Extracellular vesicles derived from live or apoptotic mesenchymal stem cells: comparison of the effects of two extracellular vesicles on liver fibrosis

Extracellular vesicles derived from live or apoptotic mesenchymal stem cells: comparison of the effects of two extracellular vesicles on liver fibrosis | 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 Extracellular vesicles derived from live or apoptotic mesenchymal stem cells: comparison of the effects of two extracellular vesicles on liver fibrosis Ruobing Ju, Siyuan Tian, Bo Li, Miao Zhang, Shuoyi Ma, Yinan Hu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6970215/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Mar, 2026 Read the published version in BMC Gastroenterology → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Mesenchymal stem cells (MSCs) have become a promising treatment of liver fibrosis which is a key process in liver diseases. Recent studies showed that transplanted mesenchymal stem cells (MSCs) undergo rapid apoptosis and the apoptotic extracellular vesicles (ApoEVs) derived from MSCs exhibited stronger immunosuppressive capability. But the effect and the mechanisms of ApoEVs in liver fibrosis remain unclear. The functional differences between ApoEVs and extracellular vesicles (EVs) have yet to be elucidated. This study aims to compare their therapeutic effects on liver fibrosis in order to optimize existing treatment strategies. Methods: ApoEVs and EVs were isolated by density gradient centrifugation and illustrated by TEM and NTA. A CCl4-induced liver fibrosis mouse model was treated with equal doses of ApoEVs and EVs. Histopathological analysis was performed on liver sections, serological indicators, fibrosis-related gene expression, macrophage polarization, and the activation status of hepatic stellate cells (HSCs) were analyzed. Subsequently, miRNA-sequencing analysis was conducted to identify potential pathway. Results: Our results demonstrated that ApoEVs had fourfold higher protein yield than EVs, and ApoEVs exhibited a significant superior ability to improve liver fibrosis. In vitro, ApoEVs enhanced macrophage polarization and suppressed HSC activation more effectively, thereby reducing the degree of fibrosis. The underlying molecular mechanism likely due to the enrichment of more miRNAs targeting the PI3K-Akt pathway in ApoEVs. Conclusion: These findings showed that ApoEVs exhibit better efficacy than EVs in treating liver fibrosis. Besides, the findings highlighted their therapeutic potential, clarified functional differences, and suggested ApoEVs as a promising strategy for liver disease treatment. Liver fibrosis mesenchymal stem cells apoptotic extracellular vesicles extracellular vesicles macrophages phenotype hepatic stellate cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Liver fibrosis or cirrhosis may result from the progression of chronic liver diseases caused by various etiologies, which is facing a significant health challenge all over the world[ 1 ]. Without effective intervention in time, the morbidity and mortality will be increased significantly because of symptoms such as portal hypertension and gastrointestinal bleeding. In China, about 300 million people suffer from these diseases. In the world, liver cirrhosis is the 11th most common cause of death and together with liver carcinoma, accounts for 3.5% of all deaths[ 2 ]. Orthotopic liver transplantation is the only curative treatment for end-stage liver diseases. But its widespread application has been limited by issues such as donor shortage and poor prognosis[ 3 ]. Therefore, many regenerative or alternative therapies are studied aiming to treat liver fibrosis or cirrhosis. In recent years, stem cell-based therapies have provided new possibilities for the treatment of liver diseases. In chronic liver diseases, mesenchymal stem cells (MSCs) play a crucial role in regulating the immune microenvironment through direct differentiation or regeneration[ 4 ]. MSCs, sourced from bone marrow, adipose tissue, or umbilical cord, offer clinical advantages such as easy to clinical translation, low immunogenicity, and strong proliferative capacity et al. Currently, numerous clinical trials have been conducted in the field of MSC therapy for liver cirrhosis to demonstrate its effectiveness[ 5 ]. However, the exact mechanism of action is still unclear. Previous studies have indicated that MSCs can affect the inflammatory phenotype of macrophages through its paracrine function, or alleviates fibrosis by degrading extracellular matrix (ECM) or reducing the activation level of hepatic stellate cells (HSCs) [ 6 – 8 ]. Meanwhile, extracellular vesicles (EVs) especially exosomes, as mediators of intercellular communication, also play a significant role in the therapeutic effects of MSCs[ 9 ]. Exosomes can alleviate liver fibrosis by influencing the differentiation phenotype of immune cells or delivering microRNAs (miRNAs) to mediate YAP down-regulation[ 10 , 11 ]. The rapid apoptosis of MSCs following infusion has been considered a major factor limiting their sustained therapeutic effects[ 12 ]. Therefore, many studies aimed at enhancing the homing ability of MSCs, improving their survival, and ensuring their retention in vivo to improving their therapeutic efficacy. However, recent studies have shown that the immunosuppressive capacity of MSCs is attenuated when their apoptosis is resistant[ 13 ]. In other words, MSCs apoptosis is a crucial component of their therapeutic function on tissue repair and inflammation regulation[ 14 , 15 ]. Apoptosis is an important form of programmed cell death. Apoptotic extracellular vesicles (ApoEVs) are formed during the process of apoptosis, through budding and fragmentation of the cell membrane. ApoEVs are a unique subtype of EVs, with diameters ranging from 50 to 5000 nm[ 16 ]. They participate in intercellular communication, signaling, and immune regulation, which are similar to EVs produced by non-apoptotic MSCs. ApoEVs contain numerous bioactive molecules including proteins (such as CD81, CD9, etc.), lipids, RNA (mRNA, miRNA, etc.). After interacting with the recipient cells, they play indispensable roles in a variety of physiological and pathological environments[ 16 , 17 ]. Current research indicates that ApoEVs can play functional roles in a variety of diseases. In the event of inflammatory injury, ApoEVs modulate the population of immune cells and macrophage inflammatory phenotypes in various diseases, including acute kidney injury, colitis, allergic pulmonary inflammation, and skin wound healing. In addition, they can be engulfed by endothelial cells or basal cells for tissue regeneration. Moreover, ApoEVs can interact directly with recipient cells, regulating activity through membrane surface ligands et al[ 18 – 20 ]. However, no studies have yet investigated ApoEVs in liver diseases, particularly in liver fibrosis. In terms of liver fibrosis treatment, previous research has mainly focused on the regulation of macrophage inflammatory phenotypes by EVs, particularly exosomes. Or focusing on the inhibition of HSCs activation, the degradation of ECM. However, the precise function of ApoEVs in liver fibrosis has not yet been explored. At the same time, there has been no comparative study on the effects of ApoEVs and EVs on liver fibrosis. Therefore, this study aims to dissect the differences between these two types of vesicles and explore their respective therapeutic efficacy. In this study, we comparatively analyzed the biological roles of ApoEVs and EVs secreted by MSCs before and after apoptosis. We evaluated the therapeutic efficacy of both types of vesicles in improving fibrosis levels in a carbon tetrachloride (CCl4)-induced mouse model. Additionally, we compared their effects on macrophages and HSCs to demonstrate potential differences in functional properties. The goal is to explore more effective therapeutic approaches for liver fibrosis. 2. Materials and Methods 2.1 Experimental animal models The male C57BL/6J mice (aged 6–8 weeks) were purchased from and housed in the Laboratory Animal Center of the Air Force Military Medical University. The mice were housed in specific pathogen-free (SPF) and ventilated conditions in the Laboratory Animal Center, with 12-hour light/dark cycles, and food and water were provided ad libitum. The animal study protocols were approved by the Animal Welfare and Ethics Committee of the Air Force Military Medical University and performed according to the “Guidelines for the Care and Use of Laboratory Animals”. The liver fibrosis mouse model was established by continuous intraperitoneal injection of 0.2 mL/20g CCl4 [20% (v/v), dissolved in olive oil] twice a week for 8 weeks. Subsequently, the liver fibrosis mice were randomly divided into groups (n = 4–6) that received phosphate buffered saline (PBS), 150 µg ApoEVs diluted in 200 µl PBS and 150 µg EVs diluted in 200 µl PBS via tail vein injection. The control group was injected with an equal volume of PBS. After treatment, the mice were observed for an additional two weeks and were continually injected with CCl4 once a week. Two weeks later, the mice were euthanized by exposing to a 60% concentration carbon dioxide (CO 2 ) asphyxiation box. The process was rapid and minimally painful. After euthanasia, liver tissues were collected for further analysis. 2.2 Induction of MSCs apoptosis Human umbilical cord-derived MSCs (hUC-MSCs) in generation 4–6 were selected and cultured in complete medium supplemented with serum substitute (EV-depleted) until they reached 80–90% confluence. Then removing the medium and MSCs were washed three times with PBS. Subsequently, the complete medium containing 250 nM staurosporine (STS) (Cell Signaling Technology, USA) was added to MSCs[ 21 ]. After 12 hours of treatment, the apoptosis of MSCs was detected by morphological observation and flow cytometry analysis. 2.3 Isolation and Characterization of ApoEVs and EVs According to the previously optimized protocol, ApoEVs were collected through differential centrifugation[ 19 , 22 , 23 ]. Briefly, collected the culture supernatant from apoptotic MSCs at the 12-hour mark of MSCs apoptosis induction. Then, centrifuged the supernatant at 800 g for 10 min at 4 ℃ to to remove some cell debris and intact cells. Following, the supernatant was further collected and centrifuged at 16,000 g for 30 min at 4℃, discarded the supernatant, and retained ApoEVs pellet. Subsequently, resuspended the pellet gently in pre-cooled PBS, centrifuged at 16,000 g for 30 min at 4 ℃. Washed twice in this method to obtain final ApoEVs. ApoEVs were quantified by measuring the protein concentration via a BCA Protein Assay Kit (GlpBio, USA). EVs derived from MSCs were isolated by differential centrifugation as previously described[ 24 ]. Briefly, MSCs (hUC-MSCs) in generation 4–6 were cultured in complete medium (EV-depleted) until they reached 70–80% confluence. After 48 hours, collected the culture supernatant and centrifuged at 300 g for 10 min to remove some cell debris and intact cells. Further collected the supernatant and centrifuged at 2000 g for 20 min to remove larger cell debris and apoptotic bodies. Then, centrifuged the supernatant at 10,000 g for 30 min at 4°C to remove micro-vesicles and other larger particles. Finally, the supernatant was ultracentrifuged at 100,000 g for 70 min at 4°C, and washed the pellet with PBS at 100,000 g for 70 min to obtain EVs. EVs were quantified by measuring the protein concentration via a BCA Protein Assay Kit (GlpBio, USA). The morphology of ApoEVs and EVs were characterized using transmission electron microscopy (TEM). Nanoparticle tracking analysis (NTA) was used to analyze the particle size distribution and concentration of both ApoEVs and EVs. For apoptotic marker detection, purified ApoEVs were characterized by Western blot using anti-Caspase-3 and anti-β-Actin antibodies. For specific EVs markers, CD9, CD81 were used as positive controls, and calnexin, GM130 were used as negative controls for Western blot analysis. 2.4 Cell Culture HUC-MSCs were provided by the National Engineering Research Center (Tianjin AmCellGene Engineering Co., Ltd, China). MSCs were cultured in mesenchymal basal medium (Dakewe Biotech Co., Ltd. China) supplemented with serum substitute at 37°C in a 5% CO2 incubator. Cells in generation 4–6 were used for subsequent experiments. RAW264.7 cells, obtained from the American Type Culture Collection (ATCC), which were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, NY) and 1% penicillin–streptomycin. Mouse bone morrow-derived macrophages (BMDMs) were obtained as previously described[ 25 ]. BMDMs were isolated from the femurs and tibias of 6-8-week-old C57BL/6J mice. Subsequently, these cells were plated at a density of 2× 10 6 cells/mL and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, plus 40 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech Inc. USA) for 7 days. Human HSC lines LX-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, NY) and 1% penicillin–streptomycin. 2.5 Cell treatment For the phenotypic induction of macrophages, the M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes were established by stimulating BMDMs or RAW 264.7 cells with 100 ng/mL LPS (Sigma, USA)/20 ng/mL IFN-γ (Peprotech, USA) and 20 ng/mL IL-4 (Peprotech, USA) for 24 h, respectively. For the phenotypic induction of HSCs, LX-2 cells were stimulated with 8 ng/mL TGF-β1 (novoprotein, China) for 48 hours to establish an activated phenotype. To investigate the effects of ApoEVs and EVs on the inflammatory phenotype of macrophages and the activation of HSCs, 20 µg of ApoEVs or EVs were added to the culture medium of BMDMs, RAW 264.7 cells or LX-2 cells, respectively. After 12 hours of treatment, the cells were collected for subsequent analyses such as Western blot or qRT-PCR. 2.6 Biochemical analysis and histological staining The serum of mice was obtained at each time point. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed using an Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) and measured with a Microplate Reader (Thermo Fisher Scientific, USA). The liver tissue samples were prepared as paraffin-embedded sections by Servicebio, China. And then, sections were stained with hematoxylin and eosin (HE) for routine histological examination or with Sirius Red and Masson for fibrosis evaluation. Further quantification and analysis of collagen fiber were assessed using Image-J software (National Institutes of Health, USA). 