The Role And Mechanism Of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase In Osteoarthritis

Objective1.To investigate the expression of protein tyrosine phosphatase SHP2 in osteoarthritis chondrocytes and cartilage tissues.2.To investigate the effects of knockdown or overexpression of SHP2 on inflammation,metabolism,apoptosis and migration of chondrocytes in vitro,and to clarify the molecular mechanism.3.To investigate the effect of adenovirus injection into the knee cavity of mice to silence SHP2 on the progression of osteoarthritis.Methods1.Detection of SHP2 expression: The knee OA of mice was induced by medial meniscus instability(DMM),and the expression of SHP2 in articular cartilage of the Sham and DMM groups was detected by tissue immunofluorescence technique.Western blot was used to detect the expression of SHP2,MMP3,MMP13 and COL2A1 in chondrocytes stimulated by IL-1β.2.Detection of chondrocyte metabolism: q PCR and Western blot were used to test the knockdown or overexpression efficiency of SHP2 in chondrocytes.The effects of SHP2 knockdown with siRNA or plasmid overexpression on IL-1β-induced chondrocyte-related indicators including catabolism(MMP3 and MMP13),cartilage anabolism(COL2A1,AGGRECAN,SOX9)and cartilage hypertrophy(COL10A1)were detected by q PCR and Western blot.3.Apoptosis detection: After the chondrocytes were transfected with siRNA or overexpressed plasmids,the apoptosis rate of chondrocytes was detected by Annexin-V-FITC/PI apoptosis detection kit and flow cytometry.4.Detection of cell proliferation: After the chondrocytes were transfected with siRNA or overexpressed plasmid,the effect of SHP2 on cell proliferation was detected by the CCK8 method.5.Cell migration detection: After chondrocytes were transfected with siRNA or overexpressed plasmids,the role of SHP2 on cell migration was tested by scarification test.6.Molecular signal channel assay: β-catenin expression and Wnt target gene expression were detected after inhibition or overexpression of SHP2,and the binding relationship between SHP2 and β-catenin was detected by immunoprecipitation(Co-IP).After the chondrocytes were transfected with siRNA or overexpressed plasmid,IL-1β was used to stimulate the chondrocytes for a short time and then the protein was extracted.The changes of MAPK and NF-κB signaling pathways were detected by Western blot.7.In vivo experiment: OA model was induced by medial meniscus instability(DMM)to investigate the effect of adenovirus injection into the knee joint to silence SHP2 on OA progression.The severity of cartilage injury was assessed by staining with Safranin O/Fast Green staining,HE and toluidine blue.Immunohistochemistry was used to evaluate the effect of SHP2 on anabolism(Aggrecan,Col2a1)and catabolism(MMP3 and MMP13)of OA articular cartilage in vivo.μCT scan was used to detect osteophyte growth and subchondral bone changes in the knee joint.Results1.The results of SHP2 immunofluorescence staining showed that the expression of SHP2 was significantly increased in the DMM group.With the increase of IL-1β concentration and time,the m RNA and protein expression levels of SHP2 gradually increased,the protein expression levels of MMP3 and MMP13 also showed an increasing trend,while the expression of COL2A1 showed a decreasing trend.2.The expression of SHP2 was obviously depressed by siRNA transfection in primary chondrocytes,while the expression of SHP2 was significantly increased by transfection of overexpressed plasmid.SHP2 knockdown inhibited the increase of IL-1β-induced catabolic genes(MMP3 and MMP13)and cartilage hypertrophy genes(COL10A1),and increased the level of cartilage matrix genes(AGGRECAN and COL2A1).In contrast,overexpression of SHP2 increased IL-1β-induced catabolism(MMP3 and MMP13)and cartilage hypertrophy(COL10A1),but inhibited the level of cartilage matrix(AGGRECAN and COL2A1).3.Annexin V-FITC/PI staining and flow cytometry results indicated that IL-1β could induce chondrocyte apoptosis,while inhibition or overexpression of SHP2 did not affect apoptosis.4.CCK8 test showed that SHP2 had no significant effect on the proliferation of chondrocytes.5.The scratch test showed that SHP2 did not affect the migration of chondrocytes.6.Western blot showed that SHP2 knockdown reduced the level of β-catenin and Wnt/β-catenin target genes,including Axin2,Lef1 and TCF4,while overexpression showed the opposite result.Co-IP results showed that SHP2 and β-catenin could combine to form a complex.7.Western blot showed that IL-1β activated MAPK and NF-κB pathways in 15-30 min.Inhibition of SHP2 inhibited the activation of MAPK and NF-κB pathways induced by IL-1β,while overexpression promoted the activation of MAPK and NF-κB pathways induced by IL-1β.8.The results of HE,toluidine blue,Safranin O/Fast Green staining,OARSI score,and μCT scan showed that the injection of adenovirus into the articular cavity to silent SHP2 could inhibit the DMM surgically induced cartilage destruction and osteophyte formation without affecting the subchondral bone.Immunostaining of cartilage with AGGRECAN,COL2A1,MMP3 and MMP13 indicated that silencing of SHP2 expression could significantly inhibit the catabolism of OA chondrocytes,enhance the anabolism,and retarded the degradation of cartilage matrix.Conclusion1.SHP2 expression is increased in primary chondrocytes after IL-1β treatment and in mouse OA cartilage.2.In vitro,SHP2 promoted catabolism and inhibited anabolism,but did not affect the proliferation,apoptosis and migration of chondrocytes.β-catenin,MAPK,NF-κB and other signaling pathways are involved in the destructive effect of SHP2 in OA.3.In vivo,intra-articular injection of sh SHP2 adenovirus could inhibit inflammatory catabolism and promote anabolism of OA chondrocytes,thereby inhibiting cartilage destruction and degradation of cartilage matrix.This suggests that SHP2 can be a candidate therapeutic target for OA.