Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages

Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages | 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 Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages Nianci Sun, Ziling Wang, Honghui Jiang, Biyao Wang, Kunhang Du, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4180160/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Extrinsic molecular mechanisms that regulate extramedullary stress erythropoiesis are still poorly understood, and the exploration of potential protective medication is needed. Materials and methods Peripheral blood parameters and BFU-E colony enumeration were measured. IHC staining was conducted to detect the proliferation of splenocytes and splenic F4/80 macrophages. The expression of β-catenin protein in RAW264.7 macrophages was assessed using immunofluorescence. The cell cycle of mouse spleen c-kit + cells was analyzed by flow cytometry assay. Detection of Ccl2, Hk2, and Pgk1 mRNA expression by RT-qPCR. Cyclin D1 protein expression was assessed using Western blotting. IL-1 and EPO levels were determined by ELISA assay. Results In the 5-FU pre-administrated mouse, ASP rescued peripheral blood parameters such as RBC counts, HGB, HCT and MCH, and the BFU-E colony enumeration in the bone marrow. Meanwhile, ASP increased cellular proliferation in the splenic red pulp and cyclin D1 expression, ASP increased macrophage chemokine Ccl2 genetic expression and the number of F4/80 macrophages in the spleen and splenic BFU-E enumeration. Furthermore, ASP facilitated glycolytic genes including Hk 2, Pgk 1, Pkm, Pdk 1 and Ldha via PI3K/Akt/HIF2α signaling in the spleen. Subsequently, ASP declined pro-proinflammatory factor IL-1β, whereas upregulating erythroid proliferation-associated genes Gdf15, Bmp4, Wnt2b, and Wnt8a . Moreover, ASP facilitated EPO/STAT5 signaling in splenic macrophages to enhance erythroid lineage Gata2, Gata1 genetic expression. Also, ASP facilitated erythroid differentiation via macrophage-mediated EpoR/STAT5 signaling. Conclusions ASP facilitate extramedullary stress erythropoiesis that suggest it might be a promising strategy for stress anemia treatment. cancer chemoprevention extramedullary hematopoiesis stress erythropoiesis splenic macrophage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Inflammation or chemotherapy/radiotherapy may cause chronic stress anemia. However, the conventional treatment for chronic anemia such as blood transfusion and recombinant EPO injection may lead to various side effects such as iron overload, hypertension, high blood viscosity, and venous thrombosis, et al [ 1 – 3 ]. During this period, extramedullary stress erythropoiesis begins as a compensatory response to the loss of erythroid output and anoxia [ 4 ]. Theoretically, in order to help maintain homeostasis until steady state conditions are restored, a wave of new erythrocytes is rapidly generated by stress erythropoiesis [ 5 ]. The strategy of varies from steady state erythropoiesis. The transient amplification of erythroid progenitors (SEPs) originates from heterogenous populations composed of the most immature CD34 + CD133 + KS cells, intermediate immature CD34 neg CD133 + KS cells, and the most mature CD34 neg CD133 neg KS cells [ 6 ]. Multifold key signaling and factors such as BMP4, GDF15, Wnt/β-catenin, and Hedgehog promote SEP proliferation [ 7 ]. However, in a new opinion, the regulation of cellular activities relies on intracellular metabolism, how these macrophage-derived signals and factors interlaced control metabolism and how alterations in metabolism leads to SEP self-renewal and proliferation remain to be elucidative and a promising therapeutic strategy for promoting stress erythropoiesis still needs exploration. Latest documents evidence that signals driving the proliferation of SEPs establish an efficient anabolic metabolism to meet the rapid cell division [ 8 ]. Just like the “Warburg effect” seen in cancer cells, aerobic glycolysis predominates in metabolic control of stress erythropoiesis proliferation, with glycolytic metabolites being shunted into anabolic pathways. For example, pathways like the pentose phosphate pathway and the serine glycine pathway, are very effective in generating nucleotides, amino acids, metabolism and regenerate NADPH levels [ 9 ]. The hypoxia-inducible factor oxygen sensing pathway reprograms cellular glucose/fatty acid metabolism under cellular defense condition [ 10 ]. HIF1α and HIF2α isoforms specifically inhibit key enzymes for fatty acid oxidation-dependent mitochondrial TCA cycle inhibition; mitochondrial complex activity; and crucial regulators of the mitochondrial contents. This inhibition reduces oxygen consumption from mitochondrial glucose and fatty acid-dependent under hypoxic conditions. This HIF-dependent damping of mitochondrial activity not only conserves oxygen during hypoxic but also reduces the formation of toxic mitochondrial reactive oxygen species (ROS), eliciting cellular tolerance to hypoxic and oxidative stress conditions [ 11 ]. Among HIFs, HIF2α is the major effector of stress-induced renal EPO producing cells and the primary inducer of EPO genes [ 12 ]. Under normoxic conditions, growth factors and cytokines play a regulatory role in the PI3K/Akt signaling pathway. Synthesis of HIFs rises when PI3K/Akt/mTOR is activated. In low oxygen levels, hypoxia-induced HIFs precede PI3K/Akt activation, however, the stability of HIFs is linked to the PI3K/Akt signaling pathway. The investigation of metabolism regulating stress erythropoiesis will be fascinating [ 13 ]. SEPs do not undergo differentiate until there is an increase in serum erythropoietin (EPO) levels. Instead of directly regulating SEPs, EPO facilitates the shift of SEPs from proliferation to differentiation by targeting splenic erythroblastic island central macrophages (EIM), which express EpoR. EPO/STAT5 signaling in macrophages stimulates the production of PGJ2, thus activating PPARg dependent repression of Wnt expression. Moreover, EPO/STAT5 signaling enhances production of prostaglandin E2 (PGE2), promoting the function of EIM in cell adhesion, phagocytosis, iron translocation, and nuclear engulfment of EIM, and the differentiation of SEPs. It has been demonstrated that depletion of EIM or inhibition of EPO/STAT5 signaling in macrophages has been shown to impair stress erythropoiesis and clearance of apoptotic cells. In murine diseases models like β-thalassemia, Sickle cell disease, and polycythemia vera, depleting macrophages has been found to enhance erythropoiesis, indicating that EIM are novel targets for therapeutic intervention. ASP (Angelica sinensis polysaccharides) is a main bioactive component of Angelica sinensis (Oliv.) Diels (Apiaceae). It has antioxidative, anti-inflammatory, anti-tumor, hematopoietic, immunoregulatory, hepatoprotective activities [ 14 , 15 ]. Our previous research demonstrated that ASP has antioxidative protective effects on BM perivascular mesenchymal progenitors, thereby maintaining hematopoietic progenitor cells in mice treated with 5-FU. Additionally, ASP has been shown to shield splenocytes from oxidative stress and apoptosis induced by 5-FU. However, it remains unclear whether ASP ameliorates stress erythropoiesis in cell defense conditions. Herein, in this study, we utilized the 5-FU-myeloablative mouse model to investigate the impact of ASP on stress anemia and its underlying mechanism. We found that ASP promoted the proliferation of splenocytes including extra-medullar erythropoiesis dependent macrophages and erythroid cells via PI3K/Akt/HIF2α-mediated glycolytic process. Additionally, ASP boosted EPO/EPOR/STAT5 signaling transduction in splenic macrophages, which could be the reason for promoting differentiation of SEPs. Intriguingly, our research suggests that ASP could be a potentially therapeutic strategy for stress anemia. 2. Material and Method 2.1 Reagent and antibody 5-FU was acquired from Sigma-Aldrich (St. Louis, USA). Angelica sinensis polysaccharides (ASP) was bought from Ciyuan Biotechnology Co. Ltd. (Shanxi, China). CD117(c-kit)-FITC was purchased from Biolegend (SanDiego, USA). EPO protein was purchased from MCE (New Jersey, USA). SYBR green I and Reverse Transcription Kit were purchased from TaKaRa (Kyoto, Japan). The following antibodies were acquired from various suppliers: \({\beta }\) - actin was procured from Proteintech (Wuhan, China); HIF2 \({\alpha }\) was purchased from Affinity (Jiangsu, China); BMP4, p-STAT5, STAT5 were obtained from ABclonal (Wuhan, China); PI3K, p-AKT were sourced from Wanleibio (Shenyang, China); cyclin D1 was obtained from CST (Danvers, USA); Ki67 was obtained from Bioss (Beijing, China); F4/80 was purchased from Affinity (Jiangsu, China). The goat-anti-rabbit immunofluorescent secondary antibody was purchased from Beyotime (Shanghai, China). 2.2 Experimental animals C57BL/6 J mice, aged 6–8 weeks, were purchased from our school’s animal experimental center (n = 60) and housed in the Specific Pathogen-Free (SPF) laboratory. The mice were randomly allocated into four distinct groups: control group, ASP group, 5-FU group, and ASP + 5-FU group. In the experimental design, the control group of mice was administered intraperitoneal injections of saline at a dosage of 10 ml/kg per day for either 7 or 10 consecutive days. The ASP group of mice was administered intraperitoneal injection of ASP at a dosage of 100 mg/kg \(\) per day for either 7 or 10 consecutive days. On the first day, the 5-FU group of mice was subjected to an initial intraperitoneal injection of 5-FU at a dosage of 150 mg/kg, followed by saline injections at a dosage of 10 ml/kg 6 hours later, and subsequently received daily saline injections for the next 6 or 9 days. In the ASP + 5-FU group, mice received a single injection of 5-FU at 150 mg/kg on the first day, followed by an intraperitoneal injection of ASP at a dosage of 100 mg/kg 6 hours later, and then continued to receive ASP injections at the same dosage for 6 and 9 days. The mice were euthanized using carbon dioxide. Repeat independent animal experiments at least 3 times. 2.3 Cell Culture RAW264.7 macrophages were cultured in DMEM-High Glucose medium (Gibco, Grand Island, USA), supplementing with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody. The RAW264.7 macrophages were randomly allocated into four groups: control group (without treatment), ASP group (100 ug/ml ASP treated for 24 hours), 5-FU group (25 ug/ml 5-FU treated for 24 hours), ASP + 5-FU group (pretreated with 25 ug/ml 5-FU for 6 hours, then cultured with 100 ug/ml ASP for 24 hours). To observe the effect of EPO on EPO/EPOR/STAT5 signaling of RAW264.7 macrophages, 5 U/ml EPO was added to the medium for 30minutes, subsequently, STAT5 and p-STAT5 were detected by western blot. 2.4 Peripheral blood parameters test Mice were anesthetized and their eyeballs were enucleated. EDTA anticoagulation tubes were utilized to gather orbital blood samples from the murine cohort for further analysis with an automated hematology analyzer. 