bc48-c7fd-435b-85cb-a2cd42e46064","added_by":"auto","created_at":"2024-05-02 03:24:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1308071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Irisin on adipogenic differentiation of BMSCs through the MST1/2-YAP pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) RT-qPCR validation of Mst1/2 overexpression efficiency; (B) Representative images and quantification of Oil Red O staining in different groups (scale bar = 100 μm); (C) Western blot analysis of MST1, MST2, and YAP expression in BMSCs during adipogenic differentiation; (D) RT-qPCR analysis of gene expression related to adipogenic differentiation in different groups; (E) Representative images and quantification of Oil Red O staining in different groups (scale bar = 100 μm); (F) Western blot analysis of MST1, MST2, and YAP expression in BMSCs during adipogenic differentiation; (G) RT-qPCR analysis of gene expression related to adipogenic differentiation in different groups; (H) RT-qPCR validation of Yap overexpression efficiency; (I) Representative images and quantification of Oil Red O staining in different groups (scale bar = 100 μm); (J) Western blot analysis of MST1, MST2, and YAP expression in BMSCs during adipogenic differentiation; (K) RT-qPCR analysis of gene expression related to adipogenic differentiation in different groups; (L) RT-qPCR validation of Mst1/2+Yap overexpression efficiency; (M) Representative images and quantification of Oil Red O staining in different groups (scale bar = 100 μm); (N) Western blot analysis of MST1, MST2, and YAP expression in BMSCs during adipogenic differentiation; (O) RT-qPCR analysis of gene expression related to adipogenic differentiation in different groups; VP: verteporfin, PLIN: PERILIPIN; * indicates difference between two groups (P\u0026lt;0.05), all experiments repeated for a minimum of 3 times.\u003c/p\u003e","description":"","filename":"Figure103.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/8321486211a96f3ab6b99a09.png"},{"id":55700901,"identity":"54b473aa-5dba-4eca-9536-8e8535650288","added_by":"auto","created_at":"2024-05-02 03:24:03","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":298253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of Irisin on the Homeostasis of the Mitochondrial Inner Membrane System during Adipogenic Differentiation of BMSCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) MitoTracker staining images (scale bars: 20 μm and 40 μm) and quantifying fluorescence intensity in each group. (B) During adipogenic induction, western blot analysis and quantification of PGC-1α expression levels in each group. (C) Ratio of mitochondrial DNA/nuclear DNA (Mito/B2M). (D) Oxygen consumption rates (OCR) measured in each group during adipogenic differentiation on day 7 after the addition of oligomycin, FCCP, rotenone, and antimycin A. (E) Quantification of OCR parameters, including basal respiration, ATP production, proton leak, maximal respiration, and spare respiration. (F) Extracellular acidification rates (ECAR) in each group. (G) Rates of ATP production from mitochondrial respiration (mito-ATP) and glycolytic respiration (glyco-ATP), with solid lines representing the ratio of total ATP production and dashed lines representing the ratio of ATP production from glycolysis. (H) DCFH-DA staining images (scale bar: 100 μm) and quantification of fluorescence intensity representing the levels of intracellular ROS generation in each group. (I) Quantification of mitochondrial membrane potential (MMP) using JC-1 staining and flow cytometry, expressed as the ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers). (J) RT-qPCR detected expression levels of uncoupling proteins UCP1, UCP2, and UCP3. (K) Expression of markers for mitochondrial inner membrane fusion detected by RT-qPCR. * indicates statistical differences (P \u0026lt; 0.05) between the two groups; all experiments were performed thrice.\u003c/p\u003e","description":"","filename":"Figure112.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/02c5920834ce6af82d235903.png"},{"id":55701732,"identity":"9f28437a-bf36-4f3a-a421-2fd6dad42de7","added_by":"auto","created_at":"2024-05-02 03:32:03","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":567799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of Irisin on the Homeostasis of the Mitochondrial Inner Membrane System via the MST1/2-YAP Signaling Pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) During adipogenic differentiation, Oxygen consumption rates were measured in each group on day 7. (B) Extracellular acidification rates (ECAR) in each group. (C) Rates of ATP production from mitochondrial respiration (mito-ATP) and glycolytic respiration (glyco-ATP), with solid lines representing the ratio of total ATP production and dashed lines representing the ratio of ATP production from glycolysis. (D) DCFH-DA staining images (scale bar: 100 μm) and quantification of fluorescence intensity representing the levels of intracellular ROS generation in each group. (E) Quantification of mitochondrial membrane potential (MMP) using JC-1 staining and flow cytometry, expressed as the ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers). (F) RT-qPCR detected expression levels of uncoupling proteins UCP1, UCP2, and UCP3. (G) During adipogenic differentiation, oxygen consumption rates were measured in each group on day 7. (H) Extracellular acidification rates (ECAR) in each group. (I) Rates of ATP production from mitochondrial respiration (mito-ATP) and glycolytic respiration (glyco-ATP), with solid lines representing the ratio of total ATP production and dashed lines representing the ratio of ATP production from glycolysis. (J) DCFH-DA staining images (scale bar: 100 μm) and quantification of fluorescence intensity representing the levels of intracellular ROS generation in each group. (K) Quantification of mitochondrial membrane potential (MMP) using JC-1 staining and flow cytometry, expressed as the ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers). (L) Expression levels of uncoupling proteins UCP1, UCP2, and UCP3, as well as markers for mitochondrial inner membrane fusion, were detected by RT-qPCR. * indicates statistical differences (P \u0026lt; 0.05) between the two groups; all experiments were performed thrice.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/dc9db559807947336654634c.png"},{"id":55700903,"identity":"893907d1-bb76-4334-871b-465b1c35106c","added_by":"auto","created_at":"2024-05-02 03:24:03","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1640257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImprovement of the Bone Marrow Phenotype in AA Mice by Irisin via the MST1/2-YAP Signaling Pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Representative images of H\u0026amp;E staining of bone marrow tissue from each group of mice (n=8) (scale bar: 100 μm) with quantification of the percentage of hematopoietic tissue in the total marrow space. (B) Oil Red O staining of femurs from each group of mice (n=8) (scale bar: 200 μm) with quantification of adipocyte area percentage. (C) Perilipin immunofluorescence staining of femurs from each group of mice (n=8) (scale bar: 200 μm) with quantification of fluorescence intensity. (D) Expression levels of adipogenic differentiation-related genes detected by RT-qPCR in the bone marrow of each group of mice (n=8). (E) MST1, MST2, and YAP expression levels were detected by Western blot and quantification in the bone marrow of each group of mice (n=8). (F) MST1, MST2, and YAP expression levels were detected by Western blot and quantification in the bone marrow of each group of mice (n=8). PLIN: PERILIPIN. * indicates statistical differences (P \u0026lt; 0.05) between the two groups.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/6e41ecfde132087dc8761649.png"},{"id":55700890,"identity":"5cfd8cc6-efec-4363-b66b-8fdf4487a869","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":365061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImprovement of the Homeostasis of the Mitochondrial Inner Membrane System in AA Mice BMSCs by Irisin via the MST1/2-YAP Signaling Pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Total ATP levels produced by mitochondrial respiration in BMSCs from each group of mice (n=8). (B) DCFH-DA staining images (scale bar: 100 μm) and quantification of fluorescence intensity representing the levels of intracellular ROS generation in BMSCs from each group of mice (n=8). (C) Quantification of mitochondrial membrane potential (MMP) using JC-1 staining and flow cytometry, expressed as the ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers) in BMSCs from each group of mice (n=8). (D) Expression levels of uncoupling proteins UCP1, UCP2, and UCP3, as well as markers for mitochondrial inner membrane fusion, were detected by RT-qPCR in BMSCs from each group of mice (n=8). * indicates statistical differences (P \u0026lt; 0.05) between the two groups.\u003c/p\u003e","description":"","filename":"Figure14.png","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/3972202ffb168abfe3947a88.png"},{"id":55701860,"identity":"c3cc287e-bb87-4c9a-aaf5-5296f4ec71c5","added_by":"auto","created_at":"2024-05-02 03:40:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9103790,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/55bac519-e49c-425d-a0a8-883ba3f61c16.pdf"},{"id":55700888,"identity":"1d7d9bea-7020-4ea0-9092-5d78e746973e","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6086917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 Quality Control, Filtering, and Principal Component Analysis of the scRNA-seq Data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Violin plots showing the number of genes (nFeature_RNA), mRNA molecules (nCount_RNA), and the percentage of mitochondrial genes (percent.mt) in each cell of the scRNA-seq data from normal donors (n=2) and AA patients (n=2). (B) Scatter plots showing the correlation between filtered data nCount_RNA and percent.mt, as well as nCount_RNA and nFeature_RNA. (C) Analysis of variance to select highly variable genes in the samples (red dots represent highly variable genes, black dots represent invariant genes). (D) Cell cycle status of each cell in the scRNA-seq data, with S.Score representing the S phase and G2M.Score representing the G2M phase. (E) p-values of the top 50 PCs obtained from PCA analysis. (F) Distribution of standard deviations of PCs, where important PCs have larger standard deviations. (G) Heatmap showing PCA's top 15 major correlated gene expressions for PC_1 - PC_6. (H) Heatmap showing the expression levels of the top 15 major correlated genes for PC_1 - PC_6 in PCA, where yellow represents upregulation and purple represents downregulation. (I) Distribution of cell distributions in PC_1 and PC_2 before batch correction, with each point representing a cell and different colors representing different samples. AA: aplastic anemia.\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/2f40617cc496077905c65f12.pdf"},{"id":55700887,"identity":"0a896ac0-43b6-4c5f-a483-57e62de91af8","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2873495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 T-SNE Clustering Tree of scRNA-seq data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: Branches represent the clustering relationship between cells, while dots represent cell clusters, with the size of the dots typically indicating cell abundance.\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/a6aeda51bafbfc7318c75538.pdf"},{"id":55700894,"identity":"51998f76-b7ba-46a1-abbd-78b2d22ae948","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10972306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3 Quality Control and PCA Dimensionality Reduction of ST data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Violin plots depicting the number of genes (nFeature_Spatial), mRNA molecule count (nCount_Spatial), and percentage of mitochondrial genes (percent.mt) in each cell of the scRNA-seq data (n=3); (B) Scatter plots showing the correlation between nCount_Spatial and percent.mt, nCount_Spatial and nFeature_Spatial, and nCount_Spatial and percent.HB in the ST data (n=3); (C) Distribution of nCount_Spatial in tissue sections of the ST data (n=3), with redder color indicating higher expression levels of nCount_Spatial in that spot; (D) Cell cycle status of each cell in the ST data, where S.Score represents the S phase and G2M.Score represents the G2M phase (n=3); (E) Visualization of the SCTransform and LogNormalize normalized results in the ST data (n=3); (F) Heatmap of the top 15 correlated genes in PC_1 - PC_6 in the PCA, where yellow represents upregulation and purple represents downregulation (n=3).\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/c40d609d29abf06eda1ec018.pdf"},{"id":55700891,"identity":"0bf3cdf3-c419-4902-978e-5f33d8ecc955","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4247911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4 Identification results of surface markers on BMSCs isolated from different mouse groups using flow cytometry.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/eebceccbc3d5eacb8b7af39a.pdf"},{"id":55700889,"identity":"c6d7eeb2-6d6a-4946-8b3c-0f1e230dcf18","added_by":"auto","created_at":"2024-05-02 03:24:02","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1752161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S5 Scatter plot depicting the correlation analysis of HR-DEGs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/4ffcb2eb333505a21a5dc736.pdf"},{"id":55701733,"identity":"2748f648-29fe-46a9-89a5-92f14e23dd02","added_by":"auto","created_at":"2024-05-02 03:32:03","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10206136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S6 Irisin affects the homeostatic regulation of the mitochondrial inner membrane in BMSCs via the MST1/2-YAP signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: (A) Oxygen consumption rates of various groups on the 7th day of adipogenic differentiation induction; (B) Extracellular acidification rate (ECAR) of various groups; (C) Rates of ATP production from mitochondrial respiration (mito-ATP) and glycolytic respiration (glyco-ATP), solid line represents the ratio of total ATP production rates between the two groups, dashed line represents the ratio of glycolytic ATP production rates between the two groups; (D) DCFH-DA staining images (scale bar = 100 μm) of BMSCs from each group and quantification of fluorescent intensity representing ROS generation levels; (E) JC-1 staining and MMP detection results using flow cytometry, along with quantitative assessment of MMP; (F) Expression levels of uncoupling proteins UCP1, UCP2, UCP3, and mitochondrial inner membrane fusion protein genes detected via RT-qPCR; (G) Oil Red O staining images (scale bar = 100 μm) and quantification of absorbance at 510 nm as a representative result; (H) Expression levels of adipogenic differentiation-related genes in each group detected via RT-qPCR; VP: verteporfin, PLIN: PERILIPIN; * indicates a difference between the two groups with a p-value \u0026lt; 0.05, and all experiments were repeated at least three times.\u003c/p\u003e","description":"","filename":"FigureS6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/be4bda12864bfabe39bad8c8.pdf"},{"id":55700900,"identity":"bfd04904-11c7-4f4c-b0ae-4421e035f6e0","added_by":"auto","created_at":"2024-05-02 03:24:03","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":16099,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4329016/v1/585ddb3cdd4ba9ccbad151c5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDeciphering the Role of the MST1/2-YAP Axis in Irisin-Treated Aplastic Anemia: Implications for Mesenchymal Stem Cell Function\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAplastic anemia (AA) is a distinct blood disorder characterized by the failure of hematopoietic function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Bone marrow, the primary site of hematopoiesis, often undergoes adipogenesis in AA, replacing normal hematopoietic cells with adipocytes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This phenomenon also contributes to reduced hematopoietic cells, leading to clinical manifestations like anemia and bleeding in affected individuals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The precise etiology of AA remains incompletely comprehended. Nonetheless, scholars posit that it potentially involves elements such as immune abnormalities, alterations in the bone marrow microenvironment, and specific genetic mutations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe bone marrow microenvironment plays a critical role in the development and differentiation of hematopoietic cells, and mesenchymal stem cells (MSCs) are considered the central cells in maintaining microenvironmental homeostasis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, in AA, mesenchymal stem cells exhibit abnormal behavior by preferentially differentiating into adipocytes, which results in bone marrow adipogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In clinical practice, bone marrow aspirates from patients with AA demonstrate reduced precursor cells for red blood cells, white blood cells, and platelets, alongside a substantial increase in adipocytes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearchers have recently discovered a molecule called Irisin, which may be associated with bone marrow adiposity while exploring treatment methods for AA [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This molecule has been demonstrated to regulate cell differentiation in various diseases, particularly by inhibiting the adipogenesis process [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the mechanism of action of Irisin and its specific functions in AA remain unclear. To tackle this issue, researchers have commenced investigating Irisin on a molecular level to discover novel treatments for AA [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBuilding upon the background above, this study aims to investigate the regulatory role of Irisin in mesenchymal stem cell differentiation and its potential impact on the development of myelodysplastic syndrome. It will be accomplished by employing cutting-edge methodologies, including single-cell sequencing, spatial transcriptomics, and transcriptome sequencing. Through this research, we aim to identify new targets for treating AA, improve therapeutic outcomes for patients, and gain insights into the regulation of MSCs in other diseases.