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目的:探讨肾气丸含药血清对转化生长因子β1(TGF-β1)诱导人肾小管上皮细胞株HK-2转分化的作用及其分子机制。方法:将细胞分为:无血清对照组,TGF-β1组(10ng/mL),空白血清组,肾气丸低、中、高剂量组。观察各组细胞形态学变化,ELISA法检测细胞上清液中Ⅰ型胶原(Col-Ⅰ)和纤维连接蛋白(FN)含量变化;荧光定量PCR法检测E-cadherin和Vimentin mRNA水平,蛋白免疫印迹法检测Smad2/3、ILK、GSK-3β等蛋白及其磷酸化水平。结果:TGF-β1刺激后,HK-2细胞由铺路石变成长梭形状,E-cadherin基因及蛋白水平下调(P<0.01),Vimentin基因及蛋白水平显著上升(P<0.01);给予肾气丸含药血清干预后,可抑制上述改变(P<0.01,P<0.05)。进一步研究发现,肾气丸能显著抑制p-Smad2/3、ILK、p-GSK-3β和β-catenin蛋白表达(P<0.05,P<0.01)。结论:肾气丸能显著抑制TGF-β1诱导HK-2细胞转分化过程,作用机制可能与其抑制TGF-β/Smads/ILK信号通路的激活有关。
Objective: To investigate the effect and its molecular mechanism of Shenqi pill containing serum on transdifferentiation of human renal tubular epithelial cell line HK-2 induced by transforming growth factor β1 (TGF-β1). Methods: The cells were divided into serum-free control group, TGF-β1 group (10ng / mL), blank serum group, and Shenqi Wan low, medium and high dose groups. The morphological changes of the cells in each group were observed. The contents of collagen Ⅰ (Col-Ⅰ) and fibronectin (FN) in the cell supernatants were detected by ELISA. The levels of E-cadherin and Vimentin mRNA were detected by quantitative real- Methods Smad2 / 3, ILK, GSK-3β and other proteins and phosphorylation levels. Results: The expression of E-cadherin gene and protein was down-regulated in HK-2 cells (P <0.01), while the expression of Vimentin gene and protein was significantly increased (P <0.01) Qi pills containing serum intervention, can inhibit the above changes (P <0.01, P <0.05). Further study found that Shenqi Pill can significantly inhibit the expression of p-Smad2 / 3, ILK, p-GSK-3β and β-catenin protein (P <0.05, P <0.01). Conclusion: Shenqi Pill can significantly inhibit the TGF-β1-induced transdifferentiation of HK-2 cells, and its mechanism may be related to the inhibition of TGF-β / Smads / ILK signaling pathway.