2.7 Flow cytometric analysis of cell phenotypes Apoptotic MSCs were detected using an apoptosis staining kit (BD Biosciences, USA). After discarding the supernatant, the cells were washed three times with PBS and resuspended in the buffer provided by the apoptosis staining kit at a density of 1 × 10 7 /mL. PE-Annexin V and 7-AAD were added according to the recommended ratio, followed by incubation at room temperature in the dark for 15 min. Then, the flow cytometric data was analyzed on a FACSVerse flow cytometer (BD Bioscience, CA, USA) and analyzed with FlowJo software (TreeStar, Ashland, OR, US). 2.8 RNA isolation and real‑time PCR analysis Total RNA was extracted from liver tissues or cells using the RNAeasy Plus Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China), and reverse transcription was generated using PrimeScript™ RT Master Mix (RR036A, Takara, Tokyo). Then, qRT-PCR was conducted by TB Green Premix Ex Taq II (DRR820A, Takara, Tokyo) and a CFX96 Touch™ real-time PCR System (Bio-Rad, CA). β-actin (Actb or ACTB) was used as an internal control for quantification. PCR primers are shown in Table S1 . 2.9 Western blot analysis Whole proteins were extracted from tissues, cells or ApoEVs using RIPA Lysis Buffer (Beyotime biotechnology, China) supplemented with proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). The protein concentration was quantified using a BCA Protein Assay Kit (GlpBio, USA). Subsequently, equal amounts of protein samples (30 µg) were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad Biotechnology, USA). The membranes were blocked in TBST buffer containing 2.5% skim milk for 30 min. Then, the membranes were incubated overnight at 4 ℃ with the following primary antibodies: anti-Caspase-3 (9662, Cell Signaling Technology, USA; diluted at 1:1000), anti-GM130 (ab52649, Abcam, UK; diluted at 1:1000), anti-calnexin (10427-2-AP, Proteintech, USA; diluted at 1:1000), anti-CD81 (ab79559, Abcam, UK; diluted at 1:1000), anti-CD9 (ab236630, Abcam, UK; diluted at 1:1000), anti-Col1a1 (ab270993, Abcam, UK; diluted at 1:1000), anti-αSMA (19245, Cell Signaling Technology, USA; diluted at 1:1000), anti-iNOS (ab15323, Abcam, UK; diluted at 1:1000), anti-Arg-1 (ab124917, Abcam, UK; diluted at 1:1000), anti-STAT3 (10253-2-AP, Proteintech, USA; diluted at 1:1000), anti-STAT6 (9362, Cell Signaling Technology, USA; diluted at 1:1000), anti- NF-κB (8242, Cell Signaling Technology, USA; diluted at 1:1000), anti-AKT (4691, Cell Signaling Technology, USA; diluted at 1:1000), and anti-β-Actin (66009-1-Ig, Proteintech, USA; diluted at 1:5000). After washing with TBS containing 0.1% Tween-20, the membranes were incubated with peroxidase-conjugated secondary antibodies (Proteintech, China) at room temperature for 1 h. And blots were visualized using an enhanced chemiluminescence kit and detected by a gel imaging system (Bio-Rad Biotechnology, USA). Further quantification and analysis of protein expression were assessed using Image-J software (National Institutes of Health, USA). 2.10 The miRNA‑sequencing and bioinformatics analysis The miRNA-sequencing was provided by LCBio Co., Ltd (Hangzhou, China). Briefly, we collected three independent ApoEVs and EVs samples. RNA samples from both typed of vesicles were extracted, sequenced, and analysed by LCBio Co., Ltd (Hangzhou, China). The purity and quantity of RNA were checked using a Bioanalyzer 2100 (Agilent, CA, USA). Small-RNA libraries were sequenced on the illumina NovaseqTM 6000 platform by LC Bio Technology CO.,Ltd. Then we analysed the differential miRNAs between ApoEVs and EVs. P =1 was set as the threshold for significantly differential expression. A total of 96 significantly differential miRNAs were identified, with 69 of them being significantly enriched in ApoEVs. Subsequently, more than 111 miRNAs associated with liver cirrhosis were identified from the Human microRNA Disease Database. Compared with the 69 differential miRNAs, resulting in 10 highly differential miRNAs that are enriched in ApoEVs and strongly associated with liver cirrhosis. To predict the genes targeted by most aboundant miRNAs, two computational target prediction algorithms (TargetScan, v5.0 and Miranda, v3.3a) were used to identify miRNA binding sites. Finally, the data predicted by both algorithms were combined and the overlaps were calculated. The gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of these most aboundant miRNAs, miRNA targets were also annotated. 2.11 Statistical analysis The data were expressed as the mean values ± standard deviation. One-way analysis of variance and t-test were performed to identify the significant differences. A P value < 0.05 was considered significant. Statistical analysis was plotted by GraphPad Prism 8.0 (GraphPad Software, CA, USA). 3. Results 3.1 Isolation and Characterization of MSC-Derived ApoEVs and EVs MSCs utilized in this study had been previously characterized and identified in our earlier literature[ 26 ]. For the induction of apoptosis, MSCs were stimulated with 250 nM STS for 12 hours, which showed characteristic cell shrinkage morphological changes under microscope (Figure S1 A). Additionally, flow cytometry analysis of dual markers 7-AAD and Annexin V-PE showed that the proportion of early apoptotic and late apoptotic cells reached up to 80% (Figure S1 B). The supernatants were collected before or after apoptosis separately from the same number of MSCs, and ApoEVs and EVs were isolated following the optimized gradient centrifugation protocol (Fig. 1 A). For the characterization of purified ApoEVs and EVs, TEM and NTA analyses were conducted. TEM analysis revealed that both ApoEVs and EVs displayed typical vesicular double-layered spherical structure (Fig. 1 B). NTA provided information on the particle size distribution of vesicles, and ApoEVs exhibited multiple peaks ranging from 100 nm to 500 nm. In contrast, EVs were smaller with a size distribution around 100 nm (Fig. 1 C). Strikingly, when comparing the protein content in ApoEVs and EVs produced by the same number of MSCs, we found that ApoEVs contained 4-fold higher protein yields than EVs per cell. Simply, an equal number of MSCs produced approximately four times more protein in ApoEVs than in EVs (Fig. 1 D). Furthermore, in protein level, Western blot analysis was utilized to detect the presence of apoptosis-specific marker cleaved-caspase 3 in ApoEVs, and identify exosome-specific markers CD9 and CD81 in EVs, whereas the non-exosomal markers calnexin and GM130 were not detected in these vesicles (Fig. 1 E, F). These results indicated that ApoEVs and EVs used in this study conform to their characteristic features respectively. 3.2 Comparison of the therapeutic effects between ApoEVs and EVs on CCl4-induced liver fibrosis mouse model To evaluate the impact of ApoEVs and EVs on liver fibrosis, we transplanted both types of vesicles into CCl4-induced mouse model of liver fibrosis. After 8 weeks of intraperitoneal CCl4 injection, we grouped the model mice as described in methods. We dissolved 150 µg ApoEVs or EVs in 200 µl PBS via tail vein injection into model mice (Fig. 2 A). Two weeks later, histopathological samples and serum were collected to assess the therapeutic effects. Histological analyses of liver tissues revealed that, compared to PBS group, ApoEVs and EVs treatment groups exhibited remarkably reduced fibrosis and ApoEVs showed a lower degree of fibrosis (Fig. 2 C). Specifically, ApoEVs treatment group exhibited a significant reduction in inflammatory cell infiltration in HE staining. Masson and Sirius Red staining revealed that after ApoEV treatment, the fibrous septa became narrower and collagen deposition decreased obviously. The quantitative analysis of the area stained by Masson and Sirius Red were conducted to accurately evaluate the therapeutic effects of two vesicles. The results indicated that ApoEVs exhibited a more significant improvement in fibrosis compared to EVs (Fig. 2 D). Meanwhile, serum parameters ALT and AST which related to liver function showed consistent findings (Fig. 2 B). Besides, Western blot analysis also confirmed that the expression levels of fibrosis-related markers Col1a1 and αSMA were lower in ApoEVs treatment group compared to EVs group (Fig. 2 E, F). In conclusion, ApoEVs exhibit superior efficacy in reducing liver fibrosis. 3.3 Modulatory effects of ApoEVs versus EVs on macrophage inflammatory phenotype transition The inflammatory state of macrophages is closely related to the progression of liver disease[ 27 ]. After stimulation by inflammasomes, macrophages can release pro-inflammatory factors to promote the progression of chronic liver injury and fibrosis[ 28 , 29 ]. Macrophages have been considered as a potential therapeutic target for fibrosis[ 30 ]. To explore the specific mechanisms of ApoEVs and EVs on the improvement of the inflammatory response in fibrosis, we investigated the regulatory effects of these two vesicles on the phenotype switching of macrophage in vivo. We isolated primary BMDMs to further assess the regulatory effects of ApoEVs and EVs on macrophages in vitro. A pro-inflammatory state in macrophages was induced using LPS/IFN-γ and then co-cultured them with ApoEVs or EVs containing 20 µg protein, respectively. Then protein and RNA samples were collected, and the expression levels of pro-inflammatory or anti-inflammatory macrophage-associated genes was analyzed using Western blot and qRT-PCR techniques. At the protein level, iNOS expression was significantly decreased in the ApoEVs and EVs groups, while the expression of Arg-1 was increased, and quantitative analysis further confirmed these results (Fig. 3 A, B). Similarly, the mRNA expression levels of pro-inflammatory macrophage-related genes including TNF-α, iNOS, and IL-23 were significantly downregulated in ApoEVs and EVs-treated groups, while regarding anti-inflammatory macrophages, the expression levels of marker genes Arg-1, CD206, and CD163 were markedly upregulated (Fig. 3 C). The same experiments were repeated on the Raw264.7 cell line, and the results were consistent with those described above (Figure S2A, B). To elucidate the mechanisms by which ApoEVs and EVs promote macrophage inflammatory phenotype switching, the activation states of canonical inflammatory pathways (JAK/STAT, PI3K-Akt, and NF-κB) were systematically analyzed. Pathway analysis revealed that both types of vesicles also inhibited the phosphorylation of STAT3, NF-κB, and AKT, while the phosphorylation of STAT6 was activated (Fig. 3 D). This further suggested that ApoEVs and EVs may participate in inhibiting the pro-inflammatory effects of macrophages by modulating the activation status of transcription factors during the inflammatory process. 3.4 Investigating the effects of ApoEVs and EVs on the activation level of HSCs HSCs are the main effector cells during liver fibrogenesis[ 31 ]. But when liver injury occurs, inflammatory factors in liver can active HSCs, causing excessive ECM deposition as a wound-healing or scarring response[ 32 ]. Therefore, the regulation of HSCs activation has been recognized as a central target for the treatment of liver fibrosis[ 33 ]. Signals from pro-fibrotic macrophages can enhance the production of TGF-β, thereby stimulating the activation of HSCs[ 34 , 35 ]. In consequence, based on the preliminary findings we considered that ApoEVs and EVs may ameliorate liver fibrosis by either promoting the secretion of anti-inflammatory factors from macrophages or directly inhibiting the activation of HSCs (Fig. 4 A). To examine the regulatory effects of ApoEVs and EVs on HSCs, human HSCs cell line LX-2 was stimulated with TGF-β1 to induce activation, then co-cultured with ApoEVs or EVs. The expression of activation-related marker genes Col1a1 and αSMA were detected at both protein and mRNA levels. Western blot analysis showed Col1a1 and αSMA were downregulated in treated groups compared to activated HSCs (Fig. 4 B, C). The same trend was observed through qRT-PCR analysis (Fig. 4 D). These results suggested that ApoEVs and EVs could directly inhibit the activation of HSCs. But they may also indirectly act on HSCs while promoting the release of anti-inflammatory cytokines from macrophages. In summary, ApoEVs and EVs could treat liver fibrosis by altering the activation state of HSCs, and ApoEVs could reduce Col1a1 and αSMA expression levels significantly. 3.5 Analyzed the potential mechanisms of ApoEVs’ superior therapeutic functions in liver fibrosis Our study demonstrated that, ApoEVs showed significant anti-inflammatory and fibrosis-reversing capabilities in CCl4-induced liver fibrosis mouse model, as well as in vitro macrophage and HSCs. To futher elucidate the therapeutic mechanism of ApoEVs and to understand the reasons behind the differences in the efficacy of ApoEVs and EVs in reversing liver fibrosis, miRNA-sequencing was employed. We examined the miRNAs contained within ApoEVs and EVs and screened miRNAs with significant differential expression between ApoEVs and EVs, and focused on the group of miRNAs that are highly expressed in ApoEVs (Fig. 5 A, B). Compared to the EVs group, ApoEVs exhibited a significant enrichment of 69 miRNAs. Through querying the HuDD database, 10 cirrhosis-associated miRNAs were identified from these 69 miRNAs (Fig. 5 C). Subsequently, the target genes of the 10 miRNAs (including let-7a-5p, let-7f-5p, miR-101-3p, miR-197-3p, miR-20a-5p, miR-381-3p, miR-411-5p, miR-455-3p, miR-92a-3p, miR-98-5p) that were significantly upregulated in ApoEVs were predicted using TargetScan and Miranda, and these genes were then further bioinformatically analyzed using GO and KEGG. In the GO analysis, the top 20 pathways of GO biological process (BP), cellular component (CC), and molecular function (MF) were displayed. These target genes could influence various biological processes to affect cellular behavior, including “protein transport”, “intracellular signal transduction”, “cell cycle”, “intracellular protein transport” et al. which were related to cell communication, cellular transport and so on (Fig. 5 D). In addition, the target genes of these 10 miRNAs were also associated with cellular components such as cytoplasm, nucleus, cytosol, and cytoskeleton et al (Fig. 5 E). Analysis of molecular functions revealed that the 10 miRNAs primarily target genes related to “ATP binding”, “hydrolase activity”, “kinase activity”, and “ubiquitin − protein transferase activity” which influenced the activity of some protein to regulate cell function (Fig. 5 F). Furthermore, KEGG pathway analysis revealed that the target genes were closely associated with MAPK signaling pathway, PI3K − Akt signaling pathway, Hippo signaling pathway, and Ras signaling pathway, which play crucial roles in regulating inflammatory responses and fibrosis (Figure G). These highly expressed miRNAs in ApoEVs and their functional analysis also explained why ApoEVs exhibit superior anti-inflammatory effects. The chord diagram illustrating the interaction analysis between miRNAs enriched in the classic inflammatory pathway PI3K-Akt and their corresponding target genes further revealed the potential characteristics of ApoEVs related to inflammation regulation, particularly in the context of liver cirrhosis-associated diseases (Fig. 5 H). In summary, the bioinformatics analysis of miRNAs in ApoEVs and EVs suggested that ApoEVs outperformed EVs in reducing fibrosis, likely due to their enriched miRNA, which mediated the modulation and activation of inflammatory pathways such as the PI3K-Akt signaling pathway. 