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eClinical research ethics statement.\u003c/b\u003e The bone marrow tissue used in this study was collected from patients diagnosed with aplastic anemia (AA) who underwent a biopsy at our hospital from January 2020 to January 2022. The study involved 5 patients with AA and 2 healthy volunteers. Before participating in this study, all participants had to sign a written informed consent form. The age range of the patients was from 43 to 65, with an average age of 54. The gathered tissue is divided into two parts. One part is promptly preserved in liquid nitrogen, while the other is fixed in 10% formaldehyde and embedded in paraffin for sectioning [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This study has been approved by the Clinical Ethics Review Committee at our hospital (KYLL-2019-023) in accordance with the Helsinki Declaration.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation and sequencing of single-cell samples.\u003c/b\u003e We will select bone marrow samples from two healthy adult donors and two patients with AA from the samples collected at our hospital. Bone marrow samples were digested using 1 mg/mL of STEMxyme1 (Worthington, LS004106) and 1 mg/mL of Dispase II (ThermoFisher Scientific, 17105041). Then, they were incubated with 2% fetal bovine serum (FBS, Gibco, 10091148) in culture medium 199 (Gibco, 11150059) at 37\u0026deg;C for 25 minutes. After digestion, the sample was filtered through a 70 \u0026micro;m cell filter (Fisher Scientific, 08-771-2) and collected in a separate tube [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOnce the samples are prepared as single-cell suspensions, the C1 Single-Cell Auto Prep System (Fluidigm, C1) is employed to capture individual cells. Once the single cells are captured, they undergo lysis within the chip to liberate mRNA. Subsequently, this mRNA is reverse-transcribed to produce complementary DNA (cDNA). The complementary DNA (cDNA) is pre-amplified in a microfluidic chip after lysis and reverse transcription, in preparation for subsequent sequencing. Library construction will be carried out on the amplified cDNA, followed by single-cell sequencing using the HiSeq 4000 Illumina platform. The sequencing parameters will include paired-end reads with a read length of 2\u0026times;75 bp and approximately 20,000 reads per cell [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eTSNE clustering analysis, cell annotation and pseudotime analysis.\u003c/b\u003e This article analyzes single-cell RNA sequencing (scRNA-seq) data using the Seurat package in the R software. Initially, a series of quality controls were conducted, with the corresponding filtering thresholds set as follows: nFeature_RNA\u0026thinsp;\u0026gt;\u0026thinsp;200, nFeature_RNA\u0026thinsp;\u0026lt;\u0026thinsp;5000, and percent.mt\u0026thinsp;\u0026lt;\u0026thinsp;10. The canonical correlation analysis (CCA) method was employed to eliminate batch effects, followed by normalization of the data using the LogNormalize function. To reduce the dimensionality of the scRNA-Seq dataset, principal component analysis (PCA) was performed on the highly variable genes using the top 2000 genes with the highest variance. Subsequently, the top 30 principal components were selected for TSNE clustering analysis. To identify major cell subpopulations, the FindClusters function provided by Seurat should be employed with the default resolution set at 0.9 (res\u0026thinsp;=\u0026thinsp;0.9). Next, the t-SNE algorithm is applied to non-linearly reduce the dimensionality of scRNA-seq sequencing data. Filtering marker genes for different cell subpopulations using the Seurat package. To further annotate the marker genes of each cell cluster, we utilized the \"SingleR\" package and loaded the reference dataset using the HumanPrimaryCellAtlasData function. We annotated the cells by combining well-known marker genes specific to cell lineages with the online resource CellMarker [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSubsequently, we conducted pseudotemporal analysis using the \"monocle\" package in the R software and examined cell communication using the \"cellchat\" package.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpatial transcriptome sequencing.\u003c/b\u003e Spatial transcriptomic analysis was conducted on the bone marrow of the remaining three patients diagnosed with AA, utilizing the 10x Genomics Visium platform. The 10 \u0026micro;m tissue sections from fresh frozen human bone marrow embedded in OCT were placed onto Visium spatial slides. It was followed by a 30-minute permeabilization step to release mRNA. The mRNA molecules attach to the barcode oligonucleotides positioned at the bottom of the slide and undergo reverse transcription following the instructions provided by the manufacturer. The cDNA libraries prepared using these samples were sequenced on the Illumina NextSeq 2000 platform, generating over 50,000 reads per position. Each library produced more than 400\u0026nbsp;million reads. We utilized the Spaceranger software (version 3.1.0, 10x Genomics) to align each Visium spatial transcriptomics slide location and acquire raw counts. Subsequently, these counts were compared to the reference data of the GRCh38 human genome.\u003c/p\u003e \u003cp\u003eThe 10x Visium spatial transcriptomics data analysis involves using the Load10X_spatial function in Seurat to integrate the raw gene expression matrix, spatial information, and tissue H\u0026amp;E images and create a Seurat object. Before conducting principal component analysis, the data was normalized using SCTransform. The dimensionality was subsequently reduced by selecting the top 30 principal components. To detect marker genes and perform differential gene expression analysis, the FindAllMarkers function in Seurat is used. To identify genes that exhibit spatial variation in situ, the FindSpatiallyVariableFeatures function was employed with default settings.\u003c/p\u003e \u003cp\u003eWe implemented spatial transcriptomics deconvolution and visualization to locate cells in the bone marrow tissue of three patients with AA. An anchor-based integration pipeline in Seurat was used to integrate the combined scRNA-seq dataset with 10x Visium spatial transcriptomics data. It enables the transfer of cell type annotations from single-cell RNA sequencing (scRNA-seq) to spatial transcriptomics. The cell type predictions in Seurat are imported into the R package SPOTlight, which provides annotations and visualizations of the cell types for each spatial location [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e\u003cb\u003eConstruction and grouping of AA mouse models.\u003c/b\u003e A total of ten C57BL/6 (B6) mice aged 213 days and 86 CB6FI mice aged eight weeks were purchased from Beijing Vitonglihua Experimental Animal Technology Co., Ltd., located in Beijing, China. The mice weigh between 20\u0026ndash;25 g and are housed in standard cages. They are subject to a 12-hour light/dark cycle that alternates regularly, and the temperature in the room is kept constant at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The mice have unrestricted access to food and water. Prior to commencing the experiment, the subjects will undergo a one-week acclimation period during which they will be exposed to adaptive feeding practices. The Institutional Animal Ethics Committee has approved this experiment and the animal use protocol(NO. 202309001).\u003c/p\u003e \u003cp\u003eFirst, we randomly divided the 86 CB6FI mice into two groups: the Control group, which comprised 16 mice, and the Model group, which included 70 mice. The construction method for the Model group AA mouse model is as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In brief, we retrieved the inguinal, axillary, and brachial lymph nodes from 10 C57BL/6 (B6) donor mice. The lymph nodes were then homogenized in Iscove's modified Dulbecco's medium (IMDM, ThermoFisher, 21056023) using a tissue grinder (Beyotime, E6600). Subsequently, they underwent washing, centrifugation, and filtration through a 70\u0026micro;m nylon mesh sieve (Labgic, 352350). The quantity of samples was determined using a Beckman Vi-Cell counter (Vi-CELL XR, USA). Subsequently, we will intravenously administer lymphocytes isolated from C57BL/6 (B6) donors, at a dosage of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e lymphocytes per 200 \u0026micro;l of PBS, into CB6FI mice matched in gender. The CB6FI receptor mice underwent 5.0 Gy total body irradiation (TBI) 2\u0026ndash;4 hours ago. A peripheral blood cell count was conducted to confirm the presence of symptoms associated with leukopenia, thereby confirming the successful construction of the model [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Control group: intraperitoneal injection of PBS. Model group: The AA model was constructed, and intraperitoneal injection of PBS was performed.\u003c/p\u003e \u003cp\u003eThe Control group will be randomly divided into two subgroups: The control group (8 mice, injected with PBS only) and the Control\u0026thinsp;+\u0026thinsp;NC-OE group (8 mice, injected with adenovirus empty vector NC-OE, which serves as the negative control for overexpression, along with PBS). Similarly, the Model group will be randomly divided into the following six subgroups: Model group (24 mice, undergoing modeling and injected with PBS), Model\u0026thinsp;+\u0026thinsp;Irisin group (14 mice, undergoing modeling and injected with Irisin), Model\u0026thinsp;+\u0026thinsp;NC-OE group (8 mice, undergoing modeling and injected with PBS and NC-OE), Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group (8 mice, undergoing modeling and injected with Irisin and NC-OE), Model\u0026thinsp;+\u0026thinsp;Mst1/2-OE group (8 mice, undergoing modeling and injected with PBS and adenovirus vector Mst1/2 overexpression Mst1/2-OE), Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group (8 mice, undergoing modeling and injected with Irisin and Mst1/2-OE).\u003c/p\u003e \u003cp\u003eThe control group received no additional treatment except for an intraperitoneal injection of PBS. Model group: Only conducting AA modeling and PBS processing. In the Model\u0026thinsp;+\u0026thinsp;Irisin group, Irisin (MCE, HY-P72534) was dissolved in PBS and injected intraperitoneally at a dose of 100 \u0026micro;g/kg per injection, once a day for a total of 14 days, starting on the 4th day after constructing the AA model [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the virus transduction was conducted in the following manner: HEK-293 cells (Procell, CL-0001) were transfected with either the adenoviral vector (Mst1/2-OE, AAV-Mst1-Mst1) carrying the mouse Mst1 and Mst2 genes, or the adenoviral empty vector (NC-OE) plasmid. The transfection was performed using the LipoFiter transfection reagent (Hanbio, HB-LF-1000). Hanbio synthesized the plasmids. After 72 hours, collect the clear liquid from the top to obtain the viral fluid. After successfully constructing the AA model, different groups of mice were injected with Mst1/2-OE or NC-OE vectors via the tail vein 4 days later. The injection was performed with a concentration of 3.5\u0026times;10\u003csup\u003e12\u003c/sup\u003e viral genomes per mouse. Control\u0026thinsp;+\u0026thinsp;NC-OE group: Only tail vein injection of NC-OE and intraperitoneal injection of PBS were performed without constructing the AA model. Experimental Group: Four days after successfully constructing the AA model, NC-OE was administered through the tail vein while PBS was injected into the peritoneal cavity. Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group: Accept model construction, NC-OE, and Irisin processing. Model\u0026thinsp;+\u0026thinsp;Mst1/2-OE group: After successfully establishing the AA model for 4 days, Mst1/2-OE was administered through a tail vein injection, followed by an intraperitoneal injection of PBS. Model: Mice treated with both Irisin and Mst1/2-OE were used to establish the AA model [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExcept for the 24 mice in the Model group and the 14 CB6FI mice in the Model\u0026thinsp;+\u0026thinsp;Irisin group, eight mice were randomly assigned to each of the remaining groups. On the second day following the administration of Irisin, all the mice were euthanized. Six mice from the Model group and six from the Model\u0026thinsp;+\u0026thinsp;Irisin group were used for transcriptome sequencing. Ten mice from the Model group were also used to extract bone marrow mesenchymal stem cells (BMSCs) for \u003cem\u003ein vitro\u003c/em\u003e mechanistic validation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBone marrow smear.\u003c/b\u003e After decapitating the mouse, expeditiously remove the sternum and utilize hemostatic forceps to grip it, facilitating the extraction of the bone marrow. Gently place the bone marrow smear onto a microscope slide pre-treated with 0.05 mL of fetal bovine serum (FBS, Hyclone), then slide it forward. The identical procedure is employed for the bone marrow obtained from normal donors and bone marrow from patients with AA, which is collected through bonemarrow aspiration (BM aspiration). Next, allow the slides to dry at room temperature and position them on a staining rack. The cells were fixed by applying a drop of methanol for 3 minutes. Then, the working solution of Wright Giemsa (Solarbio, G1021) was added. The working solution was prepared by diluting 1 part of the stock solution with 9 parts of the buffer solution. The working solution allowed the cells to be completely covered at room temperature for 20 minutes. Thoroughly rinse the glass slides with distilled water from one end to the other. The examination and observation were conducted using an OLYMPUS BX46 upright microscope. The ImageJ software was used to quantify the percentage of nucleated cells in the bone marrow. Each group has three independent perspectives [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003ePathological sections of femoral bone marrow.\u003c/b\u003e Mouse femurs were harvested, or normal donor and AA patient bone marrow were obtained through bone marrow biopsy (BM biopsy). The samples were fixed in 4% paraformaldehyde (Biosharp, BL539A) at room temperature for 48 hours. Then, rinse thrice with PBS and distilled water, each time for 20 minutes. Replace the EDTA solution (Solarbio, E1171) for decalcification every 7 days, totaling 28 days. After decalcification, rinse the femur or bone marrow using running water for 20 minutes in an embedding box. Subsequently, perform dehydration by exposing the sample to ethanol. After complete dehydration, clean with xylene. Embed the transparent mouse femur in paraffin. To obtain 4 \u0026micro;m thick sections, the paraffin blocks embedded in mouse femurs were sliced using a wax microtome (Leica, LECIA RM2235). Spread the sliced organisms onto a distillation water bath heated to 45\u0026deg;C. Then, transfer the slices onto clean glass slides, allowing them to drain and dry on a glass slide heater set at 65\u0026deg;C for one hour. De-waxing and dyeing procedures were carried out using an automated staining machine, with the dyeing time determined according to the manufacturer's instructions of the HE Staining Kit (Solarbio, G1120). Finally, seal the slices with neutral adhesive. Examine the bone marrow sections stained with hematoxylin and eosin under an inverted microscope (Leica, Leica DM IL LED) and photograph them at a magnification 200\u0026times;. The nucleus appears blue after staining, while red blood cells and bone trabeculae are stained with eosin. Hematopoietic tissues comprise granulocytes, red blood cells, megakaryocytes, and lymphocytes, whereas non-hematopoietic tissues comprise trabecular bone, cortical bone, and adipose tissue [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. ImageJ software was employed to identify and calculate the percentage area of hematopoietic tissue in the entire bone marrow interstitium. Three independent fields were quantified for each group.