苓桂术甘汤调节Sig1R抑制AngⅡ诱导的心肌细胞肥大的作用及机制  

Effect and mechanism of Linggui Zhugan Decoction in regulating Sig1R on AngⅡ-induced cardiomyocyte hypertrophy

作　　者：王翔[1] 莫佳佳 汤同娟 丁芮 黄金玲[1] WANG Xiang;MO Jia-jia;TANG Tong-juan;DING Rui;HUANG Jin-ling(Anhui University of Chinese Medicine,Hefei 230012,China;Hefei Institute of Pharmaceutical Industry Co.,Ltd.,Hefei 230601,China;Anhui Province Engineering Technology Research Center of Pharmaceutical Re-innovation,Hefei 230088,China)

机构地区：[1]安徽中医药大学,安徽合肥230012 [2]合肥医工医药股份有限公司,安徽合肥230601 [3]安徽省药物再创新工程技术研究中心,安徽合肥230088

出　　处：《中国中药杂志》2024年第3期754-762,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81973844);安徽高校自然科学研究重点项目(KJ2020A0393);安徽中医药大学青年科技英才培育项目(2021qnyc10);安徽中医药大学高层次人才支持计划项目(2022rcyb021)。

摘　　要：探讨苓桂术甘汤通过调节sigma-1受体分子(sigma-1 receptor,Sig1R)抑制血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的心肌细胞肥大的作用机制。制备苓桂术甘汤含药血清与空白血清,构建体外AngⅡ诱导H9c2心肌细胞肥大模型,将H9c2细胞分为正常组、AngⅡ模型组、20%空白血清组(20%normal rat serum,20%NSC)、20%苓桂术甘汤含药血清组。按分组将细胞用AngⅡ(1μmol·L^(-1))或AngⅡ与血清共孵育72 h后,鬼笔环肽染色检测心肌细胞表面积,微量法检测细胞Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶活性,流式细胞术检测线粒体Ca^(2+)水平,Western blot法检测细胞中心房钠尿肽(atrial natriuretic peptide,ANP)、脑钠肽(brain natriuretic peptide,BNP)、Sig1R、肌醇1,4,5-三磷酸受体2型(inositol 1,4,5-triphosphate receptor type 2,IP3R2)蛋白的表达。通过转染特异性siRNA下调Sig1R表达,观察Sig1R下调后苓桂术甘汤含药血清对AngⅡ刺激H9c2细胞后心肌细胞表面积、Na^(+)-K^(+)-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活性、线粒体Ca^(2+)、蛋白(ANP、BNP、IP3R2)表达的影响。结果显示,与正常组相比,AngⅡ组心肌细胞表面积显著增加,ANP、BNP表达显著升高(P<0.01),Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶活性显著降低(P<0.01),线粒体Ca^(2+)浓度显著降低(P<0.01),Sig1R蛋白表达显著降低、IP3R2蛋白表达显著升高(P<0.01);苓桂术甘汤含药血清干预可以显著降低心肌细胞表面积及ANP、BNP表达(P<0.05,P<0.01),升高Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶活性和线粒体Ca^(2+)浓度(P<0.01),升高Sig1R的表达(P<0.05),降低IP3R2表达(P<0.05)。而Sig1R下调后,苓桂术甘汤含药血清上述作用被逆转(P<0.01)。以上结果表明,苓桂术甘汤含药血清可以抑制AngⅡ诱导的心肌细胞肥大,且其药效作用的发挥与调节Sig1R、促进线粒体Ca^(2+)内流、恢复ATP合成、保护线粒体功能有关。This study aims to explore the mechanism of Linggui Zhugan Decoction(LGZGD)in inhibiting AngiotensinⅡ(AngⅡ)-induced cardiomyocyte hypertrophy by regulating sigma-1 receptor(Sig1R).The model of H9c2 cardiomyocyte hypertrophy induced by AngⅡin vitro was established by preparing LGZGD-containing serum and blank serum.H9c2 cells were divided into normal group,AngⅡmodel group,20%normal rat serum group(20%NSC),and 20%LGZGD-containing serum group.After the cells were incubated with AngⅡ(1μmol·L^(-1))or AngⅡwith serum for 72 h,the surface area of cardiomyocytes was detected by phalloidine staining,and the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase were detected by micromethod.The mitochondrial Ca^(2+)levels were detected by flow cytometry,and the expression levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),Sig1R,and inositol 1,4,5-triphosphate receptor type 2(IP3R2)were detected by Western blot.The expression of Sig1R was down-regulated by transfecting specific siRNA for investigating the efficacy of LGZGD-containing serum on cardiomyocyte surface area,Na^(+)-K^(+)-ATPase activity,Ca^(2+)-Mg^(2+)-ATPase activity,mitochondrial Ca^(2+),as well as ANP,BNP,and IP3R2 protein expressions.The results showed that compared with the normal group,AngⅡcould significantly increase the surface area of cardiomyocytes and the expression of ANP and BNP(P<0.01),and it could decrease the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase,the concentration of mitochondrial Ca^(2+),and the expression of Sig1R(P<0.01).In addition,IP3R2 protein expression was significantly increased(P<0.01).LGZGD-containing serum could significantly decrease the surface area of cardiomyocytes and the expression of ANP and BNP(P<0.05,P<0.01),and it could increase the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase,the concentration of mitochondrial Ca^(2+)(P<0.01),and the expression of Sig1R(P<0.05).In addition,IP3R2 protein expression was significantly decreased(P<0.05).However,aft

关 键 词：苓桂术甘汤 心肌细胞肥大 线粒体相关内质网膜 Sigma-1受体分子(Sig1R) 

分 类 号：R285.5[医药卫生—中药学]