吡格列酮诱导心肌细胞凋亡和抑制心肌细胞肥大的作用及其机制研究

Effects of Pioglitazone on Inducing Apoptosis and Inhibiting Hypertrophy of Cardiomyocytes and its Mechanism

目的:探讨吡格列酮诱导心肌细胞凋亡和抑制心肌细胞肥大的作用及其机制研究。方法:选取1-3日龄的健康新生SD大鼠,采用酶学分离心肌细胞并培养。将心肌细胞分为4组:对照组、吡格列酮10μM组、吡格列酮20μM组、阿帕替尼2μM组。采用自动细胞计数器(BioRad)计数吡格列酮和阿帕替尼对心肌细胞增殖的影响,流式细胞术检测心肌细胞凋亡率,[3H]-亮氨酸掺入法评估心肌细胞的肥厚,用相差显微镜检测心肌细胞直径。蛋白免疫印迹试验检测VEGFR-2,磷酸化VEGFR-2,蛋白激酶B(Akt),磷酸化人体抑癌基因(P53),兔抗人单克隆抗体(Bax),B淋巴细胞瘤-2(Bcl-2),哺乳动物雷帕霉素靶蛋白(mTOR)表达水平。免疫组织化学检测心肌细胞VEGFR-2、Bcl-2及Bax阳性指数。结果:吡格列酮和阿帕替尼均能抑制心肌细胞活力,其中吡格列酮以剂量依赖的方式抑制心肌细胞活力,即吡格列酮剂量越大,其抑制心肌细胞活力越强。吡格列酮或阿帕替尼治疗后,心肌细胞凋亡率显著增加,表明两者均诱导心肌细胞凋亡。吡格列酮或阿帕替尼治疗后,血管紧张素II(Ang II)诱导的[3H]-亮氨酸掺入量显著减少,心肌细胞直径减小,表明两者均抑制心肌细胞肥大。吡格列酮显著提高了新生大鼠心肌细胞中Bax和磷酸化-P53的蛋白表达,降低了mTOR、Akt、VEGFR-2、Bcl-2和磷酸化-VEGFR-2蛋白表达。与对照组比较,吡格列酮和阿帕替尼治疗后,VEGFR-2阳性指数和Bcl-2阳性指数显著降低,Bax阳性指数显著升高(P<0.05)。结论:吡格列酮通过调节VEGFR-2信号通路诱导心肌细胞凋亡,抑制心肌细胞肥大。

Objective:To investigate the effect Effects of pioglitazone on inducing apoptosis and inhibiting hypertrophy of cardiomyocytes and its mechanism.Methods:1 to 3 days old healthy newborn SD rats were selected,and cardiomyocytes were isolated and cultured by enzyme.Myocardial cells were divided into four groups:control group,pioglitazone 10μM group,pioglitazone 20μM group,and apatinib 2μM group.BioRad was used to count the effects of pioglitazone and apatinib on the proliferation of cardiomyocytes,flow cytometry was used to detect the apoptotic rate of cardiomyocytes,and the[3H]-leucine incorporation method was used to evaluate the hypertrophy of cardiomyocytes.The diameter of the cardiomyocytes was examined by a microscope.Western blot was used to detect the protein expression levels of VEGFR-2,phosphorylated VEGFR-2,Akt,phosphorylated p53,Bax,Bcl-2 and mTOR.The positive index of VEGFR-2,Bcl-2 and Bax were detected by immunohistochemistry.Results:Both pioglitazone and apatinib can inhibit the activity of myocardial cells,and pioglitazone can inhibit the activity of myocardial cells in a dose-dependent manner,that is,the greater the dose of pioglitazone,the stronger the inhibition of cardiac cell activity.The apoptosis rate of myocardial cells increased significantly after treatment with pioglitazone or apatinib,indicating that both of them induced apoptosis.After treatment with pioglitazone or apatinib,the amount of[3H]-leucine incorporation induced by Ang II was significantly reduced,and the diameter of cardiac cells decreased,indicating that both inhibited cardiomyocyte hypertrophy.Pioglitazone significantly increased the expression of Bax and phosphorylated-p53 in neonatal rat cardiomyocytes,and decreased the expression of mTOR,Akt,VEGFR-2,Bcl-2 and phosphorylated VEGFR-2.Compared with the control group,the positive index of VEGFR-2 and Bcl-2 were significantly decreased and Bax positive index increased significantly(P<0.05).Conclusion:Pioglitazone induces cardiomyocyte apoptosis and inhibits cardiomyocyte hypertrophy by regulating the VEGFR-2 signaling pathway.