20-羟二十烷四烯酸合成酶抑制剂HET0016与AngⅡ诱导心肌肥大的关系

Study on the role of 20-HETE mediated AngⅡ-induced hypertrophy in H9c2 cardiomyocytes

目的探究20-羟二十烷四烯酸(20-HETE)对血管紧张素Ⅱ(AngⅡ)诱导心肌细胞肥大的影响及机制。方法结晶紫染色检测细胞表面积;qRT-PCR检测心钠肽(ANP)和脑钠肽(BNP)表达;DHE和MitoSOX法检测超氧化物(ROS)生成;细胞色素C还原法检测NADPH氧化酶活性;JC-1探针检测线粒体膜电位;Western blot和ELISA法检测CYP4A表达及20-HETE生成。结果 AngⅡ显著增加H9c2心肌细胞表面积,上调ANP和BNP的mRNA表达。AngⅡ诱导NADPH氧化酶活性增高及ROS生成,导致线粒体膜电位下降,HET0016可阻断AngⅡ如上作用。AngⅡ可促进CYP4A表达以及20-HETE生成。结论 20-HETE激活NADPH氧化酶引起ROS生成,诱导心肌线粒体功能紊乱,参与AngⅡ诱导的心肌细胞肥大。

Objective To investigate the effects of 20 HETE on AngⅡinduced hypertrophy in H9c2 cardiomyocytes and to explore its underlying mechanisms.Methods Surface area of cardiomyocytes was measured after crystal violet staining.The mRNA expression of ANP and BNP was measured by using qRT PCR and the superoxide production by DHE and MitoSOX.NADPH oxidase activity was evaluated by cytochrome C reduction method.The fluorescent probe JC 1 was used to measure the mitochondrial membrane potential.Western blot and ELISA method were performed to evaluate the protein expression of CYP4A and the production of 20 HETE,respectively.Results Treatment of H9c2 cardiomyocytes with AngⅡsignificantly increased cell surface area and up regulated mRNA expression of ANP and BNP.Meanwhile,A ngⅡstimulated NADPH oxidase activity and increased production of reactive oxygen species(ROS)and mitochondrial superoxide,which reduced mitochondrial membrane potential.Effects of AngⅡmentioned above were significantly inhibited by co treatment with HET0016,an inhibitor of 20 HETE production.Finally,treatment of H9c2 cardiomyocytes with AngⅡsignificantly increased CYP4A expression and 20 HETE production.Conclusion 20 HETE mediates AngⅡinduced hypertrophy in H9c2 cardiomyocyte by stimulating NADPH oxidase dependent ROS generation,w hich results in mitochondrial dysfunction.