Effect Of Piezo1 SiRNA Transfection On Chondrocytes In OA Patients With Stress Model

Objective:To investigate the effect of Piezo1-si RNA on the expression of extracellular matrix of human OA chondrocytes.To explore the role of Piezo1 and p38 genes in OA chondrocytes and the mechanism of OA cartilage matrix degradation and explore new targets for gene therapy of OA.Method:Small Interfering RNA Targeting Piezo1 was designed based on the Gene Bank human FAM-38 A m RNA（XM₃61182）sequence.An mechanical tensile stress model in vitro was constructed using a Flexcell cell force machine.The experimental group was divided into 0 h mechanical tension group（Blank control group）,2 h mechanically stretched stress group,12 h mechanically stretched stress group,24 h mechanically stretched stress group,48 h mechanically stretched stress group,and transfected group（Transfected the target gene sequence,messy sequence,culture medium instead of virus solution,respectively）,and inhibitor group（specific inhibitor of p38 MAPK,SB203580）.The immunofluorescence to observe the expression of Piezo1 and p38 in chondrocytes under mechanical tensile stress.Real-time PCR was used to detect the expression of Piezo1,p38,MMP-13,TIMP1,Collagen II and Aggrecan m RNA in chondrocytes under mechanical tension stress model.In addition,the expression of Collagen II and Aggrecan were detected by western blot.Result:（1）Piezo1 protein was abundantly expressed in the nucleus of OA chondrocytes and partly expressed in cytoplasm and cell membrane;p38 was mainly expressed in the cytoplasm.（2）The expression level of 12 h mechanically stretched stress group was significantly increased compared with 2 h mechanically stretched stress group（P<0.05）.The highest expression of Piezo1 m RNA was found in 24 h mechanically stretched stress group;The expression of Piezo1 m RNA was significantly decreased in the 48 h mechanically stretched stress group compared with 24 h（P<0.05）;The expression of p38 and MMP-13 m RNA have the same expression trend as Piezo1.（3）Western blot results showed that the expression of Collagen II and Aggrecan protein decreased in the mechanical stretch stress group.Conclusion :（1）.Lentivirus-mediated Piezo1-siRNA transfection of human OA chondrocytes could effectively inhibit the expression of Piezo1,and has no significant effect on the growth state of chondrocytes.（2）.The mechanically sensitive ion channel Piezol interacts with p38 MAPK in cartilage matrix degradation mediated by mechanical stretch stress.Piezo1 induces the expression of MMP-13 via the p38 MAPK signaling pathway and accelerates the degradation of cartilage matrix and cartilage destruction.（3）.Silencing Piezo1 gene can inhibit the expression of p38,increased the expression of TIMP1 and then play a protective role for OA chondrocytes.