Study On The Mechanism Of Tanshinone ⅡA Preventing And Repairing The Brain Microvascular Endothelial Cell Damage Induced By β-Amyloid Protein

Objective:This project regarded the brain microvascular endothelial cells as research object,established a model of injured brain microvascular endothelial cells induced byβ-amyloid protein to investigate the effect of TanⅡA on the inflammatory cytokines such as VCAM-1and NF-κB,and to explore the effect and possible mechanism of TanⅡA protecting the brain microvascular endothelial cells to prevent and treat AD,which provided a certain theoretical basis for the clinical treatment of AD by TanⅡA.Methods:1 Experimental group:BMEC was subcultured,and which with good growth was carried out in experiment.The experiment was divided into 5 groups:normal group,model group,and three TanⅡA groups with low,medium and high levels.2 Cell activity detected by MTT assay:The BMEC with good growth was selected and the cell activity was detected by MTT assay.The TanⅡA groups with optimal levels and the optimal time of drug action were selected.The rats in the normal group were cultured under normal conditions.The model group was the normal group modeled with 10μmol/L Aβ_1-42.The TanⅡA groups were the model group added with different concentrations of TanⅡA,for subsequent experiments.3 The content of LDH in the cell culture medium detected by microplate method:the content of LDH in the culture medium of each group was detected by microplate method after 24hours of BMEC by TanⅡA.4 The content of interrelated factor in the cell culture medium of each group detected by ELISA:The content of VCAM-1 in the cell culture medium of each group was detected by ELISA after 24 hours of BMEC by TanⅡA.5 The level of cell interrelated protein expression in each group detected by Western Blot:The expression level of cell interrelated protein in each group were detected by Western Blot after 24 hours of BMEC by TanⅡA.Results:1 The cell activity detected by MTT assayThe results showed that:Compared with the normal group,the cell activity of model group decreased,and the difference was statistically significant（P<0.05）.Compared with the model group,the cell activity of three TanⅡA groups increased,and the difference was statistically significant（P<0.05）,which with 30μg/mL concentration had significant difference（P<0.01）,which with 10μg/mL、50μg/mL concentration had differences（P<0.05）.The TanⅡA group with 30μg/mL concentration had the best effect,and the best time of drug action is 24 hours.Therefore,the subsequent experiments take TanⅡA 10μg/mL、30μg/mL、50μg/mL concentration as the low,medium and high levels of three TanⅡA groups,and 24 hours is selected as the time of drug action.2The content of LDH in the cell culture medium detected by microplate methodThe results showed that:Compared with the normal group,the LDH secretion in the model group increased（P<0.05）.Compared with the model group,the LDH secretion in three TanⅡA groups with low,medium and high levels all decreased（P<0.05）.3 The content of VCAM-1 in the cell culture medium of each group detected by ELISAThe results showed that:Compared with the normal group,the VCAM-1 secretion in the model group increased（P<0.05）.Compared with the model group,the VCAM-1 secretion in three TanⅡA groups with low,medium and high levels all decreased（P<0.05）.And the VCAM-1 secretion in the TanⅡA groups with medium levels decreased more than that with low and high levels（P<0.01）.4 The expression level of cell interrelated protein in each group detected by Western BlotThe results showed that:（1）The expression of VCAM-1 protein:Compared with the normal group,the expression of VCAM-1 protein in model group increased（P<0.05）;Compared with the model group,the expression of VCAM-1 protein in three tanshinoneⅡA groups with low,medium and high levels all decreased（P<0.05）.And the expression of VCAM-1protein in the tanshinoneⅡA groups with medium levels decreased more than that with low and high levels（P<0.01）.（2）The expression of IKB-αprotein:Compared with the normal group,the expression of IKB-αprotein in model group decreased（P<0.05）;Compared with the model group,the expression of IKB-αprotein in three TanⅡA groups with low,medium and high levels all increased（P<0.05）.And the expression of IKB-αprotein in the tanshinoneⅡA groups with medium levels increased more than that with low and high levels（P<0.01）.（3）The expression of NF-κB p65 protein:Compared with the normal group,the expression of NF-κB p65 protein in model group increased（P<0.05）;Compared with the model group,the expression of NF-κB p65 protein in three tanshinoneⅡA groups with low,medium and high levels all decreased（P<0.05）.And the expression of NF-κB p65 protein in the tanshinoneⅡA groups with medium levels decreased more than that with low and high levels（P<0.01）.（4）The expression of NF-κB p65 phosphorylation:Compared with the normal group,the expression of NF-κB p65 phosphorylation in model group increased（P<0.05）;Compared with the model group,the expression of NF-κB p65 phosphorylation in three TanⅡA groups with low,medium and high levels all decreased（P<0.05）.Conclusion:1 TanⅡA can improve the cell viability of injured BMEC,reduce the expression of inflammatory factors,and help prevent Alzheimer’s disease.2 TanⅡA can reduce the expression of inflammatory factors in endothelial cells induced by Aβ_1-42,prevent and treat Alzheimer’s disease and inhibit NF-κB pathway may be one of its possible mechanism.