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePeripheral blood cell count.\u003c/b\u003e Mouse tail vein blood was collected on the 5th, 10th, and 15th days after establishing the AA mouse model to determine the absolute values of white blood cells (WBC), neutrophils (NEU), platelets (PLT), red blood cells (RBC), and hemoglobin (HGB) concentration in peripheral blood. It was done using a hematology analyzer (HORIBA, ABX PENTRA XL 80) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe hematopoietic colony forms experiment.\u003c/b\u003e Following the death of the mice, a semi-solid culture system employing colony-forming units (CFUs), including CFU-erythroid (CFU-E), CFU-granulocyte macrophage (CFU-GM), and CFU-megakaryocytic (CFU-MK), was utilized to extract nucleated cells from the bone marrow of the femur and subsequently adhere them onto the wells of the tissue culture plates. Bone marrow cells were cultured in discover-modified Dulbecco's medium (IMDM, Sigma-Aldrich, I3390). The culture medium consisted of 20% fetal bovine serum (FBS, Gibco, USA), 300 mg/L glutamine (Sigma-Aldrich, G7513), and either 10 \u0026micro;g/L recombinant mouse macrophage colony-stimulating factor (GM-CSF, Sigma-Aldrich, M9170) or erythropoietin (EPO, ACROBiosystems, EPO-H4214). Additionally, a viscosity carrier of 0.3% agarose was included. Cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) for the CFU-MK culture. The medium was composed of 20% fetal bovine serum, 300 mg/L of glutamine, 1% bovine serum albumin (Sigma-Aldrich, A1933), 10\u0026thinsp;\u0026minus;\u0026thinsp;5 mol/L of 2-mercaptoethanol (Sigma-Aldrich, M3148), and 10 \u0026micro;g/L of recombinant mouse thrombopoietin (Sigma-Aldrich, T4184). The experiment was repeated three times, with 10\u003csup\u003e5\u003c/sup\u003e nucleated cells per well, and cultured in a humid environment at 37\u0026deg;C with 5% CO2. Following a 5-day cultivation period, the colonies of CFU-E (\u0026ge;\u0026thinsp;8 cells) and CFU-GM (\u0026ge;\u0026thinsp;40 cells) were enumerated. As previously stated, mouse megakaryocyte cells in CFU-MK colonies were detected using acetylcholinesterase staining. The count of CFU-MK colonies with four or more cells was performed after seven days of culture [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eOrganize slice oil red o staining.\u003c/b\u003e Following decalcification and dehydration, the femur sections of the mouse were stained with a mixture of 0.21% Oil Red O (Sigma, 1320-06-5) and 100% isopropanol (Sigma, 67-63-0) for 10 minutes. The images were captured using the Olympus BX53 microscope, and the percentage of positive area of fat cells was quantified using ImageJ software. Three mice are randomly selected for each group, and three sections are extracted from each mouse. Data from three independent fields of view are analyzed for every section [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e\u003cb\u003eImmunofluorescence staining.\u003c/b\u003e Following dewaxing and dehydration, paraffin sections of the mouse femur underwent antigen retrieval at 98\u0026deg;C. Incubation was then carried out using 1% Triton X-100 (Sigma-Aldrich, X100) in PBS, followed by a 30-minute blocking step with goat serum (Solarbio). The BMSCs cells were fixed with 4% paraformaldehyde (Biosharp, BL539A) for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and then blocked with 1% BSA (Thermofisher, 37520) at room temperature for 1 hour. Mouse femoral bone slices and BMSCs cells were subjected to immunofluorescent staining using Perilipin antibody (1:200, Thermofisher, MA5-32597) or YAP antibody (1:500, Thermofisher, PA1-46189). The antibodies were incubated overnight at 4℃, followed by a 40-minute incubation at room temperature with Alexa FluorTM Plus 488 goat anti-rabbit IgG antibody (1:1000, Thermofisher, A32731). The samples were stained with 4',6-Diamidino-2-Phenylindole (DAPI) at 0.5\u0026micro;g/mL (Invitrogen, D3571). Subsequently, the stained samples were observed, and images were captured using a fluorescence microscope (Olympus FV-1000/ES). The data were analyzed using the ImageJ software. Three mice were randomly selected for each group. Three slices were taken from each mouse. The fluorescence intensity was assessed in three independent fields of view for each slice [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture.\u003c/b\u003e Bone marrow mesenchymal stem cells (BMSCs) were isolated from the bone marrow of mice by flushing the femurs and tibias of both wild-type mice and mice from different treatment groups after decapitation. The centrifugation was performed at 1000 rpm for 5 minutes. The suspension of bone marrow cells was cultured in L-DMEM (Thermo Fisher, A1443001, USA), supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin. The culture was carried out in a 10 cm\u0026sup2; culture dish. The culture medium is changed once a day for three consecutive days to remove non-adherent cells. Afterward, change the culture medium every 3 days.\u003c/p\u003e \u003cp\u003eAdditionally, to isolate human BMSCs, three-milliliter bone marrow samples were collected from patients with AA and normal donors. Bone marrow mononuclear cells were separated from these samples using Ficoll-Paque (Cytiva, 17144003, USA) through centrifugation. Subsequently, these cells were seeded in 10 cm2 culture dishes, utilizing the previously mentioned culture medium. Remove unattached cells after 48 hours and replace the culture medium every 3 days afterward. Cultured adherent BMSCs with a cell density of 80%-90% were subjected to trypsin treatment. Isolated BMSCs were characterized using flow cytometry [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry.\u003c/b\u003e Surface marker analysis was conducted on BMSCs obtained from normal donors, patients with AA, and mouse bone marrow. FITC-conjugated anti-human CD45, HLA-DR, CD34, CD105, CD44, and CD29 antibodies were used for human samples, while anti-mouse CD45, HLA-DR, CD34, CD105, CD44, and CD29 antibodies were used for mouse samples. After incubating the cells at 4\u0026deg;C in the dark for 30 minutes, wash the BMSCs twice with PBS and then centrifuge them at 2000 rpm for 5 minutes at 4\u0026deg;C. We analyzed the percentage of cells expressing these surface markers using the FACSCanto II flow cytometer (BD, USA) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eInduction of adipogenic or osteogenic differentiation in BMSCs and cellular grouping.\u003c/b\u003e Bone marrow mesenchymal stem cells (BMSCs) were seeded at a density of 2.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per well in a six-well plate. For differentiation induction, the cells were cultured in a medium comprising 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, I7018), 1 \u0026micro;M dexamethasone (Sigma-Aldrich, D4902), and 5 \u0026micro;g/mL insulin (Sigma-Aldrich, I3536). The medium for inducing adipogenic differentiation is changed every 3 days, and the induction process is carried out for 14 days [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBone marrow-derived mesenchymal stem cells (BMSCs) were seeded at a density of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well in a 6-well plate. Once the cells reached 80% confluence, they were cultured in osteogenic induction medium comprising 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.1 mM dexamethasone (Sigma-Aldrich, D4902), 10 mM β-glycerophosphate (Sigma-Aldrich, G9422), and 50 mM ascorbic acid (Sigma-Aldrich, A4403). The osteogenic induction medium is changed every 3 days [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe cell groups involved in the study were divided into five categories. These included the Normal group, AA group, Control group, Model group, and Model\u0026thinsp;+\u0026thinsp;Irisin group. The groups consisted of BMSCs isolated from clinical samples or different treatment groups of mice. BMSCs were differentiated from clinical normal donor bone marrow samples in the Normal group. BMSCs were differentiated from patient bone marrow samples with clinical AA in the AA group. In the Control group, BMSCs were differentiated from mouse bone marrow samples. In the Model group, BMSCs were differentiated from mouse bone marrow samples. In the Model\u0026thinsp;+\u0026thinsp;Irisin group, BMSCs were differentiated from mouse bone marrow samples with Model\u0026thinsp;+\u0026thinsp;Irisin treatment.\u003c/p\u003e \u003cp\u003eNext, BMSCs cells from the Model group mice will be divided into 24 treatment groups, including the PBS group, DMSO group, PBS\u0026thinsp;+\u0026thinsp;DMSO group, Irisin group, Irisin\u0026thinsp;+\u0026thinsp;DMSO group, VP group, PBS\u0026thinsp;+\u0026thinsp;VP group, Irisin\u0026thinsp;+\u0026thinsp;VP group, Rotenone group, VP\u0026thinsp;+\u0026thinsp;Rotenone group, NC-OE group, Mst1/2-OE group, Yap-OE group, Mst1/2-OE\u0026thinsp;+\u0026thinsp;Yap-OE group, PBS\u0026thinsp;+\u0026thinsp;NC-OE group, PBS\u0026thinsp;+\u0026thinsp;DMSO\u0026thinsp;+\u0026thinsp;NC-OE group, Irisin\u0026thinsp;+\u0026thinsp;NC-OE group, Irisin\u0026thinsp;+\u0026thinsp;DMSO\u0026thinsp;+\u0026thinsp;NC-OE group, Irisin\u0026thinsp;+\u0026thinsp;VP\u0026thinsp;+\u0026thinsp;NC-OE group, PBS\u0026thinsp;+\u0026thinsp;Mst1/2-OE group, Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group, Irisin\u0026thinsp;+\u0026thinsp;DMSO\u0026thinsp;+\u0026thinsp;Mst1/2-OE group, PBS\u0026thinsp;+\u0026thinsp;Yap-OE group, and Irisin\u0026thinsp;+\u0026thinsp;Yap-OE group. The differentiation-inducing medium was supplemented with PBS, DMSO, Irisin, VP (verteporfin), and Rotenone. These drugs were co-treated with BMSCs, and the duration of drug treatment coincided with the induction time.\u003c/p\u003e \u003cp\u003eIn the PBS group, DMSO group, and PBS\u0026thinsp;+\u0026thinsp;DMSO group, the differentiation-inducing culture medium was supplemented with PBS, DMSO, or PBS\u0026thinsp;+\u0026thinsp;DMSO, respectively, to induce the differentiation of BMSCs. The irisin group involved dissolving irisin in PBS at 1, 5, 10, and 20 ng/ml concentrations. The dissolved irisin was then added to the differentiation-inducing culture medium to co-treat BMSCs and determine the optimal concentration. In the Irisin\u0026thinsp;+\u0026thinsp;DMSO group, Irisin and PBS were co-administered to treat BMSCs and induce differentiation. In the VP Group, add verteporfin (verteporfin, VP; MCE, HY-B0146) dissolved in DMSO at a concentration of 0.8 \u0026micro;M to the differentiation-inducing medium for simultaneous treatment with BMSCs. PBS and VP collaborate to process BMSCs and prompt differentiation. In the Irisin\u0026thinsp;+\u0026thinsp;VP group, both Irisin and VP were included in the differentiation-inducing medium to co-treat BMSCs. In the Rotenone group, a concentration of 100 nM of the fish poison Rotenone (Rotenone, MCE, HY-B1756) dissolved in DMSO was added to the differentiation induction medium to be co-treated with BMSCs. VP\u0026thinsp;+\u0026thinsp;Rotenone group incorporated VP and Rotenone into the differentiation-inducing culture medium to co-treat with BMSCs [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the AAV-Yap1 plasmid containing the mouse Yap1 gene, synthesized by Hanheng Biotechnology, will be transfected into HEK-293 cells after packaging. It will be done following the animal grouping section, along with the Mst1/2-OE vector and NC-OE vector viral solutions for transfecting BMSCs cells. BMSCs cells in the logarithmic phase (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in a 24-well plate. Transfection occurred when the cell confluence reached 50%, with a multiplicity of infection of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e vp/cell. After 24 hours, the culture medium was replaced with an L-DMEM medium containing 10% FBS, and the medium was changed every three days [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe cell treatment after 3 days of viral transduction is as follows: In the NC-OE group, differentiation induction occurs after receiving NC-OE transfection. In the Mst1/2-OE group, differentiation induction was performed following transfection with Mst1/2 overexpression. Yap-OE group: Differentiation induction after Yap-OE transfection. In the Mst1/2-OE\u0026thinsp;+\u0026thinsp;Yap-OE group, differentiation induction was conducted following the co-transfection of Mst1/2-OE and Yap-OE. The PBS\u0026thinsp;+\u0026thinsp;NC-OE group was treated with PBS and NC-OE to induce differentiation. The PBS\u0026thinsp;+\u0026thinsp;DMSO\u0026thinsp;+\u0026thinsp;NC-OE group was treated with PBS, DMSO, and NC-OE to induce differentiation. The Irisin\u0026thinsp;+\u0026thinsp;NC-OE group received treatment with Irisin and NC-OE to induce differentiation. In the Irisin\u0026thinsp;+\u0026thinsp;DMSO\u0026thinsp;+\u0026thinsp;NC-OE group, the participants were treated with Irisin, DMSO, and NC-OE, which led to induced differentiation. The Irisin\u0026thinsp;+\u0026thinsp;VP\u0026thinsp;+\u0026thinsp;NC-OE group consisted of subjects who received Irisin, VP, and NC-OE treatments to induce differentiation. In the PBS\u0026thinsp;+\u0026thinsp;Mst1/2-OE group, cells were initially treated with PBS and then induced to differentiate by overexpressing Mst1/2 proteins. The Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group received treatment with Irisin and Mst1/2-OE to induce differentiation. Irisin\u0026thinsp;+\u0026thinsp;DMSO\u0026thinsp;+\u0026thinsp;Mst1/2-OE group: The individuals in this group received treatment with Irisin, DMSO, and Mst1/2-OE and were induced for differentiation. The PBS\u0026thinsp;+\u0026thinsp;Yap-OE group accepted both PBS and Yap-OE treatments to induce differentiation. The Irisin\u0026thinsp;+\u0026thinsp;Yap-OE group was treated with Irisin and Yap-OE to induce differentiation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eOil red O staining of BMSCs.\u003c/b\u003e After 14 days of induction for adipogenesis, the cells were fixed in a 4% paraformaldehyde solution (Biosharp, BL539A) and stained with Oil Red O solution (Sigma-Aldrich, MAK194). The dye should be dissolved in isopropanol, and the absorbance at 510 nm should be measured [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eStaining of alkaline phosphatase (ALP) and alizarin red was performed in BMSCs.\u003c/b\u003e On the seventh day of osteogenic induction, BMSCs were fixed using a 4% paraformaldehyde solution (Biosharp, BL539A) for 30 minutes. Subsequently, they were covered with a BCIP/NBT working solution (Beyotime, C3206) and incubated in the dark for 20 minutes. Observe cells under a microscope and take pictures. The protein concentration was measured using the BCA Protein Quantification Kit (ThermoFisher, 23227), and the OD values of the blank wells, standard wells, and test wells were determined at 520 nm using the alkaline phosphatase (ALP) staining kit (Jiancheng Bioengineering Research Institute, A059-2-2). Subsequently, the alkaline phosphatase activity was calculated following instructions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter 21 days of osteogenic induction, the cells were fixed using 4% paraformaldehyde (Biosharp, BL539A) and stained with 2% Alizarin Red S (Sigma-Aldrich, A5533). The Xanthein Red S should be dissolved in a solution of hexadecylpyridinium chloride (Sigma-Aldrich, 1104006) and then quantitated using spectrophotometry at a wavelength of 562nm [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot.\u003c/b\u003e Bone marrow stromal cells (BMSCs) were lysed using the RIPA total protein lysis buffer (AS1004, Wuhan Aspen Biotechnology Co., Ltd., China) after 7 days of osteogenic or adipogenic differentiation. The protein concentration was subsequently measured using the BCA protein quantification assay kit (23227, Thermo Fisher).\u003c/p\u003e \u003cp\u003eProteins were separated using SDS-PAGE and then transferred onto a PVDF membrane. The membrane was blocked with 5% BSA at room temperature for 1 hour. Afterwards, the primary antibodies were added individually, including LPL (...) Incubate the antibody at 4℃ overnight.\u003c/p\u003e \u003cp\u003eLPL, FABP4, PPARγ, CEBPα, and PERILIPIN are markers for adipogenic differentiation, whereas Runx2, ALP, OPN, and OCN are markers for osteogenic differentiation. MST1, MST2, YAP, and p-YAP are proteins associated with the MST1/2-YAP signaling pathway.\u003c/p\u003e \u003cp\u003eThe membrane was washed three times with TBST (3\u0026times;5 minutes), followed by incubation with Anti-Mouse-HRP secondary antibody (1:10000; ThermoFisher, 31430) or Goat anti-Rabbit-HRP secondary antibody (1:10000; ThermoFisher, 31460) at room temperature for 2 hours. The membrane was washed thrice with TBST (3\u0026times;5 minutes). TBST should be replaced with an appropriate ECL working solution (Millipore, WBKLS0500). The transfer membrane should be incubated at room temperature for 1 minute. Excess ECL reagent should be removed, and the membrane should be sealed with cling film. Before development and fixation, the membrane should be placed in a dark box for 5\u0026ndash;10 minutes to expose an X-ray film. The ImageJ analysis software was used to quantify the grayscale intensity of bands in Western blot images, utilizing GAPDH as the internal reference [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eRT-qPCR.