血管紧张素Ⅱ和醛固酮对肝星状细胞核因子κB信号转导通路的影响

Effects of angiotensinⅡand aldosterone on NF-κB binding activity in hepatic stellate cells

目的 探讨血管紧张素Ⅱ(AngⅡ)和醛固酮对肝星状细胞(HSC)核因子κB信号转导通路的影响。方法 体内研究部分: Wistar大鼠 60只,分为 4组,每组 15只鼠。模型组: 40% CCl4油0 .25ml/100mg皮下注射,每周 2次;培哚普利治疗组: 40% CCl4油 0 25ml/100mg皮下注射,每周2次;培哚普利 2mg/kg灌胃,每日 1次。氯沙坦治疗组: 40% CCl4油 0 .25ml/100mg皮下注射,每周2次;氯沙坦 10mg/kg灌胃,每日 1次。对照组:橄榄油皮下注射。于第 4、6周取材。Masson三色染色检测胶原,进行纤维化分级。电泳迁移率变更分析 (EMSA)检测肝组织核因子κB的DNA结合活性。Western印迹检测抑制性核因子κBα(IκBα)的表达。体外研究部分:采用HSC T6细胞株,分别予AngⅡ1μmol/L和醛固酮 1μmol/L处理 0 .5、1、2、4h,观察对核因子κBDNA结合活性的影响。另外,观察AT 1受体阻断剂irbesartan、细胞外信号调节激酶 (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(TNF-α)对核因子κBDNA结合活性的影响。Western印迹检测IκBα的表达;免疫组织化学检测核因子κB表达的核转移;逆转录 聚合酶链反应(RT PCR)检测TNF-αmRNA的表达。结果 培哚普利和氯沙坦对实验性肝纤维化具有抑制作用;

Objective To investigate the signal transduction mechanism underlying the effects of angiotensinⅡ (AngⅡ) and aldosterone (Aldo) on nuclear factor κB(NF κB) DNA binding activity. Methods Sixty male Wistar rats were randomly divided into 4 groups: model group (Mo group), injected with CCl 4 subcutaneously twice a week to establish a model of hepatic fibrosis; perindopril group (Pe group), injected with CCl 4 subcutaneously twice a week and perfused with perindopril once a day; losartan group (Lo group), injected with CCl 4 subcutaneously twice a week and perfused with losartan once a day; and control group (Nc group), injected with olive oil subcutaneously. The rats were killed in batches respectively 4 and 6 weeks after and their livers were collected to undergo Masson staining and be observed by light microscope. Electrophoretic gel mobility shift assay (EMSA) was used to detect the NF κB DNA binding activity in the liver tissues. Western blotting was used to detect the expression of IκBα in the plasma protein. Hepatic stellate cells (HSCs) T6 were cultured and preincubated for 1 h or not with U0126 (an inhibitor of MAPK/ERK kinase MEK),irbesartan(an AT 1 receptor blocker),and N acetylcysteine(NAC, an antioxidant), angiotensin converting enzyme inhibitor (ACEI), or tumor necrosis factorα(TNFα)prior to exposure to AngⅡ or Aldo for 0 5 h,1h,2h,and 4 h respectively. The binding activities of NF κB DNA were observed by EMSA. The expression of IκBα protein was detected with Western blotting. Histochemistry was used to detect the expression of NF κB p65. RT PCR was used to detect the expression of TNFα mRNA in HSC T6 cells. Results The binding activity to NF κB of the liver tissues was the strongest in the Mo group, followed by the Pe and Lo groups and Nc group. The IκBα expressions in liver tissues 4 and 6 weeks after the beginning of experiment in the Pe and Lo groups were significantly stronger than that in the Mo group (both P <0 05). 0 5 hour after the intervention of AngⅡ the DNA binding activity of the HSCs began to increase and peaked 1 hour later and then gradually decreased. The increase of NF κB activity induced by AngⅡ could be inhibited by irbesartan, ACEI and NAC pretreatment and could not be inhibited by U0126 pretreatment. Combined action of AngⅡ and TNFα significantly increased the NF κB DNA binding activity. The IκBα expression began to decrease 0.5 hour after the intervention of AngⅡ and reached the lowest value 2 hours after. The expression of IκBα protein was increased by ACEI ( P <0 05) , irbesartan and NAC (both P <0 01). EMSA showed that 0.5 hour after the intervention of Aldo the DNA binding activity began to be increased and peaked by 1 hour and then began to be decreased. NAC, but not U0126 partly inhibited the increased of NF κB activity induced by Aldo. Combined action of Aldo and TNFα significantly increased the NF κB activity. Aldo increased the expression of IκBα protein in the HSCs at different time points (all P <0 05). 0.5 hour after the AngⅡ intervention the IκBα protein expression began to decrease and reach the lowest value 1 hour later and then began to increase 2 hours later. the IκBα protein expression was significantly decreased in the NAC and NAC+ Aldo intervention groups (both P <0 05). There was no significant difference in IκBα protein expression between the Aldo intervention group and U0126+Aldo, TNFα , and Aldo+TNFα treatment groups (all P >0 05). Before stimulation, NF κB was expressed in the plasma of HSCs, however, after the stimulation of AngⅡ or Aldo for 1 hour it was expressed in the nuclei, and then transferred from the nuclei to the plasma 4 hours after the stimulation. However, little nuclear transfer was observed after pretreatment of NAC followed by AngⅡ or Aldo intervention. The TNFα mRNA expression was significantly increased in the AngⅡ and Aldo treatment groups in comparison with the control group(both P <0 05)The TNFα mRNA expression was significantl