Mechanism Of Immune-related GTPase M Alleviating Acute Liver Failure By Regulating Autophagy

Aim:Liver failure（LF）is a pathological state of severe liver injury mediated by immune inflammation.Immune-related GTPase M（IRGM）has the function of inducing autophagy and suppressing inflammation,but its specific role and the related signaling pathways remain to be explored in LF.The aim of this study was to investigate the mechanism by which IRGM attenuates immune inflammatory injury by regulating autophagy in LF.Methods:1.Clinical study:20 subjects,including 10 patients with HBV-related ACLF（HBV-ACLF）and 10 healthy controls（HCs）were included.Samples of peripheral blood and liver tissues were collected.The biochemical indexes of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,total bilirubin（TBil）,and direct bilirubin（DBil）were detected by automatic biochemical analyzer,the contents of IL-1βand TNF-αin serum were detected by ELISA,and the expressions of IRGM,NLRP3 inflammasome and autophagy-related proteins（LC3II and P62）in liver tissues were detected by Western blot.2.Animal experiment:C57BL/6 mice were given an intraperitoneal injection of D-Ga IN（500mg/kg）/LPS（10μg/kg）to establish a mouse model of ALF.C57BL/6 mice were randomly divided into three groups:control group,ALF group,and rapamycin（RAPA,4mg/kg）intervention group.To induce autophagy,rapamycin was given intraperitoneally to mice 30 min before the administration of D-Gal N/LPS.The serum levels of AST and ALT in the peripheral mice were detected by kits.HE staining was used to observe the pathological changes of liver tissues to judge a liver injury.Immunohistochemical staining was used to observe the expression and distribution of Irgm1 protein in liver.Western blot was used to detect the protein expression of Irgm1,LC3II,P62,and NLRP3 inflammasome in the liver.q RT-PCR was used to detect the m RNA expression of Irgm1,LC3II,NLRP3 inflammasome,and pro-inflammatory cytokines.3.Cell experiment:AML12 cells were used as the research object,Irgm1short hairpin RNA（sh RNA）was used to investigate the effects of downregulation of Irgm1 on autophagy and inflammation in vitro.To establish a hepatocyte injury model induced by LPS（1ug/ml）for 12h,autophagy was induced by RAPA in vitro.According to the intervention,the cells were divided into the normal control group（PBS+AML12）,model group（LPS+AML12）,Irgm1sh RNA intervention group(LPS+AML12^-Irgm1-),Control sh RNA group(LPS+AML12^NC)and RAPA intervention group(RAPA+LPS+AML12^-Irgm1-).The activities of ALT and AST in the supernatant were detected by ALT and AST kits.The m RNA expression of Irgm1,LC3II,NLRP3 inflammasome,and pro-inflammatory cytokines was detected by q RT-PCR,and the protein expressions of Irgm1,LC3II,P62,and NLRP3 were detected by Western blot.Results:1.Clinical study:1).Serum levels of ALT,AST,Tbil,and DBil were significantly elevated in HBV-ACLF（117.13±102.03,134.93±130.20,255.35±123.51,179.17±95.67）compared with healthy controls（20.53±7.57,22.43±7.79,7.66±1.93,4.11±1.52,（P<0.05）.2）.The protein levels of IRGM were decreased in liver tissue of patients with HBV-ACLF（0.30±0.32）than those of healthy controls（1.19±0.15）,（P<0.01）3).The protein levels of NLRP3,ASC and Caspase-1 were higher in liver tissue of patients with HBV-ACLF（1.48±0.21,2.84±0.51,1.62±0.37）than those of healthy controls（0.64±0.10,0.24±0.09,0.14±0.06,（P<0.05）.Serum levels of pro-inflammatory cytokines,IL-1β,and TNF-αwere significantly higher in HBV-ACLF patients（36.27±26.50,36.46±30.50）than healthy controls（8.71±1.27,5.47±0.80）.4）.The protein levels of LC3II were decreased in liver tissue of