\u003c/b\u003e The Trizol Reagent kit (Invitrogen, 10296028CN) should be utilized for cell lysis and total RNA extraction from cells or bone marrow tissue. UV-Vis spectrophotometry (ND-1000, Nanodrop, USA) was employed to evaluate RNA quality and concentration.\u003c/p\u003e \u003cp\u003eThe PrimeScript\u0026trade; RT-qPCR Kit (TaKaRa, RR037Q) was employed to measure mRNA expression levels for reverse transcription. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed using SYBR Premix Ex TaqTM (TaKaRa, RR390A) on a LightCycler 480 system (Roche Diagnostics, Pleasanton, CA, USA). GAPDH is a reference gene that functions as an internal control for mRNA. The primers utilized for amplification were designed and provided by Shanghai Universal Biotech Co., Ltd. The primer sequences can be found in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The term 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e represents the fold difference in the target gene expression between the experimental and control groups. The formula is defined as ΔΔCT\u0026thinsp;=\u0026thinsp;ΔCt experimental group - ΔCt control group, where ΔCt is calculated as the difference between the Ct values of the target gene and reference gene [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction, library construction, and sequencing.\u003c/b\u003e Total RNA was extracted from bone marrow stromal cells (BMSCs) and isolated from two groups of mice: the Model group (n\u0026thinsp;=\u0026thinsp;6) and the Model\u0026thinsp;+\u0026thinsp;Irisin group (n\u0026thinsp;=\u0026thinsp;6). The extraction was performed using a Trizol reagent (15596026, Invitrogen, USA). The concentration and purity of RNA samples were determined using a spectrophotometer instrument, specifically the Nanodrop 2000 (1011U, Nanodrop, USA). Total RNA samples meeting the following criteria are utilized for subsequent experiments: RNA Integrity Number (RIN)\u0026thinsp;\u0026ge;\u0026thinsp;7.0 and 28S:18S ratio\u0026thinsp;\u0026ge;\u0026thinsp;1.5 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCapitalBio Technology, located in Beijing, China, generated and sequenced the sequencing library. Every sample utilizes a total of 5 \u0026micro;g of RNA. We employed the Ribo-Zero Magnetic Kit (MRZE706, Epicentre Technologies) to remove ribosomal RNA (rRNA) from total RNA. The NEB Next Ultra RNA Library Prep Kit (#E7775, New England Biolabs, USA) generates Illumina-compatible sequencing libraries. Next, the RNA fragments were fragmented into 300 base pairs (bp) using the NEB Next First Strand Synthesis Reaction Buffer (5\u0026times;). The first-strand cDNA is synthesized using a reverse transcriptase primer and random primer, while the second-strand cDNA is synthesized in the reaction buffer of dUTP Mix (10\u0026times;) for the second-strand synthesis. The repair of cDNA fragments involves adding polyA tails and connecting sequencing adaptors. Following the ligation of Illumina sequencing adapters, the second cDNA strand was digested using the USER enzyme (#M5508, NEB, USA) to generate strand-specific libraries. The library DNA should be amplified, purified, and enriched through PCR. Next, the libraries were evaluated using the Agilent 2100 system and quantified using the KAPA Library Quantification Kit (KK4844, KAPA Biosystems). Lastly, we conducted paired-end sequencing using the Illumina NextSeq CN500 sequencer [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuality control of sequencing data and its alignment to a reference genome.\u003c/b\u003e The quality of the paired-end reads in the raw sequencing data was assessed using FastQC software version 0.11.8. The raw data underwent preprocessing using Cutadapt software version 1.18, which involved removing Illumina sequencing adapters and poly(A) tail sequences. Filter out reads with an N content exceeding 5% using a perl script. Using the FASTX Toolkit software version 0.0.13, we extracted reads with a base quality of 20 or higher, which accounted for 70% of the total. Repair the paired-end sequences using BBMap software. Finally, the filtered fragments of high-quality reads were aligned to the mouse reference genome using hisat2 software (version 0.7.12) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential expression gene bioinformatics analysis.\u003c/b\u003e The limma package in R was used to identify differentially expressed genes (DEGs) in the raw count matrix. DEGs were selected based on a threshold of |log fold change (FC)| \u0026gt; 1 and a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The ggplot2 package in R was used to plot the volcano plot. The clustering heat map of differentially expressed genes (DEGs) was generated using the \"pheatmap\" package in the R programming language. We conducted enrichment analysis using the R language and several packages, including the \"clusterProfiler\", \"org.Hs.eg.db\", \"org.Mm.eg.db\", \"enrichplot\", \"ggplot2\", and \"pathview\" packages. The analysis focused on Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioinformatics analysis.\u003c/b\u003e We extracted 26 mouse genes related to the Hippo signaling pathway (HRGs) from the Reactome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://reactome.org/\u003c/span\u003e\u003cspan address=\"https://reactome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Conduct a protein-protein interaction (PPI) analysis of HRGs using the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and optimize the results using Cytoscape 3.10.0 software. The Jvenn website generates Venn diagrams to identify the intersection of differentially expressed genes (HR-DEGs) associated with the Hippo pathway. The clustering heatmap, volcano plot, correlation analysis heatmap, and scatter plot for highly-regulated differentially expressed genes (HR-DEGs) were generated using the R packages \"ggplot2\" and \"pheatmap\" [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGO and KEGG are utilized to perform functional enrichment analysis of HR-DEGs. GO and KEGG analyses were conducted using the 'clusterProfiler,' 'org.Hs.eg.db,' 'enrichplot,' 'DOSE,' and 'ggplot2' packages in the R programming language [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe applied machine learning algorithms such as lasso regression, SVM-RFE, and random forest using the \"glmnet\", \"e1071\", and \"randomForest\" packages in the R programming language. The Venn diagram was plotted using the \"venn\" package in R [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eCCK-8.\u003c/b\u003e Cell viability was assessed following the guidelines provided in the CCK-8 assay kit (ab228554, Abcam, USA). Cells from each group were seeded into individual wells of 96-well plates (2500 cells per well) and cultured for 1, 3, and 5 days, respectively. At the designated time point, add 10\u0026micro;l of CCK-8 to each well. After incubating for 2 hours, the absorbance at 450 nm should be measured using an enzyme-linked immunosorbent assay reader (M1000 PRO, Tecan) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eLive-death staining.\u003c/b\u003e The LIVE/DEAD Cell Viability/Cytotoxicity Assay Kit (Invitrogen, L3224) was employed to assess cell death objectively. The assay kit offers two types of molecular probes: one that labels live cells as green based on intracellular esterase activity and another that labels dead cells red due to compromised membrane integrity. Conduct the testing in accordance with the plan provided by the manufacturer. In brief, cells were seeded in a 24-well plate and incubated overnight. Afterward, they were treated with Irisin for 3 days. Subsequently, the cells were incubated with a fluorescent dye (2.0 \u0026micro;M) for 15 minutes, and a fluorescence microscope (FV-1000/ES, Olympus) was used to capture microscopic images. The software ImageJ was employed to identify and calculate the percentage of viable and nonviable cells [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMitoTracker staining.\u003c/b\u003e After inducing adipogenic differentiation of BMSCs for 14 days, the mitochondria staining reagent, mitoTracker Green FM (Invitrogen, M7514), was applied at a concentration of 100 nM. The cells were then incubated in a cell culture incubator for 30 minutes. Next, the Hoechst 33342 Live Cell Stain (Beyotime, C1029) should be applied in a dark environment at 37\u0026deg;C for 10 minutes. Subsequently, the cells were washed twice with preheated PBS, and a fresh culture medium was added. Imaging was then promptly conducted using a Zeiss LSM 510 META confocal microscope (Zeiss). Merge and scale the original image using ImageJ software [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative analysis of mitochondrial DNA.\u003c/b\u003e Total genomic and mitochondrial DNA could be extracted using the QIAamp DNA Mini kit (Qiagen, 51304), following the manufacturer's instructions. Adjust the DNA template concentration to 10 ng/\u0026micro;l. Refer to the report by Malik et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Evaluating the amount of mitochondrial DNA through RT-qPCR for absolute quantification. We utilized mouse mitochondrial DNA primers (Mito) and mouse nuclear DNA primers (β2-microglobulin, mB2M) to amplify the corresponding products in mouse genomic DNA. The primer sequences are available in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).\u003c/b\u003e The Seahorse XF24 extracellular flux analyzer (Agilent, Seahorse XF24, 24-well plate) was employed, following the described method, to assess the oxygen consumption rate (OCR) of adipogenic-induced BMSCs after 7 days [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The OCR determination was conducted using the Seahorse XF Cell Mito Stress Test Kit (Agilent, 103672-100). The assay medium consisted of Seahorse XF DMEM culture medium (Agilent, 103680-100) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose, adjusted to pH 7.4. Sixty thousand cells were inoculated per well (0.32 cm2 growth area) into XF24 24-well culture microplates containing 500 \u0026micro;L of assay medium. The plates were incubated overnight in a humid environment at 37℃, with 95% air and 5% carbon dioxide. The culture medium should be removed before the experiment, and 500 \u0026micro;L of fresh assay medium should be added. The cells are pre-incubated in ambient air at 37\u0026deg;C for one hour. We used oligomycin at a concentration of 4 \u0026micro;g/mL to assess ATP synthesis driven by oxidative phosphorylation and respiration driven by proton leak. Following three measurement cycles, a decoupling agent, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), was added at a concentration of 5 \u0026micro;M to assess the maximum respiratory capacity. After three additional measurement cycles, 1 \u0026micro;M of Rotenone should be added to inhibit complex I and 1 \u0026micro;M of antimycin A should be used to inhibit complex III, thereby suppressing mitochondrial respiration.\u003c/p\u003e \u003cp\u003eFurthermore, the Seahorse XF Glycolysis Stress Test Kit (Agilent, 103020-100) was used to accurately measure the glycolytic rate. The cells should be replaced with XF DMEM medium (Agilent, 103575-100) supplemented with 5 mM HEPES at pH 7.4. Additionally, 1 mM pyruvate (Agilent, 103578-100), 2 mM glutamine (Agilent, 103579-100), and 10 mM glucose (Agilent, 103577-100) should be added. The cells should then be incubated in a CO2-free incubator at 37℃ for 1 hour. Before commencing the test, the culture medium should be replaced again, following the guidelines provided by the manufacturer. After establishing the baseline, 1 \u0026micro;m fisetin and antifungal A were added sequentially, along with 50 mM 2-deoxyglucose, and the response was measured. The OCR, ECAR, glycolytic proton efflux rate, and ATP production rate were determined using the Seahorse XFe96 software, version 2.6 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e\u003cb\u003eROS detection.\u003c/b\u003e The levels of reactive oxygen species (ROS) inside the cells were measured in mouse bone marrow-derived mesenchymal stem cells (BMSCs) or BMSCs induced for adipogenic differentiation for 7 days from different groups, following the guidelines provided by the manufacturer. The measurement was performed using a ROS assay kit (Beyotime, S0033S). The fluorescent probe DCFH-DA (10 mM) should be diluted 1000-fold in serum-free L-DMEM before adding it to the cells. The cells should be incubated in the dark at 37℃ for 20 minutes. After rinsing with PBS three times, we examined cell morphology using a fluorescence microscope (FV-1000/ES, Olympus). Subsequently, the cells were harvested, and the fluorescence intensity was quantified using a TriStar3 multimode reader (Berthold Technologies) with an excitation wavelength set at 488 nm and an emission wavelength of 525 nm. Normalize intracellular ROS levels to total cell number [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMitochondrial membrane potential (MMP) detection.\u003c/b\u003e After isolating bone marrow-derived mesenchymal stem cells (BMSCs) or differentiating them into adipocytes for 7 days, we assessed the mitochondrial membrane potential (MMP) using the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, C2006) [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In summary, the cells are incubated in a culture medium containing JC-1 for 30 minutes. Subsequently, they are loaded into the BD FACSMelody flow cytometer (BD) with an excitation wavelength of 480 nm and emission wavelengths of 525 nm and 590 nm [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. We analyzed 10,000 cells using FlowJo X10 software and repeated the experiment three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eATP level determination.\u003c/b\u003e The total ATP production of BMSCs in each group was quantified using the ATP assay kit (Beyotime, S0026B). Initially, the cells are inoculated into a 96-well plate. Subsequently, PBS washing, lysis, and centrifugation are performed. Subsequently, the luminescence in the supernatant was measured with the BioTek Synergy 2 microplate reader (BioTek Instruments Inc.). The MicroBCA Protein Assay Kit (ThermoFisher, 23235) was utilized to determine the protein concentration and subsequently performed normalization. Finally, the ATP content should be determined using the ATP standard curve, and the experiment should be repeated three times [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Each experiment was repeated independently at least three times, and the data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). To compare the differences between groups, we utilize either an independent samples t-test or a one-way analysis of variance. If the variance analysis results reveal differences, we will conduct Tukey's HSD post-hoc test to examine the disparities between each group. When dealing with data that is not normally distributed or exhibits heteroscedasticity, we will employ either the Mann-Whitney U or Kruskal-Wallis H test. Statistical analyses were conducted using GraphPad Prism 8.0 software [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A p-value less than 0.05 is considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eComprehensive single-cell rna sequencing analysis of bone marrow microenvironment in aplastic anemia: unraveling cellular diversity and pathological alterations.\u003c/b\u003e Aplastic anemia (AA) is a serious hematological disorder characterized by insufficient development of the bone marrow, accompanied by fatty degeneration. There are numerous unresolved issues concerning the pathogenesis and treatment options [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Single-cell RNA sequencing (scRNA-seq) could uncover alterations in the transcriptome of distinct cellular subpopulations, thus facilitating the investigation of crucial pathways that might impact the development of AA [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo examine the developmental process of AA and the alterations in the bone marrow microenvironment, single-cell RNA sequencing (scRNA-seq) was conducted on bone marrow samples from two healthy donors and two patients with AA. We conducted quality control and normalization of single-cell RNA sequencing (scRNA-seq) data using the \"Seurat\" package in the R software. After processing, the distribution of cellular RNA is presented in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA. The correlation coefficient between nCount and percent.mt is -0.13 (r = -0.13), and the correlation coefficient between nCount and nFeature is 0.74 (r\u0026thinsp;=\u0026thinsp;0.74). These findings suggest that the filtered cell data exhibits good quality and is suitable for subsequent analysis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we further analyzed the filtered cells. After filtering, we chose the top 2000 genes with high variance in gene expression for further analysis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). The cell cycle was calculated using the CellCycleScoring function (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD), and the data were subsequently normalized. Subsequently, we applied linear dimensionality reduction to the data using principal component analysis (PCA), utilizing the highly variable genes previously selected. We obtained a total of 50 principal components (PCs). A smaller p-value and a larger standard deviation suggest a greater importance of the PCs (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE-F). The results suggest that principal components PC_1-PC_30 effectively capture the information in the selected highly variable genes and hold analytical value. In this study, we provide the characteristic genes of PC_1 and PC_2, as shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG. Additionally, we present the expression heatmaps of the major correlated genes of PC_1 - PC_6, depicted in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH.