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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。
Objective To investigate the regulation of angiotensin Ⅱ (Ang Ⅱ) on rat cardiac fibroblast (CFs) transcription factor Ets-1 and its downstream pro-fibrotic factor expression and cell proliferation and related molecular mechanisms. Methods Primary cultured rat CFs were divided into control group, Ang Ⅱ treatment group and different dose groups. The effects of Ang Ⅱ on Ets-1 mRNA and protein expression were detected by real-time quantitative RT-PCR and western blotting. CFs were pretreated with angiotensin Ⅱ receptor antagonist, MAPKs, PKC and PTK inhibitors, and their effects on Ang Ⅱ-induced Ets-1, connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 -1) expression and cell proliferation. Results In CFs, Ang Ⅱ induced Ets-1 expression in a time and concentration-dependent manner (P <0.05), but not mRNA stability. Angiotensin Ⅱ type 1 receptor (AT1R) antagonist losartan, ERK inhibitor PD98059, JNK inhibitor SP600125 and PKC inhibitor BIM pretreatment significantly inhibited Ang Ⅱ-induced Ets-1 overexpression (P <0.05), down-regulated CTGF and PAI-1 protein expression, inhibited Ang Ⅱ-induced CFs proliferation (P <0.05). Conclusion Ang Ⅱ can induce the expression of Ets-1 gene in CFs through AT1R, PKC, ERK and JNK signaling pathways. Moreover, the transcription factor Ets-1 may be an important mediator of myocardial fibrosis, and its main pathway may play an important role through the regulation of CFs proliferation and the expression of fibronectin (CTGF) and PAI-1.