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- 2017
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
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Abstract:
摘要：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-T6 12、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-1 mRNA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-1 mRNA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-1 mRNA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。
ABSTRACT: Objective To investigate the effects of all-trans retinioc acid (ATRA) on proliferation of rat hepatic stellate cells (HSC-T6) and expressions of collagen Ⅰ, 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-T6s 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-T6s stimulated by TGF-β1 (5ng/mL) were treated with different concentrations of ATRA for 24h. 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. Results The 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 Smad 2/3 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-T6s 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
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