\u003c/p\u003e \u003cp\u003eFurthermore, we also illustrated the distribution of various sample cells across PC_1 and PC_2 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI). The results indicate the presence of a distinct batch effect across various samples. To mitigate batch effects among samples and enhance the accuracy of cell clustering, we employed the harmony package to correct batch variation in the sample data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The corrected results indicate that the batch effect of the samples has been successfully eliminated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we utilized the TSNE algorithm to reduce non-linear dimensionality on the initial 30 principal components (PCs). Subsequently, we conducted cluster analysis with a resolution of 0.9 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). We utilized clustering to generate 29 clusters and determine each respective cluster's marker gene expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Next, we annotated the cells by conducting a literature search and utilizing the online resource CellMarker, which provided us with identified marker genes specific to cell lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Overall, we obtained a total of 15 cell types: Hematopoietic stem and progenitor cells (HSPCs), Mesenchymal stem cells (MSCs), Adipocytes, T cells, Neutrophils, Myeloid cells, Plasmacytoid dendritic cells (pDCs), Red blood cells, Monocytes, Macrophages, B cells, Common lymphoid progenitor cells (CLPs), Plasma cells, Natural killer cells (NK cells), and Megakaryocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results above indicate that bone marrow samples from normal donors and patients with AA could be categorized into 28 clusters, comprising 15 cellular subgroups. A decrease in hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) could be observed in the bone marrow tissue of patients with AA, while the number of adipocytes increases significantly.\u003c/p\u003e \u003cp\u003e \u003cb\u003eElucidating the role of mesenchymal stem cells and adipocytes in aplastic anemia: a deep dive into cellular dynamics and intercellular communication.\u003c/b\u003e Mesenchymal stem cells (MSCs) and adipocytes play a role in the excessive differentiation of bone marrow stromal cells, thereby contributing to the occurrence of AA [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, we initially confirmed the annotation of MSCs and Adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Following this, Seurat analysis was used to calculate the proportions of different cell types in both the AA and Normal groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The results demonstrated a decrease in MSC quantity and an increase in Adipocyte quantity in the AA group compared to the Normal group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the essential role played by MSCs in differentiating into adipocytes in AA, we conducted a pseudo-temporal analysis of MSC subpopulations using the 'monocle' package. We generated plots representing the trajectories of cells changing over time and examined the disparities in their involvement in the pathogenesis of AA compared to normal conditions. The results demonstrated that, by considering the highly variable genes, MSCs could be classified into eight distinct expression patterns or branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The visualization of pseudotime results demonstrates that the trajectory of MSCs undergoes a transition from State 1 to State 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Subsequently, the cells were sorted into different groups, and the results indicated that states 3, 4, 5, 6, and 7 represented AA-specific differentiation or developmental stages during the differentiation or development process of MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Subsequently, we plotted the pseudo-temporal gene expression changes of marker genes specific to adipocytes. The results revealed an increase in marker gene expression in states 3, 4, 5, 6, and 7 over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), suggesting that adipogenesis of MSCs may be a crucial factor in the development of AA.\u003c/p\u003e \u003cp\u003eFurthermore, we employed the \"CellChat\" package to examine the intercellular communication between MSCs and Adipocyte cells. The findings revealed that the connection between MSCs and Adipocytes was stronger in the AA group compared to the Normal group, with a heightened interaction observed in the bone marrow tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). In conclusion, the excessive differentiation of mesenchymal stem cells (MSCs) into adipocytes may play a critical role in the development of AA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntegrating spatial transcriptomics and single-cell RNA sequencing to uncover the cellular landscape and intercellular interactions in aplastic anemia bone marrow.\u003c/b\u003e Before tissue dissociation, single-cell RNA sequencing (scRNA-seq) results in the loss of spatial information. Integrating spatial transcriptomics technology with scRNA-seq could compensate for the loss of spatial information caused by scRNA-seq and has wide-ranging applications in biology [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo accurately describe the distribution of various cell types in the bone marrow tissues of AA patients, we utilized the spatial transcriptomics (ST) method to analyze frozen sections of bone marrow tissues from the remaining three AA patients. This analysis aimed to provide an unbiased mapping of tissue transcript expression. The Seurat package is utilized for integrating ST sequencing data. Initially, we analyzed the count of genes (nFeature_Spatial), mRNA molecules (nCount_Spatial), and the proportion of mitochondrial genes (percent.mt) in all cells of the spatial transcriptomics (ST) dataset. The results indicate that most cells exhibit nFeature_Spatial values less than 10000, nCount_Spatial values less than 50000, and percent.mt values less than 20% (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). The correlation analysis of sequencing depth showed that, after filtering, the correlation coefficient between nCount_Spatial and percent.mt was not applicable (r\u0026thinsp;=\u0026thinsp;NA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMeanwhile, the correlation coefficient between nCount_Spatial and nFeature_Spatial was 0.96, and the correlation coefficient between nCount_Spatial and percent.HB was \u0026minus;\u0026thinsp;0.04 (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). This result suggests that the ST data is of good quality and could be utilized for further analysis. Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC depicts the distribution of nCount_Spatial on tissue sections obtained from various organizations. The CellCycleScoring function was utilized to calculate the cellular cycle of the samples (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD), and subsequently, the data were normalized (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eSubsequently, genes exhibiting high variance in gene expression were selected and the top 3000 genes with the highest variance were chosen for subsequent analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Subsequently, we conducted principal component analysis (PCA) on the selected genes with high variance to reduce the dimensionality linearly, producing a principal component analysis plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Meanwhile, we used ElbowPlot to standardize the principal components and sorted them by standard deviation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The expression heatmap of the correlated genes from principal component 1 to principal component 6 is shown here (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF). The results show that principal components 1 to 30 effectively capture the information from the selected high-variance genes and have analytical value.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing this, we used the TSNE algorithm to reduce non-linear dimensionality on the initial 30 principal components. A resolution of 0.4 was then chosen for cluster analysis. We inferred the enrichment of specific cell types in a given tissue region and annotated cells in the spatial transcriptomics (ST) data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) by quantifying the overlap between genes identified by scRNA-seq data and genes specific to a particular cell type and region. The distribution of mesenchymal stem cells (MSCs) and mature adipocytes can be observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F.\u003c/p\u003e \u003cp\u003eWe utilized the \"SPOTlight\" package in R to extract data concerning cell spatial interactions. Subsequently, we generated a circular graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) to illustrate the magnitude of intercellular interactions. A heat map (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) was also created to portray the correlations between cells. The results demonstrate an interaction between MSCs and Adipocytes, with a negative correlation observed between MSCs and Adipocytes. This finding further confirms the previous scRNA-seq results.\u003c/p\u003e \u003cp\u003eIn summary, the results above indicate a strong coexistence of MSCs and adipocytes in the bone marrow samples of AA patients, implying their contribution to the microenvironment of AA bone marrow.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnhanced adipogenic differentiation of bone marrow mesenchymal stem cells in aplastic anemia: insights from histological analysis and molecular characterization.\u003c/b\u003e To further investigate the adipogenic potential of bone marrow-derived multipotent mesenchymal stem cells (BMSCs) in AA patients and their influence on the pathogenesis of AA, Wright-Giemsa and H\u0026amp;E staining was performed on bone marrow tissues obtained from both AA patients and healthy individuals. The results revealed that patients with AA exhibited bone marrow hypoplasia and an elevated presence of fat globules compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we characterized the bone marrow mesenchymal stem cells (BMSCs) isolated from both healthy donors and patients diagnosed with aplastic anemia. The flow cytometry results revealed no differences in the expression of surface markers CD45, HLA-DR, CD34, CD29, CD105, and CD44 between the two groups of bone marrow stromal stem cells. Among them, bone marrow stromal cells (BMSCs) express positive surface antigens CD29, CD105, and CD44, while negative expression is observed for CD45, HLA-DR, and CD34 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, compared to the normal group, the number of lipid droplets in BMSCs from AA patients increased during adipocyte differentiation, indicating an enhanced ability of BMSCs to differentiate into adipocytes in AA patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In accordance with this finding, the mRNA and protein levels of adipogenic markers, including LPL, FABP4, PPARγ, CEBPα, and PERILIPIN, were notably increased in BMSCs from the AA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003eThe above results provide additional confirmation of the findings obtained from ScRNA-seq and ST techniques, suggesting that there is an increase in adiposity within the bone marrow of AA patients. Moreover, the results indicate an improved capacity of BMSCs to undergo adipocyte differentiation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin as a therapeutic agent: reversing aplastic anemia-induced bone marrow failure and marrow adiposity by regulating mesenchymal stem cell differentiation.\u003c/b\u003e Previous studies have demonstrated that Irisin has an inhibitory effect on adipocyte generation and a promoting effect on osteoblast generation during lineage-specific differentiation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The buildup of fatty cells in the bone marrow can potentially hinder the processes of hematopoiesis and osteogenesis [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Therefore, we propose a scientific hypothesis: Irisin regulates the differentiation of BMSCs into osteoblasts and inhibits their differentiation into adipocytes, thereby improving adipogenesis.\u003c/p\u003e \u003cp\u003eWe initially established a mouse model of acquired aplastic anemia (AA) to test this hypothesis. The blood routine results of peripheral blood from mice in the Model group showed a decrease in cell concentration compared to the Control group. However, the administration of Irisin in the Model\u0026thinsp;+\u0026thinsp;Irisin group reversed this change, as indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results of the hematopoietic colony formation experiment revealed a reduction in the number of hematopoietic colonies in the mice of the Model group compared to those in the Control group. However, upon Irisin treatment, the colony formation in the Model\u0026thinsp;+\u0026thinsp;Irisin group increased compared to the Model group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It suggests that Irisin greatly improves hematopoietic disorders resulting from AA and enhances the proliferation of hematopoietic cells.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe peripheral blood counts and hemoglobin concentration of WBC, NEU, PLT, and RBC in each group of mice (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWBC\u003c/p\u003e \u003cp\u003e(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNEU absolute value (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePLT\u003c/p\u003e \u003cp\u003e(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRBC\u003c/p\u003e \u003cp\u003e(10\u003csup\u003e12\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHGB\u003c/p\u003e \u003cp\u003e(g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e449.4\u0026thinsp;\u0026plusmn;\u0026thinsp;45.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e154.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e431.1\u0026thinsp;\u0026plusmn;\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e156.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e479.3\u0026thinsp;\u0026plusmn;\u0026thinsp;36.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e156.2\u0026thinsp;\u0026plusmn;\u0026thinsp;13.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e95.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e90.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e89.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.48*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e91.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e90.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.42*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e94.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003cp\u003e+Irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e263.0\u0026thinsp;\u0026plusmn;\u0026thinsp;27.2\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e107.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e333.5\u0026thinsp;\u0026plusmn;\u0026thinsp;29.4\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e120.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e414.0\u0026thinsp;\u0026plusmn;\u0026thinsp;31.3\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e147.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: WBC, white blood cell; NEU, neutrophil; PLT, platelets; RBC, red blood cell; HGB, hemoglobin; * indicates a significant difference compared to the Control group with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, △ indicates a significant difference compared to the Model group with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe number of colony-forming units of erythroid lineage (CFU-E), granulomonocytic lineage (CFU-GM), and megakaryocytic lineage (CFU-MK) in each group of mice (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCFU-E (10\u003csup\u003e5\u003c/sup\u003e cells)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCFU-GM (10\u003csup\u003e5\u003c/sup\u003e cells)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCFU-MK (10\u003csup\u003e5\u003c/sup\u003e cells)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e133.0\u0026thinsp;\u0026plusmn;\u0026thinsp;14.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e92.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e50.