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.
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。.
Keywords: apoptosis; cardiomyocyte; cell culture; cytochrome C; high glucose; p53 upregulated modulator of apoptosis.