基于Smad通路研究软肝丸对TGF-β1诱导的肝星状细胞的影响

The Influence of Liver-Softening Pills on Hepatic Stellate Cells Induced by TGF-β1 Based on Smad Pathway Study

目的:探讨软肝丸对转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的肝星状细胞增殖、凋亡和Smad通路的影响。方法:将肝星状细胞分为空白组、模型组、软肝丸低浓度组(0.17μg·L^(-1))、软肝丸中浓度(0.34μg·L^(-1))组、软肝丸高浓度(0.68μg·L^(-1))组。除空白组外,其余各组细胞采用10μg·L^(-1)TGF-β1刺激肝星状细胞24h建立肝纤维化模型。培养后,CCK-8法检测细胞活力;流式细胞术和TUNEL法分析细胞凋亡;RT-PCR和Western Blot法检测细胞中Ⅰ型胶原(collagenⅠ,Col-Ⅰ)、Col-Ⅲ、结缔组织生长因子(connective tissue growth factor,CTGF)、纤连蛋白(fibronectin,FN)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达。Western Blot法分析Smad信号通路相关蛋白表达。结果:与模型组比较,软肝丸低浓度组、软肝丸中浓度组、软肝丸高浓度组细胞活力、Col-Ⅰ、Col-Ⅲ、CTGF、FN、α-SMA和p-Smad2/3表达明显降低,细胞凋亡率、Smad7蛋白表达明显升高(P<0.05)。结论:软肝丸含药血清可抑制TGF-β1诱导的肝星状细胞增殖、活化和纤维化,促进细胞凋亡,其机制可能与调控Smad信号通路有关。

Objective:To investigate the influence of Liver-Softening Pills on cell proliferation,cell apoptosis induced by transforming growth factor-β1(TGF-β1),and Smad pathway.Methods:The hepatic stellate cells were divided into the blank group,the model group and the low-dose Liver-Softening Pills group(0.17 μg·L-1),the medium-dose Liver-Softening Pills group(0.34 μg·L-1),and the high-dose Liver-Softening Pills group(0.68 μg·L-1).Except the blank group,the other groups′ cells were treated with 10 μg·L-1 TGF-β1 to establish hepatic fibrosis model by stimulating hepatic stellate cells for 24 hours.After culture,cell viability was detected by CCK-8 method;Cell apoptosis was analyzed by flow cytometry and TUNEL;RT-PCR and Western blot were used to detect the expressions of collagen Ⅰ(Col-Ⅰ),Col-Ⅲ,connective tissue growth factor(CTGF),fibronectin(FN) and collagen α-smooth muscle actin(α-SMA).The expression of Smad pathway-related proteins was analyzed by Western Blot.Results:Compared with the model group,the cell viability,Col-Ⅰ,Col-Ⅲ,CTGF,FN,α-SMA and p-Smad2/3 expressions of the low-dose Liver-Softening Pills group,the medium-dose Liver-Softening Pills group,and the high-dose Liver-Softening Pills group were significantly decreased,while the apoptosis rate and Smad7 protein expression were significantly increased(P<0.05).Conclusion:The serum containing Liver-Softening Pills can inhibit proliferation,activation and fibrosis of hepatic stellate cells induced by TGF-β1.The mechanism may be related to the regulation of Smad signaling pathway.