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u0026thinsp;+\u0026thinsp;Irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e92.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5△\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e73.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0△\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6△\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: CFU, colony forming unit; E, erythroid; GM, granulocyte macrophage; MK, megakaryocytic; * indicates a significant difference compared to the Control group, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, △ indicates a significant difference compared to the Model group, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results of Wright-Giemsa staining showed a decrease in the number of nucleated cells in the sternum bone marrow of the Model group compared to the Control group. However, the administration of Irisin, in addition to the Model group, restored the number of nucleated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The pathological results demonstrate a substantial disparity in developmental abnormalities between the Model and Control groups. The Control group exhibited bone marrow hyperplasia, with an average area of hematopoietic tissue of 82.33% and identifiable megakaryocytes. On the other hand, the Model group displayed bone marrow underdevelopment, reduced nuclear cells, and incomplete maturation of megakaryocytes, with an average area of hematopoietic tissue of only 38.12%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, there was an increase in fatty particles in the Model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In the Model\u0026thinsp;+\u0026thinsp;Irisin group, treatment with Irisin reverses this phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This result indicates the successful establishment of an AA mouse model, in which Irisin improved bone marrow failure and marrow adiposity induced by AA.\u003c/p\u003e \u003cp\u003eIn order to further validate the inhibitory effect of Irisin on bone marrow adipogenesis, we conducted experiments on murine femurs using Oil Red O and Perilipin immunofluorescence staining. This result allowed us to investigate the changes in adipocytes within the murine bone marrow. The Oil Red O staining results revealed an increase in bone marrow adipocytes in the Model group of mice compared to the Control group. However, treatment of Model mice with Irisin resulted in a decrease in adipocytes, thereby confirming the inhibitory effect of Irisin on marrow adipogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The immunofluorescent staining results are consistent with it (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eSubsequently, we extracted bone marrow mesenchymal stem cells (BMSCs) from the femur and tibia of mice in the Control, Model, and Model\u0026thinsp;+\u0026thinsp;Irisin groups to investigate alterations in their adipogenic and osteogenic differentiation capacity. Bone marrow mesenchymal stem cells (BMSCs) were identified using flow cytometry, where they showed positive expression of surface markers CD29, CD105, and CD44, and negative expression of CD45, HLA-DR, and CD34 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Oil Red O staining revealed increased differentiation of BMSCs into adipocytes in the Model group compared to the Control group. However, in the Model\u0026thinsp;+\u0026thinsp;Irisin group, the differentiation of BMSCs into adipocytes was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In contrast, the differentiation of BMSCs into osteoblasts was reduced in the Model group mice. However, treatment with Irisin effectively reversed this phenomenon, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G. Additionally, the expression of adipogenic-related genes, including LPL, FABP4, PPARγ, CEBPα, and PERILIPIN, was up-regulated in mouse BMSCs from the Model group compared to the Control group. Conversely, the expression of osteogenic-related genes, such as Runx2, ALP, OPN, and OCN, was down-regulated. However, Irisin treatment inhibited this phenomenon, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-K.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results suggest that Irisin could alleviate AA mice's bone marrow failure and marrow adiposity. It is attributed to Irisin's ability to enhance the differentiation of AA mouse BMSCs into osteoblasts and inhibit their differentiation into adipocytes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin's role in redirecting mesenchymal stem cell fate: a transcriptomic analysis unveiling the involvement of the hippo signaling pathway.\u003c/b\u003e We collected bone marrow-derived mesenchymal stem cells (BMSCs) from mice in both the Model and Model\u0026thinsp;+\u0026thinsp;Irisin groups to investigate how Irisin enhances adipogenesis. Through transcriptome sequencing, 935 differentially expressed genes (DEGs) were identified in the Model group of mouse BMSCs treated with Irisin. Four hundred sixty-one genes were upregulated, while 474 were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional enrichment analysis on differentially expressed genes (DEGs) was conducted using the GO and KEGG databases. The results of the GO enrichment analysis revealed that the differentially expressed genes (DEGs) were primarily enriched in biological processes related to tissue remodeling and bone remodeling (Biological process, BP). Additionally, they were also enriched in cellular components such as secretory vesicles and transport vesicles (Cellular component, CC), as well as in molecular functions such as receptor-ligand activity and growth factor activity (Molecular function, MF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The KEGG enrichment analysis revealed that the differentially expressed genes (DEGs) were primarily enriched in the Hippo signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE. The validation was performed using GSEA enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Previous studies have demonstrated the involvement of the Hippo signaling pathway in regulating the differentiation fate of BMSCs toward osteogenic and adipogenic lineages [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, it is believed that Irisin has the potential to rectify the biased tendency of BMSCs towards adipocyte differentiation through the regulation of the Hippo signaling pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin's modulatory effect on the hippo signaling pathway: unraveling the mechanism behind its role in reducing adipogenesis and improving aplastic anemia.\u003c/b\u003e To further investigate the mechanism through which Irisin regulates the Hippo pathway, we initially retrieved 26 genes related to the Hippo signaling pathway (HRGs) from the Reactome database. The interconnections among these genes are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B. The HRGs should intersect with the differentially expressed genes (DEGs) identified in the Model group and Model\u0026thinsp;+\u0026thinsp;Irisin group of mouse BMSCs. This process will ultimately allow the identification of 7 differentially expressed genes (HR-DEGs) associated with the Hippo pathway. These genes include Tead1, Wwtr1 (also known as Taz), Stk3 (also known as Mst2), Yap1 (also known as Yap), Mst1 (also known as Stk4), Lats2, and Lats1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Among them, Tead1, Yap1, and Wwtr1 exhibited high expression in the samples of mouse BMSCs from the Model\u0026thinsp;+\u0026thinsp;Irisin group, whereas Stk3, Mst1, Lats2, and Lats1 showed low expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further conducted correlation analysis on HR-DEGs. Correlations were observed among HR-DEGs in the heat map of correlation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), which aligns with the findings of the correlation scatter plot (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). This result suggests that HR-DEGs play a role in regulating the Hippo signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we performed functional enrichment analysis on the 7 differentially expressed genes related to human resources. The Gene Ontology (GO) analysis revealed an enrichment of differentially expressed genes (DEGs) involved in various biological processes (BP), including Hippo signaling and regulation of the canonical Wnt signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Furthermore, these DEGs were found to be associated with specific cellular components (CC), such as the spindle pole and RNA polymerase II transcription regulator (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eH), as well as molecular functions (MF), such as protein serine/threonine kinase activity and magnesium ion binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). The KEGG analysis revealed that the HR-DEGs were enriched in pathways, including the Hippo signaling pathway, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ-K.\u003c/p\u003e \u003cp\u003eThe results suggest that Irisin can potentially mitigate adipogenesis in BMSCs and enhance AA by regulating the Hippo pathway through HR-DEGs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDeciphering the irisin-mediated modulation of lipotoxicity in BMSCs through the MST1/2-YAP axis: a comprehensive analysis in aplastic anemia.\u003c/b\u003e To further investigate the role of Irisin in improving lipotoxicity of BMSCs in AA, we utilized three different algorithms to identify potential key HR-DEGs that have the potential to mitigate BMSCs lipotoxicity. Initially, we conducted LASSO regression analysis on the 7 HR-DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Subsequently, the SVM-RFE analysis method was applied to extract feature genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Finally, we assessed the significance of genes using the random forest algorithm (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Finally, we identified three overlapping genes: Mst1, Stk3 (Mst2), and Yap1 (Yap) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). MST1/2 is the central kinase in the Hippo pathway, with YAP acting as the downstream effector. Upon activation of MST1/2, it facilitates the phosphorylation of YAP, causing its sequestration in the cytoplasm. Consequently, YAP gets inhibited, fostering adipocyte differentiation [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Thus, Irisin has the potential to enhance the impact of excessive adipogenesis in BMSCs on AA by activating the MST1/2-YAP pathway within the Hippo signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eValidation results from RT-qPCR showed downregulation of Yap1 expression in the BMSCs of mice in the Model group compared to the Control group. Conversely, the expression of Mst1 and Mst2 was upregulated in the Model group. However, the use of Irisin in the Model group effectively blocked the upregulation of Mst1 and Mst2 expression and restored the downregulated expression of Yap1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). In line with this, BMSCs isolated from AA patients' bone marrow showed upregulated expression of Mst1 and Mst2, while the expression of Yap1 was downregulated, as compared to the Normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eIn conclusion, the MST1/2-YAP pathway is involved in correcting the bias of differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) towards adipocytes and influencing the core pathway of adipogenesis in Irisin. It is accomplished by inhibiting the activation of MST1/2, promoting YAP's expression and nuclear translocation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin's modulatory effect on BMSC differentiation: inhibiting adipogenesis and enhancing osteogenesis through the MST1/2-YAP axis.\u003c/b\u003e Based on the previous bioinformatics analysis results, we continued to validate the subsequent mechanistic study of BMSCs extracted from mice with AA. CCK-8 experiments revealed that Irisin concentrations ranging from 1 to 20 ng/mL had no impact on the proliferation of BMSCs. It is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e9\u003c/span\u003eA. The results of the Live-Death staining also corroborated similar findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-C). Hence, a concentration of 20 ng/mL of Irisin was selected for the subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of staining with Oil Red O, Alizarin Red S, and ALP confirmed that Irisin inhibited the differentiation of BMSCs into adipocytes and promoted their differentiation into osteoblasts, compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e9\u003c/span\u003eD-F). Western blot analysis revealed that Irisin treatment inhibited MST1 and MST2 expression in BMSCs during osteogenic or adipogenic differentiation induction, compared to the PBS group. Additionally, Irisin treatment led to an increase in the expression level of YAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e9\u003c/span\u003eG-H). Furthermore, Irisin decreases the expression of adipogenic genes such as LPL, FABP4, PPARγ, CEBPα, and PERILIPIN while increasing the expression levels of osteogenic genes like Runx2, ALP, OPN, and OCN (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e9\u003c/span\u003eI-L). Immunofluorescence staining revealed that treatment with Irisin promoted the co-localization of YAP with the nucleus in BMSCs during the induction process of osteogenic or adipogenic differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e9\u003c/span\u003eM-N).\u003c/p\u003e \u003cp\u003e In conclusion, the ytg6results above demonstrate that Irisin inhibits the expression of MST1/2, increases YAP's expression, and facilitates YAP's nuclear localization. As a result, it effectively hinders the differentiation of BMSCs into adipocytes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin's regulatory mechanism on BMSC adipogenesis: a comprehensive study unveiling the inhibitory role of the MST1/2-YAP signaling pathway.\u003c/b\u003e To further investigate whether Irisin regulates adipogenesis of BMSCs through the MST1/2-YAP pathway, we initially constructed the MST1/2 overexpression model (Mst1/2-OE). It was achieved by overexpressing the Mst1/2 genes using adenoviral vectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). In the Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group, overexpression of Mst1/2 reversed the inhibitory effect of Irisin on the differentiation of BMSCs into adipocytes. Additionally, overexpression of Mst1/2 also blocked the upregulation of YAP expression by Irisin during the adipogenic differentiation of BMSCs compared to the Irisin\u0026thinsp;+\u0026thinsp;NC-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eB-D). This finding suggests that Irisin suppresses the adipogenic differentiation of BMSCs through the MST1/2 pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe YAP inhibitor verteporfin (VP) consistently reversed the corrective effect of Irisin on the differentiation of BMSCs into adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eE-G). In contrast, the overexpression of YAP amplified the inhibitory impact of Irisin on the differentiation process of BMSCs into adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eH-K). Moreover, the overexpression of VP and YAP did not result in changes in the expression levels of Mst1 and Mst2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eJ). This finding suggests that Irisin may potentially suppress the adipogenesis of BMSCs by interfering with the MST1/2-YAP pathway.\u003c/p\u003e \u003cp\u003eTo further validate this conclusion, we co-transfected Mst1/2-OE and Yap-OE plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eL). The results of Oil Red O staining showed that in the Mst1/2-OE\u0026thinsp;+\u0026thinsp;Yap-OE group, YAP was overexpressed and inhibited the adipocyte differentiation that was promoted by Mst1/2-OE (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eM). The western blot results indicate that the overexpression of YAP counteracted the inhibitory effect of MST1/2 overexpression on YAP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eN). Furthermore, real-time quantitative PCR (RT-qPCR) analysis demonstrated that overexpression of YAP reversed the upregulation of lipogenesis-related genes, including LPL, FABP4, PPARγ, CEBPα, and PERILIPIN induced by MST1/2 overexpression, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003eO.\u003c/p\u003e \u003cp\u003eIn summary, the results suggest that Irisin inhibits the differentiation of bone marrow mesenchymal stem cells (BMSCs) into adipocytes by suppressing the MST1/2-YAP signaling pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin mediates mitochondrial homeostasis and suppresses adipogenic differentiation in BMSCs through MST1/2-YAP signaling.\u003c/b\u003e Mitochondria play a vital role in the lineage differentiation of MSCs [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The inner mitochondrial membrane (IMM) plays a pivotal role in numerous functions, including the electron transport chain (ETC), oxidative phosphorylation (OXPHOS), energy transfer, and ion transport. Maintaining the homeostasis of the IMM is crucial for preserving normal mitochondrial function [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Studies have demonstrated that the MST1/2-YAP signaling pathway could enhance the regulation of mitochondrial function, leading to improvements in various disease states [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Hence, we propose that Irisin has the potential to enhance the homeostasis of the mitochondrial inner membrane system and suppress adipocyte differentiation of BMSCs via the MST1/2-YAP signaling pathway.