The Role And Mechanism Of Mild Hypothermia Regulated Fatty Acid Oxidation In Hepatic Ischemia-Reperfusion Injury

Objective Hepatic ischemia–reperfusion(IR)injury is a common clinical issue that can result in poor outcome and lacks effective therapies at present.Mild hypothermia(32-35°C)is a physiotherapy method,which is commonly used in neurosurgery and cardiac surgery,that has been reported to effectively attenuate ischemia-reperfusion injury.While it's protective effects are attributed to multiple mechanisms,one of which may be the regulation of fatty acid ?-oxidation(FAO).Therefore,the purposes of this study were to:(1)investigate the effects of mild hypothermia pretreatment on the normal body to evaluate the feasibility of its clinical application and to investigate the pathophysiological mechanism of mild hypothermia pretreatment alleviate hepatic ischemia-reperfusion injury,thus to lay a foundation for the subsequent studies on relevant molecular mechanisms.(2)investigate the role and underlying mechanisms of FAO in the protective effects of mild hypothermia,thus provide theoretical basis for the clinical application of mild hypothermia.Methods(1)Male C57BL/6J mice were randomly divided into 4 groups with 5 mice in each group: normal group(N),mild hypothermia pretreatment group(MH),ischemia-reperfusion injury group(IR),mild hypothermia pretreatment +ischemia-reperfusion injury group(MHP).After anesthesia,the core temperature were monitored,and mice suffered systematic mild hypothermia pretreatment for 2 h,then a longitudinal incision was made to expose the liver and free the perihepatic ligament,and the branches of the portal vein and the hepatic artery that supply the left and median lobes of the liver were occluded with an atraumatic Glover bulldog clamp for1 h,and then the clamp was removed to initiate hepatic reperfusion for 6 h,finally the blood and liver tissue samples were obtained to evaluate liver functional,morphological,cell apoptosis,mitochondrial and oxidative stress injury.(2)In addition to the above 4 groups,the newly FAO inhibitor Etomoxir +ischemia-reperfusion injury group(EIR)and the FAO agonist Leptin +ischemia-reperfusion injury group(LIR)were added in male C57BL/6J mice,also with 5 in each group,Etomoxir(5 mg/kg)and Leptin(5 mg/kg)were administered intraperitoneally 1 h before ischemia.Then,according to the first part of animal experimental methods,a model of 70% hepatic ischemia for 1 h and reperfusion for 6h was constructed,blood and liver tissue samples were collected,and liver functional,morphological,cell apoptosis,mitochondrial and oxidative stress,proteomics analysis and hepatic FAO related indicators and signal pathways were detected.Results(1)Compared with the normal group,mild hypothermia pretreatment had no significant effects on liver function,morphology,apoptosis and oxidative stress.While ischemia-reperfusion injury have a significant injury on the liver function,morphology,cell apoptosis and oxidative stress,i.e.,the serum ALT and AST level increased from 51.5±23 U/L and 87.4±39.2 U/L to 9029.8±981.8 U/L and10434.8±1094.8 U/L,respectively;the score of hepatic pathological injury increased from 0.6±0.5 to 9±1;liver cell apoptosis rate increased from 1.02%±0.31% to37.82%±3.18%;mitochondrial injury in hepatocytes and liver tissue oxidative stress index also increased significantly.Mild hypothermia can significantly inhibit ischemia-reperfusion induced injuries,i.e.,serum ALT and AST levels decreased to5071.2±776 U/L and 5735.2±1886.3 U/L,respectively;the score of hepatic pathological injury decreased to 4.2±0.8;liver cell apoptosis rate decreased to13.94%±3.67%,mitochondrial injury in hepatocytes and the index of oxidative stress in liver tissues also decreased significantly.(2)The FAO was significantly inhibited by hepatic ischemia-reperfusion injury,the expression of the rate-limiting enzyme(CPT1a)of hepatic FAO was down-regulated almost two-fold by IR,while this inhibition could be significantly reversed by mild hypothermia.Experiments with leptin and etomoxir confirmed that activation of FAO could also reduce the hepatic enzyme levels(ALT and AST reduced to 4065.6±759.7 U/L and 4632.2±1500 U/L,respectively),the score of hepatic pathological injury(decreased to 3.8±0.8),hepatocyte apoptosis(decreased to13.06%±1.92%),oxidative stress and mitochondrial injury induced by IR,which had the similar effects to mild hypothermia,while inhibition of FAO had negative effects.Furthermore,mild hypothermia and leptin could promote the phosphorylation of JAK2/STAT3 and up-regulate the ratio of BCL-2/BAX to suppress hepatocyte apoptosis.Conclusion(1)Mild hypothermia pretreatment did not damage the liver,and mild hypothermia could significantly reduce the damage of liver functional,morphological,apoptosis,mitochondrial and oxidative stress injury induced by hepatic ischemia-reperfusion injury.(2)Mitochondrial fatty acid ?-oxidation plays a vital role in the process of mild hypothermia in alleviating hepatic ischemia-reperfusion injury and mild hypothermia attenuated hepatic IR injury mainly via the up-regulation of JAK2/STAT3-CPT1a-dependent FAO.