Li Xu, Meng Ying, Huang Mao-Liang, Zhang Xiao-Lan, Zhang Zhen-Shu
Department of Emergency, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2008 Jun;28(6):963-7.
To investigate the signal transduction mechanism underlying the effects of angiotensin II (AngII) on extracellular signal-regulated kinase 1/2 (ERK1/2), early growth response-1 (EGR-1) and platelet-derived growth factor-B (PDGF-B) in hepatic stellate cells (HSCs).
HSC-T6 cells treated with AngII 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 AngII exposure, and the protein expression of phospho-P42/44 and PDGF-B were measured with Western blotting. The DNA binding activity of EGR-1 was analyzed using electrophoretic gel mobility shift assay (EMSA), and the expression of PDGF-B was detected immunohistochemically.
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 micromol/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. AngII 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.
Stimulation of the HSCs with AngII results in EGR-1 activation via the ERK1/2 pathway, leading to up-regulation of PDGF-B expression.
研究血管紧张素II（AngII）对肝星状细胞（HSCs）细胞外信号调节激酶1/2（ERK1/2）、早期生长反应因子-1（EGR-1）和血小板衍生生长因子-B（PDGF-B）作用的信号转导机制。
用AngII处理HSC-T6细胞10或30分钟后，采用蛋白质免疫印迹法检测磷酸化P42/44蛋白表达。在另一实验中，细胞在暴露于AngII之前，先在U0126（一种MAPK/ERK激酶抑制剂）、厄贝沙坦（一种AT-1受体阻滞剂）或抗氧化剂N-乙酰半胱氨酸（NAC）存在的情况下预孵育1小时，然后用蛋白质免疫印迹法检测磷酸化P42/44和PDGF-B的蛋白表达。采用电泳凝胶迁移率变动分析（EMSA）分析EGR-1的DNA结合活性，并用免疫组织化学法检测PDGF-B的表达。
AngII诱导HSC-T6中磷酸化P42/44表达，U0126或厄贝沙坦可消除该表达。NAC不抑制磷酸化P42/44表达。EMSA显示，HSC细胞暴露于AngII后，EGR-1的DNA结合活性显著增加，暴露60分钟后达到最大值，随后逐渐下降；厄贝沙坦和U0126显著抑制AngII诱导的EGR-1活性增强。1微摩尔/升和10纳摩尔/升的ACEI抑制EGR-1活性，但0.1纳摩尔/升浓度的ACEI导致EGR-1活性增强。NAC在抑制EGR-1活性方面无明显作用。AngII增加HSCs中PDGF-B蛋白水平，厄贝沙坦可抑制该作用。U0126、NAC和ACEI未减弱HSCs中PDGF-BB蛋白水平。
AngII刺激HSCs通过ERK1/2途径导致EGR-1激活，从而导致PDGF-B表达上调。
