PUMA对高糖诱导的大鼠H9C2心肌细胞凋亡的作用及机制

Effects of PUMA knockout on the apoptosis of H9C2 cardiomyocytes induced by high glucose

目的:观察促凋亡蛋白p53上调凋亡调控因子(PUMA)在高糖所致H9C2心肌细胞凋亡中的作用及机制。方法:H9C2心肌细胞随机分为对照组(使用5.5mmol/L葡萄糖作用于细胞)和高糖组(使用35 mmol/L葡萄糖作用于细胞,HG组)分别刺激6 h,12 h,24 h和48 h,每组设复孔5个,TUNEL染色检测细胞凋亡率;RT-PCR及Western blot法分别测定PUMA mRNA及蛋白表达情况;JC-1法检测线粒体膜电位;Western blot测定caspase-3表达和细胞色素c(Cyt C)释放。H9C2细胞随机分为四组,对照组、高糖(35 mmol/L)、HG+si-scramble组(使用si-scramble转染心肌细胞24 h,使用35mmol/L葡萄糖作用于细胞)和Si-PUMA组(使用si-PUMA转染心肌细胞24 h,使用35mmol/L葡萄糖作用于细胞),观察抑制PUMA表达对高糖诱导细胞凋亡率、线粒体膜电位、Cyt C的影响。结果:与对照组相比,高糖刺激心肌细胞组TUNEL染色阳性率、活化caspase-3和PUMA表达明显升高(P<0.05或P<0.01),细胞损伤和PUMA表达增高具有时间依赖性,其中高糖24 h组和48 h细胞凋亡率和PUMA表达差异无统计学意义,后续实验采用高糖刺激24 h作为作用时间。与高糖组相比,HG+si-PUMA组PUMA表达抑制、线粒体膜电位恢复、Cyt C释放减少、心肌细胞凋亡减少(P<0.05或P<0.01);HG+si-Scramble组与高糖组相比,PUMA表达、线粒体膜电位、Cyt C释放、心肌细胞凋亡均无显著性差异(P>0.05)。结论:PUMA介导高糖所致的大鼠心肌细胞凋亡,提示PUMA可能是糖尿病心肌病治疗的一个重要的靶基因。

Objective:To investigate the effects of p53 upregulated modulator of apoptosis(PUMA)on the apoptosis of H9C2 cardiomyocytes induced by high glucose and its mechanisms.Methods:H9C2 cardiomyocytes were treated with 5.5mmol/L(control group)or 35 mmol/L glucose(HG group)for 6 h,12 h,24 h or 48 h respectively to induce apoptosis,each group sets 5 multiple wells.Apoptosis was tested by TUNEL assay.PUMA mRNA was measured by RT-PCR and protein expression was measured by Western blot assay.The mitochondrial membrane potential was detected by JC-1 method.The expressions of cleaved caspase-3 and cytochrome C(Cyt C)protein in mitochondria and cytoplasm were determined by Western blot assay.H9C2 cardiomyocytes were randomly divided into four groups,control group(5.5 mmol/L),HG(35 mmol/L)group,HG+si-scramble group(si-scramble treatment for 24 h,then 35 mmol/L high glucose treatment for 24 h)and HG-si-PUMA group(si-PUMA treatment for 24 h,then 35mmol/L high glucose treatment for 24 h).Si-PUMA was transfected into cardiomyocytes and the effects of PUMA on high glucose-induced apoptosis were studied.Results:Compared with the control group,high glucose increased cardiomyocyte apoptosis and enhanced PUMA mRNA and protein expressions significantly(P<0.05 or P<0.01).Cell injury and increased PUMA expression were time-dependent and there was no significant difference between the high glucose 24 h group and the high glucose 48h group.The following experiment used high glucose 24 h as the stimulation time.The cardiomyocytes transfected with si-PUMA to inhibit PUMA expression had decreased apoptotic rate and cleaved caspase-3,increased mitochondria membrane potential and decreased Cyt C release(P<0.05 or P<0.01).There were no significant differences between the HG+si-scramble group and the high glucose group(P>0.05).Conclusion:PUMA mediates high glucose-induced cardiomyocyte apoptosis suggesting PUMA may be an important target gene of diabetic cardiomyopathy.