Li Xu, Meng Ying, Yang Xi-shan, Wu Ping-sheng, Zhang Zhen-shu
Emergency Department, Nanfang Hospital, Nanfang Medical University, Guangzhou 510515, China.
Zhonghua Gan Zang Bing Za Zhi. 2005 Aug;13(8):567-70.
It is known that intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in liver fibrogenesis. 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 extracellular signal-regulated kinase 1/2 (ERK1/2), early growth response-1 (EGR-1) and on the platelet-derived growth factor-B (PDGF-B).
In vitro, hepatic stellate cell (HSC)-T6 cell line was treated with Aldo for 10 min, 0.5 h, 1 h, 2 h and 3 h. Protein expression of phospho-p42/44 was detected by Western blot. In addition, HSC-T6 were preincubated for 1 h or not at all with U0126 (an inhibitor of the MAPK/ERK kinase), and antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expressions of phospho-p42/44 and PDGF-B were measured by Western blot. DNA biding activity of EGR-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of immunohistochemistry, expression of PDGF-B was detected.
Aldo induced phospho-p42/44 expression could be abrogated by U0126; NAC did not inhibit phospho-p42/44 expression. Gel shift study showed that stimulation of HSC by Aldo markedly increased the EGR-1 DNA binding activity, which was abrogated by U0126, reaching a maximum at 60 minutes, and then declined progressively. NAC did not reduce the EGR-1 activity. Aldo increased the PDGF-B protein level in HSC, which was not attenuated by NAC and U0126.
Stimulation of HSC by Aldo results in activation of EGR-1 via ERK1/2 pathway, leading to up-regulation of PDGF-B expression.
已知肝内肾素 - 血管紧张素 - 醛固酮系统（RAAS）在肝纤维化形成中起关键作用。醛固酮（Aldo）作为RAAS的主要效应分子，对细胞生长和纤维化形成具有局部作用。然而，Aldo对肝纤维化形成作用的信号转导机制仍有待充分阐明。本研究旨在探讨Aldo对细胞外信号调节激酶1/2（ERK1/2）、早期生长反应因子-1（EGR-1）以及血小板衍生生长因子-B（PDGF-B）作用的信号转导机制。
在体外，将肝星状细胞（HSC）-T6细胞系分别用Aldo处理10分钟、0.5小时、1小时、2小时和3小时。通过蛋白质印迹法检测磷酸化p42/44的蛋白表达。此外，在暴露于Aldo指定时间之前，HSC-T6细胞分别预先用U0126（一种MAPK/ERK激酶抑制剂）或抗氧化剂N-乙酰半胱氨酸（NAC）孵育1小时或不进行孵育。通过蛋白质印迹法检测磷酸化p42/44和PDGF-B的蛋白表达。通过电泳凝胶迁移率变动分析（EMSA）分析EGR-1的DNA结合活性。通过免疫组织化学检测PDGF-B的表达。
U0126可消除Aldo诱导的磷酸化p42/44表达；NAC不抑制磷酸化p42/44表达。凝胶迁移研究表明，Aldo刺激HSC可显著增加EGR-1的DNA结合活性，U0126可消除这种活性，在60分钟时达到最大值，然后逐渐下降。NAC不降低EGR-1活性。Aldo增加了HSC中PDGF-B蛋白水平，NAC和U0126均未使其减弱。
Aldo刺激HSC通过ERK1/2途径导致EGR-1激活，从而导致PDGF-B表达上调。
