Study On The Mechanism Of Hongjingtian Injection On Inhibiting Proliferation,Migration And Promoting Apoptosis In Vascular Smooth Muscle Cells Based On Network Pharmacology

Objective:1.Using the network pharmacological analysis,the targets and biological function of HJT on DA were studied.2.Based on the results of the network pharmacological analysis,the effects and the underlying molecular mechanism of HJT on cell proliferation,migration and apoptosis in HG-induced VSMCs was verified in vitro.Methods:1.The potential mechanisms of HJT on DA was explored through network pharmacology.By integrating multiple online databases,the targets information of five components of HJT and DA was collected,the topological parameters of the targets in the network were analyzed by Cytoscape software module,the enrichment in tissue of the targets was analyzed by Fun Rich software,and the biological process of targets was analyzed by David database.2.In order to verify the results of network pharmacology,HG-stimulated A7r5 cells served as the cell model.CCK-8 was used to screen the appropriate concentration of HJT and analyze cell proliferation;wound healing assay and Transwell assay were used to investigate cell migration;Tunel staining and Annexin V-FITC/PI assay were used to detect cell apoptosis.The effects on key targets and the AKT pathway were verified by Western blotting in experiments with the AKT inhibitor LY294002 or activator SC79.Results:1.The results of the network pharmacology showed that 279 targets for HJT,the top 50 targets for DA,and 10 candidate targets of HJT acting on DA were obtained,including VEGFA,AKT1,PTGS2,CASP3,BCL2,SERPINE1,TP53,BAX,ADRB1 and MMP9.The targets of HJT are significantly enriched in heart,kidney,blood vessels,monocytes,lymphocytes,VSMCs and other tissues and cells,and may be involved in the biological processes such as cell proliferation,migration and apoptosis and AKT signaling pathway.2.Functional experiments showed that HJT markedly suppressed the proliferation and migration and promoted the apoptosis of HG-induced VSMCs.Mechanistically,HJT significantly downregulated the expression of p AKT,MMP9,and PCNA,upregulated the expression of p53 and caspase-3 and increased the Bax/Bcl-2 ratio compared with the HG group.SC79,an AKT activator,partially reversed the inhibitory effects of HJT on HGinduced VSMCs,while LY294002,an inhibitor of AKT,showed a similar inhibitory effect as HJT,which further confirming the involvement of the AKT pathway.Conclusions:1.HJT could act on DA with multi-targets,multi-pathways and multi-cells,and its potential molecular mechanism may be related to AKT signaling pathway,involving the biological processes such as cell proliferation,migration and apoptosis.2.The experimental results verified that HJT inhibited the proliferation and migration,and promoted the apoptosis of HG-induced VSMCs partly by inhibiting the AKT pathway,which further confirms the accuracy of network pharmacological analysis.