4. Discussion Chronic liver diseases such as fatty liver disease or viral hepatitis, all involve the persistent activation of inflammatory responses as well as sustained activation of liver fibrogenesis[ 36 ]. Many types of cells, cytokines, and miRNAs are involved in the development of liver fibrosis and cirrhosis[ 37 ]. In recent years, clinical trial results have indicated that stem cell therapy holds significant potential[ 38 ]. Accumulating evidence has elucidated that a large amount of stem cells underwent apoptosis after infusion, and these apoptotic cells still possessed considerable immunomodulatory functions although the exact mechanisms have yet to be fully understood[ 18 ]. ApoEVs were emerging substances secreted during apoptosis and have shown exciting therapeutic prospects in many diseases. In our previous research, stem cells have harnessed their paracrine functions to exert potent therapeutic effects through the mediation of exosomes[ 24 ]. We aim to compare the immunomodulatory efficacies of ApoEVs and EVs which are two important vectors secreted by stem cells, with the aspiration of uncovering further refined strategies for the clinical application of stem cell therapy. In this study, we delved into the distinct characteristics of ApoEVs and EVs, unveiling their unique features and functional properties. We generated ApoEVs and EVs based on the apoptotic state of MSCs and compared their therapeutic effects on CCl4-induced fibrosis mice, as well as exploring the underlying molecular mechanisms, which provides valuable insights into the therapeutic significance of MSC apoptosis. MSCs are a type of cells with self-renewal and multi-directional differentiation potential[ 39 ]. Over the past decades, MSCs have been intensely studied for clinical applications and have yielded the encouraging pre-clinical outcomes in varied animal disease models[ 40 ]. In chronic liver diseases, MSCs play a crucial role in maintaining tissue homeostasis and regeneration, as well as modulating the immune microenvironment. However, the specific mechanisms by which MSCs exert their therapeutic effects in vivo are not yet clearly understood. Recent studies have shown that transplanted MSCs may undergo apoptosis within a short period due to the influence of disease microenvironments such as hypoxia and inflammation[ 41 ]. But cell apoptosis may not impair the immunosuppressive effects of MSCs and may even play a critical role in the treatment of diseases[ 42 ]. A growing number of studies have demonstrated that apoptotic MSCs show similar therapeutic effects to those of live MSCs[ 43 ]. To investigate the specific mechanisms that cell apoptosis exerts its effects, ApoEVs which are secreted by apoptotic MSCs have come into the spotlight of scientific inquiry. ApoEVs play a significant role in promoting immunomodulation, angiogenesis, and the regulation of homeostasis as they inherit the molecular characteristics from their parental MSCs[ 41 ]. EVs and ApoEVs are members of the “secretome” of MSCs but in different active states. A large body of evidence suggests the reparative and regenerative properties of EVs in liver diseases[ 44 ]. Currently, research on the differences between ApoEVs and EVs produced by MSCs is still very limited. Elucidating the difference of their functional properties and effects is crucial for comprehending the significance of cell apoptosis in MSCs. In the present study, we observed that ApoEVs have a larger particle size and more abundant functional proteins compared to EVs, with distinct protein markers on their surfaces. We transplanted them in CCl4-induced mouse model for comparison respectively and found that both ApoEVs and EVs can ameliorate liver fibrosis in vivo. Notably, the effect of ApoEVs was more pronounced. Subsequently, we conducted further research to explore the mechanisms underlying this phenomenon. The secretion of pro-inflammatory cytokines by macrophages and the activation of HSCs are key events that lead to fibrosis. The accumulation of pro-fibrotic macrophages also plays a significant role in the activation of HSCs. Therefore, macrophages are central to the pathogenesis of liver fibrosis and have been potential target for combating fibrosis[ 45 ]. In traditional perspectives, macrophages are functionally categorized into two major subsets: classically activated macrophages (pro-inflammatory macrophages) and alternatively activated macrophages (anti-inflammatory macrophages). Macrophages polarization is crucial for tissue repair and the resolution of inflammation[ 46 ]. Our studies confirmed that both ApoEVs and EVs have the ability to promote the transformation of macrophages towards an anti-inflammatory direction in vitro. We thought that the increased secretion of anti-inflammatory factors by macrophages controlled the progression of inflammation and may even indirectly inhibit the activation of HSCs. We will conduct further research to investigate this phenomenon. At the same time, we observed that they can directly inhibit the activation of HSCs. These results elucidated the potential mechanisms by which ApoEVs and EVs suppress inflammation and fibrosis development. And the comparative findings indicated that ApoEVs possessed remarkable capabilities to suppress the production of pro-inflammatory factors and inhibit cell activation. Comprehending the functional differences and the underlying reasons is crucial for the therapeutic potential of ApoEVs. To further explore the molecular mechanism why ApoEVs showed the superior therapeutic effects in liver fibrosis, we conducted miRNA-sequencing analysis on both ApoEVs and EVs. MiRNA is a class of small non-coding endogenous RNA molecules that regulate gene expression through translational repression or mRNA degradation. They serve as primary mediators of intercellular communication via vesicles and play a unique role in modulating the activation, infiltration, and resolution of inflammation in macrophages and HSCs[ 47 , 48 ]. We performed differential analysis on the miRNA-sequencing data of these two vesicles and screened 10 relevant miRNAs in ApoEVs (let-7a-5p, let-7f-5p, miR-101-3p, miR-197-3p, miR-20a-5p, miR-381-3p, miR-411-5p, miR-455-3p, miR-92a-3p, miR-98-5p). Then further enrichment analysis revealed that these 10 miRNAs were associated with intracellular signal transduction and protein transport, and their target genes were related to various enzyme activities. Additionally, the target genes were clustered in inflammatory pathways such as MAPK and PI3K-Akt. These results provided crucial insights into the mechanisms of ApoEVs in suppressing inflammation and cell activation. Our next step will further verify the therapeutic effects of ApoEVs-miRNAs on liver fibrosis and explore the molecular mechanisms by which ApoEVs exert their effects to offer new possibilities for disease amelioration. However, this study did not elucidate the significance of apoptotic events in MSC-based therapy and the mechanistic exploration of ApoEV-mediated regulation of macrophage inflammatory phenotypes remained insufficient. And finally, experiments to verify the specific mechanism of miRNAs was ultimately lacking. Next, we will continue to further study the mechanism of ApoEVs in liver fibrosis and clarify the important significance of apoptotic MSCs in clinical treatment. 5. Conclusions In summary, our results highlight the distinct characteristics of vesicles secreted by MSCS in different apoptotic states and explore the great potential of ApoEVs in the treatment of liver fibrosis. Notably, comparing to EVs, ApoEVs exhibited superior therapeutic effects on fibrosis, which may due to the enrichment of miRNAs associated with inflammation regulation in ApoEVs. Overall, our results suggested that ApoEVs could be a promising approach for the treatment of liver diseases and further lay a research foundation for clarifying the therapeutic mechanism of MSCs. Abbreviations MSCs mesenchymal stem cells MMPs matrix metalloproteinases ECM extracellular matrix HSCs hepatic stellate cells EVs extracellular vesicles miRNA microRNA ApoEVs apoptotic extracellular vesicles CCl4 carbon tetrachloride SPF specific pathogen-free PBS phosphate buffered saline hUC-MSCs human umbilical cord-derived MSCs STS staurosporine TEM transmission electron microscopy NTA nanoparticle tracking analysis BMDMs bone morrow-derived macrophages ALT alanine aminotransferase AST aspartate aminotransferase GO gene ontology KEGG Kyoto Encyclopedia of Genes and Genomes BP biological process CC cellular component MF molecular function. Declarations Acknowledgments We thank the Figdraw (https://www.figdraw.com) for providing graphic elements in creating the figures in this article. Author Contributions Ruobing Ju, Siyuan Tian and Bo Li performed the most of experiments, analyzed data and wrote the manuscript. Bo Li, Xia Zhou isolated and collected exosomes. Yinan Hu and Erzhuo Xia built animal models. Bo Li and Fangfang Yang contributed to flow cytometry analysis. Miao Zhang, Shuoyi Ma and Rui Su provided technical support. Ying Han, Yulong Shang and Xia Zhou designed the study and revised the manuscript. All authors read and approved the current version of the manuscript. Funding This study was funded by National Natural Science Foundation of China (No. 82270551, No. 82200680, No.82300672), Key Research and Development Program of Shaanxi province, China (No. 2024SF-GJHX-16), National Key Research and Development Program of China (No. 2020YFA0710803), Science and Technology Association youth talent lifting program of Shaanxi province, China (No.20230324) and Xijing Hospital Project (No. XJZT25ZH08). Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The animal study protocol was approved by the Animal Welfare and Ethics Committee of the Fourth Military Medical University and performed according to the “Guidelines for the Care and Use of Laboratory Animals”. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References V. Taru, G. Szabo, W. Mehal, T. Reiberger, Inflammasomes in chronic liver disease: Hepatic injury, fibrosis progression and systemic inflammation, Journal of Hepatology 81(5) (2024) 895-910. P. Ginès, A. Krag, J.G. Abraldes, E. Solà, N. Fabrellas, P.S. Kamath, Liver cirrhosis, The Lancet 398(10308) (2021) 1359-1376. S. Ling, G. Jiang, Q. Que, S. Xu, J. Chen, X. Xu, Liver transplantation in patients with liver failure: Twenty years of experience from China, Liver international : official journal of the International Association for the Study of the Liver 42(9) (2022) 2110-2116. W. Pu, H. Zhu, M. Zhang, M. Pikiolek, C. Ercan, J. Li, X. Huang, X. Han, Z. Zhang, Z. Lv, Y. Li, K. Liu, L. He, X. Liu, M.H. Heim, L.M. Terracciano, J.S. Tchorz, B. Zhou, Bipotent transitional liver progenitor cells contribute to liver regeneration, Nature genetics 55(4) (2023) 651-664. P. Liu, Y. Mao, Y. Xie, J. Wei, J. Yao, Stem cells for treatment of liver fibrosis/cirrhosis: clinical progress and therapeutic potential, Stem Cell Research & Therapy 13(1) (2022) 356. M. Wang, M. Zhang, L. Fu, J. Lin, X. Zhou, P. Zhou, P. Huang, H. Hu, Y. Han, Liver-targeted delivery of TSG-6 by calcium phosphate nanoparticles for the management of liver fibrosis, Theranostics 10(1) (2020) 36-49. L. Ma, J. Wei, Y. Zeng, J. Liu, E. Xiao, Y. Kang, Y. Kang, Mesenchymal stem cell-originated exosomal circDIDO1 suppresses hepatic stellate cell activation by miR-141-3p/PTEN/AKT pathway in human liver fibrosis, Drug Delivery 29(1) (2022) 440-453. V.N. Hieu, L.T.T. Thuy, H. Hai, N.Q. Dat, D.V. Hoang, N.V. Hanh, D.M. Phuong, T.H. Hoang, H. Sawai, Y. Shiro, M. Sato-Matsubara, D. Oikawa, F. Tokunaga, K. Yoshizato, N. Kawada, Capacity of extracellular globins to reduce liver fibrosis via scavenging reactive oxygen species and promoting MMP-1 secretion, Redox Biology 52 (2022) 102286. Z. Tian, Y. Zenan, W. Jianyu, P. Duanqing, D. Xin, H. Chang, L. Peilong, Challenges and advances in clinical applications of mesenchymal stromal cells, J Hematol Oncol (2021). W. Chen, F. Lin, X. Feng, Q. Yao, Y. Yu, F. Gao, J. Zhou, Q. Pan, J. Wu, J. Yang, J. Yu, H. Cao, L. Li, MSC-derived exosomes attenuate hepatic fibrosis in primary sclerosing cholangitis through inhibition of Th17 differentiation, Asian Journal of Pharmaceutical Sciences 19(1) (2024) 100889. F. Cheng, F. Yang, Y. Wang, J. Zhou, H. Qian, Y. Yan, Mesenchymal stem cell-derived exosomal miR-27b-3p alleviates liver fibrosis via downregulating YAP/LOXL2 pathway, Journal of Nanobiotechnology 21(1) (2023) 195. B. banimohamad-shotorbani, H. Kahroba, H. Sadeghzadeh, D.M. Wilson, H. Maadi, N. Samadi, M.S. Hejazi, H. Farajpour, B.N. Onari, M.R. Sadeghi, DNA damage repair response in mesenchymal stromal cells: From cellular senescence and aging to apoptosis and differentiation ability, Ageing Research Reviews 62 (2020) 101125. Y. Fu, B. Sui, L. Xiang, X. Yan, D. Wu, S. Shi, X. Hu, Emerging understanding of apoptosis in mediating mesenchymal stem cell therapy, Cell Death & Disease 12(6) (2021) 596. S.H.M. Pang, J. D’Rozario, S. Mendonca, T. Bhuvan, N.L. Payne, D. Zheng, A. Hisana, G. Wallis, A. Barugahare, D. Powell, J. Rautela, N.D. Huntington, G. Dewson, D.C.S. Huang, D.H.D. Gray, T.S.P. Heng, Mesenchymal stromal cell apoptosis is required for their therapeutic function, Nature communications 12(1) (2021) 6495. C. Giacomini, C. Granéli, R. Hicks, F. Dazzi, The critical role of apoptosis in mesenchymal stromal cell therapeutics and implications in homeostasis and normal tissue repair, Cellular & Molecular Immunology 20(6) (2023) 570-582. R. Lin, T. Zhang, J.