补肾活血方对人滑膜细胞Wnt/β-catenin信号通路的调控作用

Regulation effect of Bushen Huoxue Formula on Wnt/β-catenin signaling pathway of human synoviocytes

目的:观察补肾活血方对人滑膜细胞Wnt/β-catenin信号通路的调控作用。方法:采用GSK-3β选择性抑制剂SB-216763作用于正常人滑膜细胞,然后提取和检测胞核蛋白β-catenin的表达,确定激活正常人滑膜细胞的Wnt/β-catenin信号通路的最佳时间点,然后给予补肾活血方含药血清干预,采用Western Blot检测人滑膜细胞胞核蛋白β-catenin及其下游基因MMP-7和Cyclin D1的表达,酶联免疫吸附法(ELISA)检测滑膜细胞上清液中MMP-7和Cyclin D1的表达。结果:Western Blot结果显示:成功激活正常人滑膜细胞Wnt/β-catenin信号通路,与正常组比较,SB-216763干预组的人滑膜细胞核蛋白β-catenin、MMP-7和Cyclin D1的表达明显升高(P<0.05);补肾活血方含药血清干预后,滑膜细胞胞核蛋白β-catenin及其下游基因MMP-7和Cyclin D1有显著下调(P<0.05)。ELISA结果显示:与正常组滑膜细胞比较,SB-216763干预的滑膜细胞上清液中MMP-7和Cyclin D1的表达明显升高(P<0.05)。而补肾活血方干预后较SB-216763干预组明显下调(P<0.05)。结论:补肾活血方具有调控人滑膜细胞Wnt/β-catenin信号通路的作用,其结果有助于从单一通路阐释补肾活血法中药治疗膝骨关节炎的作用机制。

Objective： To observe the regulation effect of Bushen Huoxue Formula （BHF） on Wnt/β -catenin signaling pathway of human synoviocytes. Methods： Normal human synovial cells were treated by SB-216763, a selective inhibitor of GSK- 3 β. Then the nuclear proteins [3 -catenin was extracted and detected, and the timing of activation of Wnt/β -catenin signaling pathway in human synoviocytes was confirmed. Then the human synoviocytes were treated by serum containing medicine of BHF. Western Blot and ELISA were used to test the expression of β -catenin, MMP-7 and Cyclin D1. Results： The results of Western Blot showed that Wnt/β-catenin pathway was activated successfully by SB-216763. The expression of β-catenin, MMP-7 and Cyclin DI of SB-216763 treatment group was higher than the normal group respectively （P〈0.05）. After the treatment of serum containing medicine of BHF, the β -catenin, MMP-7 and Cyclin D1 were decreased significantly, compared with SB-216763 treatment group respectively （P〈0.05）. The result of ELISA showed that the expression of MMP-7 and Cyclin D1 of SB-216763 in the treatment group was increased when compared that in the normal group respectively （P〈0.05）. while after the treatment of serum containing medicine of BHF, which was decreased significantly where compared with the treatment group （P〈0.05）. Conclusion： BHF has the effect of regulating and controlling Wnt/β -catenin signaling pathway in human synoviocytes. The results of this experiment could explain the mechanism of treating knee osteoarthfitis with Bushen Huoxue Formula from a single pathway. The results of this experiment could explain the mecheanism of treating knee osteoarthritis with BHF from a single pathway.