patients with HBV-ACLF（0.41±0.10）than those of healthy controls（1.24±0.17）,and the protein levels of P62 were higher in HBV-ACLF patients（1.43±0.20）than controls（0.24±0.06）,（P<0.05）.5).The expression levels of IRGM were negatively correlated with the IL-1β,TNF-α,NLRP3,ASC,and Caspase-1 levels（r=-0.57,-0.52,-0.77,-0.82,-0.76）,（P<0.05）.The expression levels of IRGM were negatively correlated with the AST,Tbil,and DBil levels（r=-0.52,-0.88,-0.83）,（P<0.05）.The expression levels of IRGM were positively correlated with the LC3II levels（r=0.81）and were negatively correlated with the P62 levels（r=-0.89）（P<0.01）.2.Animal experiments:1).The ALF mouse model was successfully established by intraperitoneal injection of D-Ga IN/LPS.It can be seen that the liver of ALF mice is larger than that of normal mice.HE staining showed loss of cell integrity,large areas of hepatocyte necrosis,and inflammatory infiltration in the livers in the ALF group.The serum ALT and AST in ALF（930.13±74.35,907.01±60.10）were significantly elevated compared with the control group（38.52±9.20,40.31±9.06）,（P<0.01）.2).Immunohistochemistry showed that Irgm1 was mainly expressed in hepatocytes.Compared with the control group（1.38±0.18）,the expression of Irgm1 protein in the ALF group（0.70±0.11）was significantly decreased,while the expression level of the Irgm1 m RNA in ALF（0.45±0.22）was down-regulated compared to the control group（1.60±0.89）,（P<0.05）.3).The protein expression levels of NLRP3,ASC and Caspase-1 were higher in the ALF group（1.78±0.19,2.42±0.56,2.29±0.47）compared with the control group（0.47±0.15,1.12±0.36,0.49±0.11）（P<0.05）.The m RNA expression levels of NLRP3,ASC,Caspase-1 and IL-1β,TNF-αwere higher in the ALF group（9.58±3.36,67.10±25.83,58.40±23.12,8.71±1.77,10.70±4.02）compared with the control group（1.25±1.07,1.19±0.77,1.82±1.64,0.46±0.15,1.11±0.37）,（P<0.05）.4).The expression of LC3II protein in ALF（0.91±0.41）was lower than the control group（2.28±0.30）,while the expression of p62 protein in ALF（1.73±0.32）was higher than the control group（0.80±0.04）,（P<0.05）.The expression of LC3II m RNA in the ALF group（0.05±0.04）was significantly lower than that in the control group（0.83±0.25）,（P<0.01）5).The general morphology of the liver in the RAPA group tended to be normal,HE staining showed that the structure of the hepatic lobule tended to be complete,and the infiltration of inflammatory cells in the lobule and portal area was significantly less than that in the ALF group.The levels of serum ALT and AST in the RAPA group（68.54±14.10,53.53±9.68）were lower than ALF（930.13±74.35,907.01±60.10）,（P<0.01）.6).The expression of LC3II protein the in RAPA group（2.72±0.54）was higher than ALF group（0.91±0.41）,while the expression of p62 protein in the RAPA group（0.93±0.28）was lower than ALF group（1.73±0.32）,while the expression level of the LC3II m RNA in RAPA（0.38±0.22）was higher compared to ALF group（0.05±0.04）,（P<0.05）.7).The protein expression levels of NLRP3,ASC and Caspase-1 were decreased in the RAPA group（1.02±0.07,1.03±0.10,1.00±0.14）compared with the ALF group（1.78±0.19,2.42±0.56,2.29±0.47）,（P<0.05）.The m RNA expresssion levels of NLRP3,ASC,Caspase-1 and IL-1β,TNF-αwere decreaesd in RAPA group（3.54±2.10,1.90±1.09,3.19±1.33,1.34±0.15,1.50±1.12）compared with the ALF group（9.58±3.36,67.10±25.83,58.40±23.12,8.71±1.77,10.70±4.02）,（P<0.05）.3.Cell experiment:1).AML12 hepatocyte injury model was induced by LPS in vitro.Irgm1 sh RNA knockdown was successful and was verified by western blot analysis.The levels of ALT and AST in the supernatant of hepatocyte injury model group（22.20±2.91,24.09±4.40）were higher than