Protocol And Mechanism Of Fasting Improving Acute Hypoxia Tolerance In Rats

In the event of cockpit leakage or failure of oxygen supply equipment in aviation flight,pilots will suffer from acute high-altitude hypoxia,and even cause flight accidents.In addition,with the rapid development of civil aviation and railway transportation,more and more people can quickly enter the plateau area from the low altitude area.In particular,China’s Qinghai-Tibet Plateau has the state borders with many countries,the border is several thousand meters long and its average altitude is more than 4500 m.The military conflicts occur frequently on the border.If a war breaks out,it is necessary to quickly transport military troops from the plains to the plateaus in order to defeat the invaders.However,the environment in the plateau is harsh,in particular,hypobaric hypoxia can lead to acute high-altitude disease.It seriously affects human health and even threatens life safety.The combat effectiveness of soldiers would be greatly affected when rushing into the plateau.Therefore,it is of great significance to enhance acute hypoxia tolerance of pilots and soldiers before they rapidly enter the plateau.Our study found that fasting preconditioning could improve the 24-hour survival rate of rats at 7620 m,suggesting that fasting preconditioning might have a positive effect on the improvement of acute hypoxia tolerance in rats.In this study,we established a new model of fasting preconditioning that can rapidly enhance the acute hypoxia tolerance of rats.Based on this model,we clarified the scheme and effectiveness of fasting preconditioning to improve acute hypoxia tolerance of rats,and clarified the specific molecular mechanism of the protective effect of fasting preconditioning on acute hypoxia in rats.We also explored the application of relative drugs in improving acute hypoxia tolerance of rats and then elucidated their signaling pathways.Objective:1.To establish a new model that can quickly enhance acute hypoxia tolerance of rats through fasting preconditioning;2.To clarify the specific molecular mechanisms of the protective effect of fasting preconditioning on acute hypoxia in rats;3.To exploring the application of relative drugs in improving acute hypoxia tolerance of rats and then elucidated their signaling path ways.Method:1.Using hypobaric chambers to simulate an acute low-pressure hypoxia environment,and observing the survival rates of rats that are preconditioned by hypoxia and different fasting regimens after staying at 7620 m for 24 hours;2.Observing the p-AMPKαprotein expression level,autophagy flux,oxidative stress,and NF-d B protein expression and inflammation level in the myocardium of 72h-fasting rats by Western blotting,Transmission Electron Microscopy,and immunohistochemistry,etc.;3.Treatments of metformin,rapamycin or withaferin A to verify the protective effect of the above-mentioned molecular signaling pathways on acute hypobaric hypoxia in rats.Result:1.Fasting could effectively improve acute hypoxia tolerance of rats.With the extension of fasting time,acute hypoxia tolerance of rats gradually enhanced.The age of rat also affected the acute hypoxia tolerance,and the acute hypoxia tolerance of young rats was significantly higher than that of adult rats.（1）The survival rate of young rats exposed to 7620 m for 24 h was 43.3%in the control group;the survival rate was 76.7%in the 24 h-fasting group;the survival rates were 100%in both 48 h-and 72 h-fasting groups.（2）The survival rate of adult rats exposed to 7620 m for 24 h was only 13.3%;the survival rate was 63.3%in the 48 h-fasting group;the survival rate was 86.7%in the 72 h-fasting group.2.Fasting increased the expression of p-AMPKαprotein in the rat myocardium,and the young rats was higher.3.Fasting activated the autophagy flux in the rat myocardium,and the enhancement of autophagy flux in young rats was higher than that in adult rats.Western blotting indicated that fasting improved the expression of Beclin-1 and BNIP3 proteins and the LC3II/I,but the content of p62 protein in the rat myocardium was reduced.4.Fasting inhibited the anabolism of protein and lipid in the rat myocardial tissue and promoted lipid catabolism.Fasting inhibited the content of ACC1 and p-mTOR protein and improved the content of PPARαprotein.5.Fasting changed the metabolic substrates of myocardial tissue in rats.¹⁸F-FDG PET/CT imaging showed that glucose uptake in the myocardium gradually reduced from 6 h-to 72h-fasting rats.The blood glucose dropped to 3 mmol/L in 72 h-fasting rats,but nonestesterified fatty acids in the blood increased significantly,and blood ketones increased by 3 times in 72 h-fasting rats.However,blood ketones decreased to the initial concentration after acute hypoxia in the fasting group,suggesting that fasting rats might rely on ketone for energy supply during acute hypoxia.6.Fasting reduced oxidative stress in rat myocardial tissue.The MDA level was lower.7.Fasting reduced inflammatory response in the rat myocardium.Fasting reduced the expression of p-p65 protein,and reduced inflammatory factors.8.The survival rate of adult rats exposed to 7620 m for 24 h was 86.7%after treatment with withaferin A（2 mg/kg/d,i.p.）for 7 days,and was significantly higher than that of the solvent control group（13.3%）.9.Withaferin A might partially simulate the molecular mechanisms of fasting.Withaferin A increased the expression of p-AMPKαprotein,p-ULK1 protein,activated autophagy,and induced the content of p-p65 protein.Contrary to expectation,the content of mTORC1 and ACC1 protein improved in Withaferin A treatment group.Withaferin A activated autophagy through p-AMPKαprotein in the rat myocardium.Conclusion:1.Fasting effectively improved the acute hypoxia tolerance of rats.2.The acute hypoxia tolerance of juvenile rats was significantly higher than that of adult rats.3.The possible molecular mechanisms of fasting preconditioning to enhance acute hypoxia tolerance in rats were as follows:（1）activating AMPK promoted decomposition and inhibited synthesis to maintain energy homeostasis;（2）activated autophagy eliminated aged or damage mitochondrion and then reduced ROS and inflammation;（3）changes in metabolic substrates inhibited mitochondrial aerobic metabolism and fatty acid oxidation,and turned to utilize ketone metabolism with higher efficiency and less oxygen consumption;and（4）the inhibition of NF-κB signaling pathway might reduce inflammatory response in the rat myocardium.4.Withaferin A effectively improved acute hypoxia tolerance of rats by activating AMPK,activating autophagy,and inhibiting NF-κB signaling pathway.