丹参酮Ⅱ A对人肾小管上皮细胞TGF-β_1/smad信号通路的干预作用

Effect of Tanshione Ⅱ A on TGF-β_1/smad Signaling Pathway in Human Renal Epithelial Cells

目的:探讨中药单体丹参酮ⅡA抗肾间质纤维化可能的作用机制。方法:将体外正常培养的人肾小管上皮细胞(HK-2)随机分为空白对照组、转化生长因子(TGF-β1)组(TGF-β1,10 ng/ml)、丹参酮ⅡA低剂量组(丹参酮ⅡA 1μg/ml+TGF-β110 ng/ml)、丹参酮ⅡA中剂量组(丹参酮ⅡA 5μg/ml+TGF-β110 ng/ml)、丹参酮ⅡA高剂量组(丹参酮ⅡA 10μg/ml+TGF-β110 ng/ml),分别予相应的药物干预后吸取细胞上清液,用ELISA法检测Ⅲ型胶原的含量,细胞裂解后采用Western-blot法检测smad2和smad3的蛋白表达量。结果:与空白对照组比较,TGF-β1组的Ⅲ型胶原表达明显上升,加入丹参酮ⅡA后各剂量组的Ⅲ型胶原表达均下降,且呈剂量效应关系;TGF-β1组能促进smad2、smad3蛋白的表达,加入丹参酮ⅡA后各剂量组的smad2蛋白、smad3蛋白的表达量均低于TGF-β1组,但是无明显的剂量效应关系。结论:丹参酮ⅡA可以抑制TGF-β1诱导的Ⅲ型胶原的分泌,且可抑制由TGF-β1介导的smad2、smad3信号蛋白的表达,表明丹参酮ⅡA可能是通过抑制TGF-β1/smad信号通路中smad2、smad3蛋白的表达发挥其抗肾间质纤维化的作用。

Objective： To explore the mechanism of the effect of Tanshione ⅡA on renal interstitial fibrosis. Methods：Human renal tubular epithelial cells（ HK-2） cultivated in vitro were randomly divided into blank control group,TGF-β1group（ TGF-β1,10 ng/ml）,low dose Tanshione ⅡA group（ Tanshione ⅡA 1 μg/ml ＋ TGF-β110 ng /ml）,medium dose Tanshione ⅡA group（ Tanshione ⅡA 5 μg /ml ＋ TGF-β110ng /ml） and high dose Tanshione ⅡA group（ Tanshione ⅡA10 μg /ml ＋ TGF-β110 ng /ml）. Each group was intervened by corresponding measures and the cell supernatant was sucked up. Content of collagen Ⅲ was detected by ELISA and protein expressions of smad2 and smad3 were detected after cell lysis by Western blotting. Results： Compared with the blank control group,the expression of collagen Ⅲ in the TGF-β1group was increased significantly. After added with the Tanshione ⅡA,the expression of collagen Ⅲ in three Tanshione ⅡA groups were decreased,with dose-effect relationship; expressions of smad2 and smad3 protein in the TGF-β1group was significantly increased. Expressions of smad2 and smad3 protein in the Tanshione ⅡA groups were lower than those of the TGF-β1group,but without dose-effect relationship. Conclusion： Tanshione ⅡA can inhibit the secretion of collagen Ⅲ induced by TGF-β1and inhibit the expressions of smad2 and smad3 protein mediated by TGF-β1. It suggests that Tanshione ⅡA maybe act on renal interstitial fibrosis by inhibiting the expressions of smad2 and smad3 protein on TGF-β1/smad signaling pathway.