2.5 BFU-E colony forming culture In bone marrow BFU-E (erythroid burst-forming unit) colony formation assay, the femurs and tibias of the mice were extracted and the muscles and tissues were cleaned. The bone marrow mononuclear cells were separated by density gradient centrifugation using Ficoll-Paque (1.077g/mL). In splenic colony forming assay, splenocytes were treated with erythrocyte lysates to lyse the erythrocytes. Following the manufacturer’s instructions, BMMNCs and splenocytes (1 \(\times\) 10 5 ) were cultured in 35mm Petri dishes with methylcellulose medium MethoCultTM SF M3436 (StemCell, Vancouver, Canada) for 10–14 d, and then counted by microscopy. Each colony comprises no less than 50 cells. 2.6 Brilliant Cresyl Blue staining The peripheral blood of mice in each group was collected in EDTA anticoagulant tube, and was mixed 1:1 with Brilliant Cresyl Blue solution, left at room temperature for 15–20 minutes. Blood smears were prepared and observed by microscopy. 2.7 Immunohistochemistry staining SA1022 kit (BOSTER, Wuhan, China) and AR1027 kit (BOSTER, China) were used in the experiment immunohistochemistry. Paraffin sections of mouse spleen measuring 5µm were taken, antigen retrieval was performed by heating, and the sections were blocked with 5% bovine serum albumin. Tissue sections were incubated with Ki67 antibody (1: 400) and F4/80 antibody (1: 150) at 4℃ overnight. Thereafter, tissue sections were treated with secondary antibody, followed by DAB staining and counterstaining with Mayer’s hematoxylin (Solarbio, Beijing, China). Images were obtained using microscopy and analyzed by Image J. 2.8 ELISA Mouse serum and spleen homogenate were detected by IL-1 \({\beta }\) ELISA kits (QuantiCyto, Shenzhen, China) and EPO ELISA kits (MULTI SCIENCES, Hangzhou, China), respectively, according to the manufacturer’s instructions. 2.9 GO function and KEGG pathway enrichment analysis The ASP and 5-FU intersection targets were imported to the DAVID database ( https://david.ncifcrf.gov ) for GO and KEGG enrichment analysis, and sort the results by adjusted P value \(\le\) 0.05. The top 10 enriched function and top 20 enriched pathways using bioinformatics data processing platform ( http://www.bioinformatics.com.cn/ ) online tool to visualize the results. 2.10 Flow cytometry analysis After pretreatment with erythrocyte lysates, the splenocytes were adjusted to 10 6 per tube and then subjected to blocking with 5% BSA at 4°C for a duration of 30 minutes. Subsequently, incubating the cells with the primary antibody CD117 (c-kit + ) at 4 \(℃\) for 30 minutes. Later, the pre-chilled 75% ethanol was added to fix the cells at 4 ℃ overnight. The ethanol was discarded and washed with PBS after centrifugation. The cells were incubated with PI at 4 \(℃\) for 30 minutes, then centrifuged and washed twice with PBS. Cell cycle of the c-kit + cell population was analyzed by flow cytometer. 2.11 Western blot assay Splenocytes and RAW264.7 cells were prepared as mentioned previously. The total protein was extracted using pre-chilled RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China). The protein concentration was determined using a BCA assay kit (EpiZyme, Shanghai, China). Add the supernatant into the Loading Buffer and boil for 10min. Equal amounts of protein were separated using SDS-PAGE and then transferred to PVDF membrane (Beyotime, China). The membrane was blocked with 5% skimmed milk powder (dissolved in TBST). Subsequently, the membrane was incubated with corresponding primary antibodies at the appropriate dilution and refrigerated at 4 \(℃\) overnight. On next day, the membrane was washed three times with TBST lasting 10 minutes each, followed by incubation with the secondary antibodies for a duration of 1 hour at ambient temperature. The protein bands were visualized and captured using an enhanced ECL chemiluminescence detection System, and the band intensities of were analyzed using Image Lab software. 2.12 Immunofluorescence staining RAW264.7 cells were inoculated on a sterile slide inside a 24-well plate. After discarding the culture medium, the cells were rinsed with PBS and then fixed with 4% paraformaldehyde. Following this, the RAW264.7 cells were treated with 0.5% TritonX-100 (Beyotime, China) and blocked with 5% bovine serum albumin. Subsequently, the RAW264.7 cells were incubated with the primary antibody (diluted 1: 150) at 4 \(℃\) overnight. After washing with PBS, the RAW264.7 cells were incubated with goat-anti-rabbit immunofluorescent secondary antibody (diluted 1: 300) in the dark at room temperature for 2 h. DAPI was used to stain the cell nuclei for 10 min at room temperature. Finally, the images were observed and photographed. 2.13 RT-qPCR RNA was extracted using TRizol reagent (TaKaRa, Japan), followed by cDNA synthesis based on the manufacturer’s guidelines for the Reverse Transcription kit. RT-qPCR analysis was carried out on a PCR Detection System (Bio-Rad, USA), following the instructions provided by the reagent provider. The relative quantification of each gene was determined using the 2 −ΔΔCT method, with \({\beta }\) -actin serving as a reference for normalization. The primer sequences used for this analysis were as follows: β-actin (forward 5'-GGCTGTATTCCCCTCCATCG-3', reverse 5'-CCAGTTGGTAACAATGCCATGT-3'), Ccl2 (forward 5'-TTAAAAACCTGGATCGGAACCAA-3', reverse 5'-GCATTAGCTTCAGATTTACGGGT-3'), Glut1 (forward 5'-CAGTTCGGCTATAACACTGGTG-3', reverse 5'-GCCCCCGACAGAGAAGATG-3'), Hk2 (forward 5'-TGATCGCCTGCTTATTCACGG-3', reverse 5'-AACCGCCTAGAAATCTCCAGA-3'), Pgk1 (forward 5'-ATGTCGCTTTCCAACAAGCTG-3', reverse 5'-GCTCCATTGTCCAAGCAGAAT-3'), Pkm (forward 5'-GCCGCCTG GACATTGACTC-3', reverse 5'-CCATGAGAGAAATTCAGCCGAG-3'), Pdk1 (forward 5'-GGACTTCGGGTCAGTGAATGC-3', reverse 5'-TCCTGAGAAGATTGTCGGGGA-3'), Ldha (forward 5'-TGTCTCCAGCAAAGACTACTGT-3', reverse 5'-GACTGTACTTGACAATGTTGGGA-3'), Gdf15 (forward 5'-CTGGCAATGCCTGAACAACG-3', reverse 5'-GGTCGGGACTTGGTTCTGAG-3'), Bmp4 (forward 5'-TTCCTGGTAACCGAATGCTGA-3', reverse 5'-CCTGAATCTCGGCGACTTTTT-3'), Wnt2b (forward 5'-CCGACGTGTCCCCATCTTC-3', reverse 5'-GCCCCTATGTACCACCAGGA-3'), Wnt8a (forward 5'-GGGAACGGTGGAATTGTCCTG-3', reverse 5'-GCAGAGCGGATGGCATGAA-3'), Epor (forward 5'-GGGCTCCGAAGAACTTCTGTG-3', reverse 5'-ATGACTTTCGTGACTCACCCT-3'), Gata2 (forward 5'-CACCCCGCCGTATTGAATG-3', reverse 5'-CCTGCGAGTCGAGATGGTTG-3'), Gata1 ( forward 5'-TGGGGACCTCAGAACCCTTG-3', reverse 5'-GGCTGCATTTGGGGAAGTG-3'), Vcam-1 (forward 5'-AGTTGGGGATTCGGTTGTTCT- 3',reverse5'-CCCCTCATTCCTTACCACCC-3') . 2.14 Statistical analyses All statistical analyses were performed using Graph-pad Prism software (version 8.3.0, San Diego, CA, USA). Quantitative data are presented as means \(\pm\) standard deviations (SD). The statistical significance of comparison between the two groups was estimated using independent sample t-test (Student’s t-test). For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was used to assess the differences in the data. Flowjo software was utilized for the analysis of the obtained data through flow cytometry. P value less than 0.05 was considered significant. 3. Results 3.1 ASP protected erythroid lineage from 5-FU-induced damage 5-FU treatment decreased the value of peripheral blood RBC counts, HGB, HCT and MCH below the control baseline. Also, 5-FU significantly blunted the BFU-E in the bone marrow, with the ensuing unobvious anemic stress response of peripheral reticulocytes. However, ASP alleviated 5-FU-caused erythroid injury, elevating the hematologic parameters, reversing BFU-E enumeration in the bone marrow, increasing the number of reticulocytes in the blood (Fig. 1A-G). The results indicated that ASP may exert the role of chemoprevention, alleviating chemotherapeutic anemia. 3.2 ASP promoted cell proliferation in splenic red pulp To elucidate whether 5-FU-caused anemia triggers extramedullary stress erythropoiesis, the cellular proliferation of splenocytes was analyzed. Histopathology demonstrated that 5-FU treatment dramatically decreased Ki67 positive cells in the red pulp of the spleen. However, ASP restored the number of Ki67 positive splenocytes in the red pulp (Fig. 2 A, 2 B). Western blot demonstrated ASP reversed 5-FU-induced decline in cyclin D1 of splenocytes (Fig. 2 C, 2 D). F4/80 is the common marker of splenic red pulp macrophages. On the one hand, histopathologic sections showed that ASP restored the positive stain of F4/80 in splenic red pulp (Fig. 3 A, 3 B). The result was consistent with an increase in Ccl2 genetic expression of splenocytes (Fig. 3 C). On the other hand, the flow cytometry results showed that ASP abrogated 5-FU-caused G0/G1 arrest in c-kit + erythroid progenitor cells in the spleen, driving splenic c-kit + cells to phase S (Fig. 3 D-F). Moreover, colony forming assay showed that ASP promoted stress splenic BFU-E accumulation in the spleen (Fig. 3 G, 3 H). The combined results suggested that ASP may induce the migration and proliferation of splenic macrophages, thus promoting the proliferation of stress erythroid precursor cells. 3.3 ASP facilitated glycolytic process of splenocytes via PI3K/Akt signaling GO functional enrichment presented that the interaction targets of ASP and 5-FU are related to the regulation of cell proliferation, hypoxia response, positive regulation of cell growth, and positive regulation of glycolytic process (Fig. 4A). KEGG pathway analysis focused on PI3K-Akt signaling (Fig. 4B). Next, genetic expression of glucose transporter Glut 1 and glycolysis-related genes including Hk 2, Pgk 1, Pkm, Pdk 1 and Ldha were determined, and the results showed that 5-FU significantly downregulated these genes. However, ASP strengthened the genetic expression (Fig. 4C-H). Documents evidence that activated HIF is an important protein downstream of the PI3K/Akt signaling pathway, and it is linked, either directly or indirectly, to the upregulation of GLUTs and glycolytic enzymes in glycolysis and lactate production. In addition, hypoxia not only triggers the activation of the PI3K/Akt signaling pathway but also directly promotes the expression of HIF. Thus, in the study, western blotting assay was utilized to analyze PI3K/Akt/HIF2α signaling (Fig. 4I). The above results suggested that ASP was able to restore the decreased PI3K/p-Akt signaling caused by 5-FU (Fig. 4J, 4K). Under the circumstance of anemia, the group treated with 5-FU exhibited increased levels of HIF2α protein expression in comparison to control group. Interestingly, the HIF2α expression was significantly elevated by ASP in comparison to the group treated with 5-FU (Fig. 4L). The results above indicated that ASP may facilitate the switch from aerobic to anaerobic glucose metabolism by upregulation of PI3K/Akt signaling and activation of HIF2α, thus enhancing cellular tolerance to hypoxia and initiating stress erythroid cell proliferation. 