-Q. Gao, Apoptotic Vesicles of MSCs: The Natural Therapeutic Agents and Bio‐Vehicles for Targeting Drug Delivery, Small 19 (2023) e2301671. O. Q, H. W, W. B, N. L, L. Z, M. X, S. S, Apoptotic Vesicles: Therapeutic Mechanisms and Critical Issues, J Dent Res (2024). Z. Li, M. Wu, S. Liu, X. Liu, Y. Huan, Q. Ye, X. Yang, H. Guo, A. Liu, X. Huang, X. Yang, F. Ding, H. Xu, J. Zhou, P. Liu, S. Liu, Y. Jin, K. Xuan, Apoptotic vesicles activate autophagy in recipient cells to induce angiogenesis and dental pulp regeneration, Molecular Therapy 30(10) (2022) 3193-3208. B. Sui, R. Wang, C. Chen, X. Kou, D. Wu, Y. Fu, F. Lei, Y. Wang, Y. Liu, X. Chen, H. Xu, Y. Liu, J. Kang, H. Liu, R.T.K. Kwok, B.Z. Tang, H. Yan, M. Wang, L. Xiang, X. Yan, X. Zhang, L. Ma, S. Shi, Y. Jin, Apoptotic Vesicular Metabolism Contributes to Organelle Assembly and Safeguards Liver Homeostasis and Regeneration, Gastroenterology 167(2) (2024) 343-356. L. Ma, C. Chen, D. Liu, Z. Huang, J. Li, H. Liu, R.T. Kin Kwok, B. Tang, B. Sui, X. Zhang, J. Tang, X. Mao, W. Huang, S. Shi, X. Kou, Apoptotic extracellular vesicles are metabolized regulators nurturing the skin and hair, Bioactive Materials 19 (2023) 626-641. C. Zheng, B. Sui, X. Zhang, J. Hu, J. Chen, J. Liu, D. Wu, Q. Ye, X. Lei, X. Qiu, S. Liu, Z. Deng, J. Zhou, S. Liu, S. Shi, Y. Jin, Apoptotic vesicles restore liver macrophage homeostasis to counteract type 2 diabetes, Journal of Extracellular Vesicles 10 (2021). L. Yu, G. Dou, H. Kuang, L. Bao, H. Liu, Q. Ye, Z. Wang, X. Yang, L. Ren, Z. Li, H. Liu, B. Li, S. Liu, S. Ge, S. Liu, Apoptotic Extracellular Vesicles Induced Endothelial Cell-Mediated Autologous Stem Cell Recruitment Dominates Allogeneic Stem Cell Therapeutic Mechanism for Bone Repair, ACS Nano 18(12) (2024) 8718-8732. X. Zhang, J. Yang, S. Ma, X. Gao, G. Wang, Y. Sun, Y. Yu, Z. Wang, W. Tian, L. Liao, Functional diversity of apoptotic vesicle subpopulations from bone marrow mesenchymal stem cells in tissue regeneration, Journal of Extracellular Vesicles 13(4) (2024) e12434. S. Tian, X. Zhou, M. Zhang, L. Cui, B. Li, Y. Liu, R. Su, K. Sun, Y. Hu, F. Yang, G. Xuan, S. Ma, X. Zheng, X. Zhou, C. Guo, Y. Shang, J. Wang, Y. Han, Mesenchymal stem cell-derived exosomes protect against liver fibrosis via delivering miR-148a to target KLF6/STAT3 pathway in macrophages, Stem Cell Research & Therapy 13(1) (2022) 330. P.-F. Ma, C.-C. Gao, J. Yi, J.-L. Zhao, S.-Q. Liang, Y. Zhao, Y.-C. Ye, J. Bai, Q.-J. Zheng, K.-F. Dou, H. Han, H.-Y. Qin, Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice, Journal of Hepatology 67(4) (2017) 770-779. X. Zheng, X. Zhou, G. Ma, J. Yu, M. Zhang, C. Yang, Y. Hu, S. Ma, Z. Han, W. Ning, B. Jin, X. Zhou, J. Wang, Y. Han, Endogenous Follistatin-like 1 guarantees the immunomodulatory properties of mesenchymal stem cells during liver fibrotic therapy, Stem Cell Research & Therapy 13(1) (2022) 403. Z. Wang, K. Du, N. Jin, B. Tang, W. Zhang, Macrophage in liver Fibrosis: Identities and mechanisms, Int Immunopharmacol (2023). Y. Wen, J. Lambrecht, C. Ju, F. Tacke, Hepatic macrophages in liver homeostasis and diseases-diversity, plasticity and therapeutic opportunities, Cellular & Molecular Immunology 18(1) (2021) 45-56. R.F. Schwabe, I. Tabas, U.B. Pajvani, Mechanisms of Fibrosis Development in Nonalcoholic Steatohepatitis, Gastroenterology (2020). B. Simón-Codina, J. Cacho-Pujol, A. Moles, P. Melgar-Lesmes, Reprogramming macrophages to treat liver diseases, Hepatology (2024). K. Dakota R, M. Kyle S, Hepatic stellate cells in physiology and pathology, J Physiol (2022). L. Hammerich, F. Tacke, Hepatic inflammatory responses in liver fibrosis, Nature Reviews Gastroenterology & Hepatology 20(10) (2023) 633-646. T. Tsuchida, S.L. Friedman, Mechanisms of hepatic stellate cell activation, Nature Reviews Gastroenterology & Hepatology 14(7) (2017) 397-411. S. Y-Y, L. X-F, M. X-M, H. C, Z. L, L. J, Macrophage Phenotype in Liver Injury and Repair, Scand J Immunol (2016). Z. Wang, K. Du, N. Jin, B. Tang, W. Zhang, Macrophage in liver Fibrosis: Identities and mechanisms, International Immunopharmacology 120 (2023) 110357. P. Maurizio, P. Massimo, Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues, Mol Aspects Med (2018). W.-C. Zhou, Q.-B. Zhang, L. Qiao, Pathogenesis of liver cirrhosis, World journal of gastroenterology (2014). B.J. Dwyer, M.T. Macmillan, P.N. Brennan, S.J. Forbes, Cell therapy for advanced liver diseases: Repair or rebuild, Journal of Hepatology 74(1) (2021) 185-199. E. El Agha, R. Kramann, R.K. Schneider, X. Li, W. Seeger, B.D. Humphreys, S. Bellusci, Mesenchymal Stem Cells in Fibrotic Disease, Cell Stem Cell (2017). T. Zhou, Z. Yuan, J. Weng, D. Pei, X. Du, C. He, P. Lai, Challenges and advances in clinical applications of mesenchymal stromal cells, J Hematol Oncol (2021). R. Wang, J. Fu, J. He, X. Wang, W. Xing, X. Liu, J. Yao, Q. Ye, Y. He, Apoptotic mesenchymal stem cells and their secreted apoptotic extracellular vesicles: therapeutic applications and mechanisms, Stem Cell Research & Therapy 16(1) (2025) 78. Q. Ou, X. Qiao, Z. Li, L. Niu, F. Lei, R. Cheng, T. Xie, N. Yang, Y. Liu, L. Fu, J. Yang, X. Mao, X. Kou, C. Chen, S. Shi, Apoptosis releases hydrogen sulfide to inhibit Th17 cell differentiation, Cell Metab (2024). M.B. Preda, C.A. Neculachi, I.M. Fenyo, A.-M. Vacaru, M.A. Publik, M. Simionescu, A. Burlacu, Short lifespan of syngeneic transplanted MSC is a consequence of in vivo apoptosis and immune cell recruitment in mice, Cell Death Dis (2021). G. Lou, Z. Chen, M. Zheng, Y. Liu, Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases, Experimental & Molecular Medicine 49(6) (2017) e346-e346. C. Wang, C. Ma, L. Gong, Y. Guo, K. Fu, Y. Zhang, H. Zhou, Y. Li, Macrophage Polarization and Its Role in Liver Disease, Front Immunol (2021). K. Hamidzadeh, S.M. Christensen, E. Dalby, P. Chandrasekaran, D.M. Mosser, Macrophages and the Recovery from Acute and Chronic Inflammation, Annu Rev Physiol (2017). W. Yu, S. Wang, Y. Wang, H. Chen, H. Nie, L. Liu, X. Zou, Q. Gong, B. Zheng, MicroRNA: role in macrophage polarization and the pathogenesis of the liver fibrosis, Frontiers in Immunology 14 (2023). X. Wang, Y. He, B. Mackowiak, B. Gao, MicroRNAs as regulators, biomarkers and therapeutic targets in liver diseases, Gut (2021). Additional Declarations No competing interests reported. Supplementary Files Supplementaryfilenew.pdf SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 23 Mar, 2026 Read the published version in BMC Gastroenterology → Version 1 posted Editorial decision: Revision requested 19 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviews received at journal 10 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers invited by journal 06 Aug, 2025 Editor assigned by journal 05 Aug, 2025 Editor invited by journal 14 Jul, 2025 Submission checks completed at journal 12 Jul, 2025 First submitted to journal 12 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6970215","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498220087,"identity":"d553ce51-0e3b-44eb-9d80-03a0c981ee06","order_by":0,"name":"Ruobing Ju","email":"","orcid":"","institution":"The Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruobing","middleName":"","lastName":"Ju","suffix":""},{"id":498220088,"identity":"d674ec0c-40c0-40ab-bc41-cb9cea177e58","order_by":1,"name":"Siyuan Tian","email":"","orcid":"","institution":"The Fourth Military Medical 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03:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6970215/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6970215/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12876-026-04762-0","type":"published","date":"2026-03-23T16:11:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88777701,"identity":"19f0a1e9-1938-4907-93cd-f1154b61a4f3","added_by":"auto","created_at":"2025-08-11 10:14:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1355660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of apoptosis in MSCs and isolation and characterization of MSC-derived ApoEVs and EVs.\u003c/strong\u003e (A) Schematic representation of the differential centrifugation method used to isolate ApoEVs and EVs from the same number of MSCs. (B) Representative TEM images showed the morphology of ApoEVs and EVs. Scale bar, 200 nm. (C) NTA showed the size distribution of ApoEVs and EVs. (D) The ratio of protein quantification in ApoEVs to that in EVs. (E, F) Western blot analysis showed the presence of caspase3/cleaved-caspase3 in ApoEVs and CD9, CD81, calnexin and GM130 in EVs. The data in the figures represented the mean ±SD. Statistical analyses were performed by t-test. ***, p \u0026lt; 0.001. All experiments were performed three times.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/3912ff085aee082d9284247b.png"},{"id":88777703,"identity":"b58588bb-e3bf-4daf-9491-3e1782879eb3","added_by":"auto","created_at":"2025-08-11 10:14:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1637024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoEVs and EVs treatment could alleviate liver fibrosis in CCl4-induced model mice. \u003c/strong\u003e(A) Schematic diagram illustrated the establishment of an in vivo liver fibrosis model and the treatment of ApoEVs or EVs. ApoEVs or EVs were transplanted after 8 weeks of CCl4 injury, and samples were collected at 2 weeks post transplantation for subsequent analysis. (B) Changes of serum parameters including ALT and AST in different groups (n=4). (C) Histological analysis of health comparison, PBS, ApoEVs and EVs treatment by HE, Masson and Sirius Red staining in liver sections. Representative images of the staining were shown (n=4). Scale bar, 100 μm. (D) Liver fibrosis score analysis of Masson and Sirius Red-stained liver sections. The fibrotic area was presented as a percentage (n=4). (E) Western blot was used to measure the expression levels of fibrosis-related markers Col1a1 and αSMA. (F) Quantitative analysis of protein expression in Western blot(n=6). The data in the figures represented the mean ±SD. Statistical analyses were performed by One-way ANOVA with Tukey’s post hoc test. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01, ***, p \u0026lt; 0.001; ns, p \u0026gt; 0.05. All experiments were performed three times.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/b0e6b93f4c0dd24900ac9c50.png"},{"id":88777707,"identity":"ea62a791-750f-45f0-ac8f-bc5df427b21d","added_by":"auto","created_at":"2025-08-11 10:14:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1327516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoEVs and EVs promote the transition to anti-inflammatory macrophages to inhibit the inflammatory response. \u003c/strong\u003e(A) The protein expression levels of iNOS and Arg-1 using Western blot. (B) Quantitative analysis of protein expression in Western blot(n=3). (C) qRT-PCR analyses of the mRNA levels of pro-inflammatory macrophage markers (TNF-α, iNOS, IL-23) and anti-inflammatory macrophage markers (Arg-1, CD206, CD163). (D) Vesicles dephosphorylated STAT3, NF-κB, and AKT, phosphorylated STAT6. The data in the figures represented the mean ±SD. Statistical analyses were performed by One-way ANOVA with Tukey’s post hoc test. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01, ***, p \u0026lt; 0.001; ns, p \u0026gt; 0.05. All experiments were performed three times.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/c8facd8c37cee79d4b2e77c9.png"},{"id":88778200,"identity":"19538cda-8e72-4d16-9454-a198083baeaa","added_by":"auto","created_at":"2025-08-11 10:22:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":795758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoEVs and EVs inhibited the activation of HSCs. \u003c/strong\u003e(A) Schematic illustration of ApoEVs and EVs promote the transition to anti-inflammatory macrophages and inhibit HSCs activation to alleviate liver fibrosis. (B) The protein expression levels of fibrosis-related markers Col1a1 and αSMA using Western blot. (C) Quantitative analysis of protein expression in Western blot(n=3). (D) qRT-PCR analyses of the mRNA levels of Col1a1 and αSMA. The data in the figures represented the mean ±SD. Statistical analyses were performed by One-way ANOVA with Tukey’s post hoc test. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01, ***, p \u0026lt; 0.001; ns, p \u0026gt; 0.05. All experiments were performed three times.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/e22c97f2eed9db9a1c51f281.png"},{"id":88777706,"identity":"2ad1bd86-b824-45c0-8c9c-2362960db22e","added_by":"auto","created_at":"2025-08-11 10:14:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":965260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis of miRNA profiles and target genes involved in ApoEVs and EVs. \u003c/strong\u003e(A) Heatmap showed the top 100 miRNAs in ApoEVs versus EVs difference analysis. (B) Volcano Plot showed some miRNAs with significant differences in ApoEVs versus EVs. (C) Venn diagram illustrated the intersection of miRNAs differential expression between highly expressed miRNAs in ApoEVs and liver cirrhosis-associated miRNAs. (D-F) GO analysis of the target genes of the 10 significantly upregulated miRNAs in ApoEVs versus EVs: (D) Biological Process (BP), (E) Cellular Component (CC), (F) Molecular Function (MF). (G) KEGG pathway enrichment analysis for the target genes of the 10 significantly upregulated miRNAs in ApoEVs versus EVs. (H) Chordal maps of miRNAs and target mRNAs of the PI3K-Akt signaling pathway.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/eed213e25717c2ab0dba70d9.png"},{"id":105755583,"identity":"b6971be7-7f67-4cb2-b6ef-baddc15ced02","added_by":"auto","created_at":"2026-03-30 16:28:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7214329,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/fcac3e16-f397-440e-8c90-4dcc82e94e8a.pdf"},{"id":88777719,"identity":"d68b6a8d-c175-4b5c-8167-6243ce5c627b","added_by":"auto","created_at":"2025-08-11 10:14:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3007202,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfilenew.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/5588c3cec2ed457f752b5032.pdf"},{"id":88779147,"identity":"faab642b-2bea-4b94-becf-ce5e0ed600d4","added_by":"auto","created_at":"2025-08-11 10:30:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3470212,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6970215/v1/fe519578e995e4275a91dc07.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extracellular vesicles derived from live or apoptotic mesenchymal stem cells: comparison of the effects of two extracellular vesicles on liver fibrosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLiver fibrosis or cirrhosis may result from the progression of chronic liver diseases caused by various etiologies, which is facing a significant health challenge all over the world[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Without effective intervention in time, the morbidity and mortality will be increased significantly because of symptoms such as portal hypertension and gastrointestinal bleeding. In China, about 300\u0026nbsp;million people suffer from these diseases. In the world, liver cirrhosis is the 11th most common cause of death and together with liver carcinoma, accounts for 3.5% of all deaths[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Orthotopic liver transplantation is the only curative treatment for end-stage liver diseases. But its widespread application has been limited by issues such as donor shortage and poor prognosis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, many regenerative or alternative therapies are studied aiming to treat liver fibrosis or cirrhosis.