醛固酮对肝星状细胞早期生长反应因子-1信号通路调控的体外研究

Aldosterone stimulating PDGF-B expression in HSC via activation of EGR-1

目的探讨醛固酮(Aldo)对肝星状细胞(HSC)早期生长反应因子-1(EGR-1)信号传导通路的影响。方法采用HSC-T6细胞株,分别给予Aldo 1μmol/L处理10 min、30 min、1h、2h和3h, western blot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)1/2特异性阻断剂U0126、抗氧化剂N-乙酰半胱氨酸(NAC)(均预先处理60 min,再给予Aldo刺激)和肿瘤坏死因子α对磷酸化p42/44蛋白表达的影响。此外,给予Aldo、U0126和NAC处理后,用电泳迁移率分析(EMSA)检测EGR-1 DNA结合活性的变化;western blot检测血小板衍生生长因子-B(PDGF—B)蛋白的表达。免疫细胞化学检测Aldo对HSC PDGF-B蛋白表达的影响。结果Aldo可诱导磷酸化p42/44的表达,U0126可抑制磷酸化p42/44的表达。EMSA结果显示:Aldo干预HSC 30 min后EGR-1 DNA结合活性开始增加, 1h达到峰值,然后逐渐减低;U0126可显著抑制Aldo诱导的EGR-1活性增强;NAC对Aldo诱导的EGR- 1活性无抑制作用。Aldo可诱导HSC PDGF—B蛋白表达;U0126和NAC对PDGF—B表达无抑制作用。结论Aldo可经ERK1/2通路诱导HSC EGR-1活性增强。Aldo可经EGR-1通路调控PDGF-B的表达。

Objective 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）. Methods In vitro, hepatic stellate cell （HSC）-T6 cell line was treated with Aldo for 10min, 0.5h, 1h, 2h and 3h. Protein expression of phospho-p42/44 was detected by Western blot. In addition,HSC-T6 were preincubated for 1h 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. Results 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. Conclusions Stimulation of HSC by Aldo results in activation of EGR-1 via ERK1/2 pathway, leading to up-regulation of PDGF-B expression.