\u003c/p\u003e \u003cp\u003eTo confirm this hypothesis, we initially examined the impact of Irisin on mitochondrial biogenesis during the process of adipogenic differentiation in BMSCs. The results from MitoTracker staining and quantification demonstrated that the Irisin group decreased the fluorescence intensity of MitoTracker compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eA). This result suggests that Irisin plays a role in suppressing mitochondrial biogenesis during adipocyte differentiation. The Western blot results consistently demonstrated that the Irisin group suppressed the expression of PGC-1α compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eB). Moreover, the analysis of mitochondrial DNA quantification revealed that treatment with Irisin reduced the mitochondrial DNA/nuclear DNA ratio compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eC). This finding further confirms that Irisin hinders the process of mitochondrial biogenesis and enhances the quantity of mitochondria during adipogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether Irisin affects mitochondrial biogenesis and its impact on mitochondrial respiration, we observed the oxidative phosphorylation and glycolysis in BMSCs cells treated with Irisin under adipogenic differentiation conditions. The results of the mitochondrial oxygen consumption rate (OCR) detection during the adipogenic differentiation process of BMSCs demonstrated that Irisin treatment suppressed OCR, including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity, in comparison to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eD-E). The Irisin group showed a higher extracellular acidification rate (ECAR) than the PBS group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eF. These findings suggest that Irisin inhibits the mitochondrial oxidative phosphorylation activity and enhances the glycolytic pathway during adipogenic differentiation of BMSCs. Furthermore, compared to PBS, the administration of Irisin increased the production of glycolytic ATP and total ATP during adipogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eNext, we conducted additional investigations to examine the effects of Irisin on reactive oxygen species (ROS) and membrane potential. DCFH-DA staining and the obtained quantitative results demonstrated that treatment with Irisin inhibited the levels of reactive oxygen species (ROS) in cells during the adipogenic differentiation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eH). Additionally, it was observed that Irisin treatment increased the mitochondrial membrane potential (MMP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eI). These findings suggest that Irisin treatment improves mitochondrial function impairment during the adipogenic differentiation process of BMSCs. Furthermore, the mRNA expression of uncoupling proteins UCP1, UCP2, and UCP3 was observed to be downregulated in the Irisin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eJ), thus suggesting a decrease in the activity of proton leakage channels and a subsequent reduction in proton leakage. It resulted in the accumulation of protons on the mitochondrial inner membrane, increasing the mitochondrial membrane potential. This finding shows that while Irisin decreases mitochondrial respiration levels, it increases the potential across the inner membrane. Additionally, RT-qPCR analysis demonstrated a decrease in Irisin expression at the mRNA level, specifically in the mitochondrial inner membrane fusion protein OPA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e11\u003c/span\u003eK). This result suggests that Irisin facilitates the fusion of mitochondrial inner membranes, thereby inhibiting the adipogenic differentiation of BMSCs.\u003c/p\u003e \u003cp\u003eThe results above indicate that Irisin inhibits mitochondrial biogenesis, the mitochondrial respiratory chain (ETC), and reactive oxygen species (ROS) levels. Additionally, it inhibits mitochondrial inner membrane fusion. Furthermore, it increases the mitochondrial inner membrane potential to enhance the homeostasis of the mitochondrial inner membrane system. As a result, it inhibits the differentiation of BMSCs into adipocytes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin enhances mitochondrial inner membrane homeostasis and inhibits adipogenic differentiation in BMSCs via the MST1/2-YAP signaling pathway.\u003c/b\u003e To further investigate whether Irisin regulates the homeostasis of the mitochondrial inner membrane system and inhibits the differentiation of BMSCs into adipocytes through the MST1/2-YAP signaling pathway, we detected the mitochondrial OCR during BMSCs' adipogenic differentiation. It was observed that Mst1/2-OE and the YAP inhibitor VP both blocked the Irisin-mediated inhibition of OCR during the induction of adipogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eA) and reversed the upregulation of ECAR, glycolytic ATP, and total ATP production induced by Irisin (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eB-C) in comparison to the Irisin\u0026thinsp;+\u0026thinsp;DMSO\u0026thinsp;+\u0026thinsp;NC-OE group. Furthermore, the overexpression of Mst1/2 and VP antagonized the inhibitory effect of Irisin on intracellular ROS levels and the beneficial effect on mitochondrial membrane potential during adipogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eD-E). Additionally, Irisin treatment restored the suppressed proton leak channel activity and fusion of the mitochondrial inner membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eF). It suggests that Irisin may enhance the homeostasis of the mitochondrial inner membrane system via the involvement of MST1/2 and YAP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then proceeded to validate whether Irisin regulates the homeostasis of the mitochondrial inner membrane through the MST1/2-YAP pathway. The results of the OCR analysis demonstrated that Yap overexpression in the Mst1/2-OE\u0026thinsp;+\u0026thinsp;Yap-OE group effectively inhibited the increase in OCR caused by Mst1/2 overexpression. Yap overexpression also led to a decrease in ECAR, glycolytic ATP, and total ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eG-I). Simultaneously, Yap overexpression counteracted the increase in intracellular ROS levels and the decrease in mitochondrial membrane potential observed during adipogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eJ-K). The alteration in membrane potential is linked to the recovery of increased proton leak channel activity resulting from Yap overexpression in Mst1/2-OE treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eFurthermore, the overexpression of Yap in the Mst1/2-OE\u0026thinsp;+\u0026thinsp;Yap-OE group blocked the promoting effect of Mst1/2 overexpression on mitochondrial inner membrane fusion during adipogenic induction, as observed in the Mst1/2-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e12\u003c/span\u003eL). Furthermore, this finding provides additional evidence of the impact of Yap overexpression on mitochondrial homeostasis disruption. It supports the hypothesis that Irisin enhances the stability of the mitochondrial inner membrane system via the MST1/2-YAP pathway.\u003c/p\u003e \u003cp\u003eFurthermore, we investigated whether Irisin regulates the homeostasis of the mitochondrial inner membrane system to inhibit the differentiation of BMSCs into adipocytes via the MST1/2-YAP signaling pathway. We treated BMSCs with the mitochondrial respiratory chain inhibitor Rotenone to achieve this. Compared to the DMSO group, treatment with the YAP inhibitor VP enhanced mitochondrial oxidative phosphorylation activity during adipogenesis, inhibition of glycolysis, and reduction in both glycolytic and total ATP production. However, the effects of VP were attenuated by the addition of fisetin, as demonstrated in Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA-C. In contrast, YuTeng ketone mitigated the heightened VP levels during lipogenesis through the reduction of cellular ROS levels and mitochondrial membrane potential (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eD-E). The results obtained from RT-qPCR showed an upregulation of mRNA expression levels of coupling proteins UCP1, UCP2, UCP3, and mitochondrial inner membrane fusion protein OPA1 in the group treated with VP compared to the DMSO group. However, the group treated with VP\u0026thinsp;+\u0026thinsp;Rotenone showed a restoration of this change when Rotenone was administered (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eF). Furthermore, using fish triterpenes has counteracted the promoting effect of VP on the differentiation of BMSCs into adipocytes (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eG-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results above suggest that Irisin enhances the homeostasis of the mitochondrial inner membrane system by inhibiting the MST1/2-YAP signaling pathway, consequently inhibiting the differentiation of BMSCs into adipocytes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIrisin ameliorates adipogenic differentiation and mitochondrial dysfunction in BMSCs through MST1/2-YAP pathway in an AA mouse model.\u003c/b\u003e Our previous research has confirmed that Irisin plays a role in inhibiting the differentiation of BMSCs into adipocytes, leading to improvements in adiposity. To further investigate the inhibitory effect of Irisin on the differentiation of mice BMSCs into adipocytes, we intravenously injected Mst1/2-OE, which was constructed \u003cem\u003ein vitro\u003c/em\u003e, into mice to explore the progression of adipogenesis.\u003c/p\u003e \u003cp\u003eThe blood cell counts in the peripheral blood of mice were analyzed. The Model\u0026thinsp;+\u0026thinsp;NC-OE group exhibited a decrease in blood cell counts compared to the Control\u0026thinsp;+\u0026thinsp;NC-OE group. Conversely, the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group showed a notable increase in blood cell counts compared to the Model\u0026thinsp;+\u0026thinsp;NC-OE group. However, the overexpression of Mst1/2 diminished the beneficial effect of Irisin on blood cell counts according to Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The Model\u0026thinsp;+\u0026thinsp;Irisin group exhibited a notable increase in hematopoietic colony formation compared to the Model group, as supported by the experimental findings. Nevertheless, the overexpression of Mst1/2 counteracted the hematopoietic cell proliferation enhancement induced by Irisin (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This result suggests that the overexpression of Mst1/2 undermines the hematopoietic improvement facilitated by Irisin in AA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePeripheral blood WBC, NEU, PLT, RBC counts, and HGB concentration (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;8) in each group of mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWBC\u003c/p\u003e \u003cp\u003e(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNEU absolute value (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePLT\u003c/p\u003e \u003cp\u003e(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRBC\u003c/p\u003e \u003cp\u003e(10\u003csup\u003e12\u003c/sup\u003e/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHGB\u003c/p\u003e \u003cp\u003e(g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eControl\u0026thinsp;+\u0026thinsp;NC-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e449.4\u0026thinsp;\u0026plusmn;\u0026thinsp;45.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e154.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e431.1\u0026thinsp;\u0026plusmn;\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e156.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e479.3\u0026thinsp;\u0026plusmn;\u0026thinsp;36.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e156.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eModel\u0026thinsp;+\u0026thinsp;NC-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e95.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e90.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e89.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e91.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e90.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e94.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003cp\u003e+Irisin\u0026thinsp;+\u0026thinsp;NC-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e263.0\u0026thinsp;\u0026plusmn;\u0026thinsp;27.2\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e107.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e333.5\u0026thinsp;\u0026plusmn;\u0026thinsp;29.4\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e120.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e414.0\u0026thinsp;\u0026plusmn;\u0026thinsp;31.3\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e147.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eModel\u0026thinsp;+\u0026thinsp;Mst1/2-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e49.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e80.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e45.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e78.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e40.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e77.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003cp\u003e+Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e116.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e92.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e123.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e94.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e130.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e98.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: WBC, white blood cell; NEU, neutrophil; PLT, platelets; RBC, red blood cell; HGB, hemoglobin; * indicates a significant difference compared to the Control\u0026thinsp;+\u0026thinsp;NC-OE group with a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, △ indicates a significant difference compared to the Model\u0026thinsp;+\u0026thinsp;NC-OE group with a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ▲ indicates a significant difference compared to the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group with a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe number of CFU-E, CFU-GM, and CFU-MK colonies in each group of mice (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;8).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCFU-E (10\u003csup\u003e5\u003c/sup\u003e cells)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCFU-GM (10\u003csup\u003e5\u003c/sup\u003e cells)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCFU-MK (10\u003csup\u003e5\u003c/sup\u003e cells)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u0026thinsp;+\u0026thinsp;NC-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e133.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e92.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e50.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u0026thinsp;+\u0026thinsp;NC-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e92.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e73.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u0026thinsp;+\u0026thinsp;Mst1/2-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003e△\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e27.