南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (11): 1664-1671.doi: 10.12122/j.issn.1673-4254.2021.11.10

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核外p53通过AMPK/mTOR信号抑制自噬并促进热打击诱导的血管内皮细胞损伤

李 莉，邹志敏，李 琴，张 堃，苏 磊，古正涛   

  1. 南方医科大学第三附属医院创伤救治中心，广东 广州 510630；广东省骨科研究院//广东省骨科医院//广东省骨与关节退行性疾病重点实验室，广东 广州 510630；中国人民解放军南部战区总医院重症医学科，广东 广州 510010
  • 出版日期:2021-11-20 发布日期:2021-12-10

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

LI Li, ZOU Zhimin, LI Qin, ZHANG Kun, SU Lei, GU Zhengtao   

  1. Treatment Center for Traumatic Injuries, Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China; Academy of Orthopedics of Guangdong Province//Orthopedic Hospital of Guangdong Province//Guangdong Provincial Key Laboratory of Bone and Joint Degenerative Diseases, Guangzhou 510630, China; Department of Critical Medicine, General Hospital of Southern Theater Command of PLA, Guangzhou 510010, China
  • Online:2021-11-20 Published:2021-12-10

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摘要： 目的 阐明核外p53通过调控蛋白激酶（AMPK）/雷帕霉素靶体（mTOR）信号通路介导自噬抑制在热打击后血管内皮细胞（VECs）损伤的分子机制。方法 实验分为：对照组、热打击组、热打击组+Compound C组、热打击组+rapamycin组、热打击组+PFT组，并分别使用AMPK抑制剂Compound C、mTOR抑制剂（自噬激活剂）rapamycin、p53线粒体转位抑制剂PFT预处理细胞或C57BL/6小鼠，通过CCK8法检测细胞活力，Western blot观察p53线粒体移位、自噬关键蛋白LC3-Ⅱ、Beclin-1和P62以及AMPK/mTOR信号通路的激活情况，HE染色观察各组小鼠主动内皮血管病理改变，TUNEL染色观察各组小鼠动内皮血管凋亡情况。结果 与对照组相比，热打击后主动脉内皮细胞（MAECs）活力显著下降（P<0.05），热打击后小鼠主动脉血管内皮细胞肿胀、脱落，内弹力膜断裂，平滑肌排列紊乱，可见大量凋亡细胞；热打击后随着复温时间（0、3、6、9 h）延长，MAECs胞浆中p53表达逐渐减弱，而线粒体p53表达逐渐增强；热打击后（6 h）LC3-Ⅱ和Beclin-1蛋白表达被抑制，p62蛋白表达增加（P<0.05）；热打击后（6 h）AMPK的磷酸化被抑制，mTOR、4EBP1和p70S6K的磷酸化表达增加（P<0.05）。AMPK抑制剂Compound C明显抑制了热打击后MAECs中LC3-Ⅱ和Beclin-1蛋白表达，促进了p62蛋白表达，并加重了热打击后小鼠主动脉血管的损伤以及促进了凋亡的增加，而mTOR抑制剂rapamycin均呈现相反作用。p53线粒体转位抑制剂PFT促进了AMPK的磷酸化激活，并抑制mTOR、4EBP1和p70S6K的磷酸化（P<0.05）；使用PFT后促进了LC3-Ⅱ和Beclin-1的表达，抑制了P62的表达（P<0.05）；使 用PFT后明显提高了MAECs的细胞活力（P<0.05），也减轻了热打击后小鼠主动脉血管的损伤和凋亡的发生。结论 核外p53主要通过抑制AMPK活性，激活mTOR信号，继而介导细胞自噬抑制，参与热打击诱导的MAECs损伤。

关键词: 热打击；核外p53；AMPK；mTOR；血管内皮细胞；自噬

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 HS-induced 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.

Key words: heat stress; extranuclear p53; AMPK; mTOR; vascular endothelial cells; autophagy