\u003c/p\u003e\u003cp\u003eIn recent years, stem cell-based therapies have provided new possibilities for the treatment of liver diseases. In chronic liver diseases, mesenchymal stem cells (MSCs) play a crucial role in regulating the immune microenvironment through direct differentiation or regeneration[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. MSCs, sourced from bone marrow, adipose tissue, or umbilical cord, offer clinical advantages such as easy to clinical translation, low immunogenicity, and strong proliferative capacity et al. Currently, numerous clinical trials have been conducted in the field of MSC therapy for liver cirrhosis to demonstrate its effectiveness[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the exact mechanism of action is still unclear. Previous studies have indicated that MSCs can affect the inflammatory phenotype of macrophages through its paracrine function, or alleviates fibrosis by degrading extracellular matrix (ECM) or reducing the activation level of hepatic stellate cells (HSCs) [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Meanwhile, extracellular vesicles (EVs) especially exosomes, as mediators of intercellular communication, also play a significant role in the therapeutic effects of MSCs[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Exosomes can alleviate liver fibrosis by influencing the differentiation phenotype of immune cells or delivering microRNAs (miRNAs) to mediate YAP down-regulation[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe rapid apoptosis of MSCs following infusion has been considered a major factor limiting their sustained therapeutic effects[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, many studies aimed at enhancing the homing ability of MSCs, improving their survival, and ensuring their retention in vivo to improving their therapeutic efficacy. However, recent studies have shown that the immunosuppressive capacity of MSCs is attenuated when their apoptosis is resistant[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In other words, MSCs apoptosis is a crucial component of their therapeutic function on tissue repair and inflammation regulation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eApoptosis is an important form of programmed cell death. Apoptotic extracellular vesicles (ApoEVs) are formed during the process of apoptosis, through budding and fragmentation of the cell membrane. ApoEVs are a unique subtype of EVs, with diameters ranging from 50 to 5000 nm[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. They participate in intercellular communication, signaling, and immune regulation, which are similar to EVs produced by non-apoptotic MSCs. ApoEVs contain numerous bioactive molecules including proteins (such as CD81, CD9, etc.), lipids, RNA (mRNA, miRNA, etc.). After interacting with the recipient cells, they play indispensable roles in a variety of physiological and pathological environments[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Current research indicates that ApoEVs can play functional roles in a variety of diseases. In the event of inflammatory injury, ApoEVs modulate the population of immune cells and macrophage inflammatory phenotypes in various diseases, including acute kidney injury, colitis, allergic pulmonary inflammation, and skin wound healing. In addition, they can be engulfed by endothelial cells or basal cells for tissue regeneration. Moreover, ApoEVs can interact directly with recipient cells, regulating activity through membrane surface ligands et al[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, no studies have yet investigated ApoEVs in liver diseases, particularly in liver fibrosis.\u003c/p\u003e\u003cp\u003eIn terms of liver fibrosis treatment, previous research has mainly focused on the regulation of macrophage inflammatory phenotypes by EVs, particularly exosomes. Or focusing on the inhibition of HSCs activation, the degradation of ECM. However, the precise function of ApoEVs in liver fibrosis has not yet been explored. At the same time, there has been no comparative study on the effects of ApoEVs and EVs on liver fibrosis. Therefore, this study aims to dissect the differences between these two types of vesicles and explore their respective therapeutic efficacy.\u003c/p\u003e\u003cp\u003eIn this study, we comparatively analyzed the biological roles of ApoEVs and EVs secreted by MSCs before and after apoptosis. We evaluated the therapeutic efficacy of both types of vesicles in improving fibrosis levels in a carbon tetrachloride (CCl4)-induced mouse model. Additionally, we compared their effects on macrophages and HSCs to demonstrate potential differences in functional properties. The goal is to explore more effective therapeutic approaches for liver fibrosis.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental animal models\u003c/h2\u003e\u003cp\u003eThe male C57BL/6J mice (aged 6\u0026ndash;8 weeks) were purchased from and housed in the Laboratory Animal Center of the Air Force Military Medical University. The mice were housed in specific pathogen-free (SPF) and ventilated conditions in the Laboratory Animal Center, with 12-hour light/dark cycles, and food and water were provided ad libitum. The animal study protocols were approved by the Animal Welfare and Ethics Committee of the Air Force Military Medical University and performed according to the \u0026ldquo;Guidelines for the Care and Use of Laboratory Animals\u0026rdquo;.\u003c/p\u003e\u003cp\u003eThe liver fibrosis mouse model was established by continuous intraperitoneal injection of 0.2 mL/20g CCl4 [20% (v/v), dissolved in olive oil] twice a week for 8 weeks. Subsequently, the liver fibrosis mice were randomly divided into groups (n\u0026thinsp;=\u0026thinsp;4\u0026ndash;6) that received phosphate buffered saline (PBS), 150 \u0026micro;g ApoEVs diluted in 200 \u0026micro;l PBS and 150 \u0026micro;g EVs diluted in 200 \u0026micro;l PBS via tail vein injection. The control group was injected with an equal volume of PBS. After treatment, the mice were observed for an additional two weeks and were continually injected with CCl4 once a week. Two weeks later, the mice were euthanized by exposing to a 60% concentration carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) asphyxiation box. The process was rapid and minimally painful. After euthanasia, liver tissues were collected for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Induction of MSCs apoptosis\u003c/h2\u003e\u003cp\u003eHuman umbilical cord-derived MSCs (hUC-MSCs) in generation 4\u0026ndash;6 were selected and cultured in complete medium supplemented with serum substitute (EV-depleted) until they reached 80\u0026ndash;90% confluence. Then removing the medium and MSCs were washed three times with PBS. Subsequently, the complete medium containing 250 nM staurosporine (STS) (Cell Signaling Technology, USA) was added to MSCs[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. After 12 hours of treatment, the apoptosis of MSCs was detected by morphological observation and flow cytometry analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Isolation and Characterization of ApoEVs and EVs\u003c/h2\u003e\u003cp\u003eAccording to the previously optimized protocol, ApoEVs were collected through differential centrifugation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, collected the culture supernatant from apoptotic MSCs at the 12-hour mark of MSCs apoptosis induction. Then, centrifuged the supernatant at 800 g for 10 min at 4 ℃ to to remove some cell debris and intact cells. Following, the supernatant was further collected and centrifuged at 16,000 g for 30 min at 4℃, discarded the supernatant, and retained ApoEVs pellet. Subsequently, resuspended the pellet gently in pre-cooled PBS, centrifuged at 16,000 g for 30 min at 4 ℃. Washed twice in this method to obtain final ApoEVs. ApoEVs were quantified by measuring the protein concentration via a BCA Protein Assay Kit (GlpBio, USA).\u003c/p\u003e\u003cp\u003eEVs derived from MSCs were isolated by differential centrifugation as previously described[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, MSCs (hUC-MSCs) in generation 4\u0026ndash;6 were cultured in complete medium (EV-depleted) until they reached 70\u0026ndash;80% confluence. After 48 hours, collected the culture supernatant and centrifuged at 300 g for 10 min to remove some cell debris and intact cells. Further collected the supernatant and centrifuged at 2000 g for 20 min to remove larger cell debris and apoptotic bodies. Then, centrifuged the supernatant at 10,000 g for 30 min at 4\u0026deg;C to remove micro-vesicles and other larger particles. Finally, the supernatant was ultracentrifuged at 100,000 g for 70 min at 4\u0026deg;C, and washed the pellet with PBS at 100,000 g for 70 min to obtain EVs. EVs were quantified by measuring the protein concentration via a BCA Protein Assay Kit (GlpBio, USA).\u003c/p\u003e\u003cp\u003eThe morphology of ApoEVs and EVs were characterized using transmission electron microscopy (TEM). Nanoparticle tracking analysis (NTA) was used to analyze the particle size distribution and concentration of both ApoEVs and EVs. For apoptotic marker detection, purified ApoEVs were characterized by Western blot using anti-Caspase-3 and anti-β-Actin antibodies. For specific EVs markers, CD9, CD81 were used as positive controls, and calnexin, GM130 were used as negative controls for Western blot analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell Culture\u003c/h2\u003e\u003cp\u003eHUC-MSCs were provided by the National Engineering Research Center (Tianjin AmCellGene Engineering Co., Ltd, China). MSCs were cultured in mesenchymal basal medium (Dakewe Biotech Co., Ltd. China) supplemented with serum substitute at 37\u0026deg;C in a 5% CO2 incubator. Cells in generation 4\u0026ndash;6 were used for subsequent experiments.\u003c/p\u003e\u003cp\u003eRAW264.7 cells, obtained from the American Type Culture Collection (ATCC), which were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, NY) and 1% penicillin\u0026ndash;streptomycin.\u003c/p\u003e\u003cp\u003eMouse bone morrow-derived macrophages (BMDMs) were obtained as previously described[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. BMDMs were isolated from the femurs and tibias of 6-8-week-old C57BL/6J mice. Subsequently, these cells were plated at a density of 2\u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin\u0026ndash;streptomycin, plus 40 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech Inc. USA) for 7 days.\u003c/p\u003e\u003cp\u003eHuman HSC lines LX-2 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, NY) and 1% penicillin\u0026ndash;streptomycin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell treatment\u003c/h2\u003e\u003cp\u003eFor the phenotypic induction of macrophages, the M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes were established by stimulating BMDMs or RAW 264.7 cells with 100 ng/mL LPS (Sigma, USA)/20 ng/mL IFN-γ (Peprotech, USA) and 20 ng/mL IL-4 (Peprotech, USA) for 24 h, respectively.\u003c/p\u003e\u003cp\u003eFor the phenotypic induction of HSCs, LX-2 cells were stimulated with 8 ng/mL TGF-β1 (novoprotein, China) for 48 hours to establish an activated phenotype.\u003c/p\u003e\u003cp\u003eTo investigate the effects of ApoEVs and EVs on the inflammatory phenotype of macrophages and the activation of HSCs, 20 \u0026micro;g of ApoEVs or EVs were added to the culture medium of BMDMs, RAW 264.7 cells or LX-2 cells, respectively. After 12 hours of treatment, the cells were collected for subsequent analyses such as Western blot or qRT-PCR.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Biochemical analysis and histological staining\u003c/h2\u003e\u003cp\u003eThe serum of mice was obtained at each time point. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed using an Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) and measured with a Microplate Reader (Thermo Fisher Scientific, USA). The liver tissue samples were prepared as paraffin-embedded sections by Servicebio, China.\u003c/p\u003e\u003cp\u003eAnd then, sections were stained with hematoxylin and eosin (HE) for routine histological examination or with Sirius Red and Masson for fibrosis evaluation. Further quantification and analysis of collagen fiber were assessed using Image-J software (National Institutes of Health, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Flow cytometric analysis of cell phenotypes\u003c/h2\u003e\u003cp\u003eApoptotic MSCs were detected using an apoptosis staining kit (BD Biosciences, USA). After discarding the supernatant, the cells were washed three times with PBS and resuspended in the buffer provided by the apoptosis staining kit at a density of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/mL. PE-Annexin V and 7-AAD were added according to the recommended ratio, followed by incubation at room temperature in the dark for 15 min. Then, the flow cytometric data was analyzed on a FACSVerse flow cytometer (BD Bioscience, CA, USA) and analyzed with FlowJo software (TreeStar, Ashland, OR, US).