Fasting And Withaferin A Enhance Acute Hypoxic Tolerance By MTOR Inhibition

【Background】Rapid adaptation to hypoxia is a consistently relevant issue in medical research on special environments.In recent years,there has been an increasing demand for humans to enter into high altitudes.After a period of acclimation and oxygen inhalation,the human body can stay at a high altitude for a certain duration.However,the current acclimation strategy is time-consuming;additionally,it is difficult to meet the demand for rapid advances to plateau,and there is no guarantee that the human body can stay at extreme altitudes for a long period of time.The exploration of safe and effective solutions for rapid adaptation to acute hypoxia and the search for potential protective drugs are important issues that need to be resolved in the field of plateau hypoxia research.Our group found that fasting pretreatment for 72 h or treatment with withaferin A（WA）effectively improves the survival rate of SD rats kept at an altitude of 7,620 m for 24hours.However,the underlying mechanisms by which fasting pretreatment or WA allows rapid adaptation to acute hypoxia are still unclear.Insufficient ATP production（especially the lack of energy in important organs,such as the heart and brain）caused by severely reduced O₂ levels is the fundamental cause of damage and even death under acute hypoxia.Reducing the consumption of ATP and increasing the ATP supply are effective coping strategies for acute hypoxia in the body.Mammalian target of rapamycin（mTOR）is an important regulator of cell growth that affects cellular ATP production and consumption by regulating anabolism and mitochondrial quality-control mechanisms.mTOR may play an important role in rapid adaptation to acute hypoxia.This study aimed to explore the underlying mechanisms by which fasting or WA accelerates adaptation to acute hypoxia via mTOR through in vivo and in vitro experiments to provide a strong experimental basis and theoretical support for translation to humans.【Objectives】Part Ⅰ.Fasting preconditioning enhances acute hypoxic adaptability via mTOR inhibition.1.To assess the effect of fasting preconditioning on acute hypoxic injury to tissues and cells.2.To analyze the effect of fasting preconditioning on mTOR and its downstream pathways,anabolism and mitophagy.3.To evaluate the effect of fasting preconditioning on cellular ATP levels,ROS levels and mitochondrial respiratory function under acute hypoxia.4.To explore the effect of mTOR knockdown on cellular acute hypoxic tolerance via anabolism and mitophagy.Part Ⅱ.WA enhances mitochondrial biogenesis and mitophagy via mTOR inhibition to promote acute hypoxic adaptation.1.To evaluate the effect of WA on acute hypoxic injury to tissues and cells.2.To analyze the effect of WA on mTOR and the downstream anabolism pathway.3.To explore the effect of WA on mitochondrial quality-control mechanisms,including mitochondrial fission and fusion,mitochondrial biogenesis and mitophagy.4.To assess the effect of WA on cellular ATP levels,ROS levels and mitochondrial respiratory function under acute hypoxia.【Methods】1.The hypobaric hypoxic conditions experienced at 7,620 m were mimicked in an animal hypobaric chamber system,and the survival rate of SD rats was assessed after exposure to 7,620 m for 24 h.2.The cardiac function of rats was evaluated by using small-animal echocardiography.HE staining was used to observe the morphology of the myocardium and liver tissues.Tissue ATP and ROS detection kits were employed to determine ATP and ROS levels in the myocardium;additionally,Western blotting and immunohistochemistry were used to assess changes in the expression of proteins.qPCR was used to measure changes in mRNA levels,and protein localization within tissues or cells were analyzed by using immunofluorescence.Oil red O staining was used to detect hepatic lipid droplets,and transmission electron microscopy was used to observe myocardial fibers and mitochondrial morphology.3.mTOR knockdown experiments were performed with small interfering RNA（siRNA）.Flow cytometry was conducted to determine the apoptosis rate of cardiomyocytes,and the change in the mitochondrial membrane potential（MMP）was determined by using JC-1 staining.Ro-GFP and MitGO-ATeam2 were used to monitor the contents of cellular ROS and mitochondrial ATP,respectively,and a Seahorse instrument was used to measure mitochondrial respiratory function-related indicators.