透骨消痛胶囊对TNF-α诱导关节软骨细胞Rho/Rock信号通路的影响

Effects of Tougu Xiaotong Capsule on Rho/Rock Signal Transduction Pathway in Chondrocytes Induced by TNF-α

目的观察透骨消痛胶囊对TNF-α诱导体外培养大鼠关节软骨细胞Rho/Rock信号通路的影响,探讨其防治骨性关节炎的作用机制。方法采用4周龄SD大鼠膝关节软骨建立软骨细胞体外培养体系,选取第3代软骨细胞进行实验,20 ng/mL的TNF-α诱导体外培养软骨细胞的凋亡,诱导成功后,给予透骨消痛胶囊(500、100、20μg/mL)孵育24 h后,用流式细胞仪检测软骨细胞Annexin-Ⅴ观察细胞凋亡情况,采用RT-PCR和Western Blot测定各组软骨细胞RhoA、Rock1和PKN等mRNA和蛋白表达。结果 TNF-α组细胞凋亡明显增多,透骨消痛胶囊各组能减少软骨细胞的凋亡,TNF-α组的RhoA、Rock1、PKN、MKK3、ERK6、Cbfα1等mRNA和蛋白表达较空白组上升,而透骨消痛胶囊各组对RhoA/Rock信号传导通路关键信号分子RhoA、Rock1、PKN及Cbfα1等的mRNA和蛋白的表达有抑制,差异均具有统计学意义(P<0.05)。结论透骨消痛胶囊可减轻TNF-α所致的软骨细胞凋亡,其机制可能与抑制软骨细胞的RhoA/Rock信号通路有关。

Objective To observe the effects of Tougu Xiaotong capsule(TXC) on Rho / Rock signal transduction pathway in chondrocytes induced by tumor necrosis factor-α(TNF-α),and explore it’s mechanism of treating osteoarthritis. Methods Chondrocytes were derived from the cartilages of knee joints of 4-week-old male Sprague-Dawley rats. The 3rd generation of chondrocytes were treated with 20 ng·mL-1TNF-α to induce chondrocytes apoptosis,then intervened by different concentrations of TXC for 24 hours. The Annexin-Ⅴin chondrocytes was determined by flow cytometry to observe chondrocytes apoptosis,and the mRNA and protein expression of RhoA,Rock1,PKN,MKK3,ERK6 and Cbfα1 were measured by RT-PCR and Western Blot. Results Compared with blank group,the cell apoptosis and the mRNA and protein expression of RhoA,Rock1,PKN,MKK3,ERK6 and Cbfα1 in TNF-α group were increased significantly(P&lt;0.05).Compared with TNF-α group,the cell apoptosis and the mRNA and protein expression of these key signal molecules of Rho / Rock signal transduction pathway in TXC groups were respectively decreased( P&lt;0.05).Conclusion TXC can reduce the apoptosis of chondrocytes induced by TNF-α,and the mechanism may be related to its inhibitory effect on RhoA / Rock signal transduction pathway in chondrocytes.