槲皮素通过ACE2-Ang-（1-7）-Mas轴对AngⅡ诱导下大鼠心肌收缩蛋白表达的影响

贾梦楠 朱明军 王永霞 李彬 郝轩轩 王新陆 于瑞 常鑫迪 李洁薇

中圖分类号 R96;R541 文献标志码 A 文章编号 1001-0408（2021）23-2839-07

DOI 10.6039/j.issn.1001-0408.2021.23.05

摘 要 目的：探讨槲皮素（Que）通过血管紧张素转换酶2-血管紧张素-（1-7）-Mas轴[ACE2-Ang-（1-7）-Mas轴]对血管紧张素Ⅱ（AngⅡ）诱导下原代大鼠心肌收缩蛋白表达的影响。方法：取1～2 d 日龄大鼠的心肌组织，分离、培养原代心肌细胞，并构建心肌细胞ACE2基因沉默模型。实验分为12组，其中AngⅡ组、AngⅡ+小干扰RNA（siRNA）组、AngⅡ+A779组为模型组，AngⅡ+氯沙坦组为阳性对照组，AngⅡ+Que40组、AngⅡ+Que80组、AngⅡ+siRNA+Que40组、AngⅡ+siRNA+Que80组、AngⅡ+A779+Que40组、AngⅡ+A779+Que80组为实验组，另设空白组、siRNA组。其中AngⅡ终浓度为1×10-6 mol/L，siRNA终浓度为50 nmol/L，Que终浓度分别为40、80 μmol/L，A779（Mas受体拮抗剂）终浓度为1 μmol/L，氯沙坦终浓度为1×10-4 mol/L。分别检测各组心肌细胞中ACE2、Ang-（1-7）、Mas的mRNA和蛋白表达水平，以及Na+/Ca2+交换通道（NCX）、钙泵（SERCA2a）、磷蛋白（PLB）等3种心肌收缩蛋白的表达水平。结果：与AngⅡ组比较，AngⅡ+Que80组细胞中的Mas mRNA表达水平升高（P<0.05）;AngⅡ+氯沙坦组细胞中的ACE2、Mas mRNA表达水平升高（P<0.05）。与AngⅡ组比较，AngⅡ+Que40组细胞中的ACE2、Ang-（1-7）蛋白表达水平升高（P<0.05）;与AngⅡ+siRNA组比较，仅AngⅡ+siRNA+Que40组Ang-（1-7）蛋白表达水平升高（P<0.05）;与AngⅡ+A779组比较，仅AngⅡ+A779+Que40组Ang-（1-7）蛋白表达水平升高（P<0.05）。与AngⅡ组比较，AngⅡ+Que40组NCX蛋白表达水平降低（P<0.05），AngⅡ+氯沙坦组NCX蛋白表达水平降低（P<0.05）;与AngⅡ+A779组比较，AngⅡ+A779+ Que80组NCX蛋白表达水平降低（P<0.05）。结论：Que一定程度上提高了AngⅡ诱导大鼠心肌细胞模型中ACE2-Ang-（1-7）-Mas轴的表达，从而调节心肌收缩蛋白。

关键词 槲皮素;血管紧张素转换酶2-血管紧张素（1-7）-Mas轴;心肌细胞;血管紧张素Ⅱ;心肌收缩蛋白

Effects of Quercetin on the Expression of Ang Ⅱ-induced Myocardial Contractile Protein of Rats through ACE2-Ang-（1-7）-Mas Axis

JIA Mengnan1，ZHU Mingjun2，WANG Yongxia2，LI Bin2，HAO Xuanxuan1，WANG Xinlu2，YU Rui2，CHANG Xindi1，LI Jiewei1（1. Henan University of Chinese Medicine， Zhengzhou 450046， China; 2. Heart Center， the First Affiliated Hospital of Henan University of Chinese Medicine， Zhengzhou 450099， China）

ABSTRACT   OBJECTIVE： To investigate the effects of quercetin （Que） on the expression of angiotensin Ⅱ （AngⅡ）-induced myocardial contractile proteins of primary rats through angiotensin-converting enzyme 2-angiotensin-（1-7）-Mas （ACE2-Ang-（1-7）-Mas） axis. METHODS： Cardiac tissue of rats aged 1-2 d were collected， and primary cardiomyocytes were isolated and cultured. The gene silencing model of cardiomyocytes ACE2 was constructed. Experiments were divided into 12 groups. Among them， AngⅡ group， AngⅡ+ small interference RNA （siRNA） group， and AngⅡ+ A779 group were the model groups; AngⅡ+losartan group was  positive control group; AngⅡ+Que40 group， AngⅡ+Que80 group， AngⅡ+siRNA+Que40 group， AngⅡ+siRNA+Que80 group， AngⅡ+A779+Que40 group and AngⅡ+A779+Que80 group were the experimental groups; blank group and siRNA group were set up. AngⅡ concentration was 1×10-6 mol/L; siRNA final concentration was 50 nmol/L; Que concentration was 40 and 80 μmol/L; A779 （Mas receptor inhibitor） concentration was 1 μmol/L; losartan concentration was 1×10-4 mol/L. mRNA and protein expression of ACE2， Ang-（1-7） and Mas in primary cardiomyocytes were detected; the expressions of myocardial contractile proteins were also determined， such as Na+/Ca2+ exchange channel （NCX）， calcium pump （SERCA2a）， phosphoprotein （PLB）. RESULTS： Compared with AngⅡ group， mRNA expression of Mas was increased significantly in AngⅡ+Que80 group （P<0.05）; mRNA expression of ACE2 and Mas were increased significantly in AngⅡ+ losartan group （P<0.05）. Compared with AngⅡ group， the protein expression of ACE2 and Ang-（1-7） were increased significantly in AngⅡ+Que40 group （P<0.05）; compared with AngⅡ+siRNA group， the protein expression of Ang-（1-7） were increased significantly in AngⅡ+ siRNA+Que40 group （P<0.05）; compared with AngⅡ+A779 group， the protein expression of Ang-（1-7） were increased significantly in AngⅡ+A779+ Que40 group （P<0.05）. Compared with AngⅡ group， the protein expression of NCX was decreased in AngⅡ+Que40 group （P<0.05）， protein expression of NCX was reduced in AngⅡ+losartan group （P<0.05）; compared with AngⅡ+A779 group， the protein expression of NCX was decreased in AngⅡ+A779+ Que80 group （P<0.05）. CONCLUSIONS： Que improves the expression of AngⅡ-induced ACE2-Ang-（1-7）-Mas axis in cardiomyocyte model to some extent， so as to regulate myocardial contractile protein.

KEYWORDS   Quercetin; ACE2-Ang-（1-7）-Mas axis; Cardiomyocytes; Angiotensin Ⅱ; Myocardial contractile protein

心力衰竭是多种心脏疾病的终末期阶段，其主要的病理特征是心肌收缩力下降和心功能降低[1]。肾素-血管紧张素（RAS）系统是广泛存在于心血管循环的体液调节系统，其过度激活是导致心力衰竭进展的关键环节。……