全反式维甲酸对TGF-β1刺激的肝星状细胞COL1α2、MMP-2、TIMP-1以及信号通路的影响

Effects of all-trans retinioc acid on expressions of COL1α2,MMP-2,TIMP-1,and signaling pathway in TGF-β1-simulated rat hepatic stellate cells

目的观察全反式维甲酸对大鼠肝星状细胞(HSC-T6)增殖,以及经转化生长因子-β1(TGF-β1)刺激后Ⅰ型胶原α1链蛋白基因(COL1α2)、基质金属蛋白酶2(MMP-2)、金属蛋白酶组织抑制物1(TIMP-1)及Smad 2/3的表达变化,探讨其对肝纤维化的作用及其分子机制。方法不同浓度的全反式维甲酸(0.1、1、10μmol/L)分别作用HSC-T612、24、48h,采用噻唑蓝(MTT)比色实验检测HSC-T6增殖活性;另外,经TGF-β1(5ng/mL)刺激后的HSC-T6用不同浓度全反式维甲酸干预24h后,利用逆转录酶-聚合酶链反应(RT-PCR)技术测定COL1α2、MMP-2及TIMP-1mRNA的表达,细胞免疫化学染色法检测smad2/3蛋白的表达。结果全反式维甲酸能够抑制HSC-T6的增殖,而且具有浓度依赖性(P<0.05);受外源TGF-β1 5ng/mL刺激后,HSC-T6中COL1α2、MMP-2、TIMP-1 mRNA及Smad2/3蛋白表达较对照组明显增强(P<0.05),而全反式维甲酸干预能够有效降低HSC-T6受TGF-β1刺激后COL1α2、MMP-2、TIMP-1mRNA及Smad2/3蛋白的表达(P<0.05)。结论全反式维甲酸能够抑制HSC-T6的增殖,下调HSC-T6受TGF-β1刺激后COL1α2、MMP-2、TIMP-1mRNA的表达,并且可能通过抑制HSC-T6中TGF-β1的下游信号蛋白Smad 2/3的表达,影响TGF-β1/Smad信号通路,发挥其抗肝纤维化作用。

Objective To investigate the effects of all-trans retinioc acid（ATRA）on proliferation of rat hepatic stellate cells（HSC-T6）and expressions of collagenⅠ,matrix metalloproteinase-2（MMP-2）,tissue inhibitor of metalloproteinases-1（TIMP-1）and signal protein Smad2/3 in TGF-β1-simulated HSC-T6 so as to explore the impact and molecular mechanisms of ATRA on liver fibrosis in vitro.Methods Cultured HSC-T6 s were treated with different concentrations of ATRA（0.1,1,10μmol/L）for fixed time（12,24,48 hours）.After intervention time,cell proliferation was evaluated by MTT.Meanwhile,HSC-T6 s stimulated by TGF-β1（5 ng/mL）were treated with different concentrations of ATRA for 24 h.The mRNA expressions of COL1α2,MMP-2 and TIMP-1 were quantified by RT-PCR;the expression of Smad 2/3 protein was determined by cell immunochemistry.ResultsThe proliferation of hepatic stellate cells was inhibited by ATRA in a dose-dependent manner（P〈0.05）.After induced by TGF-β1,the mRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad2/3 protein were increased significantly compared with control group（P〈0.05）.However,ATRA could obviously reduce the mRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein in HSC-T6 induced by TGF-β1（P〈0.05）.Conclusion ATRA can inhibit the proliferation of HSC-T6 s and reduce the mRNA expressions of COL1α2,MMP-2 and TIMP-1 in HSC-T6 which were induced by TGF-β1.The anti-hepatic fibrosis function of ATRA may be related to its inhibition on the expression of Smad 2/3 protein in HSC-T6 to influence TGF-β1/Smad signaling pathway.