EMRE在高糖环境中的变化对心肌细胞凋亡机制的研究

Effect of High Glucose-Induced EMRE Expressions Changes on Myocardial Apoptosis

目的观察db/db糖尿病小鼠心肌细胞及高糖环境诱导下的H9c2心肌细胞中EMRE分子表达水平的变化,探究EMRE分子的表达变化对糖尿病心肌病心肌细胞凋亡的影响。方法(1)选取db/db糖尿病小鼠和野生型(WT)小鼠各6只,分别饲养18周,利用蛋白质印迹法、免疫组织化学法及实时荧光定量聚合酶链反应(RT-qPCR)法检测小鼠心肌细胞中EMRE的表达水平;小动物超声仪检测小鼠的心脏功能。(2)将购买来的H9c2心肌细胞随机分为四组,即正常组(NG,培养基中含5.5 mmol/L葡萄糖)、高糖组(HG,培养基中含33 mmol/L葡萄糖)、高糖+阴性对照组(HG+NC siRNA,培养基中含33 mmol/L葡萄糖+阴性对照siRNA)、高糖+转染EMRE组(HG+EMRE siRNA,培养基中含33 mmol/L葡萄糖+转染EMRE siRNA),培养24 h,采用蛋白质印迹法检测四组实验细胞中EMRE、凋亡蛋白caspase-3、Bax、caspase-9、细胞色素c(Cyto-c)以及凋亡拮抗蛋白MCL-1表达情况;利用流式细胞术检测细胞凋亡水平;ATP检测试剂盒检测各组细胞线粒体中ATP含量;α-酮戊二酸脱氢酶(α-KGDH)活性检测试剂盒检测其活性;活性氧(ROS)检测试剂盒检测各组细胞内ROS水平。结果(1)与WT小鼠相比,db/db糖尿病小鼠心肌中EMRE分子表达水平明显升高,左室射血分数、左心室短轴缩短率降低(P<0.01)。(2)与正常组相比,高糖组H9c2心肌细胞EMRE表达升高,蛋白质印迹法检测出caspase-3(P<0.01)、Bax(P<0.01)、caspase-9(P<0.001)和Cyto-c(P<0.001)凋亡蛋白的表达增加,凋亡拮抗蛋白MCL-1的表达下降(P<0.01),心肌细胞凋亡明显增加,线粒体ATP浓度异常减少(P<0.01),同时α-KGDH活性下降(P<0.01),而线粒体ROS生成增加(P<0.01);当下调EMRE表达后,心肌细胞凋亡水平受到明显抑制,心肌细胞线粒体ATP浓度升高(P<0.01),α-KGDH活性升高(P<0.01),ROS水平明显降低(P<0.01)。结论糖尿病小鼠心肌细胞中EMRE表达水平升高,小鼠心脏收缩功能降低。高糖环境可诱导H9c2心肌细胞EMRE的表达升高,促进心肌细胞凋亡。下调EMRE表达后,心肌细胞的凋亡水平受到抑制。

Objective To observe EMRE expressions changes in cardiomyocytes of diabetic db/db mice and H9c2 cell line induced by high glucose,and explore the effect on cardiomyocyte apoptosis and the underlying mechanism.Methods 6 diabetic(db/db)and 6 wild-type(WT)mice were feeded for 18 weeks respectively.EMRE expressions in myocardial tissues were tested by Western blot,immunohistochemical staining and RT-qPCR.Cardiac function was detected by small animal Doppler ultrasound.H9c2 cardiomyocytes were randomly divided into normal(NG,5.5 mmol/L glucose),high glucose(HG,33 mmol/L glucose),high glucose+NC siRNA(HG+siRNA negative control),high glucose+EMRE siRNA group(HG+EMRE siRNA)and cultured for 24 hours.EMRE expressions,apoptosis protein caspase-3,Bax,caspase-9,Cyto-c and anti-apoptosis protein MCL-1 were detected by Western blot and the apotosis level was tested by flow cytometry.The content of ATP in mitochondria,the activity ofα-ketoglutarate dehydrogenase(α-KGDH)and mitochondrial reactive oxygen species(ROS)were respectively measured by ATP detection,α-KGDH activity detection,and Mito-SOX^(TM) staining kits.Results Compared with WT control group,the EMRE expressions in diabetic db/db mice group were significantly increased,and left ventricular ejection fraction and short-axis shortening rate were decreased(P<0.01).Western blot showed that the caspase-3(P<0.01),Bax(P<0.001),caspase-9(P<0.001)and Cyto-c(P<0.001)expressions in HG group were up-regulated,MCL-1 down-regulated(P<0.01),cardiomyocytes apoptosis and mitochondrial ROS production increased,the mitochondrial ATP concentration andα-KGDH activity decreased.Once EMRE was down-regulated,the cardiomyocytes apotosis were significantly inhibited,the mitochondrial ATP andα-KGDH activity in cardiomyocytes increased(P<0.01)and mitochondrial ROS decreased(P<0.01).Conclusion The EMRE in cardiomyocytes of diabetic mice were up-regulated with cardiac function inhibited.High glucose may induce EMRE over-expressions and promote apoptosis of H9c2 cardiomyocytes.After EMRE interference,the cardiomyocytes apoptosis will be suppressed.