坎离颗粒抑制血管紧张素Ⅱ诱导H9c2心肌细胞肥大的作用机制探究  

Effect and Mechanism of Kanli Granule on Inhibition of AngiotensinⅡInduced Hypertrophy of H9c2 Cardiomyocytes

作　　者：李悦 王肖龙[2] 姚成增[2] LI Yue;WANG Xiaolong;YAO Chengzeng(Beijing University of Chinese Medicine Third Affiliated Hospital,Beijing 100029,China)

机构地区：[1]北京中医药大学第三附属医院,北京100029 [2]上海中医药大学附属曙光医院,上海200021

出　　处：《中西医结合心脑血管病杂志》2022年第19期3560-3566,共7页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：上海市临床重点专科(中医心病科)项目(No.shslczdzk05301);上海市卫健委项目(No.202040220);国家中医药管理局蒋梅先全国名老中医药专家传承工作室建设项目,编号:国中医药人教函〔2022〕75号;蒋梅先上海市名老中医学术经验研究工作室建设项目(No.SHGZS-202249)。

摘　　要：目的探究坎离颗粒对血管紧张素Ⅱ(AngⅡ)诱导H9c2心肌细胞肥大的干预作用及机制研究。方法体外培养H9c2心肌细胞,采用AngⅡ(100 nmol/L)刺激心肌细胞24 h、48 h和72 h,建立心肌细胞肥大模型,利用细胞免疫荧光和激光扫描共聚焦显微镜观察心肌细胞大小,实时荧光定量PCR技术(qRT-PCR)与蛋白免疫印迹(Western Blot)检测心肌细胞中心房钠尿肽(ANP)、脑钠肽(BNP)、β-肌球蛋白重链(β-MHC)、基质金属蛋白酶1(MMP-1)、基质金属蛋白酶-9(MMP-9)以及组织金属蛋白酶组织抑制因子-1(TIMP-1)的表达水平,确定AngⅡ干预的最佳作用时间。随后将H9c2心肌细胞分别与不同浓度坎离颗粒共同处理,采用CellTiter-Glo发光法检测坎离颗粒对心肌细胞活力的影响,确定坎离颗粒的安全无毒剂量区间。实验分为空白组、AngⅡ组、坎离颗粒低剂量组、坎离颗粒中剂量组和坎离颗粒高剂量组。采用qRT-PCR和Western Blot检测心肌细胞中ANP、BNP、β-MHC、MMP-1、MMP-9以及TIMP-1的蛋白表达水平,以观察坎离颗粒对血管紧张素Ⅱ诱导H9c2细胞肥大的干预作用。结果AngⅡ处理H9c2细胞48 h后,心肌肥大程度最为明显,细胞形态与体积明显增大,且细胞内ANP、BNP、β-MHC、MMP-1、MMP-9以及TIMP-1等肥大基因的mRNA和蛋白表达水平明显增加。坎离颗粒能够抑制AngⅡ诱导的心肌肥大,qRT-PCR与Western Blot法检测结果显示坎离颗粒可明显抑制AngⅡ所诱导的ANP、BNP、β-MHC的mRNA上调;并抑制心肌细胞中MMP-1、MMP-9及TIMP-1的mRNA和蛋白表达水平。结论坎离颗粒能够明显下调心肌肥大多项指标的基因与蛋白表达水平,揭示坎离颗粒通过抑制肥大因子的上调来拮抗AngⅡ引起的心肌肥大,这可能是坎离颗粒发挥治疗作用的机制之一。Objective To explore the intervention effect and mechanism of Kanli granule on angiotensinⅡ(AngⅡ)-induced hypertrophy of H9c2 cardiomyocytes.Methods H9c2 cardiomyocytes were cultured in vitro and stimulated with AngⅡ(100 nmol/L)for 24 h,48 h,and 72 h to establish the model of cardiomyocyte hypertrophy.Cellular immunofluorescence and laser scanning confocal microscopy were used to observe the size of cardiomyocytes,and real-time fluorescence quantitative PCR(qRT-PCR)and Western Blot were used to detect the atrial natriuretic peptide of cardiomyocytes atrial natriuretic peptide(ANP),brain Natriuretic Peptide(BNP),β-myosin heavy chain(β-MHC),matrix metalloproteinases 1(matrix metalloproteinases).The expression levels of 1matrix metalloproteinases(MMP-1),MMP-9 and tissue inhibitor of metalloproteinases 1(TIMP-1)were determined to determine the optimal time of AngⅡintervention.Then,H9c2 cardiomyocytes were treated with different concentrations of Kanli granules,and the effect of Kanli granules on the viability of cardiomyocytes was detected by Celltiter-Glo luminescence method,and the safe non-toxic dose interval of Kanli granules was determined.The experiment was divided into blank group,AngⅡgroup,Kanli granule low dose group,Kanli granule medium dose group,and Kanli granule high dose group.qRT-PCR and Western Blot were used to detect the protein expression levels of ANP,BNP,β-MHC,MMP-1,MMP-9,and TIMP-1 in cardiomyocytes to observe the intervention effect of Kanli granules on angiotensinⅡ-induced hypertrophy of H9c2 cells.Results H9c2 cells treated with AngⅡfor 48 h showed the most obvious myocardial hypertrophy,the morphology and volume of H9c2 cells were significantly increased,and the mRNA and protein expression levels of ANP,BNP,β-MHC,MMP-1,MMP-9,and TIMP-1 were significantly increased.Kanli granules could inhibit the myocardial hypertrophy induced by AngⅡ.The results of qRT-PCR and Western Blot showed that Kanli granules could significantly inhibit the up-regulation of ANP,BNP,andβ-MHC mRNA

关 键 词：慢性心力衰竭 心肌肥大 坎离颗粒 H9C2心肌细胞 血管紧张素Ⅱ 

分 类 号：R285[医药卫生—中药学]