柴竭抑肝纤对大鼠肝星状细胞增殖和TGF-β1/Smad信号通路的影响

Effect of Chaijie Yiganxian on HSC-T6 Cell Proliferation and TGF-β1/Smad Signaling Pathway

目的：研究保肝护肝中药复方柴竭抑肝纤（CJYGX）通过肝星状细胞治疗肝纤维化的作用机制。方法：培养肝星状细胞T6（hepatic stellate cell T6,HSC-T6）,设空白组、秋水仙碱1.0 mg·L（-1）组和200,100,50,25,12.5,6.25 mg·L（-1）质量CJYGX组,作用24 h后通过细胞计数试剂（cell counting kit-8,CCK-8）检测HSC-T6细胞的增殖情况,筛选合适的CJYGX药物浓度;实时荧光定量聚合酶链式反应（Real-time PCR）检测CJYGX（200,100,50 mg·L（-1））作用HSC-T6细胞24 h后转化生长因子-β1（TGF-β1）,信号转导蛋白Smad2,Smad3,Smad4,Smad7 mRNA表达变化;蛋白免疫印迹法（Western blot）检测CJYGX（200,100,50 mg·L（-1））作用HSC-T6细胞24 h后TGF-β1,α-平滑肌肌动蛋白（α-SMA）表达变化。结果：（1）与空白组比较,200,100,50 mg·L（-1）CJYGX对HSC-T6细胞具有显著的抑制增殖作用（P〈0.05）。（2）200,100,50 mg·L（-1）CJYGX显著下调TGF-β1,Smad2,Smad3 mRNA的表达（P〈0.05）;200,100 mg·L（-1）CJYGX显著下调Smad4 mRNA表达,并显著上调Smad7 mRNA表达（P〈0.05）。（3）200,100 mg·L（-1）CJYGX显著下调HSC-T6细胞TGF-β1和α-SMA蛋白表达（P〈0.05）。结论：CJYGX能抑制HSC-T6细胞的增殖,其作用机制与TGF-β1/Smad通路有关。

Objective： To study the protective mechanism of Chaijie Yiganxian（ CJYGX） on the liver fibrosis through hepatic stellate cells（ HSC-T6） and transforming growth factor-β1/signal transduction protein（ TGF-β1/Smad） signaling pathway. Method： Different concentrations of colchicine（ 1. 0 mg·L（-1）） and CJYGX（ 200,100,50,25,12. 5,6. 25 mg·L（-1）） were added to the culture medium,and the proliferation of HSC-T6 was detected by cell counting kit-8（ CCK-8） after 24 hours. The appropriate drug concentration that can inhibit the cell proliferation was screened out according to the CCK-8 results. The mRNA expressions of TGF-β1,Smad2,Smad3,Smad4 and Smad7 in HSC-T6 cells treated with CJYGX（ 200,100,50 mg·L（-1）） were detected by Real-time PCR,and the protein expressions of TGF-β1 and α-SMA also were detected by Western blot. Result： Compared with the blank group,CJYGX（ 200,100,50 mg·L（-1）） had a significantly inhibitory effect on HSC-T6 cells（ P〈 0. 05）.CJYGX（ 200,100,50 mg·L（-1）） can significantly down-regulate the mRNA expressions of TGF-β1,Smad2 and Smad3（ P〈 0. 05）. Meanwhile,CJYGX（ 200,100 mg·L（-1）） can significantly down-regulate Smad4 mRNA expression and up-regulated Smad7 mRNA expression（ P〈 0. 05）. CJYGX（ 200,100 mg·L（-1）） can significantly down-regulate the protein expressions of TGF-β1 and α-SMA in HSC-T6 cells（ P〈 0. 05）. Conclusion： CJYGX can inhibit HSC-T6 cell proliferation,and its mechanism is related to TGF-β1/Smad signaling pathway.