siRNA-Piezo1通过ERK1/2信号通路抑制滑膜细胞增殖和炎性因子表达的相关机制

Mechanism research on siRNA-Piezo1 inhibiting cell proliferation of synoviocytes and expression of inflammatory factors through ERK1/2 signaling pathway

目的:探究siRNA-Piezo1通过抑制Piezo1表达、阻断ERK1/2信号通路后,对滑膜细胞增殖情况和炎性因子表达的影响及相关机制.方法:以类风湿关节炎(RA)患者和截肢患者为研究对象,手术过程中获取滑膜组织.利用免疫组化染色法检测滑膜组织中Piezo1蛋白的表达量.分离并培养人关节炎成纤维样滑膜细胞(RAFLS),通过Flexcell 4000 T设备构建体外力学RAFLS细胞模型,根据干预情况分成空白对照组,siRNA-Piezo1组、siRNA-Piezo1+PD98059组、24 h应力组、siRNA-Piezo1+24 h应力组、siRNA-Piezo1+PD98059+24 h应力组、48 h应力组、siRNA-Piezo1+48 h应力组和siRNA-Piezo1+PD98059+48 h应力组.RT-PCR方法检测Piezo1和EKR1/2的变化,CCK-8试剂盒检测增殖情况,ELISA方法观察关节液和细胞上清液中白介素1β(IL-1β)、白介素6(IL-6)和肿瘤坏死因子α(TNF-α)的表达.结果:免疫组化的结果显示,RA患者滑膜组织中Piezo1蛋白的表达量要明显高于正常对照者(P<0.05).荧光显微镜下观察,慢病毒转染RAFLS的效率大于90%,RT-PCR结果显示,siRNA-Piezo1干扰序列转染RAFLS后,Piezo1 mRNA的表达量明显降低(P<0.05).CCK-8测定结果显示,24 h应力组和48 h应力组RAFLS细胞的增殖率均明显高于空白对照组(P<0.05);siRNA-Piezo1+24 h应力组和siRNA-Piezo1+48 h应力组RAFLS细胞的增殖率分别明显低于24 h应力组和48 h应力组(P<0.05);siRNA-Piezo1+PD98059+24 h应力组和siRNA-Piezo1+PD98059+48 h应力组RAFLS的细胞增殖率分别明显低于24 h应力组和48 h应力组(P<0.05)及siRNA-Piezo1+24 h应力组和siRNA-Piezo1+48 h应力组(P<0.05).ELISA测定结果显示,24 h应力组和48 h应力组细胞上清液的IL-1β、IL-6和TNF-α的表达量均明显高于空白对照组(P<0.05);siRNA-Piezo1+24 h应力组和siRNA-Piezo1+48 h应力组细胞上清液的IL-1β、IL-6和TNF-α的表达量分别明显低于24 h应力组和48 h应力组(P<0.05);siRNA-Piezo1+PD98059+24 h应力组和siRNA-Piezo1+PD98059+48 h应力组细胞上清液的IL-1β、IL-6和TNF-α的表达量分别明显低于siRNA-Piezo1+24 h应力组和siRNA-Piezo1+PD98059+48 h应力组(P<0.05).RT-PCR测定结果显示,24 h应力组和48 h应力组细胞Piezo1和ERK1/2的mRNA的相对表达量均明显高于空白对照组(P<0.05);siRNA-Piezo1+PD98059+24 h应力组和siRNA-Piezo1+PD98059+48 h应力组细胞ERK1/2的mRNA的相对表达量分别明显低于siRNA-Piezo1+24 h应力组和siRNA-Piezo1+48 h应力组(P<0.05).结论:ERK1/2可能为Piezo1的下游信号分子,通过ERK1/2信号通路阻断力学信号传导,可抑制RAFLS细胞增殖和炎性因子的释放.

Objective:To explore the mechanism of siRNA-Piezo1 to inhibit synovial cell proliferation and inflammatory factor release by inhibiting Piezo1 protein expression and the ERK1/2 signaling pathway.Methods:The rheumatoid arthritis(RA)patients and the amputees patients was treated as the research objects,whose synovium tissues was obtained during operation.The expression of Piezo1 protein in synovium was detected by immunohistochemistry.Rheumatoid arthritis fibroblast-like synoviocytes(RAFLS)were isolated and cultured,and a mechanical RAFLS cell model in vitro was constructed by Flexcell 4000T equipment.According to the interventions,it was divided into blank control group,siRNA-Piezo1 group,siRNA-Piezo1+PD98059 group,24 h stress group,siRNA-Piezo1+PD98059+24 h stress group,48 h stress group,siRNA-Piezo1+48 h stress group and siRNA-Piezo1+PD98059+48 h stress group.The RT-PCR was used to observe the mRNA expression of Piezo1 and ERK1/2.The expression levels of IL-1β,IL-6 and TNF-αin the synovial fluid of RA patients and normal controls were detected by ELISA.CCK-8 kit was used to detect the cell proliferation.Results:The results of immunohistochemistry showed that the expression of Piezo1 protein in synovium of RA patients was significantly higher than that of normal controls(P<0.05).The results of RT-PCR showed that the expression of Piezo1 mRNA in RAFLS cells decreased significantly(P<0.05)after transfected by siRNA-Piezo1.The results of CCK-8 showed that the proliferation rates of RAFLS in 24 h stress group and 48 h stress group were significantly higher than that in the blank control group(P<0.05),those in siRNA-Piezo1+24 h stress group and siRNA-Piezo1+48 h stress group were significantly lower than that in the 24 h stress group and 48 h stress group respectively(P<0.05),and those in siRNA-Piezo1+PD98059+24 h stress group and siRNA-Piezo1+PD98059+48 h stress group were significantly lower than that in the 24 h stress group and 48 h stress group(P<0.05),as well as that in siRNA-Piezo1+24 h stress group and siRNA-Piezo1+48 h stress group respectively(P<0.05).The results of ELISA showed that the expression of IL-1β,IL-6 and TNF-αin the supernatant of 24 h stress group and 48 h stress group was significantly higher than that of the blank control group(P<0.05).The expression of IL-1β,IL-6 and TNF-αin supernatant of siRNA piezo1+24 h stress group and siRNA Piezo1+48 h stress group were significantly lower than that of 24 h stress group and 48 h stress group(P<0.05).The expression of IL-1β,IL-6 and TNF-αin the supernatant of siRNA-Piezo1+PD98059+24 h stress group and siRNA-Piezo1+PD98059+48 h stress group were significantly lower than that of siRNA-Piezo1+24 h stress group and siRNA Piezo1+PD98059+48 h stress group(P<0.05).The results of RT-PCR showed that the relative expressions of Piezo1 and ERK1/2 mRNA in 24 h stress group and 48 h stress group were significantly higher than that in the blank control group(P<0.05).The relative expressions of ERK1/2 mRNA in siRNA-Piezo1+PD98059+24 h stress group and siRNA-Piezo1+PD98059+48 h stress group were significantly lower than that in siRNA-Piezo1+24 h stress group and siRNA-Piezo1+48 h stress group respectively(P<0.05).Conclusion:ERK1/2 may be the downstream signal molecule of Piezo1,and blocking the mechanical signal transmission through ERK1/2 signaling pathway may may inhibit the proliferation of RAFLS cells and the release of inflammatory factors.