Control（8.03±0.97,9.59±1.71）,（P<0.05）.The protein expression levels of NLRP3,ASC,and Caspase-1 were higher in the hepatocyte injury model group（1.05±0.08,0.99±0.05,0.49±0.01）compared with the control group（0.25±0.04,0.52±0.02,0.24±0.01）,（P<0.05）.The m RNA expression levels of NLRP3,ASC and Caspase-1 were higher in the hepatocyte injury model group（6.16±0.74,6.30±0.71,5.32±0.88）compared with the control group（1.16±0.76,1.13±0.66,1.15±0.74）,（P<0.05）.2).The LC3II protein in Irgm1 sh RNA intervention group（0.42±0.02）was significantly lower than that in the hepatocyte injury model group（1.14±0.10）and Control group（0.71±0.05）,while p62 protein in Irgm1sh RNA intervention group（1.11±0.23）was significantly higher than that in hepatocyte injury model group（0.07±0.01）and Control groups（0.35±0.07）,（P<0.05）.The m RNA expression levels of LC3II were decreased in the Irgm1 sh RNA intervention group（0.25±0.10）compared with the hepatocyte injury model group（1.19±0.07）and control group（1.00±0.08）,（P<0.01）.3).The levels of ALT and AST in the supernatant of Irgm1 sh RNA intervention group（71.66±9.01,89.05±6.80）were higher than hepatocyte injury model group（22.20±2.91,24.09±4.40）and Control group（8.03±0.97,9.59±1.71）,（P<0.01）.4).The protein expression levels of NLRP3,ASC and Caspas-1 were higher in the Irgm1sh RNA intervention group（3.73±0.21,2.07±0.05,2.09±0.07）compared with the hepatocyte injury model group（1.05±0.08,0.99±0.05,0.49±0.01）and control group（0.25±0.04,0.52±0.02,0.24±0.01）,（P<0.05）.The m RNA expression levels of NLRP3,ASC and Caspase-1 were higher in the Irgm1sh RNA intervention group（25.51±4.17,37.71±5.07,9.65±1.21）compared with the hepatocyte injury model group（6.16±0.74,6.30±0.71,5.32±0.88）and control group（1.16±0.76,1.13±0.66,1.15±0.74）,（P<0.01）.The m RNA expression levels of IL-1βand TNF-αwere higher in the Irgm1 sh RNA intervention group（14.18±5.28,16.01±1.01）compared with the hepatocyte injury model group（1.25±0.52,1.56±0.27）and control group（1.10±0.57,1.11±0.63）,（P<0.01）.5).After RAPA intervention,the protein expression levels of LC3II were higher in the RAPA group（5.57±0.56）compared with the Irgm1 sh RNA intervention group（0.42±0.02）,the protein expression levels of P62 were decreased in the RAPA group（0.06±0.06）compared with the Irgm1 sh RNA intervention group（1.11±0.23）,（P<0.01）.The m RNA expression levels LC3II were higher in the RAPA group（11.58±1.57）compared with the Irgm1 sh RNA intervention group（0.25±0.10）,（P<0.01）.6).The serum ALT and AST in RAPA group（11.04±1.94,11.14±1.71）were significantly decreased compared with the Irgm1 sh RNA intervention group（71.66±9.01,89.05±6.80）,（P<0.01）.7).The protein expression levels of NLRP3,ASC and Caspase-1 were decreaesd in the RAPA group（0.97±0.14,0.91±0.11,0.54±0.06）compared with the Irgm1 sh RNA intervention group（3.73±0.21,2.07±0.05,2.09±0.07）,（P<0.01）.The m RNA expression levels of NLRP3,ASC and Caspase-1 were decreaesd in the RAPA group（1.20±0.60,1.74±0.92,3.78±0.30）compared with the Irgm1 sh RNA intervention group（25.51±4.17,37.71±5.07,9.65±1.21）,（P<0.01）.The m RNA expression levels of IL-1βand TNF-αwere decreased in the RAPA group（0.28±0.28,0.26±0.25）compared with the Irgm1 sh RNA intervention group（14.18±5.28,16.01±1.01）,（P<0.01）.Conclusion:1.IRGM/Irgm1 and autophagy is suppressed in liver during the procession of LF,which is related to the degree of inflammatory injury in liver tissue.2.IRGM/Irgm1 plays a protective role on hepatocytes by inhibiting the activation of NLRP3 inflammasome pathway to reduce the immune inflammatory response of LF.3.IRGM/Irgm1 attenuates inflammation by regulating autophagy in LF.