| Objective:To explore the mechanism of Bushen Tianjing Fang(BSTJF)as an adjuvant Immunosuppressive therapy(IST)to improve the bone marrow microenvironment and promote the proliferation of hematopoietic stem cells in AA mice by regulating the level of inflammatory cytokines and inhibiting the transduction of Fas/FasL signaling pathway.Method:Sublethal dose of 60Coy-ray irradiation combined with immune mediations made mouse animal models of bone marrow suppression aplastic anemia(AA).IST combined with Bushen Tianjing Fang was used for intervention.Anti-thymocyte globulins(ATG)combined with Cyclosporine A(CsA)as an IST intervention,using TPO-R receptor agonist Eltrombopag(EP)combined with ATG and CsA as positive control,the changes of routine hematological parameters were analyzed.The morphology and composition of bone marrow cells were observed by HE staining,and the effects of Bushen Tianjing Fang on routine hematological parameters and bone marrow histopathologic changes of AA mice were studied.Flow cytometry was used to detect the changes in the number and apoptosis rate of hematopoietic stem cells(HSCs)in bone marrow cells,and ELISA was used to detect the contents of Interferon-gamma(IFN-γ),Tumor necrosis factor-alpha(TNF-α)inflammatory cytokines and apoptosis-related factors Fas and FasL in peripheral blood,Wes was used to detect the expression of Fas protein in spleen Treg cells,and to to explore the inhibitory effect of Bushen Tianjing Fang on inflammatory injury and apoptosis of HSCs by interfering the expression of inflammatory cytokines.Results:l Compared with the normal group,the levels of red blood cells,white blood cells and platelets in the peripheral blood of the model group were significantly decreased,and the hematopoietic tissue in the bone tissue was significantly reduced,indicating that the sublethal dose of 60Co γ-ray irradiation combined with immune-mediated AA mouse model was successfully established.2 Blood routine test showed that compared with the normal group,the White blood cells(WBC)in the peripheral blood of each model group were significantly reduced 7 days after modeling(P<0.01);After 7 days of intervention,the white blood cells,red blood cells(RBC),platelets(PLT)and plateletcrit(PCT)were significantly decreased in all groups(P<0.01).After 21 days of intervention,the number of white blood cells in each group was significantly decreased(P<0.01).The red blood cell,hemoglobin(HGB),mean corpuscular volume(MCV),red blood cell volume distribution width-variable coefficient(RDW-CV),platelet(PLT),mean platelet volume(MPV),plateletcrit and platelet distribution width(PDW)in model group were significantly decreased(P<0.01).After 28 days of treatment,the three lines of peripheral blood cells in the model group were significantly reduced(P<0.01).Compared with model group,after 7 days of medication intervention,WBC and HGB were increased in two treatment groups(P>0.05),and the difference was not statistically significant.RDW-CV,PLT and MPV of the two treatment groups were significantly increased(P<0.01),RBC and HCT of the positive control group were significantly increased(P<0.01),and RBC and HCT of the Chinese medicine group were significantly increased(P<0.05).After 21 days of intervention,RBC,RDW-CV,PLT,MPV and PCT in the two treatment groups were significantly increased(P<0.01),HGB content was increased to a certain extent(P<0.05),and the mean corpuscular hemoglobin concentration(MCHC)in the positive control group was decreased(P<0.05).After 28 days of intervention,WBC,RBC,HGB,HCT,mean corpuscular hemoglobin(MCH),RDW-CV and PCT of the two treatment groups were significantly increased(P<0.01),PLT of the positive control group was increased(P<0.05),PLT of the Chinese medicine group was also increased(P>0.05),but the difference was not statistically significant.3 HE staining showed that the bone marrow space of mice in the normal group was larger,and the cell development and distribution were normal.In model group,hematopoietic tissue was significantly reduced,non-hematopoietic tissue was increased,and bone marrow space was reduced.After treatment,the pathological changes of bone tissue of mice in each administration group were relieved to a certain extent,bone marrow space was enlarged,bone marrow was hyperplasia to a certain extent,and hematopoietic tissue was increased.4 Flow cytometry showed that compared with normal group,the number of hematopoietic stem cells in model group was significantly decreased(P<0.01),and the apoptosis rate of hematopoietic stem cells was increased(P<0.05).Compared with model group,the number of hematopoietic stem cells in positive control group was increased(P>0.05),but the difference was not statistically significant.The number of hematopoietic stem cells in the IST combined with BSTJF group was increased(P<0.05),and the apoptosis rate of hematopoietic stem cells in the positive control group and the IST combined with BSTJF group was decreased(P<0.05).5 Compared with the normal group,the contents of IFN-γ,Fas and FasL in the model group were significantly increased(P<0.01),and the contents of TNF-α were significantly increased(P<0.05).Compared with model group,the contents of inflammatory cytokines IFN-γ,TNF-α and apoptosis-related factors Fas and FasL were significantly decreased in positive control group and IST combined with BSTJF group(P<0.01).6 Wes detection of Fas protein expression in mouse spleen Treg cells showed that Fas protein expression in the model group was increased compared with the normal group,and Fas protein expression in the two medication groups was decreased compared with the model group,but the differences were not statistically significant(P>0.05).Conclusion:Bushen Tianjing Fang as an adjuvant therapy for IST may interfere with the expression of inflammatory factors,reduce inflammatory injury and apoptosis of HSCs by inhibiting the transduction of Fas/FasL signaling pathway,promote the proliferation of HSCs and proliferation and differentiation of peripheral blood cells,and improve the hematopoietic function of bone marrow. |
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