Mechanism Of TRPM7 Mediated M1 Polarization Of Synovial Macrophages In Knee Osteoarthritis And Intervention Effect Of Xibining Decoction

Objective:To explore the molecular mechanism between the transient potential receptor channel TRPM7 and knee osteoarthritis(KOA)synovial macrophage M1 polarization,and the interventional effect of Xibining Decoction.Method:1)The DMM method was used to destabilize the mouse joints.The level of inflammatory factor IL-1? and TNF-? in serum was detected by ELISA.Double immunofluorescence staining is used to label different phenotypes of macrophages specific markers to assess whether there is synovial macrophages polarization in the KOA model of mechanical stress disorder.Evaluation of synovitis and Krenn score by HE staining,and evaluation of cartilage degeneration and OARSI score by using Safranin O&Fast green staining.2)In vitro,macrophages were subjected to cyclic mechanical stretch of 0%,7%and 12%at 5Hz.The polarization markers of M1 or M2 macrophages,iNOS and IL-1? or CD206 and Arg-1,were detected by Western blot and PCR.NS8593,a selective inhibitor of TRPM7,was administrated with 12%cyclic mechanical stretch at 5Hz.iNOS and IL-1? or CD206 and Arg-1 were detected in bothe protein and gene level.3)Pretreat macrophages with small interfering RNA of TRPM7,and then mechanically stimulate macrophages to induce their polarization,then examine TRPM7,total ERK1/2 and phosphorylated ERK1/2,total P65 and phosphorylated P65 and I?B? expression changes in protein level.4)Using TRPM7 selective inhibitor,HE staining,Sirius red staining and Safranin O fast green staining were used to observe synovial inflammation,fibrosis and cartilage degeneration in KOA mice,and to score synovial Krenn and cartilage OARSI respectively,using immunofluorescence Double staining technique to label F4/80 and iNOS or CD206 and other macrophage specific markers,ELISA to detect serum levels of inflammatory factors IL-1? and TNF-?,Western Blot to detect protein expression of TRPM7,total ERK1/2 and phosphorylated ERK1/2,total P65 and phosphorylated P65 and I?B? in synovial tissue.Western Blot and PCR were used to detect changes in the expression of proteins and genes related to cartilage tissue matrix degradation.5)The KOA mouse model was established by the DMM method.The TRPM7 inhibitor group was the positive control group,and the Xibining group was the observation group.The synovial inflammation,fibrosis and cartilage degeneration of KOA mice were observed by HE staining,Sirius red staining and Safranin O fast green staining.Serum levels of inflammatory factors IL-1? and TNF-? were detected by ELISA,and protein and gene expression changes related to cartilage tissue matrix degradation were detected by Western Blot and PCR.6)DMM method established KOA mouse model,using immunofluorescence double staining technology to label F4/80 and iNOS or CD206,observe the polarized phenotype of synovial macrophages in the KOA model,Western Blot to detect protein expression of TRPM7,total ERK1/2 and phosphorylated ERK1/2,total P65 and phosphorylated P65 and IKBa in synovial tissue.Result:1)In the KOA mouse model established by the DMM method,the expression of IL-1? and TNF-? in synovial tissue increased,the infiltration of synovial macrophages increased,the ratio of iNOS-positive cells was significantly up-regulated(P<0.05),while the ratio of CD206-positive cells decreased(P<0.05).2)After 7%of cyclic mechanical stretch intervenes in macrophages,compared with the 0%group,the expression of CD206 and Arg-1 protein was up-regulated,and the expression of iNOS and IL-1? protein was down-regulated(P<0.05);while the 12%group,ompared with the 0%group,the expression of iNOS and IL-1? protein was significantly up-regulated,and the expression of CD206 and Arg-1 protein was down-regulated(P<0.05);after the application of TRPM7 inhibitors,the up-regulation trend of iNOS and IL-1? wasdecreased,and CD206 and Arg-1 decrease trend was partially reversed(P<0.05).3)12%cyclic mechanical stretch can increase the protein expression of TRPM7,increase the phosphorylation of ERK and P65,and down-regulate the expression of I?B?(P<0.05).After the application of TRPM7 siRNA,the phosphorylation of ERK and P65 was decreased,and the expression of I?B? was decreased(P<0.05).4)Compared with mice in the sham group,the pathological scores of synovium and cartilage in KOA mice were significantly increased,and TRPM7,ERK and P65 phosphorylation in synovial tissue were significantly increased(P<0.05);while the mice were treated wiht selective inhibitor of TRPM7,the pathological score was significantly decreased,the expression of MMP-3 and MMP-13 proteins and genes and Adamts-4 and Adamts-5 genes in cartilage were significantly down-regulated,and TRPM7,phosphorylated ERK and P65 were also significantly down-regulated in synovium(P<0.05).5)Compared with the model group of mice,in the Xibining gourp,mice' serum IL-1? and TNF-? were significantly down-regulated,synovial and cartilage pathological scores were reduced,and the proteins and genes of MMP-3 and MMP-13,and Adamts-4 and Adamts-5 genes in the cartilage were significantly down-regulated(P<0.05).6)Compared with the model group of mice,Xibining Decoction significantly reduced the ratio of M1 type macrophages in synovial tissue of mice,and also reduced TRPM7,ERK 1/2,phosphorylated ERK1/2,phosphorylated P65 proten expression in synovial tissue(P<0.05).Conclusion:1)There is macrophage polarization in the KOA model induced by DMM method,and abnormal mechanical stretch can stimulate synovial macrophage polarization in vivo.2)Cyclic mechanical stretch induces macrophage polarization by activating TRPM7-ERK and NF-?B signaling pathways.3)Xibining decoction can effectively improve the synovial inflammation,synovial fibrosis and cartilage degeneration in KOA mice.4)The therapeutic effect of Xibining decoction is achieved by inhibiting the activation of TRPM7-ERK and NF-?B signaling pathways,thereby inhibiting synovial macrophage Ml polarization.