Mechanisms Of Massage Mediating Chondrocyte Apoptosis In Knee Osteoarthritis Through Piezo1/JAK2 Signaling Pathway

Objective:Through the Establishment of rat model of knee osteoarthritis to observe the correlation between Piezo1,JAK2 and chondrocyte apoptosis;Through the intervention of massage manipulation to explore the mechanism of massage intervention on chondrocyte apoptosis in knee osteoarthritis through Piezo 1/JAK2 signaling pathway.Methods:Experiment 1:50 adult male rats were randomly divided into sham operation group(10 rats)and operation group(40 rats).The chondrocyte morphology,Mankin's score,TUNEL dyeing positive region,protein expression of Piezo1?JAK2 and Caspase3 of knee osteoarthritis rats at different stages(operation time,7d,30d,60d,90d)was detected after successful modeling.Experiment 2:(1)Transfection experiment:30 adult male rats were randomly divided into5 groups,they were Sham operation group,KOA group,KOA+Piezo1 Lentivirus overexpression group,KOA+Piezo1 Lentivirus silencing group and KOA+empty vector group.No treatment in sham operation group.KOA group only modeled.The other three groups were injected with Piezo 1 gene silencing lentivirus vector,Piezo 1 gene overexpression lentivirus vector and lentivirus(empty vector)0.01 ml(viral dose 10¹¹/ml)respectively.Seven days later,the cartilage tissue of knee joint was sacrificed and Piezo 1 expression level was detected.(2)Intervention experiment:As experimental animals,48 Male rats were randomly divided into 8 groups.they were Sham operation group,KOA group,KOA+Piezo1 Lentivirus overexpression group,KOA+Piezo1 Lentivirus silencing group,KOA+Piezo1 inhibitor group,KOA+Massage Group,KOA+Piezo1Lentivirus Overexpression+Massage group and KOA+Piezo1 Lentivirus Overexpression+JAK2 inhibitor group.Massage intervention,Piezo1 specific inhibitor intervention and JAK2 specific inhibitor intervention were performed on the 7th day after re-modeling.Mankin's score and TUNEL dyeing positive region were calculated.The expression of Piezo1 and p-JAK2 in articular cartilage was detected by Western Blot.Results:1.Establishment of an animal model of knee osteoarthritis in rats and correlation between Piezo1,JAK2,Caspase3 and chondrocyte apoptosis:(1)Along with time,the positive areas of TUNEL staining in articular cartilage also increased.(2)As the course of disease progressed,the mankin's score of KOA rats increased gradually.It is suggested that the degree of pathological damage of knee joint in rats is gradually aggravated and KOA is gradually progressing.(3)With the development of osteoarthropathy,the expression of Piezo1 protein in knee cartilage of KOA rats also increased,accompanied by the increase of apoptosis-related protein Caspase3 and the activation of JAK.2.Therapeutic effect and molecular mechanism of Chinese massage on knee osteoarthritis in rats:(1)We found that the overexpression of Piezo1 protein was more serious than that of KOA alone.The silencing of Piezo1 protein,Piezo1inhibitor and JAK2 phosphorylation inhibitor AG490 could reverse and alleviate the degree of joint injury.At the same time,the injury of KOA joint was significantly reduced by massage intervention.Pathological score of articular cartilage in massage.By comparing Pathological score of articular cartilage in massage and the overexpression of Piezo1 protein with the overexpression of Piezo1 protein,the results showed that massage intervention could significantly reduce the degree of articular cartilage damage.By observing the TUNEL staining results of articular cartilage sections in each group,we found the same trend.(2)In KOA rats,Piezo1 Lentivirus overexpression group could significantly increase Piezo1 expression and JAK2 activation.Lentivirus silencing reduced Piezo1 expression and JAK2 activation.Piezo1inhibitor has little effect on Piezo1 protein expression.Massage intervention can significantly reduce Piezo1 protein and JAK2 activation.In addition,inhibition of Piezo1 protein activation can significantly reduce JAK2activation.The expression of Piezo1 protein and phosphorylation of JAK2 in lentivirus overexpression group were also significantly reduced,and lower than that of KOA group.In comparison with the overexpression of Piezo1 protein,intraperitoneal injection of JAK2 inhibitor could significantly reduce the apoptosis of articular chondrocytes in KOA rats with piezo1overexpression,and then,delay the injury of articular cartilage.This suggests that Piezo 1 may play a pathogenic role in the development of KOA through the activation of JAK2.Conclusions:1.With the progress of KOA,the pathological damage of knee joint cartilage is progressively aggravated,and its pathological basis is progressive apoptosis of articular chondrocyte.2.Piezo1/JAK2/Caspase3 may be involved in the process of pathological apoptosis of knee chondrocytes.3.Piezo1 plays an important role in the development of KOA through JAK2-induced apoptosis of articular chondrocytes.4.Massage intervention can inhibit the apoptosis of knee chondrocyte and delay the progress of KOA by down-regulating Piezo 1 protein expression and inhibiting the activation of Piezo1/JAK2 signal transduction pathway.