Effect Of Wnt/β-catenin In Regulating Pyroptosis Of Chondrocytes In Knee Osteoarthritis

Objective To investigate the mechanism of verapamil and MCC950 in regulating pyroptosis of chondrocytes in knee osteoarthritis by the Wnt/β-catenin signaling pathway,so as to further clarify the role and mechanism of Wnt/β-catenin signaling pathway in regulating cell pyroptosis in chondrocytes of knee osteoarthritis.Methods 1: Clinical experiment: In this study,fresh tibial plateau specimens of 10 patients with total knee arthroplasty for knee osteoarthritis were first randomly selected.Specimens were divided into mild,moderate and severe groups based on the results of safraninO-fast green staining and hematoxylin-eosin staining,and cartilage degeneration was assessed according to the OARSI rating developed by the International Association for the Study of Arthritis.Immunofluorescence and immunohistochemical staining were used to detect the expression of Wnt/β-catenin signaling pathway correlation factors GSK-3β,β-catenin,and coketosis related factors caspase-1,NLRP3 and GSDMD in articular cartilage.2: Animal experiment: 72 6-week-old C57BL/6J mice were used to establish a mouse knee OA model by DMM method.2.1: Observation of verapamil treatment:(1)DMM groups were set up separately;(2)Model group;(3)Verapamil intra-articular injection group,each group set up 2weeks and 8 weeks of observation cycles,and the degree of knee joint degeneration in mouse knee cartilage specimens was evaluated by OARSI grading by safranin-O-fast green staining and hematoxylin-eosin staining,and the expression of Wnt/β-catenin signaling pathway correlation factors GSK-3β and β-catenin,and conodyosis related factors caspase-1,NLRP3 and GSDMD were detected by immunofluorescence and immunohistochemical staining.Transmission electron microscopy was used to observe the morphological changes of chondrocytes.2.2: MCC950 treatment observation:(1)DMM group was set up separately;(2)Model group;(3)MCC950 intraperitoneal injection group,each group set up 2 weeks and 8weeks of observation cycles,and the safranin-O-fast green staining and hematoxylin-eosin staining was used to evaluate the degree of knee joint degeneration in mouse knee cartilage specimens by OARSI rating,and the expression of Wnt/β-catenin signaling pathway correspondents GSK-3β and β-catenin,and the pyrozoan related factors caspase-1,NLRP3 and GSDMD were detected by immunofluorescence and immunohistochemical staining.Transmission electron microscopy was used to observe the morphological changes of chondrocytes.Results 1.Clinical experiments: Compared with early articular cartilage,safranin-O-fast green staining and hematoxylin-eosin staining showed that the loss of proteoglycans in advanced articular cartilage gradually increased,cartilage loss gradually worsened,the injury gradually broke through the tidal line and gradually exposed calcified cartilage,and immunohistochemistry showed that Wnt/β-catenin signaling pathway and pyroptosis-related proteins such as β-catenin,GSDMD,Caspase1,NLRP3,and immunohistochemistry showed that Wnt/-catenin signaling pathway and pyroptosis-related proteins in early,middle and late articular cartilage showed that the patients were gradually increased.The expression of GSK3βand others increases gradually,and the expression of these proteins reaches its highest in advanced articular cartilage.2.Animal experiments: Compared with the DMM group,the rate of cartilage damage,caspase-1,GSK-3β,NLRP3,GSDMD,β-catenin expression and pyroptosis rate of chondrocytes in the model group increased significantly.Compared with the model group,injection of MCC950 could inhibit cartilage tissue damage,inhibit the activation of NLRP3,reduce the expression of caspase-1,NLRP3 and GSDMD,inhibit the pyroptosis of chondrocytes,and reduce the expression of GSK-3β and β-catenin.Verapamil injection could inhibit the expression and nuclear translocation of β-catenin,reduce the expression of GSK-3βand β-catenin and inhibit cartilage degeneration,while the expression of caspase-1,NLRP3,GSDMD and chondrocytes pyroptosis decreased.Compared with the verapamil group,the expression of caspase-1,NLRP3,GSDMD and other proteins in the MCC950 treatment group was significantly reduced,but the expression of caspase-1,NLRP3,GSDMD and other proteins was not significantly reduced.The expression of GSK-3β and β-catenin in the verapamil treatment group was significantly reduced,and the expression of caspase-1,NLRP3,GSDMD and other proteins was also significantly reduced.Conclusion Wnt/β-catenin signaling pathway can reduce articular cartilage degeneration in knee osteoarthritis by regulating cell pyroptosis.