3.4 ASP facilitated the activity of splenic macrophages in stress erythropoiesis Documents evidence that splenic macrophages produce canonical Wnt ligands and Gdf15, Bmp4 signals that promote SEP proliferation, whereas ERO/STAT5-dependent signaling in macrophages can alter the splenic niche to promote erythroid differentiation. This study found that ASP could alleviate spleen-derived proinflammatory factor IL-1β induced by 5-FU (Fig. 5 A). Simultaneously, ASP significantly upregulated proliferation-associated genetic expression in splenocytes, including Gdf15, Bmp4, Wnt2b, and Wnt8a (Fig. 5 B-D) Also, immunofluorescence results showed that ASP restored 5-FU-declined β-catenin signaling in macrophages (Fig. 5 E), indicating ASP promoted the production of SEP proliferation signals in splenic macrophages. Moreover, it was found that after 5-FU treatment, concomitant with increased serum EPO, EpoR expression in splenocytes increased (Fig. 6 A, 6 B). Although no significance of EpoR was found between 5-FU group and ASP + 5-FU group, ASP boosted p-STAT5 signals in splenocytes (Fig. 6 C, 6 D), and provoking high expression of erythroid differentiation-related genes Gata2 and Gata1 in splenocytes (Fig. 6 E). These data were consistent with the in vitro experiment which ASP facilitated the expression of Epor, Bmp4, and adhesion molecule Vcam-1 in RAW 264.7 macrophages (Fig. 6 F-I). Interestingly, it was evidenced that ASP dramatically enhanced p-STAT5 signals in macrophages response to ectogenic EPO stimulation (Fig. 6 J, 6 K). These results suggested that ASP facilitated EPO/STAT5 signaling in splenic macrophages, thus enhancing SEP differentiation. To sum up, it indicated that ASP may alter the number and activity of splenic niche macrophages, orchestrating an appropriate environment for stress erythropoiesis. 4. Discussion Inflammation or chemotherapy/radiotherapy may cause chronic stress anemia, which is a clinically intractable disease currently [ 16 , 17 ]. Chemotherapy-caused stress anemia called chemotherapy-induced anemia (CIA) [ 18 ]. 5-FU has myeloablative effects, which is commonly used in the establishment of myeloablative inductive mouse model. In this research, we elucidated a protective mechanism of ASP against chemotherapy-induced anemia caused by 5-FU, which is related to extra-medullar stress erythropoiesis. It was found that ASP significantly resumed peripheral blood RBC counts, HGB, HCT and MCH parameters. Although on the seventh day following administration of 5-FU treatment, BFU-E in bone marrow has still been impaired, with the ensuing unobvious anemic stress response of peripheral reticulocytes. However, interestingly, ASP enhanced chemokine Ccl2 expression, which can attract monocytes to the spleen, the main extra-medullar erythropoiesis site, turning into erythroblastic island central macrophage (EIM). Subsequently, ASP promoted the proliferation of F4/80 + EIM, thus abrogating 5-FU-caused G0/G1 arrest in c-kit + erythroid progenitor cells in the spleen, driving splenic c-kit + cells to phase S, dramatically increasing splenic stress BFU-E ultimately. Documents demonstrate that the canonical Wnt/β-catenin signaling pathway originating from macrophages promotes SEP proliferation. This signaling, combined with previously identified factors (including SCF, BMP4, GDF15), drives effective proliferation of SEPs at the initial phage of stress erythropoiesis [ 19 , 20 ]. As expected, in the current study, ASP increased level of Gdf15 and Bmp4 in the splenic niche. Also, ASP elevated macrophage-derived Wnt8a, Wnt2b, β-catenin, and Bmp4. These results suggested ASP may promote the activity of splenic macrophages facilitating SEP expansion. Lately, Baiye Ruan et al shed light on the metabolic process involved in stress erythropoiesis, emphasizing the role of glycolytic metabolism in the initial SEP expansion stage. Stress erythropoiesis initiation relies on inflammatory signals [ 21 ]. In macrophages, inflammatory signals lead to “broken TCA cycle”. Citrate and succinate exit the mitochondria to aid in anabolic metabolism [ 22 ]. Exported citrate is transformed into oxaloacetate and acetyl-CoA. Histone acetyltransferases use acetyl-CoA to sustain glycolytic enzymes expression and for lipogenesis, which helps in promoting cell proliferation [ 23 ]. Succinate can block the activity of proline hydroxylases (PHDs) responsible for regulating the stability of hypoxia-inducible factors (HIFs), leading to an increase in hypoxia-inducible glycolysis. Additionally, succinate hinders the differentiation of stress erythroid progenitor cells by obstructing alterations in DNA and histone methylation. Previous data indicate that GDF15 signaling can increase the expression of PDK1 and PDK3 enzymes. PDK1 and PDK3 enzymes play a role in limiting the entry of pyruvate into the tricarboxylic acid cycle [ 24 ]. Moreover, GDF15 enhances the levels of HIF1α and Glut1, thereby augmenting the glycolysis of SEPs. In this study, proinflammatory factor IL-1β significantly increased following the administration of 5-FU treatment. However, ASP alleviated IL-1β levels somehow, which may be the reason that ASP suppressing oxidative stress and apoptosis, antagonizing 5-FU-induced spleen injury and dysfunction [ 25 ]. Surprisingly, it was also found that concomitant with the proliferation of splenic macrophages and c-kit + erythroid progenitor cells, ASP promoted the genetic expression of glucose transporter1 ( Glut1 ), rate-limiting enzymes of glycolysis such as hexokinase2 ( Hk 2) and pyruvate kinase M ( PkM ), and key glycolytic genes, including phosphoglycerate kinase ( Pgk ), lactate dehydrogenase A ( LdhA ), and pyruvate dehydrogenase kinase ( Pdk -1), indicating ASP facilitates glycolysis of splenocytes. These proliferative signals and ultimate metabolism alteration of the extra-medullar niche may be the one fascinating mechanism of enhanced stress erythropoiesis after ASP treatment. GO functional enrichment analysis presented that the interaction targets of ASP and 5-FU are related to cell regulation of proliferation, response to hypoxia, positive regulation of cell growth, and positive regulation of glycolytic process. KEGG pathway enrichment analysis focused on the PI3K-Akt signaling. Documents demonstrate that Glycogen synthase kinase 3β (GSK-3β) is an important downstream molecule regulated by Akt. Akt cause GSK-3β phosphorylated to inhibit GSK-3β activity [ 26 ]. Reduced glycogen synthesis leads to accumulation of cyclin D1, resulting in cell cycle advancement and proliferation. Therefore, glycogen synthesis reduction and glycolysis may be crucial links in regulating cell proliferation [ 27 ]. Moreover, PI3K/Akt pathway regulates the expression of fructose 2, 6-bisphosphatase (PFKFB2) and enhances glycolysis. HIFs are regulated by downstream mTOR of PI3K/Akt signaling pathway [ 28 ]. The expression of genes involved in erythropoiesis and glycolytic metabolism, which are regulated by HIFs, is enhanced in hypoxic environments. Acute hypoxic responses are associated with HIF1α, while chronic hypoxia responses are linked to HIF2α [ 29 ]. Knock down HIF2α gene seriously impaired erythroid progenitors in the bone marrow and spleen. This study found that ASP strengthened PI3K, p-Akt, accumulating cyclin D1. Consistent with it, ASP significantly elevated HIF2α in the niche, also the erythroid differentiation genes Gata1 and Gata2. The results above hinted that under hypoxic conditions, ASP may improve glycolysis and stress erythroid proliferation via PI3K/Akt/HIF2α signals. The next of stress erythropoiesis is erythroid differentiation, which is dependent on EIM. Li et al surprisingly high lightened the expression of erythropoietin receptor (EpoR) on EIM [ 30 ]. The dependency of erythropoiesis on EPO/EpoR led to the initial belief that the expression of EpoR was limited to erythroid lineage [ 31 ]. However, growing evidence indicate that EpoR is widely expressed in various non-erythroid cells. EpoR signaling in macrophages has been found to impact the splenic niche, promoting stress erythroid differentiation [ 32 ]. Epor −/− or Stat5 −/− in macrophages impaired stress erythropoiesis, indicating Epo/EpoR/Stat5 plays a vital role in stress erythropoiesis [ 33 ]. Day 7 following 5-FU treatment in our study, concomitant with an increase in serum EPO, ASP dramatically increased the activity of STAT5 dramatically strengthened in the ASP-treated spleen niche. Consistent with it, ASP resumed the expression of EpoR on RAW 264.7 macrophages, activating STAT5 signal response to ectogenic EPO stimulation. Also, ASP restored cellular adhesion molecule Vcam-1 expression on macrophages, which is indispensable for erythroid differentiation. These results suggested that ASP may improve stress erythropoiesis via macrophage-mediated EPO/STAT5 signaling. In another study, we demonstrated ASP enhanced macrophage-dependent iron homeostasis and transfer, and nuclear engulfment, which may be associated with the EIM dependent EPO/STAT5 signaling. Conclusion The results suggest that ASP may improve glycolysis after 5-FU treatment, promoting the activity of splenic macrophages and the expansion of erythroid progenitor cells. Also, ASP facilitate erythroid differentiation via macrophage-mediated EpoR/STAT5 signaling. Therefore, it may be a promising strategy for stress anemia. Declarations Acknowledgements The authors appreciate the helpful suggestions provided by Honghui Jiang and for Kunhang Du and Cheng Wang addressing software-related issues. Author contributions YL and LW designed the study and wrote the manuscript. YW contributed to revising the manuscript. Data analysis and figures preparation were performed by NS. HJ, BW, ZW, KD, CH, TY and CW contributed to the validation and interpretation of the results. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (Grant number 81873103) and Natural Science Foundation of Chongqing (Grant number cstc2021jcyj-msxmX0669). Data availability The datasets used and/or analyzed during the current study can be made available upon reasonable request from the corresponding author. Conflict of Interest The authors declare that they have no conflict of interests regarding the publication of this paper. Ethical statements and consent to participate The animal experimental procedures were carried out and approved by the Ethics Committee of Chongqing Medical University under the approval number IACUC-CQMU-2022–0026. This article contains any studies with animals performed by any of authors. All authors agreed to participate in the study. 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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-4180160","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284838229,"identity":"005b6efe-a5d0-47ca-838a-91327c57e031","order_by":0,"name":"Nianci Sun","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nianci","middleName":"","lastName":"Sun","suffix":""},{"id":284838231,"identity":"d09e0a53-e2e7-4959-b000-6de3ad8fca36","order_by":1,"name":"Ziling Wang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ziling","middleName":"","lastName":"Wang","suffix":""},{"id":284838232,"identity":"2523653e-e938-4336-a4c8-76766ace8c02","order_by":2,"name":"Honghui Jiang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Honghui","middleName":"","lastName":"Jiang","suffix":""},{"id":284838233,"identity":"a65550f5-b9f4-4a55-bed8-e0423458415b","order_by":3,"name":"Biyao Wang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Biyao","middleName":"","lastName":"Wang","suffix":""},{"id":284838234,"identity":"bf52d03f-ed21-491c-9d6f-a4ce12d55b65","order_by":4,"name":"Kunhang Du","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kunhang","middleName":"","lastName":"Du","suffix":""},{"id":284838235,"identity":"29afb8fb-f4f9-4319-9a4f-80c0ea414968","order_by":5,"name":"Caihong Huang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Caihong","middleName":"","lastName":"Huang","suffix":""},{"id":284838236,"identity":"77419726-5b65-4b22-b5de-2447ea518bb7","order_by":6,"name":"Cheng Wang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Wang","suffix":""},{"id":284838237,"identity":"02f8d959-65b3-4b34-9f9a-d1ee42cbef7f","order_by":7,"name":"Ting Yang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Yang","suffix":""},{"id":284838238,"identity":"323718b5-6664-49eb-a3c4-5ebda27460f3","order_by":8,"name":"Yaping Wang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Wang","suffix":""},{"id":284838239,"identity":"9dee550b-ea66-4900-b422-4c6839c093a8","order_by":9,"name":"Yafei Liu","email":"","orcid":"","institution":"Chongqing University Jiangjin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yafei","middleName":"","lastName":"Liu","suffix":""},{"id":284838240,"identity":"ebcaf9cf-1f76-4356-a394-27bcc6199226","order_by":10,"name":"Lu Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACPghlA+WyEaEFqiaNdC2HSdEikWP4ueDXeXm+a2cMGD6UHWbgn91AUIux9My+24Yzb+cYMM44d5hB4s4BQlpyN0jz9txOMABqYeZtO8xgIJFAUMvm37w95yBa/hKpZZs0z48DEC2MRGnhef/NmrchGeiXtIKDPefSeSRuENDCz56WfJvnj5083+3kjQ9+lFnL8c8goAUMGNuAxAEwYuAhQj0I/IFoGQWjYBSMglGAFQAAp2ZAXp0GUYEAAAAASUVORK5CYII=","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Lu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-03-28 07:02:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4180160/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4180160/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53696628,"identity":"807fed0a-8a5f-4ac3-9e53-49f8d9d9197c","added_by":"auto","created_at":"2024-03-29 03:52:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":713867,"visible":true,"origin":"","legend":"\u003cp\u003eASP protected erythroid lineage from 5-FU-induced injury on the day 7 after 5-FU treatment. (A-D) ASP reversed 5-FU-induced decline in RBC, hemoglobin, hematocrit, and mean corpuscular hemoglobin in the peripheral blood (n=3). (E) The ratio of reticulocytes in the blood smear was presented as the histogram. Data is expressed as means ± SD (n=3). (F) BMMNCs were cultured in methylcellulose media MethoCultTM SF M3436 for 10 days, BFU-E frequency was scored and presented as the histogram (n=5). (G) The top panel is the representative images showing the reticulocytes in the blood smear stained by Brilliant Cresyl Blue. Scale bar is 10 µm. The following images show the representative pictures of BFU-E in bone marrow under the microscope. Scale bar is 100 µm. * P \u0026lt;0.05; ** P \u0026lt;0.01; *** P \u0026lt;0.001; **** P \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4180160/v1/3b466326d12c8fdce0a042bd.png"},{"id":53696626,"identity":"34029a31-5b3a-48fc-81b7-d58a0fd27eb8","added_by":"auto","created_at":"2024-03-29 03:52:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":692545,"visible":true,"origin":"","legend":"\u003cp\u003eASP promoted cell proliferation in splenic red pulp on the day 7 after 5-FU treatment. (A) Representative immunohistochemistry images showing the Ki67 nuclear protein expression in the red pulp of spleen. The scale bar represents 10 µm. (B) The histogram represents the average optical density of Ki67 nuclear protein in the red pulp of spleen. Data was processed utilizing the Image J software (n=3). (C-D) Representative band and histogram of cyclin D1 protein in splenocytes were detected through western blotting assay. β-actin was used as the loading control and the band intensity was assessed using Image Lab software. Data are showed as means ± SD (n=5). * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05; ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01; ****\u003cem\u003e P \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4180160/v1/69ddf7eae9179f7599873506.png"},{"id":53696625,"identity":"a37fa952-e995-4109-aaa5-a74428260ce5","added_by":"auto","created_at":"2024-03-29 03:52:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":364802,"visible":true,"origin":"","legend":"\u003cp\u003eASP facilitated the proliferation of splenic F4/80 macrophages and c-kit\u003csup\u003e+\u003c/sup\u003e cell. (A-B) Representative images showing F4/80 protein expression in the red pulp of spleen examined by immunohistochemistry. \u0026nbsp;Scale bar is 100 µm. The average optical density of F4/80 protein was assessed utilizing Image J, and the results were displayed in the histogram. The results are showed as means ±\u0026nbsp;SD (n=3). (C) Histogram showing relative expression of Ccl2 mRNA in splenocytes on day 7 and day10 after 5-FU treatment, with β-actin serving as a loading control (n=3). (D) Schematic diagram of the gating strategy for flow cytometry, showing the cell cycle analysis of c-kit\u003csup\u003e+\u003c/sup\u003e cell in the spleen. (E-F) Representative flow cytometry images and histograms showing the cell cycle distribution of splenic c-kit\u003csup\u003e+\u003c/sup\u003e cell population. Data are showed as means ±\u0026nbsp;SD (n=3). (G-H) Splenocytes were cultured for 10 days in methylcellulose culture media MethoCult\u003csup\u003eTM\u003c/sup\u003e SF M3436. BFU-E frequency was scored and presented as the histogram. The results are showed as means ±\u0026nbsp;SD (n=5). The scale bar represents 100 µm. *\u003cem\u003e P \u003c/em\u003e\u0026lt;0.05; ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01; *** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001; **** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001; ns indicates no statistical significance.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4180160/v1/d94174cd28fa699557453296.png"},{"id":53696627,"identity":"17e6c9ea-3bfc-478d-9416-3193eb6b37cb","added_by":"auto","created_at":"2024-03-29 03:52:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":351692,"visible":true,"origin":"","legend":"\u003cp\u003eASP facilitated glycolytic process of splenocytes via PI3K/Akt signaling on the day 7 after 5-FU treatment. (A) Bioenrichment function hinted that one biological process of ASP and 5-FU intersection targets is related with cellular proliferation, response to hypoxia, and glycolytic process. (B) KEGG enrichment analysis indicated that one biological effect of ASP and 5-FU intersection targets are correlated with PI3K/AKT signaling. (C-H) The glycolytic related genetic expression including Glut1, Hk2, Pgk1, Pkm, Pdk1, Ldha was tested by RT-qPCR and shown by the histogram. β-actin serving as a reference for normalization (n=3). (I-L) PI3K/Akt/HIF2α signaling was testified by western blotting assay. The representative protein bands of PI3K, p-AKT, HIF2α of splenocytes and the corresponding histogram. The bands intensity was analyzed through the utilization of Image Lab software. β-actin serving as a reference for normalization (n=3). All data are showed as means ± SD. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05; ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01; *** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001; **** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001; ns indicates no statistical significance.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4180160/v1/28fc19b280ffcdca4241ee3b.png"},{"id":53696630,"identity":"f17709a9-e63e-4e59-9843-1681148e15bb","added_by":"auto","created_at":"2024-03-29 03:52:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":528749,"visible":true,"origin":"","legend":"\u003cp\u003eASP facilitated the activity of splenic macrophages in stress erythropoiesis on the day 7 after 5-FU treatment. (A) The pro-inflammation factor IL-1β in Spleen homogenate was detected by ELISA assay (n=3). (B-C). Relative genetic expression of proliferation-related genes during stress erythropoiesis Gdf15 and Bmp4 in splenocytes and the results are presented as the histogram. β-actin serving as a reference for normalization (n=3). (D) Histograms show the relative genetic expression of erythroblastic island central macrophage proliferation-related genes Wnt2b and Wnt8a in splenocytes on day 7 and day 10 after 5-FU treatment respectively. (E) Representative images of immunofluorescence detection of β-catenin activity in RAW 264.7 macrophages. Scale bar represents 20 µm. All data are showed as means ± SD. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05; ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01; ****\u003cem\u003e P \u003c/em\u003e\u0026lt;0.0001; ns indicates no statistical significance.