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 RNA isolation and real‑time PCR analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from liver tissues or cells using the RNAeasy Plus Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China), and reverse transcription was generated using PrimeScript\u0026trade; RT Master Mix (RR036A, Takara, Tokyo). Then, qRT-PCR was conducted by TB Green Premix Ex Taq II (DRR820A, Takara, Tokyo) and a CFX96 Touch\u0026trade; real-time PCR System (Bio-Rad, CA). β-actin (Actb or ACTB) was used as an internal control for quantification. PCR primers are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Western blot analysis\u003c/h2\u003e\u003cp\u003eWhole proteins were extracted from tissues, cells or ApoEVs using RIPA Lysis Buffer (Beyotime biotechnology, China) supplemented with proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). The protein concentration was quantified using a BCA Protein Assay Kit (GlpBio, USA). Subsequently, equal amounts of protein samples (30 \u0026micro;g) were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad Biotechnology, USA). The membranes were blocked in TBST buffer containing 2.5% skim milk for 30 min. Then, the membranes were incubated overnight at 4 ℃ with the following primary antibodies: anti-Caspase-3 (9662, Cell Signaling Technology, USA; diluted at 1:1000), anti-GM130 (ab52649, Abcam, UK; diluted at 1:1000), anti-calnexin (10427-2-AP, Proteintech, USA; diluted at 1:1000), anti-CD81 (ab79559, Abcam, UK; diluted at 1:1000), anti-CD9 (ab236630, Abcam, UK; diluted at 1:1000), anti-Col1a1 (ab270993, Abcam, UK; diluted at 1:1000), anti-αSMA (19245, Cell Signaling Technology, USA; diluted at 1:1000), anti-iNOS (ab15323, Abcam, UK; diluted at 1:1000), anti-Arg-1 (ab124917, Abcam, UK; diluted at 1:1000), anti-STAT3 (10253-2-AP, Proteintech, USA; diluted at 1:1000), anti-STAT6 (9362, Cell Signaling Technology, USA; diluted at 1:1000), anti- NF-κB (8242, Cell Signaling Technology, USA; diluted at 1:1000), anti-AKT (4691, Cell Signaling Technology, USA; diluted at 1:1000), and anti-β-Actin (66009-1-Ig, Proteintech, USA; diluted at 1:5000).\u003c/p\u003e\u003cp\u003eAfter washing with TBS containing 0.1% Tween-20, the membranes were incubated with peroxidase-conjugated secondary antibodies (Proteintech, China) at room temperature for 1 h. And blots were visualized using an enhanced chemiluminescence kit and detected by a gel imaging system (Bio-Rad Biotechnology, USA). Further quantification and analysis of protein expression were assessed using Image-J software (National Institutes of Health, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 The miRNA‑sequencing and bioinformatics analysis\u003c/h2\u003e\u003cp\u003eThe miRNA-sequencing was provided by LCBio Co., Ltd (Hangzhou, China). Briefly, we collected three independent ApoEVs and EVs samples. RNA samples from both typed of vesicles were extracted, sequenced, and analysed by LCBio Co., Ltd (Hangzhou, China). The purity and quantity of RNA were checked using a Bioanalyzer 2100 (Agilent, CA, USA). Small-RNA libraries were sequenced on the illumina NovaseqTM 6000 platform by LC Bio Technology CO.,Ltd. Then we analysed the differential miRNAs between ApoEVs and EVs. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log\u003csub\u003e2\u003c/sub\u003e(foldchange)|\u0026gt;=1 was set as the threshold for significantly differential expression. A total of 96 significantly differential miRNAs were identified, with 69 of them being significantly enriched in ApoEVs. Subsequently, more than 111 miRNAs associated with liver cirrhosis were identified from the Human microRNA Disease Database. Compared with the 69 differential miRNAs, resulting in 10 highly differential miRNAs that are enriched in ApoEVs and strongly associated with liver cirrhosis. To predict the genes targeted by most aboundant miRNAs, two computational target prediction algorithms (TargetScan, v5.0 and Miranda, v3.3a) were used to identify miRNA binding sites. Finally, the data predicted by both algorithms were combined and the overlaps were calculated. The gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of these most aboundant miRNAs, miRNA targets were also annotated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe data were expressed as the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way analysis of variance and t-test were performed to identify the significant differences. A P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. Statistical analysis was plotted by GraphPad Prism 8.0 (GraphPad Software, CA, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Isolation and Characterization of MSC-Derived ApoEVs and EVs\u003c/h2\u003e\u003cp\u003eMSCs utilized in this study had been previously characterized and identified in our earlier literature[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For the induction of apoptosis, MSCs were stimulated with 250 nM STS for 12 hours, which showed characteristic cell shrinkage morphological changes under microscope (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Additionally, flow cytometry analysis of dual markers 7-AAD and Annexin V-PE showed that the proportion of early apoptotic and late apoptotic cells reached up to 80% (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The supernatants were collected before or after apoptosis separately from the same number of MSCs, and ApoEVs and EVs were isolated following the optimized gradient centrifugation protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eFor the characterization of purified ApoEVs and EVs, TEM and NTA analyses were conducted. TEM analysis revealed that both ApoEVs and EVs displayed typical vesicular double-layered spherical structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). NTA provided information on the particle size distribution of vesicles, and ApoEVs exhibited multiple peaks ranging from 100 nm to 500 nm. In contrast, EVs were smaller with a size distribution around 100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Strikingly, when comparing the protein content in ApoEVs and EVs produced by the same number of MSCs, we found that ApoEVs contained 4-fold higher protein yields than EVs per cell. Simply, an equal number of MSCs produced approximately four times more protein in ApoEVs than in EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, in protein level, Western blot analysis was utilized to detect the presence of apoptosis-specific marker cleaved-caspase 3 in ApoEVs, and identify exosome-specific markers CD9 and CD81 in EVs, whereas the non-exosomal markers calnexin and GM130 were not detected in these vesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). These results indicated that ApoEVs and EVs used in this study conform to their characteristic features respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Comparison of the therapeutic effects between ApoEVs and EVs on CCl4-induced liver fibrosis mouse model\u003c/h2\u003e\u003cp\u003eTo evaluate the impact of ApoEVs and EVs on liver fibrosis, we transplanted both types of vesicles into CCl4-induced mouse model of liver fibrosis. After 8 weeks of intraperitoneal CCl4 injection, we grouped the model mice as described in methods. We dissolved 150 \u0026micro;g ApoEVs or EVs in 200 \u0026micro;l PBS via tail vein injection into model mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Two weeks later, histopathological samples and serum were collected to assess the therapeutic effects.\u003c/p\u003e\u003cp\u003eHistological analyses of liver tissues revealed that, compared to PBS group, ApoEVs and EVs treatment groups exhibited remarkably reduced fibrosis and ApoEVs showed a lower degree of fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Specifically, ApoEVs treatment group exhibited a significant reduction in inflammatory cell infiltration in HE staining. Masson and Sirius Red staining revealed that after ApoEV treatment, the fibrous septa became narrower and collagen deposition decreased obviously. The quantitative analysis of the area stained by Masson and Sirius Red were conducted to accurately evaluate the therapeutic effects of two vesicles. The results indicated that ApoEVs exhibited a more significant improvement in fibrosis compared to EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Meanwhile, serum parameters ALT and AST which related to liver function showed consistent findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Besides, Western blot analysis also confirmed that the expression levels of fibrosis-related markers Col1a1 and αSMA were lower in ApoEVs treatment group compared to EVs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). In conclusion, ApoEVs exhibit superior efficacy in reducing liver fibrosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Modulatory effects of ApoEVs versus EVs on macrophage inflammatory phenotype transition\u003c/h2\u003e\u003cp\u003eThe inflammatory state of macrophages is closely related to the progression of liver disease[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After stimulation by inflammasomes, macrophages can release pro-inflammatory factors to promote the progression of chronic liver injury and fibrosis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Macrophages have been considered as a potential therapeutic target for fibrosis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo explore the specific mechanisms of ApoEVs and EVs on the improvement of the inflammatory response in fibrosis, we investigated the regulatory effects of these two vesicles on the phenotype switching of macrophage in vivo. We isolated primary BMDMs to further assess the regulatory effects of ApoEVs and EVs on macrophages in vitro. A pro-inflammatory state in macrophages was induced using LPS/IFN-γ and then co-cultured them with ApoEVs or EVs containing 20 \u0026micro;g protein, respectively. Then protein and RNA samples were collected, and the expression levels of pro-inflammatory or anti-inflammatory macrophage-associated genes was analyzed using Western blot and qRT-PCR techniques. At the protein level, iNOS expression was significantly decreased in the ApoEVs and EVs groups, while the expression of Arg-1 was increased, and quantitative analysis further confirmed these results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Similarly, the mRNA expression levels of pro-inflammatory macrophage-related genes including TNF-α, iNOS, and IL-23 were significantly downregulated in ApoEVs and EVs-treated groups, while regarding anti-inflammatory macrophages, the expression levels of marker genes Arg-1, CD206, and CD163 were markedly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The same experiments were repeated on the Raw264.7 cell line, and the results were consistent with those described above (Figure S2A, B). To elucidate the mechanisms by which ApoEVs and EVs promote macrophage inflammatory phenotype switching, the activation states of canonical inflammatory pathways (JAK/STAT, PI3K-Akt, and NF-κB) were systematically analyzed. Pathway analysis revealed that both types of vesicles also inhibited the phosphorylation of STAT3, NF-κB, and AKT, while the phosphorylation of STAT6 was activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This further suggested that ApoEVs and EVs may participate in inhibiting the pro-inflammatory effects of macrophages by modulating the activation status of transcription factors during the inflammatory process.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Investigating the effects of ApoEVs and EVs on the activation level of HSCs\u003c/h2\u003e\u003cp\u003eHSCs are the main effector cells during liver fibrogenesis[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. But when liver injury occurs, inflammatory factors in liver can active HSCs, causing excessive ECM deposition as a wound-healing or scarring response[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, the regulation of HSCs activation has been recognized as a central target for the treatment of liver fibrosis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Signals from pro-fibrotic macrophages can enhance the production of TGF-β, thereby stimulating the activation of HSCs[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In consequence, based on the preliminary findings we considered that ApoEVs and EVs may ameliorate liver fibrosis by either promoting the secretion of anti-inflammatory factors from macrophages or directly inhibiting the activation of HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTo examine the regulatory effects of ApoEVs and EVs on HSCs, human HSCs cell line LX-2 was stimulated with TGF-β1 to induce activation, then co-cultured with ApoEVs or EVs. The expression of activation-related marker genes Col1a1 and αSMA were detected at both protein and mRNA levels. Western blot analysis showed Col1a1 and αSMA were downregulated in treated groups compared to activated HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). The same trend was observed through qRT-PCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggested that ApoEVs and EVs could directly inhibit the activation of HSCs. But they may also indirectly act on HSCs while promoting the release of anti-inflammatory cytokines from macrophages. In summary, ApoEVs and EVs could treat liver fibrosis by altering the activation state of HSCs, and ApoEVs could reduce Col1a1 and αSMA expression levels significantly.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Analyzed the potential mechanisms of ApoEVs\u0026rsquo; superior therapeutic functions in liver fibrosis\u003c/h2\u003e\u003cp\u003eOur study demonstrated that, ApoEVs showed significant anti-inflammatory and fibrosis-reversing capabilities in CCl4-induced liver fibrosis mouse model, as well as in vitro macrophage and HSCs. To futher elucidate the therapeutic mechanism of ApoEVs and to understand the reasons behind the differences in the efficacy of ApoEVs and EVs in reversing liver fibrosis, miRNA-sequencing was employed. We examined the miRNAs contained within ApoEVs and EVs and screened miRNAs with significant differential expression between ApoEVs and EVs, and focused on the group of miRNAs that are highly expressed in ApoEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Compared to the EVs group, ApoEVs exhibited a significant enrichment of 69 miRNAs. Through querying the HuDD database, 10 cirrhosis-associated miRNAs were identified from these 69 miRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Subsequently, the target genes of the 10 miRNAs (including let-7a-5p, let-7f-5p, miR-101-3p, miR-197-3p, miR-20a-5p, miR-381-3p, miR-411-5p, miR-455-3p, miR-92a-3p, miR-98-5p) that were significantly upregulated in ApoEVs were predicted using TargetScan and Miranda, and these genes were then further bioinformatically analyzed using GO and KEGG. In the GO analysis, the top 20 pathways of GO biological process (BP), cellular component (CC), and molecular function (MF) were displayed. These target genes could influence various biological processes to affect cellular behavior, including \u0026ldquo;protein transport\u0026rdquo;, \u0026ldquo;intracellular signal transduction\u0026rdquo;, \u0026ldquo;cell cycle\u0026rdquo;, \u0026ldquo;intracellular protein transport\u0026rdquo; et al. which were related to cell communication, cellular transport and so on (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In addition, the target genes of these 10 miRNAs were also associated with cellular components such as cytoplasm, nucleus, cytosol, and cytoskeleton et al (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Analysis of molecular functions