[Extranuclear p53 suppresses autophagy through AMPK/mTOR signaling to promote heat stress-induced vascular endothelial cell damage].

OBJECTIVE: To explore the role of extranuclear p53-mediated autophagy suppression by regulating AMPK/mTOR signaling pathway in heat stress (HS)-induced injury of mouse aortic endothelial cells (MAECs). METHODS: Primary cultures of MAECs were pretreated with compound C (an AMPK inhibitor), rapamycin (a mTOR inhibitor) or pifithrin-α (PFT, a selective p53 inhibitor) for 1 h before exposure to HS (43 ℃) for 2 h. The changes in cell viability at different time points after HS were examined using CCK-8 assay, and the protein expressions of P53, LC3-II, Beclin-1, p62 and the AMPK/mTOR signaling proteins were detected using Western blotting. In the animal experiment, C57 mice were pretreated with compound C, rapamycin or PFT and exposed to a high temperature at 40 ℃ to induce HS. The pathological changes in the aorta of the mice were observed with HE staining, and cell apoptosis was detected using TUNEL staining. RESULTS: In cultured MAECs, the cell viability was significantly reduced (P < 0.05) and the mitochondrial fraction of p53 increased while its cytoplasmic fraction decreased progressively over time following HS. HS significantly lowered the expressions of LC3-II and Beclin-1, increased p62 level, suppressed AMPK phosphorylation, and increased mTOR phosphorylation and the expressions of its downstream proteins at 6 h after the exposure (P < 0.05). Pretreatment with compound C significantly inhibited LC3-II and Beclin- 1 expression, enhanced p62 expression, and aggravated HS-induced cell injury and apoptosis in MAECs; rapamycin treatment produced the opposite effects (P < 0.05). PFT treatment significantly enhanced the viability of MAECs and alleviated HSinduced injury and apoptosis; PFT also significantly promoted activation of AMPK phosphorylation, inhibited mTOR phosphorylation and its downstream proteins (P < 0.05), enhanced the expressions of LC3-II and Beclin 1, and inhibited p62 expression in the MAECs (P < 0.05). In C57 mice, HS resulted in swelling, shedding and apoptosis of aortic vascular endothelial cells. Pretreatment with compound C obviously aggravated HS-induced vascular injury and endothelial cell apoptosis, while pretreatment with either rapamycin or PFT significantly alleviated these injuries. CONCLUSION: Autophagy inhibition mediated by extranuclear p53 via inhibiting AMPK activity and activating mTOR signaling participates in HS-induced injury of MAECs.