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4180160/v1/d6210eb18dd5a7fb88e14830.png"},{"id":53696629,"identity":"6c48a5bb-3931-46ec-a7e2-896b0027035e","added_by":"auto","created_at":"2024-03-29 03:52:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":291213,"visible":true,"origin":"","legend":"\u003cp\u003eASP facilitated erythroid differentiation via EPO/STAT5 signaling-regulated splenic macrophage. (A) EPO in serum was analyzed by ELISA assay (n=3). (B) The histogram of relative expression of Epor mRNA in splenocytes. β-actin serving as a reference for normalization (n=3). (C-D) Representative protein bands of p-STAT5 and STAT5 in splenocytes. The activity of STAT5 was measured and STAT5 is served as an internal control for p-STAT5. β-actin serving as a reference for normalization (n=5). (E) The histogram showing the relative genetic expression of erythrocyte-related genes Gata2, Gata1 in the spleen by RT-qPCR analysis. β-actin serving as a reference for normalization (n=3). (F-G) The histogram of mRNA expression related to Vcam-1 and Epor in the RAW 264.7 macrophages. β-actin serving as a reference for normalization (n=3). (H-I) Representative image and histogram of BMP4 protein bands in the RAW 264.7 macrophages. Band intensity was calculated through the utilization of Image Lab software. β-actin serving as a reference for normalization (n=3). (J-K) The activity of STAT5 was analyzed in RAW 264.7 macrophages with the addition of ectogenic EPO. The representative image and histogram of p-STAT5 and STAT5 protein in RAW 264.7 macrophages with the addition of EPO. STAT5 served as the internal control for p-STAT5, with β-actin serving as a reference for normalization (n=5). The intensity of the bands was determined through the utilization of Image Lab software. All data are showed as means ± SD. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05; ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01; *** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001; **** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4180160/v1/f24b782d4138698d95c9d521.png"},{"id":53939032,"identity":"fd91b7b0-46ef-4c20-aad8-388b6eb35d1f","added_by":"auto","created_at":"2024-04-02 12:52:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2938513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4180160/v1/95280153-7457-4dc9-808a-a2537e288fc6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInflammation or chemotherapy/radiotherapy may cause chronic stress anemia. However, the conventional treatment for chronic anemia such as blood transfusion and recombinant EPO injection may lead to various side effects such as iron overload, hypertension, high blood viscosity, and venous thrombosis, et al [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During this period, extramedullary stress erythropoiesis begins as a compensatory response to the loss of erythroid output and anoxia [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Theoretically, in order to help maintain homeostasis until steady state conditions are restored, a wave of new erythrocytes is rapidly generated by stress erythropoiesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The strategy of varies from steady state erythropoiesis. The transient amplification of erythroid progenitors (SEPs) originates from heterogenous populations composed of the most immature CD34\u003csup\u003e+\u003c/sup\u003eCD133\u003csup\u003e+\u003c/sup\u003eKS cells, intermediate immature CD34\u003csup\u003eneg\u003c/sup\u003eCD133\u003csup\u003e+\u003c/sup\u003eKS cells, and the most mature CD34\u003csup\u003eneg\u003c/sup\u003eCD133\u003csup\u003eneg\u003c/sup\u003eKS cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Multifold key signaling and factors such as BMP4, GDF15, Wnt/β-catenin, and Hedgehog promote SEP proliferation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, in a new opinion, the regulation of cellular activities relies on intracellular metabolism, how these macrophage-derived signals and factors interlaced control metabolism and how alterations in metabolism leads to SEP self-renewal and proliferation remain to be elucidative and a promising therapeutic strategy for promoting stress erythropoiesis still needs exploration.\u003c/p\u003e \u003cp\u003eLatest documents evidence that signals driving the proliferation of SEPs establish an efficient anabolic metabolism to meet the rapid cell division [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Just like the \u0026ldquo;Warburg effect\u0026rdquo; seen in cancer cells, aerobic glycolysis predominates in metabolic control of stress erythropoiesis proliferation, with glycolytic metabolites being shunted into anabolic pathways. For example, pathways like the pentose phosphate pathway and the serine glycine pathway, are very effective in generating nucleotides, amino acids, metabolism and regenerate NADPH levels [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The hypoxia-inducible factor oxygen sensing pathway reprograms cellular glucose/fatty acid metabolism under cellular defense condition [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. HIF1α and HIF2α isoforms specifically inhibit key enzymes for fatty acid oxidation-dependent mitochondrial TCA cycle inhibition; mitochondrial complex activity; and crucial regulators of the mitochondrial contents. This inhibition reduces oxygen consumption from mitochondrial glucose and fatty acid-dependent under hypoxic conditions. This HIF-dependent damping of mitochondrial activity not only conserves oxygen during hypoxic but also reduces the formation of toxic mitochondrial reactive oxygen species (ROS), eliciting cellular tolerance to hypoxic and oxidative stress conditions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among HIFs, HIF2α is the major effector of stress-induced renal EPO producing cells and the primary inducer of EPO genes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Under normoxic conditions, growth factors and cytokines play a regulatory role in the PI3K/Akt signaling pathway. Synthesis of HIFs rises when PI3K/Akt/mTOR is activated. In low oxygen levels, hypoxia-induced HIFs precede PI3K/Akt activation, however, the stability of HIFs is linked to the PI3K/Akt signaling pathway. The investigation of metabolism regulating stress erythropoiesis will be fascinating [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSEPs do not undergo differentiate until there is an increase in serum erythropoietin (EPO) levels. Instead of directly regulating SEPs, EPO facilitates the shift of SEPs from proliferation to differentiation by targeting splenic erythroblastic island central macrophages (EIM), which express EpoR. EPO/STAT5 signaling in macrophages stimulates the production of PGJ2, thus activating PPARg dependent repression of Wnt expression. Moreover, EPO/STAT5 signaling enhances production of prostaglandin E2 (PGE2), promoting the function of EIM in cell adhesion, phagocytosis, iron translocation, and nuclear engulfment of EIM, and the differentiation of SEPs. It has been demonstrated that depletion of EIM or inhibition of EPO/STAT5 signaling in macrophages has been shown to impair stress erythropoiesis and clearance of apoptotic cells. In murine diseases models like β-thalassemia, Sickle cell disease, and polycythemia vera, depleting macrophages has been found to enhance erythropoiesis, indicating that EIM are novel targets for therapeutic intervention.\u003c/p\u003e \u003cp\u003eASP (Angelica sinensis polysaccharides) is a main bioactive component of Angelica sinensis (Oliv.) Diels (Apiaceae). It has antioxidative, anti-inflammatory, anti-tumor, hematopoietic, immunoregulatory, hepatoprotective activities [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Our previous research demonstrated that ASP has antioxidative protective effects on BM perivascular mesenchymal progenitors, thereby maintaining hematopoietic progenitor cells in mice treated with 5-FU. Additionally, ASP has been shown to shield splenocytes from oxidative stress and apoptosis induced by 5-FU. However, it remains unclear whether ASP ameliorates stress erythropoiesis in cell defense conditions.\u003c/p\u003e \u003cp\u003eHerein, in this study, we utilized the 5-FU-myeloablative mouse model to investigate the impact of ASP on stress anemia and its underlying mechanism. We found that ASP promoted the proliferation of splenocytes including extra-medullar erythropoiesis dependent macrophages and erythroid cells via PI3K/Akt/HIF2α-mediated glycolytic process. Additionally, ASP boosted EPO/EPOR/STAT5 signaling transduction in splenic macrophages, which could be the reason for promoting differentiation of SEPs. Intriguingly, our research suggests that ASP could be a potentially therapeutic strategy for stress anemia.\u003c/p\u003e"},{"header":"2. Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagent and antibody\u003c/h2\u003e \u003cp\u003e5-FU was acquired from Sigma-Aldrich (St. Louis, USA). Angelica sinensis polysaccharides (ASP) was bought from Ciyuan Biotechnology Co. Ltd. (Shanxi, China). CD117(c-kit)-FITC was purchased from Biolegend (SanDiego, USA). EPO protein was purchased from MCE (New Jersey, USA). SYBR green I and Reverse Transcription Kit were purchased from TaKaRa (Kyoto, Japan). The following antibodies were acquired from various suppliers: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\beta }\\)\u003c/span\u003e\u003c/span\u003e\u003cem\u003e-\u003c/em\u003eactin was procured from Proteintech (Wuhan, China); HIF2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\alpha }\\)\u003c/span\u003e\u003c/span\u003e was purchased from Affinity (Jiangsu, China); BMP4, p-STAT5, STAT5 were obtained from ABclonal (Wuhan, China); PI3K, p-AKT were sourced from Wanleibio (Shenyang, China); cyclin D1 was obtained from CST (Danvers, USA); Ki67 was obtained from Bioss (Beijing, China); F4/80 was purchased from Affinity (Jiangsu, China). The goat-anti-rabbit immunofluorescent secondary antibody was purchased from Beyotime (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental animals\u003c/h2\u003e \u003cp\u003eC57BL/6 J mice, aged 6\u0026ndash;8 weeks, were purchased from our school\u0026rsquo;s animal experimental center (n\u0026thinsp;=\u0026thinsp;60) and housed in the Specific Pathogen-Free (SPF) laboratory. The mice were randomly allocated into four distinct groups: control group, ASP group, 5-FU group, and ASP\u0026thinsp;+\u0026thinsp;5-FU group. In the experimental design, the control group of mice was administered intraperitoneal injections of saline at a dosage of 10 ml/kg per day for either 7 or 10 consecutive days. The ASP group of mice was administered intraperitoneal injection of ASP at a dosage of 100 mg/kg\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\)\u003c/span\u003e\u003c/span\u003eper day for either 7 or 10 consecutive days. On the first day, the 5-FU group of mice was subjected to an initial intraperitoneal injection of 