Role of cytoplasmic p53-mediated suppression of autophagy in heat stress-induced injury of cultured mouse aortic endothelial cells
Objective: To explore the association of cytoplasmic p53 with autophagy and apoptosis of primary aortic endothelial cells (MAECs) exposed to heat stress.
Methods: Cultured mouse MAECs were exposed to heat stress induced by incubation at 43 ℃ for 2 h, with the cells in routine culture condition (37 ℃, 5% CO2) as the control group. All the cells were further incubated for 1, 3, 6 or 9 h at 37 ℃ before treatment with the autophagy inhibitor 3-MA (5 mmol/L), the autophagy inducer rapamycin (20 μmol/L), or the p53 inhibitor PFT (10 μmol/L) for 1 h. After the treatments, the cell viability was measured with CCK8 method, cell apoptosis analyzed by flow cytometry, and the mitochondrial membrane potential detected with flow cytometry with JC-1 staining; the subcellular localization of p53 and the autophagy- associated protein LC3-Ⅱ was detected with immunofluorescence staining, and their protein expressions were analyzed using Western blotting.
Results: Compared with the control cells, MAECs exposed to heat stress showed significantly decreased viability (P < 0.05). At 6 h after the exposure, the cells exhibited significantly decreased mitochondrial membrane potential with increased apoptotic rate (P < 0.05). The cytoplasmic fraction of p53 expression decreased and its mitochondrial fraction increased gradually with time within 6 h after heat stress. Treatment with 3- MA further decreased the mitochondrial membrane potential and significantly increased the apoptotic rate of the exposed cells (P < 0.05), while rapamycin obviously reversed these heat stress-induced cell injuries (P < 0.05). PFT significantly enhanced the expression of LC3-Ⅱ and also inhibited heat stress-induced mitochondrial membrane potential reduction and cell apoptosis (P < 0.05).
Conclusions: Heat stress induces mitochondrial damage and apoptosis in MAECs possibly in relation with mitochondrial translocation of cytoplasmic p53 to result in autophagy inhibition.