OBJECTIVES: The effect of p53 upregulated modulator of apoptosis (PUMA) in hypoxia/reoxygenation-induced cardiomyocyte injuries in rats was investigated. METHODS: PUMA-targeting (si-PUMA) and scramble siRNAs were designed and transfected into primarily rat cardiomyocytes in vitro. RESULTS: RT-PCR and Western blot analysis showed that 50 nmol/l of si-PUMA can specifically inhibit PUMA expression. MTT assay and lactate dehydrogenase activity detection showed that the cell survival rate in the si-PUMA group was enhanced and that the lactate dehydrogenase enzymatic activity dramatically decreased compared with the control group (p < 0.01). Spectrophotometry, as well as annexin V and propidium iodide staining, combined with flow cytometry, revealed that caspase-3 activity in the si-PUMA group was downregulated and the apoptotic rate was decreased (p < 0.01). RT-PCR also showed that Bax expression was downregulated and Bcl-2 expression was upregulated in the si-PUMA group, compared with the control group (p < 0.05). si-PUMA protects cardiomyocytes from apoptosis. CONCLUSION: PUMA mediates hypoxia/reoxygenation-induced cardiomyocyte apoptosis, which can be a potential target of gene therapy for ischemia/reperfusion cardiomyocyte injuries.
OBJECTIVES: The effect of p53 upregulated modulator of apoptosis (PUMA) in hypoxia/reoxygenation-induced cardiomyocyte injuries in rats was investigated. METHODS: PUMA-targeting (si-PUMA) and scramble siRNAs were designed and transfected into primarily rat cardiomyocytes in vitro. RESULTS: RT-PCR and Western blot analysis showed that 50 nmol/l of si-PUMA can specifically inhibit PUMA expression. MTT assay and lactate dehydrogenase activity detection showed that the cell survival rate in the si-PUMA group was enhanced and that the lactate dehydrogenase enzymatic activity dramatically decreased compared with the control group (p < 0.01). Spectrophotometry, as well as annexin V and propidium iodide staining, combined with flow cytometry, revealed that caspase-3 activity in the si-PUMA group was downregulated and the apoptotic rate was decreased (p < 0.01). RT-PCR also showed that Bax expression was downregulated and Bcl-2 expression was upregulated in the si-PUMA group, compared with the control group (p < 0.05). si-PUMA protects cardiomyocytes from apoptosis. CONCLUSION: PUMA mediates hypoxia/reoxygenation-induced cardiomyocyte apoptosis, which can be a potential target of gene therapy for ischemia/reperfusion cardiomyocyte injuries.
Authors: Boris Sabirzhanov; Oleg Makarevich; James Barrett; Isabel L Jackson; Alan I Faden; Bogdan A Stoica Journal: Int J Mol Sci Date: 2020-05-24 Impact factor: 5.923