【Results】Part Ⅰ.Fasting preconditioning enhances acute hypoxic adaptability via mTOR inhibition.1.Fasting preconditioning significantly improved the survival rate of rats after exposure to 7,620 m for 24 h,reduced hypoxic damage to the myocardium and liver tissues and improved the cardiac function of rats under acute hypoxia.In addition,fasting preconditioning significantly reduced ROS production and restored ATP generation in the myocardium in rats under acute hypoxia.Moreover,the Western blotting results indicated that fasting pretreatment obviously decreased the levels of protein synthesis-related and lipogenesis-related proteins and increased the expression of autophagy-related proteins and BNIP3 in rat tissues under acute hypoxia.Immunofluorescence and transmission electron microscopy showed that fasting preconditioning significantly enhanced mitochondrial autophagic flux in the myocardium.2.The results of the cell experiments showed that the simulated fasting cardiomyocytes exhibited restored MMP and reduced apoptosis rate after exposure to acute hypoxia for 24 h compared with control hypoxic cardiomyocytes.The levels of protein synthesis-related and lipogenesis-related proteins were significantly reduced in simulated fasting cardiomyocytes subjected to acute hypoxia for 24 h.Furthermore,the levels of BNIP3 and other autophagy-related proteins in cells subjected to simulated fasting were increased,and the colocalization of BNIP3 and LC3 fluorescence and Tom20and LAMP1 fluorescence was obviously increased.Analysis of mitochondrial respiratory function-related indicators showed that simulated fasting significantly improved the mitochondrial oxidative phosphorylation capacity of cardiomyocytes,further reduced ROS production and restored ATP generation under acute hypoxia.3.Cellular mTOR knockdown experiments showed that mTOR knockdown significantly reduced the expression of protein synthesis-related and lipogenesis-related proteins,enhanced BNIP3-mediated mitophagy and maintained the MMP of cardiomyocytes under acute hypoxia.It also reduced apoptosis and ROS production in cardiomyocytes,effectively improving mitochondrial oxidative phosphorylation capacity and increasing ATP production under acute hypoxia.Part Ⅱ.WA enhances mitochondrial biogenesis and mitophagy via mTOR inhibition to promote acute hypoxic adaptation.1.WA reduced acute hypoxic damage to tissues,maintained cardiac function,reduced myocardial ROS production and restored ATP reserves under acute hypoxia,ultimately improving the survival rate of rats exposure to 7,620 m for 24 h.The Western blotting results showed that WA significantly decreased protein synthesis but had no significant effect on lipogenesis in the myocardium.Moreover,electron microscopy and qPCR showed that WA significantly increased the number of myocardial mitochondria under acute hypoxia.Further examinations found that WA significantly increased the expression of BNIP3 and other autophagy-related proteins,and increased the expression of mitochondrial biogenesis-related proteins（PGC-1α,NRF2 and TFAM）but had no significant effect on the expression of mitochondrial fission and fusion-related proteins.2.In vitro experiments showed that compared with control hypoxic cells,WA significantly reduced apoptosis and enhanced mitochondrial biogenesis and mitophagy in cardiomyocytes under acute hypoxia.Furthermore,WA increased the number of cellular mitochondria,reduced cellular ROS production,maintained the MMP,enhanced mitochondrial respiratory function and increased mitochondrial ATP generation under acute hypoxia.【Conclusions】1.Fasting for 72 h enhances acute hypoxic tolerance.mTOR is the key factor for fasting preconditioning in protecting against acute hypoxia.Fasting pretreatment inhibits mTOR activity.Firstly,mTOR inhibition reduces anabolism,which correspondingly prevents the unnecessary consumption of cellular ATP in hypoxic environments.Secondly,mTOR inhibition enhances BNIP3-mediated mitophagy to preemptively remove damaged or dysfunctional mitochondria,thereby reducing the production of cellular ROS and improving mitochondrial oxidative phosphorylation ability to increase the ATP supply under acute hypoxia.Thus,it enables the body to quickly adapt to acute hypoxia.2.WA can be used as an effective drug to improve acute hypoxic tolerance.WA decreases myocardial protein synthesis to reduce the unnecessary consumption of ATP to a certain extent,thus playing a role in improving hypoxic tolerance.More importantly,WA plays a regulatory role in mitochondrial quality-control mechanisms,specifically enhancing PGC-1α-mediated mitochondrial biogenesis and BNIP3-mediated mitophagy in the myocardium and cardiomyocytes.Furthermore,it removes damaged mitochondria by mitophagy to reduce hypoxic injury.Additionally,by activating PGC-1α-mediated mitochondrial biogenesis,a sufficient number of mitochondria are ensured to maintain effective mitochondrial respiratory function to generate ATP in acute hypoxic environments.Regulation of mitophagy and mitochondrial biogenesis by WA may be an important mechanism for improving acute hypoxic tolerance.