revealed that the 10 miRNAs primarily target genes related to \u0026ldquo;ATP binding\u0026rdquo;, \u0026ldquo;hydrolase activity\u0026rdquo;, \u0026ldquo;kinase activity\u0026rdquo;, and \u0026ldquo;ubiquitin\u0026thinsp;\u0026minus;\u0026thinsp;protein transferase activity\u0026rdquo; which influenced the activity of some protein to regulate cell function (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Furthermore, KEGG pathway analysis revealed that the target genes were closely associated with MAPK signaling pathway, PI3K\u0026thinsp;\u0026minus;\u0026thinsp;Akt signaling pathway, Hippo signaling pathway, and Ras signaling pathway, which play crucial roles in regulating inflammatory responses and fibrosis (Figure G). These highly expressed miRNAs in ApoEVs and their functional analysis also explained why ApoEVs exhibit superior anti-inflammatory effects. The chord diagram illustrating the interaction analysis between miRNAs enriched in the classic inflammatory pathway PI3K-Akt and their corresponding target genes further revealed the potential characteristics of ApoEVs related to inflammation regulation, particularly in the context of liver cirrhosis-associated diseases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In summary, the bioinformatics analysis of miRNAs in ApoEVs and EVs suggested that ApoEVs outperformed EVs in reducing fibrosis, likely due to their enriched miRNA, which mediated the modulation and activation of inflammatory pathways such as the PI3K-Akt signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eChronic liver diseases such as fatty liver disease or viral hepatitis, all involve the persistent activation of inflammatory responses as well as sustained activation of liver fibrogenesis[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Many types of cells, cytokines, and miRNAs are involved in the development of liver fibrosis and cirrhosis[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In recent years, clinical trial results have indicated that stem cell therapy holds significant potential[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Accumulating evidence has elucidated that a large amount of stem cells underwent apoptosis after infusion, and these apoptotic cells still possessed considerable immunomodulatory functions although the exact mechanisms have yet to be fully understood[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. ApoEVs were emerging substances secreted during apoptosis and have shown exciting therapeutic prospects in many diseases. In our previous research, stem cells have harnessed their paracrine functions to exert potent therapeutic effects through the mediation of exosomes[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We aim to compare the immunomodulatory efficacies of ApoEVs and EVs which are two important vectors secreted by stem cells, with the aspiration of uncovering further refined strategies for the clinical application of stem cell therapy. In this study, we delved into the distinct characteristics of ApoEVs and EVs, unveiling their unique features and functional properties. We generated ApoEVs and EVs based on the apoptotic state of MSCs and compared their therapeutic effects on CCl4-induced fibrosis mice, as well as exploring the underlying molecular mechanisms, which provides valuable insights into the therapeutic significance of MSC apoptosis.\u003c/p\u003e\u003cp\u003eMSCs are a type of cells with self-renewal and multi-directional differentiation potential[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Over the past decades, MSCs have been intensely studied for clinical applications and have yielded the encouraging pre-clinical outcomes in varied animal disease models[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In chronic liver diseases, MSCs play a crucial role in maintaining tissue homeostasis and regeneration, as well as modulating the immune microenvironment. However, the specific mechanisms by which MSCs exert their therapeutic effects in vivo are not yet clearly understood. Recent studies have shown that transplanted MSCs may undergo apoptosis within a short period due to the influence of disease microenvironments such as hypoxia and inflammation[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. But cell apoptosis may not impair the immunosuppressive effects of MSCs and may even play a critical role in the treatment of diseases[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. A growing number of studies have demonstrated that apoptotic MSCs show similar therapeutic effects to those of live MSCs[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. To investigate the specific mechanisms that cell apoptosis exerts its effects, ApoEVs which are secreted by apoptotic MSCs have come into the spotlight of scientific inquiry. ApoEVs play a significant role in promoting immunomodulation, angiogenesis, and the regulation of homeostasis as they inherit the molecular characteristics from their parental MSCs[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. EVs and ApoEVs are members of the \u0026ldquo;secretome\u0026rdquo; of MSCs but in different active states. A large body of evidence suggests the reparative and regenerative properties of EVs in liver diseases[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Currently, research on the differences between ApoEVs and EVs produced by MSCs is still very limited. Elucidating the difference of their functional properties and effects is crucial for comprehending the significance of cell apoptosis in MSCs. In the present study, we observed that ApoEVs have a larger particle size and more abundant functional proteins compared to EVs, with distinct protein markers on their surfaces. We transplanted them in CCl4-induced mouse model for comparison respectively and found that both ApoEVs and EVs can ameliorate liver fibrosis in vivo. Notably, the effect of ApoEVs was more pronounced. Subsequently, we conducted further research to explore the mechanisms underlying this phenomenon.\u003c/p\u003e\u003cp\u003eThe secretion of pro-inflammatory cytokines by macrophages and the activation of HSCs are key events that lead to fibrosis. The accumulation of pro-fibrotic macrophages also plays a significant role in the activation of HSCs. Therefore, macrophages are central to the pathogenesis of liver fibrosis and have been potential target for combating fibrosis[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In traditional perspectives, macrophages are functionally categorized into two major subsets: classically activated macrophages (pro-inflammatory macrophages) and alternatively activated macrophages (anti-inflammatory macrophages). Macrophages polarization is crucial for tissue repair and the resolution of inflammation[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our studies confirmed that both ApoEVs and EVs have the ability to promote the transformation of macrophages towards an anti-inflammatory direction in vitro. We thought that the increased secretion of anti-inflammatory factors by macrophages controlled the progression of inflammation and may even indirectly inhibit the activation of HSCs. We will conduct further research to investigate this phenomenon. At the same time, we observed that they can directly inhibit the activation of HSCs. These results elucidated the potential mechanisms by which ApoEVs and EVs suppress inflammation and fibrosis development. And the comparative findings indicated that ApoEVs possessed remarkable capabilities to suppress the production of pro-inflammatory factors and inhibit cell activation.\u003c/p\u003e\u003cp\u003eComprehending the functional differences and the underlying reasons is crucial for the therapeutic potential of ApoEVs. To further explore the molecular mechanism why ApoEVs showed the superior therapeutic effects in liver fibrosis, we conducted miRNA-sequencing analysis on both ApoEVs and EVs. MiRNA is a class of small non-coding endogenous RNA molecules that regulate gene expression through translational repression or mRNA degradation. They serve as primary mediators of intercellular communication via vesicles and play a unique role in modulating the activation, infiltration, and resolution of inflammation in macrophages and HSCs[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We performed differential analysis on the miRNA-sequencing data of these two vesicles and screened 10 relevant miRNAs in ApoEVs (let-7a-5p, let-7f-5p, miR-101-3p, miR-197-3p, miR-20a-5p, miR-381-3p, miR-411-5p, miR-455-3p, miR-92a-3p, miR-98-5p). Then further enrichment analysis revealed that these 10 miRNAs were associated with intracellular signal transduction and protein transport, and their target genes were related to various enzyme activities. Additionally, the target genes were clustered in inflammatory pathways such as MAPK and PI3K-Akt. These results provided crucial insights into the mechanisms of ApoEVs in suppressing inflammation and cell activation. Our next step will further verify the therapeutic effects of ApoEVs-miRNAs on liver fibrosis and explore the molecular mechanisms by which ApoEVs exert their effects to offer new possibilities for disease amelioration.\u003c/p\u003e\u003cp\u003eHowever, this study did not elucidate the significance of apoptotic events in MSC-based therapy and the mechanistic exploration of ApoEV-mediated regulation of macrophage inflammatory phenotypes remained insufficient. And finally, experiments to verify the specific mechanism of miRNAs was ultimately lacking. Next, we will continue to further study the mechanism of ApoEVs in liver fibrosis and clarify the important significance of apoptotic MSCs in clinical treatment.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, our results highlight the distinct characteristics of vesicles secreted by MSCS in different apoptotic states and explore the great potential of ApoEVs in the treatment of liver fibrosis. Notably, comparing to EVs, ApoEVs exhibited superior therapeutic effects on fibrosis, which may due to the enrichment of miRNAs associated with inflammation regulation in ApoEVs. Overall, our results suggested that ApoEVs could be a promising approach for the treatment of liver diseases and further lay a research foundation for clarifying the therapeutic mechanism of MSCs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emesenchymal stem cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMMPs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ematrix metalloproteinases\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eextracellular matrix\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHSCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehepatic stellate cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEVs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eextracellular vesicles\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emiRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emicroRNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eApoEVs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eapoptotic extracellular vesicles\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCCl4\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecarbon tetrachloride\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSPF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003especific pathogen-free\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphate buffered saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ehUC-MSCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehuman umbilical cord-derived MSCs\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estaurosporine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etransmission electron microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNTA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enanoparticle tracking analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMDMs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebone morrow-derived macrophages\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ealanine aminotransferase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003easpartate aminotransferase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egene ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebiological process\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecellular component\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emolecular function.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Figdraw (https://www.figdraw.com) for providing graphic elements in creating the figures in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRuobing Ju, Siyuan Tian and Bo Li performed the most of experiments, analyzed data and wrote the manuscript. Bo Li, Xia Zhou isolated and collected exosomes. Yinan Hu and Erzhuo Xia built animal models. Bo Li and Fangfang Yang contributed to flow cytometry analysis. Miao Zhang, Shuoyi Ma and Rui Su provided technical support. Ying Han, Yulong Shang and Xia Zhou designed the study and revised the manuscript. All authors read and approved the current version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by National Natural Science Foundation of China (No. 82270551, No. 82200680, No.82300672),\u0026nbsp;Key Research and Development Program of Shaanxi province, China (No. 2024SF-GJHX-16), National Key Research and Development Program of China (No. 2020YFA0710803), Science and Technology Association youth talent lifting program of Shaanxi province, China (No.20230324) and Xijing Hospital Project (No. XJZT25ZH08).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was approved by the Animal Welfare and Ethics Committee of the Fourth Military Medical University and performed according to the \u0026ldquo;Guidelines for the Care and Use of Laboratory Animals\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eV. Taru, G. Szabo, W. Mehal, T. Reiberger, Inflammasomes in chronic liver disease: Hepatic injury, fibrosis progression and systemic inflammation, Journal of Hepatology 81(5) (2024) 895-910.\u003c/li\u003e\n\u003cli\u003eP. Gin\u0026egrave;s, A. Krag, J.G. Abraldes, E. Sol\u0026agrave;, N. Fabrellas, P.S. Kamath, Liver cirrhosis, The Lancet 398(10308) (2021) 1359-1376.\u003c/li\u003e\n\u003cli\u003eS. Ling, G. Jiang, Q. Que, S. Xu, J. Chen, X. Xu, Liver transplantation in patients with liver failure: Twenty years of experience from China, Liver international : official journal of the International Association for the Study of the Liver 42(9) (2022) 2110-2116.