沙库巴曲缬沙坦对缺氧心肌细胞线粒体动力系统和细胞凋亡的影响

Effects of sacubitril valsartan on mitochondrial dynamics and cell apoptosis in hypoxic cardiomyocytes

目的验证沙库巴曲缬沙坦(S/V)能否通过调节线粒体动力系统改善缺氧大鼠胚胎心肌细胞(H9c2)凋亡水平,发挥心脏保护作用。方法培养H9c2心肌细胞,建立糖氧剥夺模型(OGD),将细胞分为对照组、造模组、药物组。对照组正常培养心肌细胞,造模组采用OGD建模,药物组采用OGD建模后加用S/V 20μmol/L干预处理,每组重复5遍。采用流式细胞术检测细胞凋亡及活性氧(ROS),JC-1检测线粒体膜电位,蛋白质免疫印迹法(WB)检测线粒体融合蛋白1(Mfn1)、线粒体融合蛋白2(Mfn2)、动力相关蛋白1(Drp1)、线粒体分裂蛋白1(Fis1)、细胞色素C(CytC)、B细胞淋巴瘤2(Bcl-2)、Bcl-2关联X蛋白(Bax)及含半胱氨酸天冬氨酸蛋白水解酶3(Caspase-3)表达情况。采用GraphPad Prism 8统计软件进行数据分析,多组间比较采用单因素方差分析,两两比较采用LSD-t检验。结果H9c2心肌细胞建立OGD模型,经S/V处理后,光镜下心肌细胞形态学明显改善;流式细胞技术分析结果显示S/V明显降低细胞内ROS水平,抑制心肌细胞凋亡(P<0.05);荧光显微镜分析结果显示S/V明显改善线粒体膜电位水平(P<0.05);WB结果显示S/V可明显提升Mfn2、Mfn1、Bcl2蛋白表达水平,降低Drp1、Fis1、CytC、Bax及Caspase-3蛋白表达水平(P<0.05)。结论S/V可能通过促进线粒体融合、抑制线粒体分裂调节线粒体稳态,减少ROS生成,减轻心肌细胞凋亡。

Objective To verify whether sacubitril valsartan(S/V)can ameliorate the apoptosis level of hypoxic H9c2 cardiomyocytes by regulating mitochondrial dynamic system,and exert cardioprotective effects.Methods H9c2 cardiomyocytes were cultured and established a glucose oxygen deprivation model(OGD).The cells were divided into three groups:control group(CON),model group(OGD),S/V group(S/V).Apoptosis and reactive oxygen species(ROS)were detected by flow cytometry,mitochondrial membrane potential was detected by JC-1,and mitochondrial fusion protein 1(Mfn1),mitochondrial fusion protein 2(Mfn2),dynamin related protein 1(Drp1),mitochondrial fission protein 1(FIS1),cytochrome c(CytC),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax)and cysteinyl aspartate specific proteinase(Caspase-3)expression were detected by Western blotting(WB).GraphPad Prism 8 statistics was used for data analysis,multiple groups comparisons were conducted using one-way analysis of variance,and LSD-t test was performed for pairwise multiple comparisons.Results H9c2 cardiomyocytes were used to establish OGD cell model,and the morphology of cardiomyocytes was signifcantly improved by S/V treatment under light microscope.Flow cytometry analysis showed that S/V significantly reduced the level of intracellular ROS and inhibited cardiomyocyte apoptosis(P<0.05).Fluorescence microscope analysis showed that S/V significantly improved the level of mitochondrial membrane potential(P<0.05);WB showed S/V significantly increase the protein expression levels of Mfn2,mfn1 and Bcl-2,and reduce the protein expression levels of Drp1,FIS1,CytcC,Bax and Caspase-3(P<0.05).Conclusion S/V may regulate mitochondrial homeostasis,reduce ROS production and cardiomyocyte apoptosis by promoting mitochondrial fusion and inhibiting mitochondrial division.