The Potential Role And Underlying Mechanisms Of Caspase Recruitment Domain 6 In Hepatic Ischemia/Reperfusion Injury

Background and purpose:Liver disease is the leading cause of death worldwide.Up to 25% of the world’s population currently has risk factors for liver disease,including viral infections,alcohol abuse and non-alcoholic hepatitis(NASH).High prevalence causes an increase in the incidence of primary liver cancer,causing more than 800,000 deaths each year.Hepatic ischemia-reperfusion(I/R)is one of the most common tissue injuries in liver tumor resection and liver transplantation.The severity of liver damage is determined by the complex interaction between aseptic inflammation and hepatocyte death during ischemia/reperfusion(I/R).Caspase-collecting domain protein 6(CARD6)plays an important role in NF-κB activation and regulates inflammatory responses,but whether it is involved in the regulation of hepatic ischemia/reperfusion injury is unclear.This study is intended to elucidate whether CARD6 is involved in the regulation of the pathophysiological processes of liver I/R injury and to explore potential molecular mechanisms.Methods:Part Ⅰ: The expression of CARD6 after ischemia/reperfusion injury was detected in human,mouse liver tissue and cell samples.Quantitative PCR and Western Blot were applied to detect the expression of CARD6 in human liver transplantation samples,in mouse hepatic ischemia-reperfusion injury model established by clamping the blood vessels in the left and left middle leaves(8-week-old,male,C57BL/6 wild-type mice),and in mouse primary hepatocyte hypoxia/reoxygenation model(Hepatocyte H/R).Part Ⅱ: The in vivo and in vitro hepatic I/R model was established using the Card6-HKO and Card6-HTG and the corresponding control mice.In vivo,mice with hepatocyte-specific CARD6 knockout(Card6-HKO)and overexpression(hepatocyte-specific CARD6 transgenic,Card6-HTG)were constructed.Eight-week-old,male,Card6-HKO and Card6-HTG and corresponding control mice were randomly divided into sham operation group(Sham group)and operation group(I/R group,0h,1h,3h,6h,12h),24h).At the corresponding time points,blood was collected from the iliac vein,serum was separated,and the levels of liver enzymes(ALT,AST)and inflammatory factors(TNF-α,IL-1β,IL-6,MCP-1,and CXCL-1)in the serum were measured.The abdominal cavity was cut,the mouse liver was photographed,and then a part was placed in a formaldehyde solution for pathological analysis,and the other part was placed at-80 °C for molecular biological detection.The severity of hepatic ischemia/reperfusion injury in mice was evaluated by H&E staining(paraffin liver tissue section,5 μm)histopathological examination.Furthermore,quantitative PCR and Western Blot were used to detect the m RNA and protein expression levels of genes related to inflammation and apoptosis in mouse liver tissues.Flow cytometry was used to detect non-parenchymal inflammatory infiltration in the liver.For the in vitro experiments,primary hepatocytes were isolated from 6-8 weeks old,male,Card6-HKO and Card6-HTG and corresponding control mice.Hepatocyte H/R model was established,and the supernatant of cell culture was taken to detect the severity of inflammation.The level of lactate dehydrogenase(LDH)toxicity test was used to detect the damage of primary hepatocytes,and Western Blot was used to detect the expression levels of primary hepatocyte inflammation and apoptosis-related genes after injury.Part Ⅲ: To identify and validate the possible mechanisms by which CARD6 plays a regulatory role in hepatic ischemia/reperfusion injury.Western Blot was used to detect changes in JNK/P38 signaling pathway in vivo and in vitro and to lock the upstream key regulatory kinase ASK1.Co-immunoprecipitation and GST-Pulldown was used to confirm the direct interaction between CARD6 and ASK1.Further possible sites of action between CARD6 and ASK1 were determined by truncating the CARD6 and ASK1 sequences.Part Ⅳ: Hepatic I/R model was established in mice injected with Ad-CARD6 and Ad-CARD6 mutant adenovirus.Phenotypic correlation analysis was performed to confirm