The Role Of Extranuclear P53 And AMPK/mTOR Signals In The Heat Stress-induced Autophagy And Apoptosis Of Endothelial Cells

BACKGROUNDHeat stroke is a disease with high morbidity and mortality.During heat stroke,endothelial cells are one of the earliest cells to undergo morphological and functional changes.Previous studies have suggested that p53 not only induces apoptosis,but also has dual regulation on autophagy.Nuclear p53 can induce autophagy,while extranuclear p53 inhibits autophagy.Studies have shown that activation of AMP-activated protein kinase(AMPK)negatively regulates mTOR through the AMPK/mTOR signaling pathway and other factors involved in autophagy to induce autophagy.However,the role of extranuclear p53 and AMPK/mTOR in vascular endothelial cell apoptosis after heat stress has not been reported.Method and PurposeWe established a heat stress mouse aortic endothelial cell(MAEC)model and observed the relationship between extracellular p53,AMPK/mTORand autophagy and apoptosis after heat-stress in mice aortic endothelial cell(MAEC),and clarified the death pattern and molecular mechanism of vascular endothelial cells after heat stress.Results1.Heat stress induces cell viability decrease,mitochondrial damage and apoptosisThe cells in the control group(37℃)and the heat stress group(0,1,3,6,and 9h)were collected and detected by the CCK8 method.The cell viability after heat stress showed a time-dependent decline after rewarming 0 h.JC-1 staining and flow cytometry was used to quantitatively analyze the changes in mitochondrial membrane potential after heat stress.It was found that heat stress induced a significant decrease of mitochondrial membrane potential in MAEC cells.Using Annexin V-FITC/PI staining,flow cytometry analysis showed that heat stress caused a significant increase in apoptosis of MAEC cells.It was shown that during the heat stress,the mitochondria of the cells were damaged,eventually induced apoptosis-based cell death patterns,and initiated the activation of endogenous apoptotic pathways.2.Effect of autophagy inhibition/activation on apoptosis of MAEC cells after heat stressTo clarify the impact of heat stress on autophagy in MAEC cells,we observed the expression of autophagy-associated protein LC3-I after heat stress MAEC cells.We found that LC3-I was expressed at 1 h,the expression to the peak at 3h,decreased significantly at 6h and increased at 9h.To further clarify the effect of autophagy on the mitochondrial apoptosis pathway of endothelial cells after heat stress,we used a cell autophagy inhibitor 3-MA and an autophagy inducer rapamycin to pretreat cells.The results showed that autophagy inhibitor 3-MA further promoted the heat stress-induced decrease in mitochondrial membrane potential and apoptosis,while the autophagy inducer rapamycin significantly reversed the mitochondrial membrane potential decrease and apoptosis induced by heat stress.3.Extranuclear p53 and AMPK/mTOR signals are involved in the regulation of autophagy and promote heat stress-induced apoptosisWe confirmed that mitochondrial translocation occurred in the cytoplasmic p53 after heat stress was detected by p53 subcellular localization in MAEC cells after heat stress.Further,by pre-treating cells with p53 activity inhibitor PFT,it was found that PFT significantly promoted the expression of autophagy-associated protein LC3-‖,and it also significantly inhibited the heat stress-induced reduction of mitochondrial membrane potential,suggesting that the extracellular p53 mediates autophagy inhibition,which in turn activates mitochondrial apoptotic signals.The AMPK inhibitor Compound C and the mTOR inhibitor rapamycin were used to pretreat MAEC cells.The AMPK inhibitor Compound C significantly inhibited heat stress-induced LC3-‖ protein expression,whereas the mTOR inhibitor rapamycin showed the opposite effect.This result suggests that AMPK/mTOR signaling also involved in mediating the process of thermal stress-induced autophagy.CONCLUSIONThis study preliminary confirmed that extranuclear p53 and AMPK/mTOR signals are involved in the regulation of autophagy in heat stress vascular endothelial cells.Insufficient autophagy promotes mitochondrial damage,activates mitochondrial apoptosis signals,and ultimately mediates vascular endothelial cells undergo extensive apoptosis.