基于网络药理学、分子对接及实验验证探究右归丸治疗骨关节炎作用机制OA

Objective To explore the mechanism of Yougui Pills in treating osteoarthritis through tonifying kidney and bone method based on network pharmacology and animal experiment.Methods The active components and their targets in the Yougui Pills were obtained from the TCMSP database;GeneCards database was used to obtain disease targets;after the intersection of drugs and disease targets,protein interaction networks and drug-component-target networks were constructed to obtain core targets;GO and KEGG pathway enrichment analysis was performed on intersecting targets,and molecular docking was ultimately performed to demonstrate the stability of the key components and core targets.The rat model of osteoarthritis was established by bilateral knee joint injection of sodium iodoacetate solution.The rats were randomly divided into control group,model group,positive drug group and Yougui Pills group,and were intervened for 14 days.HE staining was used to observe the morphology of rat knee cartilage.qPCR was used to detect the mRNA expressions of NF-κB p65,NF-κB p50,MMP-13,ADAMTS-5 and IL-6 in cartilage tissue.Western blot was used to detect the protein expressions of NF-κB p65,NF-κB p50,p-p38,MMP-13,IKKβ and COX-2 in cartilage tissue.Immunohistochemistry was used to detect the protein positive expressions of p-p38,MMP-13 and COX-2 in cartilage tissue.Results Network pharmacology results showed that there were 127 active components in Yougui Pills,corresponding to 190 targets.There were a total of 1 266 related targets for osteoarthritis,46 intersecting targets of drugs and diseases,and core targets such as INS,IL10,IFNG,PTGS2,IL1A,etc.KEGG pathway enrichment analysis yielded 20 pathways,mainly involving tumor cell signaling pathway,NF-κB signaling pathway,PI3K-AKT signaling pathway,cytokine interaction signaling pathway,neural deformation pathway,etc.Animal experiments showed that compared with the control group,typical cartilage damage was observed in the knee joint tissue sections of the model group,with a loss of bone surface structure and disordered arrangement of chondrocytes;a large number of chondrocytes exhibited vacuolar degeneration,and some nuclei underwent nuclear condensation and fragmentation;the phenomenon of cell clustering could be observed.The expressions of NF-κB p65,NF-κB p50,MMP-13,ADAMTS-5 and IL-6 mRNA significantly increased,the expressions of NF-κB p65,NF-κB p50,p-p38,MMP-13,IKKβ and COX-2 proteins in the cartilage tissue of the model group increased(P<0.05).Compared with the model group,Yougui Pills group showed significant improvement in inflammatory damage to chondrocytes,the expressions of MMP-13,ADAMTS-5 and IL-6 mRNA decreased,and the expressions of NF-κB p65,NF-κB p50,p-p38,MMP-13,IKKβ and COX-2 proteins in the cartilage tissue decreased(P<0.05).Conclusion Yougui Pills can inhibit the NF-κB signaling pathway through tonifying kidney and bone method,thereby reducing the expression levels of ADAMTS-5,MMP-13 and COX-2,alleviating inflammation and destruction of knee joint cartilage,and treating osteoarthritis.