L Li1,2, Z Zou1,2, Q Li1,2, K Zhang1,2, L Su3, Z Gu1,2. 1. Treatment Center for Traumatic Injuries, Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China. 2. Academy of Orthopedics of Guangdong Province//Orthopedic Hospital of Guangdong Province//Guangdong Provincial Key Laboratory of Bone and Joint Degenerative Diseases, Guangzhou 510630, China. 3. Department of Critical Medicine, General Hospital of Southern Theater Command of PLA, Guangzhou 510010, China.
Abstract
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.
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.
Authors: Jeffery Ho; Jun Yu; Sunny H Wong; Lin Zhang; Xiaodong Liu; Wai T Wong; Czarina C H Leung; Gordon Choi; Maggie H T Wang; Tony Gin; Matthew T V Chan; William K K Wu Journal: Autophagy Date: 2016-05-12 Impact factor: 16.016