Ang(1-7)对AngⅡ诱导的肝星状细胞Mas受体、TGF-β1/Smad3、CTGF的影响

Effects of Ang(1-7) on Mas receptor,TGF-β1/Smad3,CTGF induced by AngⅡin hepatic stellate cell

目的体外培养大鼠肝星状细胞(HSC-T6),观察血管紧张素(1-7)(Ang1-7)对血管紧张素Ⅱ(AngⅡ)诱导的肝星状细胞Mas受体、转化生长因子β1(TGF-β1)、Smad3、结缔组织生长因子(CTGF)表达情况的影响,进而探讨Ang(1-7)在肝纤维化进程中的作用及可能的作用机制,为临床治疗肝纤维化提供理论依据。方法体外培养大鼠肝星状细胞(HSC-T6),分为正常对照组、不同浓度AngⅡ处理组、AngⅡ+Ang(1-7)组、AngⅡ+Ang(1-7)+Mas受体拮抗剂A779组。培养24 h后,流式细胞仪(Annexin V/PI)双染法检测各组细胞凋亡率;同时采用Real-time PCR及Western Blotting检测肝星状细胞中Mas受体、TGF-β1、Smad3及CTGF mRNA及蛋白的表达。结果 (1)与正常对照组相比,AngⅡ处理组肝星状细胞凋亡活性增加,TGF-β1、Smad3、CTGF、Mas受体mRNA及蛋白表达量较正常对照组增加(P<0.05)。(2)与AngⅡ处理组相比,AngⅡ+Ang(1-7)组肝星状细胞的凋亡率增加,而TGF-β1、Smad3、CTGF mRNA表达降低,Mas受体表达增加(P<0.05)。(3)与AngⅡ+Ang(1-7)组相比,AngⅡ+Ang(1-7)+Mas受体拮抗剂A779组,肝星状细胞的凋亡率、TGF-β1、Smad3、CTGF mRNA表达降低(P<0.05),Mas受体表达增加(P<0.05)。结论 (1)AngⅡ可诱导大鼠肝星状细胞活化,促使TGF-β1、Smad3、CTGF表达增加。(2)Ang(1-7)可促使AngⅡ诱导的肝星状细胞凋亡而发挥抗肝纤维化作用。(3)与AngⅡ+Ang(1-7)组相比,AngⅡ+Ang(1-7)+Mas受体拮抗剂A779组,肝星状细胞凋亡率、Mas受体表达下降(P<0.05),TGF-β1、Smad3、CTGF mRNA表达增加(P<0.05)。

Objective To observe the effects of angiotensin（ Ang）（ 1-7） on the expressions of Mas receptor,transforming growth factor-β1（ TGF-β1）,Smad3,connective tissue growth factor（ CTGF） in the hepatic stellate cells HSC-T6 which induced by AngⅡ,and to discuss the role of Ang（ 1-7） and its possible mechanism in the process of liver fibrosis and then provide theoretical basis for clinical treatment of liver fibrosis. Methods Rat hepatic stellate cells（ HSC-T6） were divided into six groups： normal control group,three concentrations Ang Ⅱ treatment groups,Ang Ⅱ ＋ Ang（ 1-7） group,Ang Ⅱ ＋ Ang（ 1-7） ＋ Mas receptor antagonist A779 group. When cultered 24 hours,we used flow cytometry instrument（ Annexin V / PI staining） to test the apoptosis rates and at the same time we detected the expressions of Mas receptor,TGF-β1,Smad3,CTGF mRNA and protein by Real-time PCR and Western Blotting. Results（ 1） Compared with normal control group,the apoptosis activity increased,and the expressions of mRNA and protein of TGF-β1,Smad3,CTGF,Mas receptor increased in Ang Ⅱ treatment group（ P〈0. 05）.（ 2） Compared with Ang Ⅱ treatment group,the apoptosis rate increased,and the expressions of TGF-β1,Smad3,CTGF mRNA reduced（ P〈0. 05）,the expression of Mas receptor increased in AngⅡ ＋ Ang（ 1-7） group（ P〈0. 05）.（ 3） Compared with AngⅡ ＋ Ang（ 1-7） group,the apoptosis rate,TGF-β1,Smad3,CTGF mRNA expression reduced,Mas receptor expression increased in AngⅡ ＋ Ang（ 1-7） ＋ A779 group（ P〈0. 05）. Conclusion（ 1） AngⅡcan induce the activation of rat hepatic stellate cell and prompt the expression of TGF-β1,Smad3,CTGF.（ 2） Ang（ 1-7）can increase the apoptosis of hepatic stellate cells induced by AngⅡand play a role of anti-liver fibrosis.（ 3）Ang（ 1-7） exerts its antagonism role mainly through combination with Mas receptor,and its mechanism may be related to lower TGF-β1 / Smad3 signaling pathway.