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

doi:10.12047/j.cjap.6338.2022.094

Abstract

Abstract: 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.

Key words: p53 upregulated modulator of apoptosis, high glucose, cardiomyocyte, cell culture, apoptosis, cytochrome C

CLC Number:

R542.2+2

GUO Jia, LI Zhi-dong, XIAO Chuan-shi, BIAN Yun-fei. Effects of PUMA knockout on the apoptosis of H9C2 cardiomyocytes induced by high glucose[J]. CJAP, 2022, 38(5): 504-509.

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