鬼针草总黄酮对肝纤维化大鼠肝星状细胞TGF-β1信号传导通路的影响

Effect of TFB on TGF-β1 signaling pathway in HSC of liver fibrosis rats

目的研究外源性转移生长因子-β1(transforming growth factor betal,TGF-β1)对肝星状细胞(hepatic stellate cells,HSC)的激活作用,观察鬼针草总黄酮(total flavonoids of Bidens Bipinnata L,TFB)对HSC中smad2/7和Ⅰ型胶原mRNA和蛋白表达的影响,以探讨TFB抗肝纤维化的作用及分子机制。方法采用Collagenase Ⅳ胶原酶原位肝灌注法分离HSC,MTT法观察TFB对TGF-β1刺激下HSC增殖的作用,ELISA法检测HSC自分泌TGF-β1和产生Ⅰ型胶原的影响;RT-PCR法观察分析TFB对TGF-β1刺激下HSC中smad2/7和Ⅰ型胶原基因表达的影响;Westernblot法观察TFB对TGF-β1刺激下HSC中smad2蛋白表达的影响。结果TGF-β1明显促进HSC增殖、自分泌TGF-β1和产生胶原,促进HSCsmad2、Ⅰ型胶原mRNA及smad2蛋白的表达,明显抑制smad7mRNA表达(P<0.05),TFB作用后,TGF-β1刺激的HSC中smads2mRNA、蛋白和Ⅰ型胶原mRNA表达受到抑制,smad7mRNA表达明显上调。结论TFB抗肝纤维化作用可能与其阻断HSCTGF-β1通路进而阻断HSC的活化、增殖有关。

Aim To investigate the proliferation of HSCs stimulated by exogenous TGF-β1 （transforming growth factor betal）,observe the effect of TFB（total flavonoids of Bidens Bipinnata L.）on smad2/7,typeⅠcollagen mRNA and protein expression of HSCs and study the protective effect and molecular mechanism of TFB on hepatic fibrosis.Methods HSCs were isolated with collagenase Ⅳ perfusion in situ and density gradient centrifugation.The effect of TFB on cell proliferation was observed by MTT colormetric assay.The auto-secretion of TGF-β1 and synthesis of type Ⅰ collagen were measured by enzyme-linked immuneadsordent assay （ELISA）.Moreover,the expression of smad2/7,typeⅠcollagen mRNA and protein was measured by semi-quantitative RT-PCR and Western blot methods respectively.Results TFB could markedly inhibit the proliferation of HSCs of liver fibrosis rats stimulated by TGF-β1 and production of TGF-β1 and type Ⅰ collagen.In addition,TFB treatment could significantly down-regulate smad2 and type Ⅰ collagen mRNA expression and up-regulated smad7 mRNA expression of HSCs Smad2 protein expression of HSCs stimulated by TGF-β1 was also down-regulated by TFB.Conclusion TFB has the protective effect against hepatic fibrosis by inhibiting the activation of TGF-β1 signaling pathway and suppressing the HSC proliferation.