5-FU at a dosage of 150 mg/kg, followed by saline injections at a dosage of 10 ml/kg 6 hours later, and subsequently received daily saline injections for the next 6 or 9 days. In the ASP\u0026thinsp;+\u0026thinsp;5-FU group, mice received a single injection of 5-FU at 150 mg/kg on the first day, followed by an intraperitoneal injection of ASP at a dosage of 100 mg/kg 6 hours later, and then continued to receive ASP injections at the same dosage for 6 and 9 days. The mice were euthanized using carbon dioxide. Repeat independent animal experiments at least 3 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell Culture\u003c/h2\u003e \u003cp\u003eRAW264.7 macrophages were cultured in DMEM-High Glucose medium (Gibco, Grand Island, USA), supplementing with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody. The RAW264.7 macrophages were randomly allocated into four groups: control group (without treatment), ASP group (100 ug/ml ASP treated for 24 hours), 5-FU group (25 ug/ml 5-FU treated for 24 hours), ASP\u0026thinsp;+\u0026thinsp;5-FU group (pretreated with 25 ug/ml 5-FU for 6 hours, then cultured with 100 ug/ml ASP for 24 hours). To observe the effect of EPO on EPO/EPOR/STAT5 signaling of RAW264.7 macrophages, 5 U/ml EPO was added to the medium for 30minutes, subsequently, STAT5 and p-STAT5 were detected by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Peripheral blood parameters test\u003c/h2\u003e \u003cp\u003eMice were anesthetized and their eyeballs were enucleated. EDTA anticoagulation tubes were utilized to gather orbital blood samples from the murine cohort for further analysis with an automated hematology analyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 BFU-E colony forming culture\u003c/h2\u003e \u003cp\u003eIn bone marrow BFU-E (erythroid burst-forming unit) colony formation assay, the femurs and tibias of the mice were extracted and the muscles and tissues were cleaned. The bone marrow mononuclear cells were separated by density gradient centrifugation using Ficoll-Paque (1.077g/mL). In splenic colony forming assay, splenocytes were treated with erythrocyte lysates to lyse the erythrocytes. Following the manufacturer\u0026rsquo;s instructions, BMMNCs and splenocytes (1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e5\u003c/sup\u003e) were cultured in 35mm Petri dishes with methylcellulose medium MethoCultTM SF M3436 (StemCell, Vancouver, Canada) for 10\u0026ndash;14 d, and then counted by microscopy. Each colony comprises no less than 50 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Brilliant Cresyl Blue staining\u003c/h2\u003e \u003cp\u003eThe peripheral blood of mice in each group was collected in EDTA anticoagulant tube, and was mixed 1:1 with Brilliant Cresyl Blue solution, left at room temperature for 15\u0026ndash;20 minutes. Blood smears were prepared and observed by microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunohistochemistry staining\u003c/h2\u003e \u003cp\u003eSA1022 kit (BOSTER, Wuhan, China) and AR1027 kit (BOSTER, China) were used in the experiment immunohistochemistry. Paraffin sections of mouse spleen measuring 5\u0026micro;m were taken, antigen retrieval was performed by heating, and the sections were blocked with 5% bovine serum albumin. Tissue sections were incubated with Ki67 antibody (1: 400) and F4/80 antibody (1: 150) at 4℃ overnight. Thereafter, tissue sections were treated with secondary antibody, followed by DAB staining and counterstaining with Mayer\u0026rsquo;s hematoxylin (Solarbio, Beijing, China). Images were obtained using microscopy and analyzed by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 ELISA\u003c/h2\u003e \u003cp\u003eMouse serum and spleen homogenate were detected by IL-1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\beta }\\)\u003c/span\u003e\u003c/span\u003e ELISA kits (QuantiCyto, Shenzhen, China) and EPO ELISA kits (MULTI SCIENCES, Hangzhou, China), respectively, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 GO function and KEGG pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eThe ASP and 5-FU intersection targets were imported to the DAVID database ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) for GO and KEGG enrichment analysis, and sort the results by adjusted \u003cem\u003eP\u003c/em\u003e value \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e0.05. The top 10 enriched function and top 20 enriched pathways using bioinformatics data processing platform ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) online tool to visualize the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Flow cytometry analysis\u003c/h2\u003e \u003cp\u003eAfter pretreatment with erythrocyte lysates, the splenocytes were adjusted to 10\u003csup\u003e6\u003c/sup\u003e per tube and then subjected to blocking with 5% BSA at 4\u0026deg;C for a duration of 30 minutes. Subsequently, incubating the cells with the primary antibody CD117 (c-kit\u003csup\u003e+\u003c/sup\u003e) at 4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e for 30 minutes. Later, the pre-chilled 75% ethanol was added to fix the cells at 4 ℃ overnight. The ethanol was discarded and washed with PBS after centrifugation. The cells were incubated with PI at 4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e for 30 minutes, then centrifuged and washed twice with PBS. Cell cycle of the c-kit\u003csup\u003e+\u003c/sup\u003e cell population was analyzed by flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Western blot assay\u003c/h2\u003e \u003cp\u003eSplenocytes and RAW264.7 cells were prepared as mentioned previously. The total protein was extracted using pre-chilled RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China). The protein concentration was determined using a BCA assay kit (EpiZyme, Shanghai, China). Add the supernatant into the Loading Buffer and boil for 10min. Equal amounts of protein were separated using SDS-PAGE and then transferred to PVDF membrane (Beyotime, China). The membrane was blocked with 5% skimmed milk powder (dissolved in TBST). Subsequently, the membrane was incubated with corresponding primary antibodies at the appropriate dilution and refrigerated at 4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e overnight. On next day, the membrane was washed three times with TBST lasting 10 minutes each, followed by incubation with the secondary antibodies for a duration of 1 hour at ambient temperature. The protein bands were visualized and captured using an enhanced ECL chemiluminescence detection System, and the band intensities of were analyzed using Image Lab software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were inoculated on a sterile slide inside a 24-well plate. After discarding the culture medium, the cells were rinsed with PBS and then fixed with 4% paraformaldehyde. Following this, the RAW264.7 cells were treated with 0.5% TritonX-100 (Beyotime, China) and blocked with 5% bovine serum albumin. Subsequently, the RAW264.7 cells were incubated with the primary antibody (diluted 1: 150) at 4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e overnight. After washing with PBS, the RAW264.7 cells were incubated with goat-anti-rabbit immunofluorescent secondary antibody (diluted 1: 300) in the dark at room temperature for 2 h. DAPI was used to stain the cell nuclei for 10 min at room temperature. Finally, the images were observed and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 RT-qPCR\u003c/h2\u003e \u003cp\u003eRNA was extracted using TRizol reagent (TaKaRa, Japan), followed by cDNA synthesis based on the manufacturer\u0026rsquo;s guidelines for the Reverse Transcription kit. RT-qPCR analysis was carried out on a PCR Detection System (Bio-Rad, USA), following the instructions provided by the reagent provider. The relative quantification of each gene was determined using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method, with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\beta }\\)\u003c/span\u003e\u003c/span\u003e-actin serving as a reference for normalization. The primer sequences used for this analysis were as follows: \u003cem\u003eβ-actin\u003c/em\u003e (forward 5'-GGCTGTATTCCCCTCCATCG-3', reverse 5'-CCAGTTGGTAACAATGCCATGT-3'), \u003cem\u003eCcl2\u003c/em\u003e (forward 5'-TTAAAAACCTGGATCGGAACCAA-3', reverse 5'-GCATTAGCTTCAGATTTACGGGT-3'), \u003cem\u003eGlut1\u003c/em\u003e(forward 5'-CAGTTCGGCTATAACACTGGTG-3', reverse 5'-GCCCCCGACAGAGAAGATG-3'), \u003cem\u003eHk2\u003c/em\u003e (forward 5'-TGATCGCCTGCTTATTCACGG-3', reverse 5'-AACCGCCTAGAAATCTCCAGA-3'), \u003cem\u003ePgk1\u003c/em\u003e (forward 5'-ATGTCGCTTTCCAACAAGCTG-3', reverse 5'-GCTCCATTGTCCAAGCAGAAT-3'), \u003cem\u003ePkm\u003c/em\u003e (forward 5'-GCCGCCTG GACATTGACTC-3', reverse 5'-CCATGAGAGAAATTCAGCCGAG-3'), \u003cem\u003ePdk1\u003c/em\u003e (forward 5'-GGACTTCGGGTCAGTGAATGC-3', reverse 5'-TCCTGAGAAGATTGTCGGGGA-3'), \u003cem\u003eLdha\u003c/em\u003e (forward 5'-TGTCTCCAGCAAAGACTACTGT-3', reverse 5'-GACTGTACTTGACAATGTTGGGA-3'), \u003cem\u003eGdf15\u003c/em\u003e (forward 5'-CTGGCAATGCCTGAACAACG-3', reverse 5'-GGTCGGGACTTGGTTCTGAG-3'), \u003cem\u003eBmp4\u003c/em\u003e (forward 5'-TTCCTGGTAACCGAATGCTGA-3', reverse 5'-CCTGAATCTCGGCGACTTTTT-3'), \u003cem\u003eWnt2b\u003c/em\u003e (forward 5'-CCGACGTGTCCCCATCTTC-3', reverse 5'-GCCCCTATGTACCACCAGGA-3'), \u003cem\u003eWnt8a\u003c/em\u003e (forward 5'-GGGAACGGTGGAATTGTCCTG-3', reverse 5'-GCAGAGCGGATGGCATGAA-3'), \u003cem\u003eEpor\u003c/em\u003e (forward 5'-GGGCTCCGAAGAACTTCTGTG-3', reverse 5'-ATGACTTTCGTGACTCACCCT-3'), \u003cem\u003eGata2\u003c/em\u003e (forward 5'-CACCCCGCCGTATTGAATG-3', reverse 5'-CCTGCGAGTCGAGATGGTTG-3'), \u003cem\u003eGata1\u003c/em\u003e ( forward 5'-TGGGGACCTCAGAACCCTTG-3', reverse 5'-GGCTGCATTTGGGGAAGTG-3'), \u003cem\u003eVcam-1\u003c/em\u003e (forward 5'-AGTTGGGGATTCGGTTGTTCT-\u003cem\u003e3',reverse5'-CCCCTCATTCCTTACCACCC-3')\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using Graph-pad Prism software (version 8.3.0, San Diego, CA, USA). Quantitative data are presented as means \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003e standard deviations (SD). The statistical significance of comparison between the two groups was estimated using independent sample t-test (Student\u0026rsquo;s t-test). For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was used to assess the differences in the data. Flowjo software was utilized for the analysis of the obtained data through flow cytometry. \u003cem\u003eP\u003c/em\u003e value less than 0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 ASP protected erythroid lineage from 5-FU-induced damage\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e5-FU treatment decreased the value of peripheral blood RBC counts, HGB, HCT and MCH below the control baseline. Also, 5-FU significantly blunted the BFU-E in the bone marrow, with the ensuing unobvious anemic stress response of peripheral reticulocytes. However, ASP alleviated 5-FU-caused erythroid injury, elevating the hematologic parameters, reversing BFU-E enumeration in the bone marrow, increasing the number of reticulocytes in the blood (Fig.