Activated AXL Protects Against Hepatic Ischemia Reperfusion Injury By Upregulating SOCS-1 Expression

Background:Hepatic ischemia reperfusion（I/R）injury is a major factor affecting perioperative morbidity and mortality in liver transplantation and hepatectomy.Our previous study have found that the expression of AXL was up-regulated in the liver suffered from ischemia-reperfusion injury.through proteomic studies.As we know,AXL,a member of the TAM family,is a receptor tyrosine kinase involved in immune regulation and cells apoptosis in multiple organs.However,the role of AXL in hepatic I/R injury and the underlying mechanisms remain to be elucidated.Purpose:To study the effect and mechanism of AXL on liver ischemia-reperfusion injury,and to find new potential targets for clinical prevention and treatment of liver ischemia-reperfusion injury.Methods:In order to study the role of AXL in hepatic ischemia-reperfusion injury,we collected 3 normal human liver samples and 9 liver transplant donor liver samples to verify the differential expression of AXL in normal liver tissue and donor liver tissue.By constructing a mouse model of partial warm ischemia-reperfusion injury,we used western blotting to verify the expression of AXL and p-AXL at each reperfusion injury time point.Then,mice were pretreated with AXL activator recombinant mouse Gas6 protein and AXL specific inhibitor R428 to construct a mouse model of warm ischemia-reperfusion injury.Western blotting,RT-q PCR,ELISA,immunohistochemistry and TUNEL staining were used to evaluate the inflammatory response and apoptosis of mice in each treatment group after ischemia-reperfusion injury.We used mouse liver primary cells to perform after oxygen glucose deprivation and reoxygenation experiment to simulate the process of liver ischemia-reperfusion injury in vitro and detected the degree of apoptosis of primary liver cells by flow cytometry.Finally,we used adeno-associated virus to specifically knock down SOCS-1 expression in mouse liver tissue to further evaluate the role of AXL in hepatic ischemia-reperfusioninjury.Result:1.AXL upregulation and p-AXL downregulation in liver I/R injury.Western blotting experiments showed that the level of AXL protein in liver tissue of liver transplant donors was significantly increased,while the level of phosphorylated AXL（p-AXL）protein was significantly decreased.In the mouse liver I/R model,AXL gradually increased and peaked at 0-6 hours after I/R,and gradually decreased after 6 hours.In contrast,p-AXL decreased to a nadir within 0-6 hours after I/R injury,and then gradually increased within 24 hours.2.Activated AXL protects against liver I/R injuryMice were pretreated with different doses of recombinant mouse Gas6 protein,and the levels of serum transaminases ALT/AST in the Gas6 pretreatment group were significantly decreased after I/R injury in mice.HE staining showed that the area of liver damage in the Gas6 pretreatment group was reduced.3.Activated AXL ameliorates inflammation in liver I/R injuryWestern blotting,RT-q PCR and ELISA experiments confirmed that Gas6 pretreatment of mice could significantly reduce the hepatic inflammatory response,and the levels of inflammatory factors IL-1β,IL-6 and TNF-α were significantly decreased.LY6 G staining showed that the degree of neutrophil infiltration in the liver of mice in the Gas6 pretreatment group was significantly decreased after liver I/R.4.Activated AXL improves hepatocytes apoptosis in hepatic I/R injury.Western blotting and RT-q PCR experiments showed that Gas6 pretreatment reduced the degree of hepatocyte apoptosis after I/R,and the expression of pro-apoptotic proteins BAX and cleaved-caspase3 decreased,while the expression of anti-apoptotic protein Bcl-2 increased.TUNEL staining also confirmed that Gas6 pretreatment reduced hepatocyte apoptosis.5.Activated AXL activation aggravates liver I/R injury.The levels of ALT and AST in the R428 pretreatment group increased,and the area of hepatic I/R injury increased.After R428 pretreatment,the expressions of inflammatory factors IL-1β,IL-6 and TNF-α were increased,the expressions of BAX and c-Caspase3 were increased,and the expression of Bcl-2 was decreased in liver I/R injury.Meanwhile,the results of R428+rm Gas6 showed that R428 reversed the effect of rm Gas6 in reducing inflammatory response and hepatocyte apoptosis in liver I/R injury.Apoptosis of hepatocytes was increased after R428 pretreatment using apoptosis flow cytometry.6.Activated AXL inhibits the TLR4/MYD88/NF-κB pathway by upregulating SOCS1Western blotting confirmed that Gas6 pretreatment up-regulated the expression of SOCS-1 and decreased the expression of TLR4,MYD88,TRAF6 and p-P65.R428 pretreatment inhibits SOCS-1 expression and activates the TLR4/MYD88/NF-κB signaling axis.7.The protective effect of SOCS1-mediated activated AXL on liver I/R injurySpecific knockdown of SOCS-1 in mouse liver using adeno-associated virus.The SOCS1 knockdown group had a higher level of hepatic I/R serum transaminase and a larger area of liver injury.Meanwhile,western blotting confirmed that SOCS-1 knockdown group exhibited aggravated inflammation and increased apoptosis,and increased levels of TLR4,MYD88,TRAF6 and p-P65.Conclusion:AXL activation protects the liver from I/R injury by upregulating SOCS-1 and inhibiting the TLR4/MYD88/NF-κB signaling axis.Targeting AXL may be a novel therapeutic strategy to improve liver I/R injury.