Sfrp4 Repression Of Wnt Signaling Alleviates Osteoarthritis Via Targeting Articular Chondrocytes

Purpose:At present,the early treatment effect of osteoarthritis(OA)is limited,and only joint replacement surgery can be used in the late stage,which brings a heavy burden to the family and society.Therefore,there is an urgent need for drugs that can stop and reverse the pathological process of OA.The search for disease-modifying osteoarthritis drug(DMOAD)has also become the focus and difficulty of current OA research.More and more studies show that Wnt signaling pathway is closely related to the progression of OA.Some progress has also been made in the research of treating OA with Wnt pathway inhibitors as the target.Secreted Frizzed Related Protein 4(Sfrp4)belongs to the secreted frizzed related protein family,which can directly bind to Wnt protein,thus inhibiting Wnt signal pathway and exerting many biological effects.As an inhibitor of Wnt pathway,Sfrp4 protein is expected to play a role in the treatment of OA.However,there is no research on the relationship between Sfrp4 protein and OA.Therefore,it is innovative to study the role of Sfrp4 protein in the treatment of OA.This project deeply explored the mechanism of the anti-OA effect of Sfrp4 protein through regulating articular cartilage.The completion of the study will hopefully provide a new idea and method for the treatment of OA.Methods:(1)Chondrocytes were extracted from mouse articular cartilage and cultured in vitro.IL-1β treatment was used to simulate the osteoarthritis environment,and cells were treated with different concentrations of Sfrp4 protein and Wnt pathway agonist SKL2001.western-blot and qRT-PCR were used to verify the changes in sfrp4 expression in different cartilage environments.(2)Mouse chondrocytes were cultured in vitro and IL-1β was used to stimulate chondrocytes to mimic the arthritic environment.Different concentrations of sfrp4 and SKL2001 were applied to different groups of cells to detect changes in cell activity.(3)In vitro cultured mouse chondrocytes were stimulated with IL-1β to mimic the arthritic environment,and different concentrations of sfrp4 and SKL2001 were applied to different groups of cells to detect changes in NO and PGE2.(4)In vitro culture of mouse chondrocytes,stimulation of chondrocytes with IL-1β to mimic arthritic environment,application of different concentrations of sfrp4 and SKL2001 to different groups of cells,detection of iNOS and COX-2 expression in WB.(5)In vitro cultured mouse chondrocytes were stimulated with IL-1β to mimic the arthritic environment,different concentrations of sfrp4 and SKL2001 were applied to different groups of cells,and the expression of MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5 were measured by qRT-PCR.(6)Mouse chondrocytes were cultured in vitro,and IL-1β was used to stimulate chondrocytes to simulate an arthritic environment.Different concentrations of sfrp4 and SKL2001 were applied to different groups of cells for 24 h.Afterwards,chondrocytes were collected and Cleaved Caspase-3 apoptosis index was measured by WB,and the apoptosis rate was detected by flow cytometry.(7)Mouse chondrocytes were cultured in vitro and IL-1β was used to stimulate chondrocytes to simulate an arthritic environment.Different concentrations of sfrp4 and SKL2001 were applied to different groups of cells for 24 hours,and protein was extracted to detect changes in the Wnt/β-catenin pathway.(8)Animal experiment:3010-week-old wild-type C57BL/6 mice were randomly and equally divided into "control group","OA/Vehicle group" and "OA+Sfrp4 group"."After 8 weeks,the knee joint tissues of the above mice were collected and stained with paraffin,and the OARSI scores were compared.Results:(1)Sfrp4 protein expression was reduced in IL-1β-treated chondrocytes.(2)The experimental drug treatment regimen had no significant effect on chondrocytes activity.(3)Sfrp4 protein decreased IL-1β-induced NO and PGE2 in mouse chondrocytes.(4)Sfrp4 protein inhibited IL-1β-induced expression of iNOS and COX-2 in mouse chondrocytes.(5)Sfrp4 protein inhibited IL-1β-induced cartilage matrix degradation in mice.(6)Sfrp4 protein inhibited IL-1β-mediated apoptosis in mouse chondrocytes.(7)Sfrp4 protein inhibited IL-1β-induced upregulation of Wnt/β-catenin signaling pathway.(8)In a mouse OA model,Srfp4 protein inhibited the progression of osteoarthritis pathology.Conclusion:Sfrp4 can achieve anti-osteoarthritis effects by inhibiting the classical Wnt/β-catenin signaling pathway,reducing inflammatory factor production,alleviating articular cartilage matrix degradation and inhibiting chondrocyte apoptosis.This project provides insight into the mechanism by which Sfrp4 protein exerts its anti-OA effects through the regulation of articular cartilage.The completion of the study is expected to provide a new idea and method for the treatment of OA.