that CARD6 gene plays a regulatory role in hepatic ischemia/reperfusion injury through interacting with ASK1.In vivo and in vitro I/R model were established in mice infected with different doses of Ad-CARD6 and Ad-CARD6 mutant adenoviruses Ad-Card6-m(281-480)and the expression of ASK1 was detected by Western Blot.Serum was separated,and the level of liver enzymes(ALT,AST)in the serum was used to evaluate the severity of liver injury.The liver tissue was taken and H&E pathological staining was performed to evaluate the severity of liver histological damage in mice.Further,the changes of JNK/P38 signaling pathway in the liver tissue of mice after ischemia/reperfusion injury were detected by Western Blot.Results:Part Ⅰ: Compared with normal human liver tissue,the expression of CARD6 in liver tissue of liver transplantation was significantly down-regulated.In the hepatic ischemia/reperfusion injury model of Hepatic I/R and Hepatocyte H/R in vitro,the m RNA and protein expression levels of CARD6 gradually decreased with the ischemia/reperfusion or hypoxia/reoxygenation time.Part Ⅱ: In hepatic I/R and primary hepatocytes H/R model from Card6-HKO and Card6-HTG and corresponding control mice,the following results were found: 1)In sham-operated group,there was no significant difference for tissue damage evaluated by serum liver enzymes and liver histology.However,after ischemia/reperfusion injury,the serum liver enzyme levels(ALT,AST)in Card6-HKO mice were significantly higher than those in the control mice.Histological damage was significantly heavier in Card6-HKO mice than that in the control group.However,compared with the control group,Card6-HTG showed a significant improvement in liver enzyme levels and liver histological damage.2)Compared with the control group,increased serum inflammatory cytokines,inflammatory infiltration and liver cell death were found in the Card6-HKO after I/R injury,as determined by the expression of inflammatory factors,flow cytometry,immunofluorescence staining,q PCR and Western Blot in serum and primary cell supernatants.However,the level of serum inflammatory factors in Card6-HTG mice was significantly inhibited,and inflammation infiltration and liver cell death were significantly improved in liver tissues.Part Ⅲ: 1)Western Blot analysis of JNK/P38 signaling pathway confirmed that CARD6 knockout can promote phosphorylation of ASK1 and promote the activation of JNK/P38 signaling pathway,while CARD6 overexpression significantly inhibits the activation of the JNK/P38 signaling pathway.2)Through Co-IP and GST pulldown experiments,CARD6 was found to interact with ASK1.The truncation of the CARD6 gene sequence at more than three loci suggests that only the 280-481 amino acid stretch of CARD6 can interact with ASK1.The truncation of the ASK1 gene sequence at multiple sites suggests that the catalytic binding domain of ASK1 can interact with CARD6.4)Co-IP further confirmed that the mutant of CARD6(amino acid 281-480 deletion)lost its ability to interact with ASK1.Part Ⅳ: After overexpression of CARD6 and Card6-m(281-480)genes in WT mice we found that: 1)the ASK1 phosphorylation was inhibited after overexpression of CARD6 in a dose-dependent manner.While,CARD6 mutants lost the effect of inhibiting ASK1 phosphorylation.2)CARD6 gene can significantly improve liver injury after ischemia/reperfusion.CARD6 mutant failed to prevent liver damage after ischemia/reperfusion.3)CARD6 mutant overexpression in mouse liver could not effectively inhibit the activation of JNK/P38 signaling pathway and improve inflammatory infiltration and liver cell death as CARD6 overexpression.Conclusion:Through the mouse Hepatic I/R model and the primary hepatocyte H/R model to simulate the process of hepatic ischemia/reperfusion injury,we found that CARD6 plays an important protective role against hepatic ischemia/reperfusion injury.The mechanism of the protective effects is that CARD6 directly interacts with ASK1,inhibits the activation of ASK1/JNK/P38 signaling pathway,and improves inflammatory infiltration and hepatocyte death after hepatic ischemia/reperfusion injury.