\u0026nbsp;1A-G). The results indicated that ASP may exert the role of chemoprevention, alleviating chemotherapeutic anemia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 ASP promoted cell proliferation in splenic red pulp\u003c/h2\u003e \u003cp\u003eTo elucidate whether 5-FU-caused anemia triggers extramedullary stress erythropoiesis, the cellular proliferation of splenocytes was analyzed. Histopathology demonstrated that 5-FU treatment dramatically decreased Ki67 positive cells in the red pulp of the spleen. However, ASP restored the number of Ki67 positive splenocytes in the red pulp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Western blot demonstrated ASP reversed 5-FU-induced decline in cyclin D1 of splenocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). F4/80 is the common marker of splenic red pulp macrophages. On the one hand, histopathologic sections showed that ASP restored the positive stain of F4/80 in splenic red pulp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The result was consistent with an increase in \u003cem\u003eCcl2\u003c/em\u003e genetic expression of splenocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). On the other hand, the flow cytometry results showed that ASP abrogated 5-FU-caused G0/G1 arrest in c-kit\u003csup\u003e+\u003c/sup\u003e erythroid progenitor cells in the spleen, driving splenic c-kit\u003csup\u003e+\u003c/sup\u003e cells to phase S (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Moreover, colony forming assay showed that ASP promoted stress splenic BFU-E accumulation in the spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). The combined results suggested that ASP may induce the migration and proliferation of splenic macrophages, thus promoting the proliferation of stress erythroid precursor cells.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 ASP facilitated glycolytic process of splenocytes via PI3K/Akt signaling\u003c/h2\u003e \u003cp\u003eGO functional enrichment presented that the interaction targets of ASP and 5-FU are related to the regulation of cell proliferation, hypoxia response, positive regulation of cell growth, and positive regulation of glycolytic process (Fig.\u0026nbsp;4A). KEGG pathway analysis focused on PI3K-Akt signaling (Fig.\u0026nbsp;4B). Next, genetic expression of glucose transporter \u003cem\u003eGlut\u003c/em\u003e1 and glycolysis-related genes including \u003cem\u003eHk\u003c/em\u003e2, \u003cem\u003ePgk\u003c/em\u003e1, \u003cem\u003ePkm, Pdk\u003c/em\u003e1 and \u003cem\u003eLdha\u003c/em\u003e were determined, and the results showed that 5-FU significantly downregulated these genes. However, ASP strengthened the genetic expression (Fig.\u0026nbsp;4C-H). Documents evidence that activated HIF is an important protein downstream of the PI3K/Akt signaling pathway, and it is linked, either directly or indirectly, to the upregulation of GLUTs and glycolytic enzymes in glycolysis and lactate production. In addition, hypoxia not only triggers the activation of the PI3K/Akt signaling pathway but also directly promotes the expression of HIF. Thus, in the study, western blotting assay was utilized to analyze PI3K/Akt/HIF2α signaling (Fig.\u0026nbsp;4I). The above results suggested that ASP was able to restore the decreased PI3K/p-Akt signaling caused by 5-FU (Fig.\u0026nbsp;4J, 4K). Under the circumstance of anemia, the group treated with 5-FU exhibited increased levels of HIF2α protein expression in comparison to control group. Interestingly, the HIF2α expression was significantly elevated by ASP in comparison to the group treated with 5-FU (Fig.\u0026nbsp;4L). The results above indicated that ASP may facilitate the switch from aerobic to anaerobic glucose metabolism by upregulation of PI3K/Akt signaling and activation of HIF2α, thus enhancing cellular tolerance to hypoxia and initiating stress erythroid cell proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 ASP facilitated the activity of splenic macrophages in stress erythropoiesis\u003c/h2\u003e \u003cp\u003eDocuments evidence that splenic macrophages produce canonical Wnt ligands and Gdf15, Bmp4 signals that promote SEP proliferation, whereas ERO/STAT5-dependent signaling in macrophages can alter the splenic niche to promote erythroid differentiation. This study found that ASP could alleviate spleen-derived proinflammatory factor IL-1β induced by 5-FU (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Simultaneously, ASP significantly upregulated proliferation-associated genetic expression in splenocytes, including \u003cem\u003eGdf15, Bmp4, Wnt2b, and Wnt8a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D) Also, immunofluorescence results showed that ASP restored 5-FU-declined β-catenin signaling in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), indicating ASP promoted the production of SEP proliferation signals in splenic macrophages. Moreover, it was found that after 5-FU treatment, concomitant with increased serum EPO, EpoR expression in splenocytes increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Although no significance of EpoR was found between 5-FU group and ASP\u0026thinsp;+\u0026thinsp;5-FU group, ASP boosted p-STAT5 signals in splenocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), and provoking high expression of erythroid differentiation-related genes \u003cem\u003eGata2\u003c/em\u003e and \u003cem\u003eGata1\u003c/em\u003e in splenocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These data were consistent with the \u003cem\u003ein vitro\u003c/em\u003e experiment which ASP facilitated the expression of Epor, Bmp4, and adhesion molecule Vcam-1 in RAW 264.7 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-I). Interestingly, it was evidenced that ASP dramatically enhanced p-STAT5 signals in macrophages response to ectogenic EPO stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). These results suggested that ASP facilitated EPO/STAT5 signaling in splenic macrophages, thus enhancing SEP differentiation. To sum up, it indicated that ASP may alter the number and activity of splenic niche macrophages, orchestrating an appropriate environment for stress erythropoiesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eInflammation or chemotherapy/radiotherapy may cause chronic stress anemia, which is a clinically intractable disease currently [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Chemotherapy-caused stress anemia called chemotherapy-induced anemia (CIA) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 5-FU has myeloablative effects, which is commonly used in the establishment of myeloablative inductive mouse model. In this research, we elucidated a protective mechanism of ASP against chemotherapy-induced anemia caused by 5-FU, which is related to extra-medullar stress erythropoiesis. It was found that ASP significantly resumed peripheral blood RBC counts, HGB, HCT and MCH parameters. Although on the seventh day following administration of 5-FU treatment, BFU-E in bone marrow has still been impaired, with the ensuing unobvious anemic stress response of peripheral reticulocytes. However, interestingly, ASP enhanced chemokine \u003cem\u003eCcl2\u003c/em\u003e expression, which can attract monocytes to the spleen, the main extra-medullar erythropoiesis site, turning into erythroblastic island central macrophage (EIM). Subsequently, ASP promoted the proliferation of F4/80\u003csup\u003e+\u003c/sup\u003e EIM, thus abrogating 5-FU-caused G0/G1 arrest in c-kit\u003csup\u003e+\u003c/sup\u003e erythroid progenitor cells in the spleen, driving splenic c-kit\u003csup\u003e+\u003c/sup\u003e cells to phase S, dramatically increasing splenic stress BFU-E ultimately.\u003c/p\u003e \u003cp\u003eDocuments demonstrate that the canonical Wnt/β-catenin signaling pathway originating from macrophages promotes SEP proliferation. This signaling, combined with previously identified factors (including SCF, BMP4, GDF15), drives effective proliferation of SEPs at the initial phage of stress erythropoiesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As expected, in the current study, ASP increased level of Gdf15 and Bmp4 in the splenic niche. Also, ASP elevated macrophage-derived Wnt8a, Wnt2b, β-catenin, and Bmp4. These results suggested ASP may promote the activity of splenic macrophages facilitating SEP expansion.\u003c/p\u003e \u003cp\u003eLately, Baiye Ruan et al shed light on the metabolic process involved in stress erythropoiesis, emphasizing the role of glycolytic metabolism in the initial SEP expansion stage. Stress erythropoiesis initiation relies on inflammatory signals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In macrophages, inflammatory signals lead to “broken TCA cycle”. Citrate and succinate exit the mitochondria to aid in anabolic metabolism [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Exported citrate is transformed into oxaloacetate and acetyl-CoA. Histone acetyltransferases use acetyl-CoA to sustain glycolytic enzymes expression and for lipogenesis, which helps in promoting cell proliferation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Succinate can block the activity of proline hydroxylases (PHDs) responsible for regulating the stability of hypoxia-inducible factors (HIFs), leading to an increase in hypoxia-inducible glycolysis. Additionally, succinate hinders the differentiation of stress erythroid progenitor cells by obstructing alterations in DNA and histone methylation. Previous data indicate that GDF15 signaling can increase the expression of PDK1 and PDK3 enzymes. PDK1 and PDK3 enzymes play a role in limiting the entry of pyruvate into the tricarboxylic acid cycle [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, GDF15 enhances the levels of HIF1α and Glut1, thereby augmenting the glycolysis of SEPs. In this study, proinflammatory factor IL-1β significantly increased following the administration of 5-FU treatment. However, ASP alleviated IL-1β levels somehow, which may be the reason that ASP suppressing oxidative stress and apoptosis, antagonizing 5-FU-induced spleen injury and dysfunction [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Surprisingly, it was also found that concomitant with the proliferation of splenic macrophages and c-kit\u003csup\u003e+\u003c/sup\u003e erythroid progenitor cells, ASP promoted the genetic expression of glucose transporter1 (\u003cem\u003eGlut1\u003c/em\u003e), rate-limiting enzymes of glycolysis such as hexokinase2 (\u003cem\u003eHk\u003c/em\u003e2) and pyruvate kinase M (\u003cem\u003ePkM\u003c/em\u003e), and key glycolytic genes, including phosphoglycerate kinase (\u003cem\u003ePgk\u003c/em\u003e), lactate dehydrogenase A (\u003cem\u003eLdhA\u003c/em\u003e), and pyruvate dehydrogenase kinase (\u003cem\u003ePdk\u003c/em\u003e-1), indicating ASP facilitates glycolysis of splenocytes. These proliferative signals and ultimate metabolism alteration of the extra-medullar niche may be the one fascinating mechanism of enhanced stress erythropoiesis after ASP treatment.