血管紧张素1-7对血管紧张素Ⅱ诱导的SD大鼠心室成纤维细胞的影响

目的 在血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)存在情况下,探讨血管紧张素1-7(angiotensin1-7,Ang1-7)对SD大鼠心室成纤维细胞的影响.方法 采用新生1～3d的SD乳鼠,用胰蛋白酶和Ⅱ型胶原酶消化心室,采用差速贴壁法获取成纤维细胞,将细胞完全随机化分组为对照组(不予处理)、AngⅡ组、Ang1-7组、AngⅡ+Ang1-7组(先用Ang1-7预处理30 min,再加入AngⅡ处理).采用免疫荧光鉴定细胞,CCK-8检测细胞增殖,Western blot检测细胞Rac1、Rad、gp91 phox(Nox2)、P65、结缔组织生长因子(connective tissue growth factor,CTGF)蛋白的表达,实时荧光定量PCR测定Ⅰ型、Ⅲ型胶原蛋白(Col Ⅰ、ColⅢ)和纤维连接蛋白(fibronectin) mRNA的表达.结果 AngⅡ能够促进SD大鼠心室成纤维细胞增殖,Ang1-7能减弱AngⅡ的促增殖作用(P＜0.05);与对照组比较,AngⅡ组Rac1、Nox2、P65、CTGF表达增加,Rad表达下降,Col Ⅰ、ColⅢ、fibronectin转录增加(P＜0.05);与AngⅡ组比较,AngⅡ+Ang1-7组Rac1、Nox2、P65、CTGF表达减少,Rad表达升高,Col Ⅰ、ColⅢ、fibronectin转录下降(P ＜0.05);Ang1-7组上述各项指标与对照组比较,差异无统计学意义(P＞0.05).结论 AngⅡ存在时,Ang1-7对心室成纤维细胞具有保护作用.Ang1-7通过调节Rac1、Rad及下游蛋白的表达,能够抑制成纤维细胞合成细胞外基质.

Effects of angiotensin1-7 on rat ventricular fibroblasts induced by angiotensin Ⅱ

Objective To investigate the effects of angiotensin1-7 (Ang1-7) on rat ventricular fibroblasts induced by angiotensin Ⅱ (Ang Ⅱ).Methods The ventricles from 1 ～3-day-old Sprague-Dawley rats were digested with trypsin and collagenase type 2.The isolated ventricular fibroblasts were randomly divided into 4 groups:control group,Ang Ⅱ group,Ang1-7 group,and Ang Ⅱ + Ang1-7 group.The control group,Ang Ⅱ group and Ang1-7 group were treated with serum free DMEN,Ang Ⅱ and Ang1-7,respectively.The Ang Ⅱ + Ang1-7 group was pre-incubated with Ang1-7 for 30 min and then treated with Ang Ⅱ.Fibroblasts were identified by immunofluorescence assay.CCK-8 assay was used to measure the proliferation of fibroblasts.Western blot assay was used to detect the expression of Rac1,Rad,gp91phox (Nox2),P65 and connective tissue growth factor (CTGF).Real-time PCR was performed to quantify the mRNA expression of collagen Ⅰ (Col Ⅰ),collagen Ⅲ (Col Ⅲ) and fibronectin.Results Ang Ⅱ could promote proliferation of fibroblasts which could be inhibited by Ang1-7 (P ＜ 0.05).Compared with the control group,the expression levels of Rac1,Nox2,P65 and CTGF were increased and that of Rad was decreased in the Ang Ⅱ group (P ＜ 0.05),while the mRNA levels of Col Ⅰ,Col Ⅲ and fibronectin were increased in the Ang Ⅱ group (P ＜ 0.05).Compared with the Ang Ⅱ group,the expression levels of Rac1,Nox2,P65 and CTGF were decreased and that of Rad was increased in the Ang Ⅱ + Ang1-7 group (P ＜0.05),while the mRNA levels of Col Ⅰ,Col Ⅲ and fibronectin were decreased in the Ang Ⅱ + Ang1-7 group (P ＜ 0.05).Conclusion With the existance of Ang Ⅱ,Ang1-7 plays a protective role in the ventricular fibroblasts.It inhibits the production of extracellular matrix through regulating Racl,Rad and downstream proteins.