| Objective The purpose of this study was to evaluate the therapeutic effect of epimedin C(EPIc)on aplastic anemia(AA)mice through in vivo and in vitro experiments,and to explore the effect of epimedin C on bone marrow mesenchymal stem cells(BMSCs)and the possible mechanism of action,so as to provide more theoretical basis for epimedin C in the treatment of AA and to find more effective treatment programs for AA.Methods In the first part,SPF grade ICR mice were randomly divided into group A(blank control group),Group B(model control group),group C(EPIc treatment group),group D(Cs A treatment group)and group E(combined treatment group).Except group A,the other 4 groups were given Busulfan suspension for 10 days to establish aplastic anemia mouse model.The 5 groups of mice were treated with normal saline(group A,group B),EPIc(group C),Cs A(group D)and EPIc combined with Cs A(group E)for 10 days,then the eyeball was removed and blood was collected from both sides of the femur.Peripheral blood cells and bone marrow nucleated cells(BMNC)were counted,and bone marrow morphological changes were observed under the microscope by HE staining.The levels of cytokines(IFN-γ,IL-6,CXCL-12)were determined by ELISA.In the second part,EPIc treated mouse BMSCs(groups A ～ F)with different concentrations(10,20,30,40,50ng/m L)were used.CCK-8 was used to detect the effects of EPIc on BMSCs proliferation.Western blot analysis was performed to determine the effects of EPIc on the expression of Runx2,Osterix and PPAR-γ in BMSCs.Results Part I 1.General state of mice in each group: compared with group A,after treatment,mice in group B were listless,mucous membrane was pale,hair was sparse and scattered,and back bowing was obvious;Compared with group B,group C,D and E were more active in spirit,and their mucous membranes were rudder than before.2.Comparison of peripheral blood cell counts of mice in each group:compared with group A,WBC,ANC,RBC,Hb and PLT counts in group B and WBC and PLT counts in groups C,D and E were significantly decreased.Compared with group B,the RBC and Hb counts of group C and D and the WBC,ANC,RBC,Hb and PLT counts of group E were significantly increased.3.Comparison of pathological morphology of bone marrow in each group: Group A had active bone marrow hyperplasia;In group B,bone marrow hyperplasia was severely reduced and a large number of fat vacuoles were observed.In group C,D and E,myelodysplasia was improved to varying degrees,nucleated cells increased and fat vacuoles decreased gradually.4.Comparison of BMNC count among groups: The BMNC count in groups B,C and D was significantly lower than that in group A,and the BMNC count in groups C,D and E was higher than that in group B to varying degrees.5.Comparison of serum cytokine levels in groups B,C,D and E showed that IFN-γ in groups B,C,D and E was higher than that in group A,and that in group E was lower than that in groups B and C.IL-6 in group B,C and D was significantly higher than that in group A,and decreased in group E than that in group B,C and D.CXCL-12 in group B was significantly lower than that in group A,higher in groups C and D than that in group B,and higher in group E than that in groups A,B and C.Part Ⅱ 1.EPIc at a certain concentration(10,20,30,40,50ng/m L)could promote the proliferation of BMSCs(P < 0.05).Its proliferative effect was gradually enhanced with the increase of drug concentration,and gradually stabilized with the extension of treatment time.2.EPIc at 20,30,40 and 50ng/m L significantly increased Runx2 expression compared with the control group,and EPIc at 40 and 50ng/m L significantly promoted Runx2 expression compared with EPIc at 20 and 30ng/m L.EPIc at 40 and 50ng/m L significantly increased the expression of Osterix.50ng/m L EPIc increased the expression of PPAR-γ.Conclusion 1.There is no significant difference between EPIc and Cs A in promoting blood image recovery and regulating cytokines(INF-γ,IL-6,CXCL-12).Cs A is better than EPIc in reducing bone marrow adipose and promoting proliferation of bone marrow nucleated cells.2.Compared with EPIc and Cs A alone,the combination of EPIc and Cs A can better promote the recovery of blood image in AA mice,reduce bone marrow fat,reduce the levels of inflammatory factors(INF-γ and IL-6)in vivo,increase the secretion of stromal cell derived factors(CXCL-12),promote the proliferation of bone marrow nucleated cells,and have more significant therapeutic effect.3.A certain concentration of EPIc can promote the proliferation of BMSCs,and its proliferative effect is positively correlated with drug concentration.4.EPIc can promote the expression of Runx2 and Osterix in BMSCs,and increase the expression of PPAR-γ at high concentrations,which can affect the differentiation of BMSCs into osteoblasts and adipocytes. |
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