The Role And Mechanism Of TRPM7 In Chondrocyte Apoptosis In Rheumatoid Arthritis

Articular cartilage damage with subsequent impairment of joint function is a common feature of articular diseases,in particular,rheumatoid arthritis and osteoarthritis.Rheumatoid arthritis is an autoimmune disease of unknown etiology that can cause inflammation of the synovium,bone destruction,erosion,and cartilage damage.While articular cartilage injury mediated by chondrocyte apoptosis is a known major pathological feature of arthritis,the specific mechanisms remain unclear at present.TRPM7 is a receptor potential channel that can participate in many pathophysiological processes by mediating divalent cations.Some studies show that TRPM7 plays an important role in apoptosis,and inhibition or silencing of TRPM7 could effectively reduce apoptosis.IHH is a secreted protein expressed by prehypertrophic chondrocytes,and belongs to a type of HH.Studies have shown that IHH is highly expressed in cartilage damage and promotes the occurrence and development of arthritis.Silencing TRPM7 could significantly reduce the expression of IHH,but it is still unclear whether TRPM7 could play a role in arthritis and articular cartilage damage by regulating IHH.To this end,we will use the in vitro cultured rat chondrocytes and the in vivo AA rat model as objects to carry out the following research:1.The effect of blocking or silencing TRPM7 on the apoptosis of rat articular chondrocytesMTT analysis and LDH analysis were used to observe the effect of SNP on rat articular chondrocyte viability and cytotoxicity at different concentrations and at different times,and to observe the effect of blocking or silencing TRPM7 on SNP-induced chondrocyte viability;Hoechst 33258 staining was used,flow Cytometry was used to detect the effect of blocking or silencing TRPM7 on SNP-induced chondrocyte apoptosis;Rhodamine 123 staining was used to detect the effect of blocking or silencing TRPM7 on SNP-induced mitochondrial membrane potential;Western blot was used to detect the changes of related apoptotic proteins;DCFH-DA staining and flow cytometric detection of the effect of 2-APB on the expression of ROS induced by SNP.The effects of 2-APB on the expression of TRPM7,IHH,MMP-13,IL-6,ADAMTS-5 were detected by western blot and realtime quantitative PCR.The results show that SNP can reduce chondrocyte viability in a time-dependent and concentration-dependent manner.Blocking or silencing TRPM7 can reverse SNP-induced articular chondrocyte damage,apoptosis,and decrease in mitochondrial membrane potential;and significantly reverse SNP-induced TRPM7,IHH,and MMP-13,IL-6,ADAMTS-5 increase.The results suggest that TRPM7 may play an important role in chondrocyte apoptosis in articular rats.2.The effect of activated IHH on chondrocyte apoptosis protected by 2-APB MTT analysis was used to observe the effect of activated IHH on the viability of 2-APBprotected chondrocytes;Hoechst33258 staining and flow cytometry were used to detect the effect of activated IHH on 2-APB-protected chondrocyte apoptosis;Rhodamine 123 staining was used to detect mitochondria the influence of membrane potential changes;the changes of related apoptotic proteins were detected by western blot.The results show that activation of IHH can reverse the increased chondrocyte viability protected by 2-APB,reverse the chondrocyte apoptosis and the increase of mitochondrial membrane potential that 2-APB reduces.The results suggest that in the apoptosis of rat articular chondrocytes,TRPM7 can regulate apoptosis by regulating IHH.3.Blocking the effect of TRPM7 on articular cartilage damage in AA rats During the experiment for 12-26 days,evaluate the model arthritis score and foot swelling analysis every two days,and use it to detect the effect of blocking TRPM7 on rat paw swelling;On the 26 th day of the experiment,imaging analysis was used to observe the effect of blocking TRPM7 on joints.HE staining and Safranin O staining were used to observe the effect of blocking TRPM7 on articular cartilage damage;immunohistochemistry was used to detect IL-6,MMP-13,ADAMTS-5 expression;detect the changes of related apoptosis proteins by western blot.The results show that blocking TRPM7 can reduce rat paw swelling,reduce articular cartilage damage,reduce the expression of IL-6,MMP-13,ADAMTS-5,and reduce articular cartilage apoptosis.The results suggest that blocking TRPM7 can reduce the damage of articular cartilage in AA rats.In summary,the results of this study suggest that TRPM7 can regulate apoptosis by regulating IHH,and blocking TRPM7 can reduce articular cartilage damage,which strengthens people’s understanding of TRPM7 and also provides a new potential target for the treatment of RA.