| Objective To explore the effect of sepsis on differentiation of hematopoietic stem cells and progenitor cells,and observe the intervention effect of Xuebijing injection(XBJ)on the differentiation of hematopoietic stem cells in bone marro w of sepsis mice.Methods The effect of XBJ and its main component Angelica sinensis on the differentiation of hematopoietic stem cells in bone marrow of normal mice was analyzed by colony-forming assay and real-time quantitative PCR analysis.A mouse model of sepsis was established by intraperitoneal injection of lipopolysaccharide(LPS)from escherichia coli,and XBJ was administered to the mouse model of sepsis:XBJ was injected into the tail vein of the XBJ group(n=20)20 mL/kg daily for 4 consecutive days.The normal control group(n=20)and the model group(n=20)were given the same amount of normal saline daily for 4 consecutive days).Flow cytometry was used to analyze the hematopoietic stem cells and progenitor cells in bone marrow,and the immune cells in spleen of septic mice,with and without XBJ injection,including Lin-sca-1+ c-kit+(LSK)cells,long-term hematopoietic stem cells and T lymphocyte subsets.The effect of XBJ on mRNA expression of hematopoietic stem cell related transcription factors and signaling pathway in septic mice was analyzed by real-time quantitative PCR,including GATA2,RUNX1,GFilB,LSD1,etc.Peripheral blood of septic patients in clinic and healthy volunteers were collected and the hematopoietic stem cells,progenitor cells and immune cells,including CD34+,HSC,MPP and T lymphocyte subsets,were analyzed by flow cytometryResults(1)The experimental results of colony formation assay showed that:In the colony of XBJ group,the cell morphology changed obviously,and the number of protrusions on the cell surface increased.Real-time quantitative PCR results showed that the expressions of Runx1,GFi1B and FOS genes were significantly down-regulated and the expressions of CD5 genes were significantly up-regulated at all three concentrations of XBJ(all P<0.05).At the concentration of 1mg/ml and 10mg/ml,the expression of GATA2,LSD1,CD71,CD90 and CD123 were significantly down-regulated(all P<0.05)upon XBJ treatment.CD11b gene expression was significantly down-regulated at the concentration of 0.1mg/ml and lmg/ml(all P<0.05)upon XBJ treatment.The expression of CD133 gene was significantly up-regulated at the concentration of 10mg/ml(P<0.05)upon XBJ treatment.In the wine angelica sinensis from three places,the expressions of GATA2,Runx1,GFilB and LSD1 genes were significantly down-regulated(all P<0.05);among the three raw angelica sinensis,Yanjing Shuangqiao showed a significant up-regulation effect on the expression of GATA2,Runx1 and GFi1B genes(all P<0.01),and Guangzhou Zhixin had a significant up-regulation effect on the expression of GATA2 and Runx1 genes(all P<0.0001),Shandong Boyuan significantly increased the expression of GFi1B gene(P<0.01).(2)Before LPS injection,there was no statistically significant difference in weight among all groups(all P>0.05).24h-48h after LPS injection,the body weight of the LPS group and LPS+XBJ group significantly reduced,and 72h-96h after LPS injection,the weight of the two groups of mice gradually increased,and the body weight at all four time points were reduced compared to the normal control group(both P<0.01).The body weight of the mice in the LPS+XBJ group was significantly higher than that in the LPS group at 24 h after LPS injection(P<0.05).The results of flow cytometry analysis showed that the number of hematopoietic stem cells in the bone marrow of the LPS group was significantly increased than that in the normal control group(P<0.01),while the number of hematopoietic stem cells in the LPS+XBJ group was significantly reduced compare to that in the LPS group(P<0.01).Compared to the normal control group,the number of LT-HSC in the LPS group and LPS+XBJ group was increased significantly,while the number of MPP cells decreased significantly(both P<0.01).The decrease of MPP in the XBJ group was more significant than that in the LPS group(P<0.01).The number of hematopoietic progenitor cells in the LPS group was significantly increased than that in the normal control group(P<0.01),while the number of hematopoietic progenitor cells in LPS+XBJ group was not statistically significant compared to the normal control group(P>0.05).The number of MEP in the LSP group was significantly increased compared to that in the normal control group(P<0.05).Upon the administration of XBJ,the number of MEP was decreased significantly compared to the LPS group(P<0.01).The number of myeloid-derived suppressor cells(MDSC)in the bone marrow and spleen in the LSP group was significantly increased compared to that in the normal control group,while the number of T cells and B cells was significantly decreased(both P<0.05).The number of MDSCs,T cells,and B cells in LPS+XBJ group was not statistically significant compared to the normal control group(all P>0.05).The results of real-time quantitative PCR showed that the expressions of GATA2,GFi1B,Runx1,FOS,and LSD1 in the LPS group were significantly down-regulated,and the expression of PU.1 genes was up-regulated(all P<0.01)compared to the normal control group.After the treatment of XBJ,FOS was significantly down-regulated,and LSD1 was significantly up-regulated(both P<0.05)compared to the LPS group.(3)General data and basic vital signs:the age difference between the healthy control group and the case group was not statistically significant(P>0.05),and the temperature and systolic blood pressure of the case group were significantly higher than those of the control group(both P<0.05).Blood tests showed that the values of the healthy control group were normal,and the white blood cell count,neutrophil percentage,and neutrophil count of the case group were higher than those of the control group on day 1 and day 4.(Both P<0.05),and the percentage of neutrophils on day 7 of the case group was significantly lower than that on day 1(P<0.05);the percentage of lymphocytes and lymphocyte count in the case group on day 1 and day 4 were lower than those in the control group Group(both P<0.05),and the percentage of lymphocytes in the case group on day 7 was significantly higher than that on day 1(P<0.05).Peripheral blood flow test results showed that compared with the healthy control group,the proportion of CD34+cells,hematopoietic progenitor cells(CD34+CD38+)and CMP in case group on day 4 were significantly increased(all P<0.05);meanwhile,the proportion of T cells and CD4+T cells in case group on day 1 and day 4 were significantly lower than that of the control group(both P<0.01),and the proportion of T cells and CD4+T cells in the case group on day 7 was significantly higher than that of on day 1(both P<0.05);The proportion of CD8+T cells in the case group on day 1,4 and 7 were significantly lower than that in the control group(all P<0.05),and compared with the case groups on day 1 and day 4,the proportion of CD8+T cells in the case group on day 7 increased significantly(all P<0.05).Conclusion 1.Xuebijing injection and its main component Angelica sinensis can promote the differentiation and maturation of bone marrow cells by affecting the expression of transcription factors of bone marrow cells in vitro.2.Abnormal proliferation of hematopoietic stem cells in septic mice results in decreased immune function.Xuebijing injection can improve the immune function by inhibiting the abnormal proliferation of hematopoietic stem cells in septic mice,and the mechanism may be related to the up-regulation of LSD1 and other factors.3.Hematopoietic stem cells proliferated abnormally in septic patients,while the number of lymphocytes(T cells)decreased. |
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