Exploring The Mechanism Of Action Of Bushen Huoxue Prescription In Intervening Chondrocyte Apoptosis In Knee Osteoarthritis Based On Wnt/β-catenin Signaling Pathwa

Objective: Based on the network pharmacology,to investigate the possible mechanism of Bushen Huoxue Recipe intervene Chondrocyte apoptosis in knee osteoarthritis By modulating the Wnt /β-catenin signaling pathway and to verify by cell experiments.Method:1.Search TCMSP and BATMAN-TCM databases to obtain the effective chemical components and targets of each drugs in the formula,GEO,Gnen Card,OMIM 3 databases,Intersection between drug target and disease target to obtain a common target,Subsequently,the "component-disease target network map" and protein interaction network analysis were constructed through STRING database and Cytoscape 3.7.2 software,Line GO,KEGG enrichment analysis with the help of Metascape database;2.Cell experiment: the effect of CCK 8and Edu staining on the proliferation by human normal chondrocytes;the apoptosis by flow cytometry,Cleaved-casapse-3,Bcl-2,Bax,and pathway proteins Wnt 3a,β-catenin,GSK-3β;and the effects of TNF-α,IL-6 and IL-1βwere measured by ELISA.Result: Network pharmacology:In this study,the main active ingredients and target genes of TCM were analyzed by TCMSP,and 4706 drug target genes;3849 osteoarthritis-related genes were identified by Gene Cards,and 174 differential genes were identified in osteoarthritis based on chip GSE46750.Taking intersection significant significantly in 48 drug target genes.Metascape Analysis found that 48 differential gene sets were mainly significantly correlated with the regulation of Wnt signaling pathway,endochondral osteogenesis and skeletal dysplasia,ossification,overview of skeletal system morphogenesis,proinflammatory and profibrotic mediators,IL-17 signaling pathway,rheumatoid arthritis,TNF signaling pathway,inflammatory response,and modulation of neuroinflammatory response.Compound-main active substance and its drug target gene network diagram: including 66 nodes and 357 edges.Network diagram of major active ingredient-drug target genes and their enriched pathway: including 67 nodes and302 edges;Experimental part:1.Cell proliferation was measured by CCK8 Human normal chondrocytes C28 / I2 were divided into Blank,LPS,LPS + BSHXF groups,after LPS treatment,chondrocyte viability was diminished and chondrocyte viability was restored after BSHXF treatment;2.Cell proliferation was detected by EDU staining LPS treatment inhibited chondrocyte proliferation,and restored chondrocyte proliferation capacity after BSHXF treatment;3.Apoptosis was detected by flow cytometry Group: Blank group,LPS group,LPS +BSHXF group LPS induced the apoptosis of chondrocytes and inhibited its apoptosis after BSHXF treatment;4.Western blot for Cleaved-Caspase-3,Bcl-2 and Bax Group: Blank group,LPS group,LPS + BSHXF group LPS treatment promoted the expression of the pro-apoptosisrelated proteins Cleaved-Caspase-3 and Bax and inhibited the anti-apoptotic protein Bcl-2,and the protein level of Bcl-2 increased after BSHXF treatment decreased Cleaved-Caspase-3 and Bax;5.The TNF-α,IL-6,and IL-1 β were detected by ELISA,LPS treatment promoted the expression of the inflammatory cytokines TNF-α,IL-6,and IL-1β,which was inhibited by BSHXF treatment.Conclusion: 1.The results of the network pharmacology are basically consistent with the experimental verification results,and the network pharmacology is of certain guiding significance for our analysis of the deep molecular mechanism of TCM compound;2.Bushen Huoxue Recipe can reduce the apoptosis induced by LPS,thus inhibiting the inflammatory response and playing an anti-KOA effect;3.Bushen Huoxue Recipe prescription plays an antiKOA effect by inhibiting Wnt/ β-catenin signaling pathway and alleviating chondrocyte apoptosis and inflammation induced by LPS.