| Objective:To study the therapeutic effect of Busuishengxuewan on aplastic anemia,and to explore part of its mechanism of action from the perspective of regulating bone marrow microenvironment,so as to provide empirical evidence for its in-depth development and clinical rational use.Methods:Experiment1:Effect of Busuishengxuewan on aplastic anemia model mice and their effect on bone marrow microenvironmentKunming male mice were randomly divided into normal group,model group,Bupai Shengxue Wan low-dose,medium-dose and high-dose groups(2.25,4.50,9.00 g·kg-1)and positive drug group(2.70 g·kg-1),with 15 mice in each group.Except for the normal group,the other groups were injected with 10 mg·kg-1and 20 mg·kg-1by alternating intraperitoneal injection for 12 consecutive days to prepare aplastic anemia mouse model.At the same time,the mice in each group were given the drug by intragastric administration according to the dose for 30 consecutive days.Peripheral blood images were detected in each group.Swiss-giemsa method was used to observe the mouse bone marrow smear and calculate the number of bone marrow nucleated cells.HE staining was used to observe the pathological changes of bone marrow.endothelial growth factor（VEGF）、Thrombopoietin（TPO）、Vascular cell adhesion molecule-1（VCAM-1）content in mouse serum and bone marrow were compared by ELISA,;Immunohistochemical assay was performed to detect the protein expression of mouse bone marrow microvascular density（MVD）,VEGF and Notch-1.The relative expressions of VEGF and Notch-1 in mouse bone marrow were detected by Western Blot.Experiment2:Effect of serum containing Busuishengxuewan on VEGF-Notch-1signaling pathway in mouse Bone marrow stromal cells（BMSCs）Forty SD rats were randomly divided into blank serum group and drug-containing serum group,with 20rats in each group.The rats were given distilled water and Busui shengxuewan(9.00g·kg-1),respectively,for 7 days.1 h after the last administration,The rats were anesthetized,blood was collected from abdominal aorta under sterile conditions,and drug-containing serum was obtained by centrifugation.BMSCs were divided into blank serum group,drug-containing serum group,DAPT group,Jagged1 group,DAPT+drug-containing serum group and Jagged1+drug-containing serum group.CCK-8 method was used to screen the optimal concentration of Busui Shengxue pills in the intervention of mouse bone marrow mesenchymal stem cells.The cell proliferation of each group was detected by CCK-8 method.The contents of VEGF,TPO and VCAM-1 in the supernatant were determined by ELISA.The relative expressions of VEGF and Notch-1 were detected by Western Blot.Results:Experiment 1:Compared with normal group,the contents of peripheral red blood cells（RBC）,white blood cells（WBC）,hemoglobin（HGB）and platelets（PLT）in model group were significantly decreased.The marrow cavity is empty;VEGF,TPO and VCAM-1 contents in serum and bone marrow were decreased.Bone marrow MVD decreased significantly,VEGF and Notch-1 protein expression decreased significantly.After 30 days of continuous administration,compared with the model group,the indexes of peripheral blood of mice in the administration group were increased to varying degrees.Bone marrow hematopoietic function improved obviously;VEGF,TPO and VCAM-1 contents in serum and bone marrow showed an increasing trend.Bone marrow MVD increased;The expression of VEGF and Notch-1 protein showed an upward trend.Experiment 2:20%concentration containing the drug serum action for 24 h was selected as the optimal concentration and time of action.After administration of 10μM DAPT and 10μM Jagged1,the contents of VEGF and VCAM-1 in the cell supernatant of the DAPT group decreased significantly compared with the blank serological group,and the relative expression of VEGF and Notch-1 protein decreased significantly.The VEGF in the cell supernatant of the Jagged1 group was significantly increased,and the relative expression of Notch-1 protein was significantly increased.Compared with the drug-containing serum group,the content of VEGF in cell supernatant in the DAPT+drug-containing serum group was reduced,and the relative expression of VEGF and Notch-1 protein was significantly reduced.The relative expression of VEGF and Notch-1 protein in the Jagged1+drug-containing serum group was significantly increased.Compared with the Jagged1 group,the relative expression of VEGF and Notch-1 protein in the Jagged1+drug-containing serum group was significantly increased.Conclusion:Busuishengxuewan has obvious prevention and treatment effects on aplastic anemia model mice,and the improvement of bone marrow hematopoietic microenvironment through VEGF-Notch-1 pathway may be part of its mechanism... |
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