\u003c/p\u003e \u003cp\u003eGO functional enrichment analysis presented that the interaction targets of ASP and 5-FU are related to cell regulation of proliferation, response to hypoxia, positive regulation of cell growth, and positive regulation of glycolytic process. KEGG pathway enrichment analysis focused on the PI3K-Akt signaling. Documents demonstrate that Glycogen synthase kinase 3β (GSK-3β) is an important downstream molecule regulated by Akt. Akt cause GSK-3β phosphorylated to inhibit GSK-3β activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Reduced glycogen synthesis leads to accumulation of cyclin D1, resulting in cell cycle advancement and proliferation. Therefore, glycogen synthesis reduction and glycolysis may be crucial links in regulating cell proliferation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, PI3K/Akt pathway regulates the expression of fructose 2, 6-bisphosphatase (PFKFB2) and enhances glycolysis. HIFs are regulated by downstream mTOR of PI3K/Akt signaling pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The expression of genes involved in erythropoiesis and glycolytic metabolism, which are regulated by HIFs, is enhanced in hypoxic environments. Acute hypoxic responses are associated with HIF1α, while chronic hypoxia responses are linked to HIF2α [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Knock down HIF2α gene seriously impaired erythroid progenitors in the bone marrow and spleen. This study found that ASP strengthened PI3K, p-Akt, accumulating cyclin D1. Consistent with it, ASP significantly elevated HIF2α in the niche, also the erythroid differentiation genes Gata1 and Gata2. The results above hinted that under hypoxic conditions, ASP may improve glycolysis and stress erythroid proliferation via PI3K/Akt/HIF2α signals.\u003c/p\u003e \u003cp\u003eThe next of stress erythropoiesis is erythroid differentiation, which is dependent on EIM. Li et al surprisingly high lightened the expression of erythropoietin receptor (EpoR) on EIM [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The dependency of erythropoiesis on EPO/EpoR led to the initial belief that the expression of EpoR was limited to erythroid lineage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, growing evidence indicate that EpoR is widely expressed in various non-erythroid cells. EpoR signaling in macrophages has been found to impact the splenic niche, promoting stress erythroid differentiation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Epor\u003csup\u003e−/−\u003c/sup\u003e or Stat5\u003csup\u003e−/−\u003c/sup\u003e in macrophages impaired stress erythropoiesis, indicating Epo/EpoR/Stat5 plays a vital role in stress erythropoiesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Day 7 following 5-FU treatment in our study, concomitant with an increase in serum EPO, ASP dramatically increased the activity of STAT5 dramatically strengthened in the ASP-treated spleen niche. Consistent with it, ASP resumed the expression of EpoR on RAW 264.7 macrophages, activating STAT5 signal response to ectogenic EPO stimulation. Also, ASP restored cellular adhesion molecule Vcam-1 expression on macrophages, which is indispensable for erythroid differentiation. These results suggested that ASP may improve stress erythropoiesis via macrophage-mediated EPO/STAT5 signaling. In another study, we demonstrated ASP enhanced macrophage-dependent iron homeostasis and transfer, and nuclear engulfment, which may be associated with the EIM dependent EPO/STAT5 signaling.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThe results suggest that ASP may improve glycolysis after 5-FU treatment, promoting the activity of splenic macrophages and the expansion of erythroid progenitor cells. Also, ASP facilitate erythroid differentiation via macrophage-mediated EpoR/STAT5 signaling. Therefore, it may be a promising strategy for stress anemia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e The authors appreciate the helpful suggestions provided by Honghui Jiang and for Kunhang Du and Cheng Wang addressing software-related issues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e YL and LW designed the study and wrote the manuscript. YW contributed to revising the manuscript. Data analysis and figures preparation were performed by NS. HJ, BW, ZW, KD, CH, TY and CW contributed to the validation and interpretation of the results. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by the National Natural Science Foundation of China (Grant number 81873103) and Natural Science Foundation of Chongqing (Grant number cstc2021jcyj-msxmX0669).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The datasets used and/or analyzed during the current study can be made available upon reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors declare that they have no conflict of interests regarding the publication of this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statements and consent to participate\u003c/strong\u003e The animal experimental procedures were carried out and approved by the Ethics Committee of Chongqing Medical University under the approval number IACUC-CQMU-2022\u0026ndash;0026. This article contains any studies with animals performed by any of authors. All authors agreed to participate in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u0026nbsp;\u003c/strong\u003eAll authors agreed to the publication of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Razeq H, Hashem H (2020) Recent update in the pathogenesis and treatment of chemotherapy and cancer induced anemia. Crit Rev Oncol Hematol. 145:102837. doi: https://doi.org10.1016/j.critrevonc.2019.102837\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Vasconcellos JF, Meier ER, Parrow N, Editorial (2023) Stress erythropoiesis. Front Physiol 14:1165315. doi: https://doi.org10.3389/fphys.2023.1165315\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFibach E, Rachmilewitz EA (2017) Iron overload in hematological disorders. 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Exp Hematol. 91:10\u0026ndash;21. doi: https://doi.org10.1016/j.exphem.2020.09.185\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Wang S, Liu D, Gao C, Han Y, Guo X et al (2021) EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells. Blood. 138:1986-97. doi: https://doi.org10.1182/blood.2021011410\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao C, Prabhu KS, Paulson RF (2018) Monocyte-derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia. Blood. 132:2580-93. doi: https://doi.org10.1182/blood-2018-06-856831\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026oacute;thov\u0026aacute; Z, Tomc J, Debeljak N, Sol\u0026aacute;r P (2021) STAT5 as a Key Protein of Erythropoietin Signalization. Int J Mol Sci. 22. doi: https://doi.org10.3390/ijms22137109\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cancer chemoprevention, extramedullary hematopoiesis, stress erythropoiesis, splenic macrophage","lastPublishedDoi":"10.21203/rs.3.rs-4180160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4180160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eExtrinsic molecular mechanisms that regulate extramedullary stress erythropoiesis are still poorly understood, and the exploration of potential protective medication is needed.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003ePeripheral blood parameters and BFU-E colony enumeration were measured. IHC staining was conducted to detect the proliferation of splenocytes and splenic F4/80 macrophages. The expression of β-catenin protein in RAW264.7 macrophages was assessed using immunofluorescence. The cell cycle of mouse spleen c-kit\u003csup\u003e+\u003c/sup\u003e cells was analyzed by flow cytometry assay. Detection of Ccl2, Hk2, and Pgk1 mRNA expression by RT-qPCR. Cyclin D1 protein expression was assessed using Western blotting. IL-1 and EPO levels were determined by ELISA assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the 5-FU pre-administrated mouse, ASP rescued peripheral blood parameters such as RBC counts, HGB, HCT and MCH, and the BFU-E colony enumeration in the bone marrow. Meanwhile, ASP increased cellular proliferation in the splenic red pulp and cyclin D1 expression, ASP increased macrophage chemokine \u003cem\u003eCcl2\u003c/em\u003e genetic expression and the number of F4/80 macrophages in the spleen and splenic BFU-E enumeration. Furthermore, ASP facilitated glycolytic genes including \u003cem\u003eHk\u003c/em\u003e2, \u003cem\u003ePgk\u003c/em\u003e1, \u003cem\u003ePkm, Pdk\u003c/em\u003e1 and \u003cem\u003eLdha\u003c/em\u003e via PI3K/Akt/HIF2α signaling in the spleen. Subsequently, ASP declined pro-proinflammatory factor IL-1β, whereas upregulating erythroid proliferation-associated genes \u003cem\u003eGdf15, Bmp4, Wnt2b, and Wnt8a\u003c/em\u003e. Moreover, ASP facilitated EPO/STAT5 signaling in splenic macrophages to enhance erythroid lineage \u003cem\u003eGata2, Gata1\u003c/em\u003e genetic expression. Also, ASP facilitated erythroid differentiation via macrophage-mediated EpoR/STAT5 signaling.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eASP facilitate extramedullary stress erythropoiesis that suggest it might be a promising strategy for stress anemia treatment.\u003c/p\u003e","manuscriptTitle":"Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 03:52:15","doi":"10.21203/rs.3.rs-4180160/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23c972d2-1dee-46a2-bf8b-979bd60ae281","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-05T02:59:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-29 03:52:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4180160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4180160","identity":"rs-4180160","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}