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003e▲\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: CFU stands for colony forming unit; E represents erythroid; GM represents granulocyte macrophage; MK represents megakaryocytic; * indicates a significant difference compared to the Control\u0026thinsp;+\u0026thinsp;NC-OE group with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, △ indicates a significant difference compared to the Model\u0026thinsp;+\u0026thinsp;NC-OE group with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ▲ indicates a significant difference compared to the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAdditional HE staining showed that the overexpression of Mst1/2 in the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group blocked the rescuing effect of Irisin on bone marrow hypoplasia compared to the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group. This result also reduced nucleated cell count, impaired megakaryocyte development, increased lipid droplets, and decreased hematopoietic tissue area in Model mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e13\u003c/span\u003eA). These findings suggest that the overexpression of Mst1/2 undermines the positive effects of Irisin on bone marrow failure and excessive marrow adiposity induced by AA. To further confirm the inhibitory effect of Irisin on adipogenesis in the bone marrow of the mouse model through overexpression of Mst1/2, we performed experiments on the femurs of mice. We utilized Oil Red O and Perilipin immunofluorescence staining to examine alterations in adipocytes within the bone marrow. The Oil red O staining results indicated an increase in the number of adipocytes in the mouse bone marrow of the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group compared to the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e13\u003c/span\u003eB). Immunofluorescence staining results supported these findings, suggesting that the overexpression of Mst1/2 in the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group inhibited the suppressive effect of Irisin on adipocytes in the mouse bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e13\u003c/span\u003eC). The expression of lipogenic-related genes in mouse bone marrow was determined using RT-qPCR. The findings indicated that Mst1/2 overexpression mitigated the downregulation of LPL, FABP4, PPARγ, CEBPα, and PERILIPIN gene expression by Irisin (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e13\u003c/span\u003eD). These results suggest that the overexpression of Mst1/2 counteracted Irisin's inhibitory effect on bone marrow failure and marrow adipogenesis in the mouse model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, Western blot analysis of bone marrow samples showed that the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group exhibited a reversal in the upregulation of MST1 and MST2, as well as YAP downregulation, which was induced by AA treatment, compared to the Model\u0026thinsp;+\u0026thinsp;NC-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e13\u003c/span\u003eE). Nevertheless, the upregulation of YAP expression induced by Irisin was reversed upon overexpression of Mst1/2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e13\u003c/span\u003eF). This finding suggests that Irisin activation occurs through the inhibition of the MST1/2-YAP pathway, suppressing the differentiation of mouse BMSCs into adipocytes and improving adipose tissue function.\u003c/p\u003e \u003cp\u003eTo further validate the improvement of mitochondrial inner membrane homeostasis by Irisin through the MST1/2-YAP pathway, we inhibited the differentiation of BMSCs into adipocytes and evaluated its impact on acidosis (AA) in a mouse model. Subsequently, BMSCs were isolated from the bone marrow of mice in each group. The detection of total ATP and ROS levels exhibited the following results: compared to the Control\u0026thinsp;+\u0026thinsp;NC-OE group, the Model\u0026thinsp;+\u0026thinsp;NC-OE group showed a decrease in ATP levels and an increase in ROS levels. These findings suggest that AA induces damage to the respiratory chain and energy conversion function of BMSCs' mitochondria and impairs the homeostasis of the mitochondrial inner membrane. Compared to the Model\u0026thinsp;+\u0026thinsp;NC-OE group, the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group showed a rescue in the decrease of ATP levels and an increase in ROS levels caused by AA in BMSCs after Irisin treatment. It improved the damage to the mitochondrial respiratory chain and energy conversion function in the BMSCs of model mice.\u003c/p\u003e \u003cp\u003eNevertheless, the rescuing effect of Irisin on mitochondrial inner membrane homeostasis was hindered by the overexpression of Mst1/2, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e14\u003c/span\u003eA-B. Furthermore, treatment with Irisin ameliorated the reduced mitochondrial membrane potential in BMSCs induced by AA. However, when comparing the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;NC-OE group to the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group, overexpression of Mst1/2 in the latter group counteracted the favorable effects of Irisin on the mitochondrial membrane potential in the experimental mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e14\u003c/span\u003eC). Irisin treatment restores the upregulation of mRNA expressions of uncoupling proteins UCP1, UCP2, and UCP3, which AA induces. This restoration inhibits the increased activity of mitochondrial inner membrane proton leak channels caused by AA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, the beneficial effect was hindered by the overexpression of Mst1/2 in the Model\u0026thinsp;+\u0026thinsp;Irisin\u0026thinsp;+\u0026thinsp;Mst1/2-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e14\u003c/span\u003eD). Furthermore, Irisin treatment in BMSCs decreased the mRNA expression of OPA1, a protein involved in mitochondrial inner membrane fusion, compared to the Model\u0026thinsp;+\u0026thinsp;NC-OE group. This result suggests that Irisin can potentially enhance AA-induced mitochondrial inner membrane fusion. However, based on this premise, the expression of Mst1/2 reversed this alteration (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e14\u003c/span\u003eD). The results mentioned above confirm that Irisin improves damage to the homeostasis of the mitochondrial membrane system in model mice BMSCs by inhibiting the activation of the MST1/2-YAP pathway. In summary, Irisin enhances the homeostasis of the mitochondrial inner membrane system by inhibiting the activation of the MST1/2-YAP pathway. It also prevents the differentiation of BMSCs into adipocytes and relieves AA-induced bone marrow failure and marrow adiposity, ultimately mitigating acidosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAplastic anemia (AA) is a condition marked by the dysfunction of hematopoiesis, resulting in the abnormal production of red blood cells, white blood cells, and platelets in the bone marrow [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Previous studies have shown that patients with AA undergo adipogenesis in their bone marrow, where hematopoietic cells are replaced by adipocytes [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. This study further elucidated that lipidization results from the excessive differentiation of mesenchymal stem cells (MSCs) into adipocytes. Previous studies have identified irisin as a hormone linked to fat and metabolism [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. This study revealed that Irisin is involved in fat metabolism and influences the differentiation direction of MSCs. Irisin could delay the differentiation of MSCs into adipocytes by inhibiting the activation of the MST1/2-YAP signaling pathway. This slowdown or mitigation of the progression of AA occurs as a result.\u003c/p\u003e \u003cp\u003eThe MST1/2-YAP signaling pathway regulates cell proliferation, apoptosis, and differentiation [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. This study confirms that the signaling pathway also has a regulatory role in differentiating MSCs into adipocytes. In comparison to previous studies, we have provided additional clarification regarding the involvement of Irisin in this pathway, subsequently influencing the differentiation of MSCs. Mitochondria play a vital role in cellular energy production and regulation. This study indicates, for the first time, that the differentiation of MSCs could be influenced by modulating the homeostasis of the mitochondrial inner membrane system. Compared to previous studies focused on mitochondrial function and cell fate decisions, this study offers a fresh and distinct understanding of the mitochondrial function of MSCs.\u003c/p\u003e \u003cp\u003eThis study employed two advanced techniques, single-cell sequencing and spatial transcriptomics, enabling us to examine the differentiation characteristics of MSCs at the single-cell level and accurately determine their specific location within the bone marrow tissue. Compared to previous research methods that rely on group averages, these two techniques offer more extensive and precise information [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Considering the role of Irisin in inhibiting MSC differentiation into adipocytes and improving mitochondrial inner membrane system homeostasis, it can potentially become a promising therapeutic target for AA. However, additional clinical research is required to ascertain Irisin's precise mechanisms, dosage, treatment timing, and potential side effects. Additionally, future studies could investigate additional potential factors that regulate MSC differentiation.\u003c/p\u003e \u003cp\u003eBased on the results above, the following preliminary conclusions could be drawn: Irisin can increase the expression of Yap and facilitate its nuclear translocation by suppressing the expression of Mst1/2. This action restores the stability of the mitochondrial inner membrane system in adriamycin-damaged mesenchymal stem cells, preventing their differentiation into adipocytes and subsequently ameliorating adriamycin-induced nephropathy (Fig.\u0026nbsp;15). This study revealed the therapeutic mechanism of Irisin in the treatment of AA, offering novel insights into the pathogenesis of this condition. Unfortunately, because of time and budget limitations, we could not validate the mechanism using clinical samples. It undermined the reliability and scientific validity of our conclusions. Henceforth, we will persist in exploring clinical mechanisms.\u003c/p\u003e \u003cp\u003eBone marrow fatty infiltration is a prominent characteristic of acquired aplastic anemia (AA) [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. This study uncovered how the excessive differentiation of mesenchymal stem cells (MSCs) into adipocytes contributes to AA, offering novel insights into the mechanistic understanding of the disease. Previous research has demonstrated that Irisin can enhance the homeostasis of the mitochondrial inner membrane system by inhibiting the activation of the Mst1/2-YAP signaling pathway. Consequently, this inhibition leads to the suppression of mesenchymal stem cell differentiation into adipocytes, offering a promising new approach for treating AA. Advanced techniques, such as single-cell sequencing, spatial transcriptomics, and transcriptome sequencing, have been utilized to provide methodological references for future studies. The intervention of Irisin could reverse adipogenesis in AA bone marrow, thereby restoring hematopoietic function and providing practical clinical benefits to patients.\u003c/p\u003e \u003cp\u003eThis study primarily focused on AA model mice. However, it is important to consider that the physiological differences between humans and mice may impact the translation of research findings to clinical settings. While this study primarily investigates the MST1/2-YAP signaling pathway, it is important to note that other intricate signaling pathways may also regulate the differentiation of mesenchymal stem cells. The study utilized Irisin as a treatment; however, the potential side effects and long-term safety in the human body have yet to be definitively established. Additional clinical trials are required to assess the effectiveness of Irisin in the treatment of AA and to evaluate its safety and efficacy in humans. This study could offer guidance in finding potential drugs capable of regulating the MST1/2-YAP signaling pathway. Given the strong correlation between the mechanisms addressed in this study and the hematopoietic function of the bone marrow, additional research should be undertaken to examine their potential therapeutic applications in other hematopoietic disorders. This study aims to investigate the precise regulation of the MST1/2-YAP signaling pathway by Irisin and contribute to a more solid theoretical foundation for its clinical application.\u003c/p\u003e \u003cp\u003eThis research has profound implications as it introduces new directions and methods for thalassemia treatment. However, further clinical studies are necessary to validate its effectiveness and safety in humans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that supports the findings of this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by our Clinical Ethics Committee and complied with the Declaration of Helsinki (KYLL-2019-023). This experimental program and animal use protocol were approved by our institutional animal ethics committee (NO. 202309001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXL and HL designed the study. BG collated the data, designed and developed the database, carried out data analyses and produced the initial draft of the manuscript. DK contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by SDU-KI Collaborative Research Project of Qilu Medical College of Shandong University (Project No. SDU-KI-2019-15) and Natural Science Foundation of Shandong Province (Project No. ZR2023MH341).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication was obtained from the participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoore CA, Krishnan K. Aplastic Anemia. In: StatPearls. Treasure Island (FL): StatPearls Publishing; July 17, 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeZern AE, Churpek JE. Approach to the diagnosis of aplastic anemia. Blood Adv. 2021;5(12):2660\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/bloodadvances.2021004345\u003c/span\u003e\u003cspan address=\"10.1182/bloodadvances.2021004345\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Liu H. Pathogenesis of aplastic anemia. Hematology. 2019;24(1):559\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/16078454.2019.1642548\u003c/span\u003e\u003cspan address=\"10.1080/16078454.2019.1642548\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJavan MR, Saki N, Moghimian-Boroujeni B. 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Mol Med Rep. 2022;25(1):27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/mmr.2021.12543\u003c/span\u003e\u003cspan address=\"10.3892/mmr.2021.12543\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-hematology-and-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"exho","sideBox":"Learn more about [Experimental Hematology \u0026 Oncology](http://ehoonline.biomedcentral.com)","snPcode":"40164","submissionUrl":"https://submission.nature.com/new-submission/40164/3","title":"Experimental Hematology \u0026 Oncology","twitterHandle":"@SN_Oncology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Irisin, Aplastic Anemia, Mesenchymal Stem Cells, MST1/2-YAP Pathway, Gene Expression Analysis, Mitochondrial Homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-4329016/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4329016/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAplastic anemia (AA) is a debilitating hematological disorder characterized by bone marrow failure. Recent advancements in mesenchymal stem cell (MSC) research have highlighted potential therapeutic avenues, particularly through the modulation of cellular pathways influenced by novel agents like Irisin. This study investigates Irisin's effects on MSCs in the context of AA using advanced techniques such as single-cell sequencing and spatial transcriptomics. Irisin administration in AA model mice significantly altered gene expression in MSCs, particularly affecting 935 genes associated with the Hippo signaling pathway, notably the MST1/2-YAP axis. These changes were linked to decreased adipogenic differentiation and enhanced mitochondrial membrane system homeostasis. In vitro experiments supported these findings, showing Irisin's capability to inhibit the MST1/2-YAP signaling pathway and suppress adipogenesis in bone marrow stem cells (BMSCs). Corresponding in vivo studies demonstrated that Irisin treatment not only downregulated Mst1 and Mst2 but also upregulated Yap expression. Importantly, these molecular alterations led to reduced bone marrow adiposity and improved hematopoietic function in AA mice, showcasing Irisin's potential as an effective treatment option. The study underscores the critical role of the MST1/2-YAP pathway in mediating Irisin's therapeutic effects, suggesting promising strategies for AA management through targeted MSC pathway modulation.\u003c/p\u003e","manuscriptTitle":"Deciphering the Role of the MST1/2-YAP Axis in Irisin-Treated Aplastic Anemia: Implications for Mesenchymal Stem Cell Function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 03:23:56","doi":"10.21203/rs.3.rs-4329016/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-04-27T02:50:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-26T11:54:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental Hematology \u0026 Oncology","date":"2024-04-26T10:15:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-hematology-and-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"exho","sideBox":"Learn more about [Experimental Hematology \u0026 Oncology](http://ehoonline.biomedcentral.com)","snPcode":"40164","submissionUrl":"https://submission.nature.com/new-submission/40164/3","title":"Experimental Hematology \u0026 Oncology","twitterHandle":"@SN_Oncology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2a35bdd6-7089-4500-adb9-5bb561eba137","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-02T03:23:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 03:23:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4329016","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4329016","identity":"rs-4329016","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}