醛固酮对肝星状细胞激活蛋白- 1通路调控的体外研究

目的 探讨醛固酮（Aldo）对肝星状细胞（HSC）激活蛋白-1（AP—1）信号转导通路的影响。方法 采用HSCT6细胞株，分别予Aldo 1μmol／L处理10、30、60、120、180min，蛋白质印迹法检测磷酸化P42／44蛋白的表达。另外，观察细胞外信号调节激酶（ERK）特异性抑制剂-U0126，抗氧化剂-乙酰半胱氨酸（NAC）（均先预处理60min，再予Aldo刺激）和肿瘤坏死因子α（TNFα）对磷酸化P42／44蛋白表达的影响。Aldo在干预30、60、120、240min后，电泳迁移率变更分析（EMSA）检测AP-1 DNA结合活性的变化；予U0126和NAC干预后，EMSA检测AP-1 DNA结合活性；逆转录聚合酶链反应检测α1-1型前胶原基因的表达。结果 Aldo可诱导磷酸化P42／44的表达，并呈时间依赖性，10min达到峰值，之后逐渐减低。U0126可抑制磷酸化P42／44的表达。Aldo可增强HSC的AP-1的DNA结合活性。U0126可以显著抑制Aldo诱导的AP—1的活化，NAC部分抑制Aldo诱导的AP-1的活化。Aldo可诱导α1-1型前胶原mRNA的表达。U0126和NAC可显著抑制Aldo诱导的α1-1型前胶原mRNA表达增强。结论 Aldo可经ERK通路诱导HSC AP-1结合活性增强。Aldo可经AP-1通路调控α1-1型前胶原基因的表达。

Aldosterone stimulates α 1-(1) procollagen mRNA expression in HSC via activation of ERK1/2 and AP-1

OBJECTIVE: It has been known that the intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis in livers. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on the signal passageway of active protein-1 (AP-1). METHODS: In vitro, HSCs-T6 cell line was treated with Aldo for 10 min, 30 min, 60 min, 120 min and 180 min, and protein expression of Phospho-P42/44 was detected by Western blot. In addition, HSCs-T6 cell line was preincubated for 60 min or not with U0126 (an inhibitor of the MAPK/ERK kinase), and also with antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expression of Phospho-P42/44 was measured by Western blot. DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, expression of alpha1(1) procollagen mRNA was detected. RESULTS: Aldo stimulated HSC via extracellular signal-regulated kinase (ERK1/2) pathway. Time course experiments showed that Aldo induced Phospho-P42/44 expression, which was abrogated by U0126, reaching a maximum at 10 minutes, and then declined progressively. NAC inhibited the Phospho-P42/44 expression. EMSA showed that stimulation of HSC by Aldo markedly increased AP-1 DNA binding activity. U0126 markedly reduced AP-1 DNA binding activity induced by Aldo; NAC partly decreased AP-1 activity induced by Aldo. Aldo up-regulated expression of alpha1(1) procollagen mRNA, which was attenuated by U0126 and NAC. CONCLUSION: Stimulation of HSC by Aldo results in activation of AP-1 via ERK1/2 pathway, leading to up-regulation of AP-1 target gene alpha1(1) procollagen mRNA expression.