大黄■虫丸对TGF-β1诱导A549细胞Rho/ROCK信号通路表达的影响

Effects of Dahuang Zhechong Pills on the expression of Rho/ROCK signal pathway in A549 cells after TGF-β1 induction

目的:观察大黄■虫丸含药血清对转化生长因子-β1(TGF-β1)诱导后A549细胞RhoA/ROCK信号通路表达情况的影响,探讨其防治特发性肺纤维化(IPF)的作用机制。方法:正常培养并取对数生长期A549细胞,TGF-β1对其诱导48 h,电子显微镜下观察A549细胞的形态学变化;选择吡非尼酮作为阳性对照药,RT-PCR法检测TGF-β1、ROCK1、CollagenⅢ mRNA的表达;Western Blot法检测p-RhoA、ROCK1、α-SMA蛋白的表达。结果:成功获取发生上皮细胞-间充质转化(EMT)的A549细胞;与模型组比较,各给药组CollagenⅢ mRNA表达均显著降低(P<0.05,P<0.01);与模型组比较,除大黄■虫丸大剂量组ROCK1 mRNA下调不明显外,其他各实验组TGF-β1 mRNA、p-RhoA蛋白、ROCK1 mRNA及其蛋白的表达均出现不同程度下调(P<0.01)。结论:大黄■虫丸可通过不同程度下调TGF-β1介导RhoA/ROCK1信号通路相关的EMT程度,起到抗纤维化作用,且与其剂量密切相关。

Objective: To observe the effects of Dahuang Zhechong Pills-containing serum on the expression of RhoA/ROCK signaling pathway in A549 cells after transforming growth factor-β1(TGF-β1) induction, and to explore its prevention and treatment of idiopathic pulmonary fibrosis(IPF) mechanism of action. Methods: Normally grow and take A549 cells in logarithmic growth phase, TGF-β1 intervened for 48 h, observe the morphological changes of A549 cells under electron microscope;Choose pirfenidone as a positive control drug, RT-PCR method to detect the mRNA expression of TGF-β1, ROCK1 and CollagenⅢ;Western Blot was used to detect the protein expression of p-RhoA, ROCK1 and α-smooth muscle actin(α-SMA).Results: A549 cells with EMT were successfully obtained;Compared with the model group, the expression of CollagenⅢ mRNA in each administration group was significantly decreased(P<0.05, P<0.01);Compared with the model group, the expression of ROCK1 mRNA was not significantly down-regulated in the large-dose Dahuang Zhechong Pills group, but TGF-β1 mRNA,p-Rho A protein, ROCK1 mRNA and its protein in all other experimental groups were down-regulated to varying degrees(P<0.01).Conclusion: Dahuang Zhechong Pills can play an anti-fibrotic effect by down-regulating the EMT degree related to RhoA/ROCK1signaling pathway mediated by TGF-β1 to different degrees, and it is closely related to its dose.