摘要
目的探讨血管紧张素Ⅱ(AngⅡ)和醛固酮(Aldo)对肝星状细胞(HSC)细胞外调节蛋白激酶(ERK)和激活蛋白1(AP1)信号转导通路的影响。方法采用HSCT6细胞株,分别予AngⅡ1μmol/L和Aldo1μmol/L处理,免疫Western印迹检测磷酸化P42/44蛋白水平。另外,观察AngⅡ1型受体(AT1受体)阻断剂伊贝沙坦(irbesartan),细胞外调节蛋白激酶(ERK)特异性抑制剂U0126,抗氧化剂乙酰半胱氨酸(NAC)、血管紧张素转化酶抑制剂(ACEI)和肿瘤坏死因子α(TNFα)对磷酸化P42/44蛋白水平的影响。电泳迁移率变更分析(EMSA)检测AP1DNA结合活性的变化;反转录聚合酶链反应检测α1Ⅰ型前胶原基因的表达。结果AngⅡ和Aldo可诱导磷酸化P42/44蛋白水平的变化,与阴性对照组的比值达4.3±0.1、4.0±0.1,并呈时间依赖性,10min达到峰值后逐渐减低。U0126可抑制磷酸化P42/44蛋白水平。irbesartan可抑制AngⅡ诱导的磷酸化P42/44蛋白水平。AngⅡ和Aldo可增强HSC的AP1的DNA结合活性。irbesartan和ACEI可显著抑制AP1的活性;U0126和NAC可以显著抑制AngⅡ诱导的AP1的活化,部分抑制Aldo诱导的AP1的活化。AngⅡ和Aldo可诱导α1Ⅰ型前胶原mRNA的表达。irbesartan、U0126和NAC可显著抑制AngⅡ诱导的α1Ⅰ型前胶原mRNA表达增强;U0126和NAC可显著抑制Aldo诱导的α1Ⅰ型前胶原mRNA表达增强。结论AngⅡ和Aldo可经ERK通路诱导HSCAP1结合活性增强。AngⅡ和Aldo可经AP1通路调控α1Ⅰ型前胶原基因的表达。
Objective To investigate the signal transduction mechanism underlying the effects of angiotensinⅡ (AngⅡ) and aldosterone (Aldo) on the signal passageway of active protein-1 (AP-1).Methods In vitro, Hepatic stellate cells (HSCs) of the line HSC-T6 were cultured and treated with AngⅡ or Aldo, the principal effector molecules of the renin-angiotensin-aldosterone system (RAAS) for 10, 30, 60, 120, and 180 minutes respectively. The protein expression of phospho-P42/44 was detected by Western blotting. In addition, HSC-T6 cells were preincubated for 60 min with U0126, an inhibitor of MAPK/ERK kinase, irbesartan, an AT-1 receptor blocker, N-acetylcysteine (NAC), antioxidant, angiotensin converting enzyme inhibitor (ACEI), or tumor necrosis factor α (TNFα) prior to exposure to AngⅡ or Aldo. Then the protein expression of phospho-P42/44 was measured by Western blotting. The DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, the mRNA expression of α1 (Ⅰ) procollagen was detected. Results The levels of phopho-ERK1/2 protein increased after the treatment of AngⅡ and Aldo at all time points and both peaked 10 minutes after (both P<0.01). The levels of phopho-ERK1/2 protein of the irbesartan+ AngⅡ and U0126+ AngⅡ groups were significantly lower than that of the AngⅡ group (both P<0.01). The level of phopho-ERK1/2 protein of the AngⅡ group was lower than that of the TNFα group, however, was especially significantly lower than that of the AngⅡ+ TNFα group (P<0.01) . The level of phopho-ERK1/2 protein of the U0126+ Aldo group was significantly lower than that of the Aldo group (P<0.01). The phopho-ERK1/2 protein level of the NAC+ Aldo group was not significantly different from that of the Aldo group (P>0.05). The phopho-ERK1/2 protein level of the Aldo group was lower than that of the TNFα group, however, was especially significantly lower than that of the Aldo+ TNFα group (P<0.01) .The AP-1 DNA binding protein increased after the treatment
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第26期1831-1835,共5页
National Medical Journal of China
基金
国家自然科学基金项目(30270610)
