血管紧张素Ⅱ对肝星状细胞早期生长反应因子-1通路的调控作用

Angiotensin Ⅱstimulates platelet-derived growth factor-B expression in hepatic stellate cells by activating EGR-1

目的探讨血管紧张素Ⅱ(AngⅡ)对肝星状细胞(HSC)早期生长反应因子-1(EGR-1)信号转导通路的影响。方法采用HSC-T6细胞株,分别予AngⅡ1μmol/L处理10min、30min,Western blot检测磷酸化P42/44蛋白的表达。另外,观察AT-1受体阻滞剂-irbesartan、ERK1/2特异性阻断剂-U0126、抗氧化剂-乙酰半胱氨酸(NAC)(均预处理60min,再予AngⅡ刺激)和TNFα对磷酸化P42/44蛋白表达的影响。此外,予AngⅡ、irbesartan、U0126、NAC和ACEI处理后,电泳迁移率变更分析(EMSA)检测EGR-1DNA结合活性的变化;Westernblot检测血小板衍生生长因子-B(PDGF-B)蛋白的表达。免疫组化检测AngⅡ对HSCPDGF-B蛋白表达的影响。结果AngⅡ可诱导磷酸化P42/44的表达。U0126和irbesartan可抑制磷酸化P42/44的表达。EMSA结果显示:AngⅡ干预HSC0.5h后,EGR-1DNA结合活性开始增加,1h达到峰值,然后逐渐减低。U0126和irbesartan处理后可显著抑制AngⅡ诱导的EGR-1活性增强。较高浓度的ACEI可抑制EGR-1的活性,当ACEI浓度为0.1nmol/L时EGR-1的活性增强。NAC对EGR-1的活性无抑制作用。AngⅡ可诱导HSCPDGF-B蛋白表达,irbesartan可抑制AngⅡ诱导的PDGF-B表达。U0126、NAC和ACEI对PDGF-B表达无抑制作用。结论AngⅡ可经ERK1/2通路诱导HSCEGR-1活性增强。AngⅡ可经EGR-1通路调控PDGF-B的表达。

Objective To investigate the signal transduction mechanism underlying the effects of angiotensin Ⅱ （AngⅡ） on extracellular signal-regnlated kinase 1/2 （ERK1/2）, early growth response-1 （EGR-1） and platelet-derived growth factor-B （PDGF-B） in hepatic stellate cells （HSCs）. Methods HSC-T6 cells treated with AnglI for 10 or 30 min were examined for phospho-P42/44 protein expression using Western blotting. In another experiment, the cells were preincubated for 1 h in the presence of U0126 （an inhibitor of the MAPK/ERK kinase）, irbesartan （an AT-1 receptor blocker）, or antioxidant-N-acetylcysteine （NAC） prior to AnglI exposure, and the protein expression ofphospho-P42/44 and PDGF-B were measured with Western blotting. The DNA binding activity of EGR-1 was analyzedusing electrophoretic gel mobility shift assay （EMSA）, and the expression of PDGF-B was detected immunohistochemically. Results AngⅡ induced phospho-P42/44 expression in HSC-T6, which was abrogated by U0126 or irbesartan. NAC did not inhibit phospho-P42/44 expression. EMSA showed that AngⅡ exposure of the HSC cells markedly increased EGR-1 DNA binding activity, reaching the maximum after 60 min of exposure followed by progressive declination; irbesartan and U0126 significantly suppressed AngⅡ-induced EGR-1 activity enhancement. ACEI at 1 μmol/L and 10 nmol/L inhibited EGR-1 activity, but ACEI at the concentration of 0.1 nmol/L resulted in enhanced EGR-1 activity. NAC showed no obvious effect in suppressing EGR-1 activity. AnglI increased PDGF-B protein level in the HSCs, the effect of which was inhibited by irbesartan. U0126, NAC and ACEI did not attenuate PDGF-BB protein level in the HSCs. Conclusion Stimulation of the HSCs with AngⅡ results in EGR-1 activation via the ERK 1/2 pathway, leading to up-regulation of PDGF-B expression.