\u003c/li\u003e\n\u003cli\u003eW. Pu, H. Zhu, M. Zhang, M. Pikiolek, C. Ercan, J. Li, X. Huang, X. Han, Z. Zhang, Z. Lv, Y. Li, K. Liu, L. He, X. Liu, M.H. Heim, L.M. Terracciano, J.S. Tchorz, B. Zhou, Bipotent transitional liver progenitor cells contribute to liver regeneration, Nature genetics 55(4) (2023) 651-664.\u003c/li\u003e\n\u003cli\u003eP. Liu, Y. Mao, Y. Xie, J. Wei, J. Yao, Stem cells for treatment of liver fibrosis/cirrhosis: clinical progress and therapeutic potential, Stem Cell Research \u0026amp; Therapy 13(1) (2022) 356.\u003c/li\u003e\n\u003cli\u003eM. Wang, M. Zhang, L. Fu, J. Lin, X. Zhou, P. Zhou, P. Huang, H. Hu, Y. Han, Liver-targeted delivery of TSG-6 by calcium phosphate nanoparticles for the management of liver fibrosis, Theranostics 10(1) (2020) 36-49.\u003c/li\u003e\n\u003cli\u003eL. Ma, J. Wei, Y. Zeng, J. Liu, E. Xiao, Y. Kang, Y. Kang, Mesenchymal stem cell-originated exosomal circDIDO1 suppresses hepatic stellate cell activation by miR-141-3p/PTEN/AKT pathway in human liver fibrosis, Drug Delivery 29(1) (2022) 440-453.\u003c/li\u003e\n\u003cli\u003eV.N. Hieu, L.T.T. Thuy, H. Hai, N.Q. Dat, D.V. Hoang, N.V. Hanh, D.M. Phuong, T.H. Hoang, H. Sawai, Y. Shiro, M. Sato-Matsubara, D. Oikawa, F. Tokunaga, K. Yoshizato, N. Kawada, Capacity of extracellular globins to reduce liver fibrosis via scavenging reactive oxygen species and promoting MMP-1 secretion, Redox Biology 52 (2022) 102286.\u003c/li\u003e\n\u003cli\u003eZ. Tian, Y. Zenan, W. Jianyu, P. Duanqing, D. Xin, H. Chang, L. Peilong, Challenges and advances in clinical applications of mesenchymal stromal cells, J Hematol Oncol (2021).\u003c/li\u003e\n\u003cli\u003eW. Chen, F. Lin, X. Feng, Q. Yao, Y. Yu, F. Gao, J. Zhou, Q. Pan, J. Wu, J. Yang, J. Yu, H. Cao, L. Li, MSC-derived exosomes attenuate hepatic fibrosis in primary sclerosing cholangitis through inhibition of Th17 differentiation, Asian Journal of Pharmaceutical Sciences 19(1) (2024) 100889.\u003c/li\u003e\n\u003cli\u003eF. Cheng, F. Yang, Y. Wang, J. Zhou, H. Qian, Y. Yan, Mesenchymal stem cell-derived exosomal miR-27b-3p alleviates liver fibrosis via downregulating YAP/LOXL2 pathway, Journal of Nanobiotechnology 21(1) (2023) 195.\u003c/li\u003e\n\u003cli\u003eB. banimohamad-shotorbani, H. Kahroba, H. Sadeghzadeh, D.M. Wilson, H. Maadi, N. Samadi, M.S. Hejazi, H. Farajpour, B.N. Onari, M.R. Sadeghi, DNA damage repair response in mesenchymal stromal cells: From cellular senescence and aging to apoptosis and differentiation ability, Ageing Research Reviews 62 (2020) 101125.\u003c/li\u003e\n\u003cli\u003eY. Fu, B. Sui, L. Xiang, X. Yan, D. Wu, S. Shi, X. Hu, Emerging understanding of apoptosis in mediating mesenchymal stem cell therapy, Cell Death \u0026amp; Disease 12(6) (2021) 596.\u003c/li\u003e\n\u003cli\u003eS.H.M. Pang, J. D\u0026rsquo;Rozario, S. Mendonca, T. Bhuvan, N.L. Payne, D. Zheng, A. Hisana, G. Wallis, A. Barugahare, D. Powell, J. Rautela, N.D. Huntington, G. Dewson, D.C.S. Huang, D.H.D. Gray, T.S.P. Heng, Mesenchymal stromal cell apoptosis is required for their therapeutic function, Nature communications 12(1) (2021) 6495.\u003c/li\u003e\n\u003cli\u003eC. Giacomini, C. Gran\u0026eacute;li, R. Hicks, F. Dazzi, The critical role of apoptosis in mesenchymal stromal cell therapeutics and implications in homeostasis and normal tissue repair, Cellular \u0026amp; Molecular Immunology 20(6) (2023) 570-582.\u003c/li\u003e\n\u003cli\u003eR. Lin, T. Zhang, J.-Q. Gao, Apoptotic Vesicles of MSCs: The Natural Therapeutic Agents and Bio‐Vehicles for Targeting Drug Delivery, Small 19 (2023) e2301671.\u003c/li\u003e\n\u003cli\u003eO. Q, H. W, W. B, N. L, L. Z, M. X, S. S, Apoptotic Vesicles: Therapeutic Mechanisms and Critical Issues, J Dent Res (2024).\u003c/li\u003e\n\u003cli\u003eZ. Li, M. Wu, S. Liu, X. Liu, Y. Huan, Q. Ye, X. Yang, H. Guo, A. Liu, X. Huang, X. Yang, F. Ding, H. Xu, J. Zhou, P. Liu, S. Liu, Y. Jin, K. Xuan, Apoptotic vesicles activate autophagy in recipient cells to induce angiogenesis and dental pulp regeneration, Molecular Therapy 30(10) (2022) 3193-3208.\u003c/li\u003e\n\u003cli\u003eB. Sui, R. Wang, C. Chen, X. Kou, D. Wu, Y. Fu, F. Lei, Y. Wang, Y. Liu, X. Chen, H. Xu, Y. Liu, J. Kang, H. Liu, R.T.K. Kwok, B.Z. Tang, H. Yan, M. Wang, L. Xiang, X. Yan, X. Zhang, L. Ma, S. Shi, Y. Jin, Apoptotic Vesicular Metabolism Contributes to Organelle Assembly and Safeguards Liver Homeostasis and Regeneration, Gastroenterology 167(2) (2024) 343-356.\u003c/li\u003e\n\u003cli\u003eL. Ma, C. Chen, D. Liu, Z. Huang, J. Li, H. Liu, R.T. Kin Kwok, B. Tang, B. Sui, X. Zhang, J. Tang, X. Mao, W. Huang, S. Shi, X. Kou, Apoptotic extracellular vesicles are metabolized regulators nurturing the skin and hair, Bioactive Materials 19 (2023) 626-641.\u003c/li\u003e\n\u003cli\u003eC. Zheng, B. Sui, X. Zhang, J. Hu, J. Chen, J. Liu, D. Wu, Q. Ye, X. Lei, X. Qiu, S. Liu, Z. Deng, J. Zhou, S. Liu, S. Shi, Y. Jin, Apoptotic vesicles restore liver macrophage homeostasis to counteract type 2 diabetes, Journal of Extracellular Vesicles 10 (2021).\u003c/li\u003e\n\u003cli\u003eL. Yu, G. Dou, H. Kuang, L. Bao, H. Liu, Q. Ye, Z. Wang, X. Yang, L. Ren, Z. Li, H. Liu, B. Li, S. Liu, S. Ge, S. Liu, Apoptotic Extracellular Vesicles Induced Endothelial Cell-Mediated Autologous Stem Cell Recruitment Dominates Allogeneic Stem Cell Therapeutic Mechanism for Bone Repair, ACS Nano 18(12) (2024) 8718-8732.\u003c/li\u003e\n\u003cli\u003eX. Zhang, J. Yang, S. Ma, X. Gao, G. Wang, Y. Sun, Y. Yu, Z. Wang, W. Tian, L. Liao, Functional diversity of apoptotic vesicle subpopulations from bone marrow mesenchymal stem cells in tissue regeneration, Journal of Extracellular Vesicles 13(4) (2024) e12434.\u003c/li\u003e\n\u003cli\u003eS. Tian, X. Zhou, M. Zhang, L. Cui, B. Li, Y. Liu, R. Su, K. Sun, Y. Hu, F. Yang, G. Xuan, S. Ma, X. Zheng, X. Zhou, C. Guo, Y. Shang, J. Wang, Y. Han, Mesenchymal stem cell-derived exosomes protect against liver fibrosis via delivering miR-148a to target KLF6/STAT3 pathway in macrophages, Stem Cell Research \u0026amp; Therapy 13(1) (2022) 330.\u003c/li\u003e\n\u003cli\u003eP.-F. Ma, C.-C. Gao, J. Yi, J.-L. Zhao, S.-Q. Liang, Y. Zhao, Y.-C. Ye, J. Bai, Q.-J. Zheng, K.-F. Dou, H. Han, H.-Y. Qin, Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice, Journal of Hepatology 67(4) (2017) 770-779.\u003c/li\u003e\n\u003cli\u003eX. Zheng, X. Zhou, G. Ma, J. Yu, M. Zhang, C. Yang, Y. Hu, S. Ma, Z. Han, W. Ning, B. Jin, X. Zhou, J. Wang, Y. Han, Endogenous Follistatin-like 1 guarantees the immunomodulatory properties of mesenchymal stem cells during liver fibrotic therapy, Stem Cell Research \u0026amp; Therapy 13(1) (2022) 403.\u003c/li\u003e\n\u003cli\u003eZ. Wang, K. Du, N. Jin, B. Tang, W. Zhang, Macrophage in liver Fibrosis: Identities and mechanisms, Int Immunopharmacol (2023).\u003c/li\u003e\n\u003cli\u003eY. Wen, J. Lambrecht, C. Ju, F. Tacke, Hepatic macrophages in liver homeostasis and diseases-diversity, plasticity and therapeutic opportunities, Cellular \u0026amp; Molecular Immunology 18(1) (2021) 45-56.\u003c/li\u003e\n\u003cli\u003eR.F. Schwabe, I. Tabas, U.B. Pajvani, Mechanisms of Fibrosis Development in Nonalcoholic Steatohepatitis, Gastroenterology (2020).\u003c/li\u003e\n\u003cli\u003eB. Sim\u0026oacute;n-Codina, J. Cacho-Pujol, A. Moles, P. Melgar-Lesmes, Reprogramming macrophages to treat liver diseases, Hepatology (2024).\u003c/li\u003e\n\u003cli\u003eK. Dakota R, M. Kyle S, Hepatic stellate cells in physiology and pathology, J Physiol (2022).\u003c/li\u003e\n\u003cli\u003eL. Hammerich, F. Tacke, Hepatic inflammatory responses in liver fibrosis, Nature Reviews Gastroenterology \u0026amp; Hepatology 20(10) (2023) 633-646.\u003c/li\u003e\n\u003cli\u003eT. Tsuchida, S.L. Friedman, Mechanisms of hepatic stellate cell activation, Nature Reviews Gastroenterology \u0026amp; Hepatology 14(7) (2017) 397-411.\u003c/li\u003e\n\u003cli\u003eS. Y-Y, L. X-F, M. X-M, H. C, Z. L, L. J, Macrophage Phenotype in Liver Injury and Repair, Scand J Immunol (2016).\u003c/li\u003e\n\u003cli\u003eZ. Wang, K. Du, N. Jin, B. Tang, W. Zhang, Macrophage in liver Fibrosis: Identities and mechanisms, International Immunopharmacology 120 (2023) 110357.\u003c/li\u003e\n\u003cli\u003eP. Maurizio, P. Massimo, Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues, Mol Aspects Med (2018).\u003c/li\u003e\n\u003cli\u003eW.-C. Zhou, Q.-B. Zhang, L. Qiao, Pathogenesis of liver cirrhosis, World journal of gastroenterology (2014).\u003c/li\u003e\n\u003cli\u003eB.J. Dwyer, M.T. Macmillan, P.N. Brennan, S.J. Forbes, Cell therapy for advanced liver diseases: Repair or rebuild, Journal of Hepatology 74(1) (2021) 185-199.\u003c/li\u003e\n\u003cli\u003eE. El Agha, R. Kramann, R.K. Schneider, X. Li, W. Seeger, B.D. Humphreys, S. Bellusci, Mesenchymal Stem Cells in Fibrotic Disease, Cell Stem Cell (2017).\u003c/li\u003e\n\u003cli\u003eT. Zhou, Z. Yuan, J. Weng, D. Pei, X. Du, C. He, P. Lai, Challenges and advances in clinical applications of mesenchymal stromal cells, J Hematol Oncol (2021).\u003c/li\u003e\n\u003cli\u003eR. Wang, J. Fu, J. He, X. Wang, W. Xing, X. Liu, J. Yao, Q. Ye, Y. He, Apoptotic mesenchymal stem cells and their secreted apoptotic extracellular vesicles: therapeutic applications and mechanisms, Stem Cell Research \u0026amp; Therapy 16(1) (2025) 78.\u003c/li\u003e\n\u003cli\u003eQ. Ou, X. Qiao, Z. Li, L. Niu, F. Lei, R. Cheng, T. Xie, N. Yang, Y. Liu, L. Fu, J. Yang, X. Mao, X. Kou, C. Chen, S. Shi, Apoptosis releases hydrogen sulfide to inhibit Th17 cell differentiation, Cell Metab (2024).\u003c/li\u003e\n\u003cli\u003eM.B. Preda, C.A. Neculachi, I.M. Fenyo, A.-M. Vacaru, M.A. Publik, M. Simionescu, A. Burlacu, Short lifespan of syngeneic transplanted MSC is a consequence of in vivo apoptosis and immune cell recruitment in mice, Cell Death Dis (2021).\u003c/li\u003e\n\u003cli\u003eG. Lou, Z. Chen, M. Zheng, Y. Liu, Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases, Experimental \u0026amp; Molecular Medicine 49(6) (2017) e346-e346.\u003c/li\u003e\n\u003cli\u003eC. Wang, C. Ma, L. Gong, Y. Guo, K. Fu, Y. Zhang, H. Zhou, Y. Li, Macrophage Polarization and Its Role in Liver Disease, Front Immunol (2021).\u003c/li\u003e\n\u003cli\u003eK. Hamidzadeh, S.M. Christensen, E. Dalby, P. Chandrasekaran, D.M. Mosser, Macrophages and the Recovery from Acute and Chronic Inflammation, Annu Rev Physiol (2017).\u003c/li\u003e\n\u003cli\u003eW. Yu, S. Wang, Y. Wang, H. Chen, H. Nie, L. Liu, X. Zou, Q. Gong, B. Zheng, MicroRNA: role in macrophage polarization and the pathogenesis of the liver fibrosis, Frontiers in Immunology 14 (2023).\u003c/li\u003e\n\u003cli\u003eX. Wang, Y. He, B. Mackowiak, B. Gao, MicroRNAs as regulators, biomarkers and therapeutic targets in liver diseases, Gut (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Liver fibrosis, mesenchymal stem cells, apoptotic extracellular vesicles, extracellular vesicles, macrophages phenotype, hepatic stellate cells","lastPublishedDoi":"10.21203/rs.3.rs-6970215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6970215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Mesenchymal stem cells (MSCs) have become a promising treatment of liver fibrosis which is a key process in liver diseases. Recent studies showed that transplanted mesenchymal stem cells (MSCs) undergo rapid apoptosis and the apoptotic extracellular vesicles (ApoEVs) derived from MSCs exhibited stronger immunosuppressive capability. But the effect and the mechanisms of ApoEVs in liver fibrosis remain unclear. The functional differences between ApoEVs and extracellular vesicles (EVs) have yet to be elucidated. This study aims to compare their therapeutic effects on liver fibrosis in order to optimize existing treatment strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e ApoEVs and EVs were isolated by density gradient centrifugation and illustrated by TEM and NTA. A CCl4-induced liver fibrosis mouse model was treated with equal doses of ApoEVs and EVs. Histopathological analysis was performed on liver sections, serological indicators, fibrosis-related gene expression, macrophage polarization, and the activation status of hepatic stellate cells (HSCs) were analyzed. Subsequently, miRNA-sequencing analysis was conducted to identify potential pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eOur results demonstrated that ApoEVs had fourfold higher protein yield than EVs, and ApoEVs exhibited a significant superior ability to improve liver fibrosis. In vitro, ApoEVs enhanced macrophage polarization and suppressed HSC activation more effectively, thereby reducing the degree of fibrosis. The underlying molecular mechanism likely due to the enrichment of more miRNAs targeting the PI3K-Akt pathway in ApoEVs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e These findings showed that ApoEVs exhibit better efficacy than EVs in treating liver fibrosis. Besides, the findings highlighted their therapeutic potential, clarified functional differences, and suggested ApoEVs as a promising strategy for liver disease treatment.\u003c/p\u003e","manuscriptTitle":"Extracellular vesicles derived from live or apoptotic mesenchymal stem cells: comparison of the effects of two extracellular vesicles on liver fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 10:14:48","doi":"10.21203/rs.3.rs-6970215/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-19T15:34:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T10:10:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-10T06:19:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178126297987852081911608316701100247517","date":"2025-08-08T14:21:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321238049817941002073078291327399776581","date":"2025-08-06T13:53:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-06T08:50:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-05T12:56:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-14T07:32:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-12T09:19:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Gastroenterology","date":"2025-07-12T09:15:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6bbd4622-2678-4ed1-8865-c7991edae624","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:21:40+00:00","versionOfRecord":{"articleIdentity":"rs-6970215","link":"https://doi.org/10.1186/s12876-026-04762-0","journal":{"identity":"bmc-gastroenterology","isVorOnly":false,"title":"BMC Gastroenterology"},"publishedOn":"2026-03-23 16:11:57","publishedOnDateReadable":"March 23rd, 2026"},"versionCreatedAt":"2025-08-11 10:14:48","video":"","vorDoi":"10.1186/s12876-026-04762-0","vorDoiUrl":"https://doi.org/10.1186/s12876-026-04762-0","workflowStages":[]},"version":"v1","identity":"rs-6970215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6970215","identity":"rs-6970215","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}