血管紧张素Ⅱ对心脏成纤维细胞Ets-1表达的影响及机制研究

Regulation of angiotensin Ⅱ in Ets-1 expression and its mechanism in cardiac fibroblasts

目的研究血管紧张素Ⅱ(Ang Ⅱ)对大鼠心脏成纤维细胞(CFs)转录因子Ets-1及其下游促纤维化因子表达和细胞增殖的调节及相关的分子机制。方法将原代培养的大鼠CFs分为对照组,Ang Ⅱ处理不同时间组以及不同剂量组,采用实时定量RT-PCR及western blotting实验技术检测Ang Ⅱ对Ets-1mRNA及蛋白表达的影响。用Ang Ⅱ受体拮抗剂、MAPKs、PKC及PTK抑制剂预处理CFs,测定其对Ang Ⅱ诱导的Ets-1、结缔组织生长因子(CTGF)、纤溶酶原激活物抑制因子-1(PAI-1)表达及细胞增殖的影响。结果在CFs中,Ang Ⅱ可呈时间及浓度依赖性诱导Ets-1表达(P<0.05),对其mRNA稳定性则无显著影响。血管紧张素Ⅱ1型受体(AT1R)拮抗剂losartan、ERK抑制剂PD98059、JNK抑制剂SP600125及PKC抑制剂BIM预处理可显著抑制Ang Ⅱ诱导Ets-1过表达(P<0.05),下调CTGF及PAI-1蛋白表达,抑制Ang Ⅱ诱导的CFs增殖(P<0.05)。结论 Ang Ⅱ通过AT1R及PKC,ERK、JNK信号通路介导诱导CFs Ets-1基因的表达。而且,转录因子Ets-1可能是心肌纤维化过程的一个重要介导因素,其发挥作用的主要途径可能是通过参与调控CFs增殖及促纤维化因子CTGF及PAI-1的表达。

AIM To investigate the role of Ets-1 in profibrotic actions of angiotensin II （AngII） in cardiac fibroblasts （CFs）. METHODS Primary cultured CFs were divided into the following groups： control, AngII treated for different time and different dose. Expression levels of Ets-1 mRNA and protein were examined using real-time RT-PCR and Western blotting. CFs were pretreated with AngII receptor blocker, inhibitors of MAPKs, PKC, and PTK. AngII-induced cell proliferation and expression of Ets-1. Connective tissue growth factor （CTGF） and plasminogen activator inhibitor-1 （PAI-1） were examined using MTT, real-time RT-PCR and Western blotting. RESULTS In growth-arrested CFs, AnglI induced Ets-1 expression in a time- and concentration-dependent manner （ P 〈 0.05 ）. Pretreatment with Ang II type 1 receptor （ATIR） blocker losartan, PKC inhibitor BIM, ERK inhibitor PD98059, or JNK inhibitor SP600125 partly inhibited this induction （P 〈 0.05 ） accompanied by impaired cell proliferation and production of PAI-1 and CTGF protein （P 〈0. 05）, the two downstream targets of Ets-1. CONCLUSION AnglI induces Ets-1 expression through AT1R and PKC, ERK and JNK signaling pathways. Ets-1 may be an important mediator in cardiac fibrosis process by regulating CF proliferation and expression of CTGF and PAI-1.