Thioredoxin Interacting Protein-NLRP3 Inflammasome Pathway Involved In Liver Failure And Being Regulated By Endoplasmic Reticulum Stress

Background:Liver failure is a severe syndrome with a variety of causes,including viruses,drugs,hepatotoxicity,alcohol,and genetic factors.The pathogenesis of hepatic failure is mainly due to the disorders caused by the mentioned factors or decompensation of the liver function.The mortality of liver failure in extremely high,and the clinical features include coagulation dysfunction,jaundice,hepatic encephalopathy,ascites,etc.Epidemiological data shows that the main cause of liver failure is HBV infection in China and the most common subtype of liver failure is acute on chronic liver failure(ACLF).The role of NLRP3 inflammasome in various liver diseases has received increasing attention.NLRP3 inflammasome consists of the NOD-like receptor NLRP3,the adaptor molecule ASC and the effector molecule pro-Caspase-1.When the NLRP3 forms a complex with the adaptor molecule ASC,pro-Caspase-1 is activated to become mature form(Caspase-1)and eventually leads to activation of NLRP3 inflammasome and release of IL-18and IL-1β.At present,several studies have confirmed that the activation of NLRP3inflammasomeplaysanimmunoregulatoryrolein acetaminophen-induced acute liver injury,but currently the role and specific regulation factors of NLRP3 inflammasome in the pathogenesis of liver failure is still not clear.The activation of NLRP3 inflammasome is a two-steps process:Firstly,bacterial signals including lipopolysaccharide can upregulate the expression of NLRP3 through an NF-κB-dependent pathway,followed by a second-step process that can trigger the activation of NLRP3 through multiplepathways.Oneoftheimportantpathwaysisthe thioredoxin-interacting protein(TXNIP)related pathway.Under a variety of precipitating stimuli,TXNIP disassociates from the protein complex and binds to NLRP3,triggering its activation,and finally regulating the activation process of NLRP3 inflammasome.However,the role of this phenomenon in liver failure has not been fully studied yet.There is a close relationship between ERS and TXNIP-NLRP3 inflammasome pathways.Endoplasmic reticulum stress(ERS)is able to induce the maturation and release of pro-IL-1βby activating the NF-κB pathway,and this process relies on the TXNIP-mediated activation of the NLRP3 inflammasome.Our team has also confirmed that ERS can play an important role in the pathogenesis of acute hepatic failure by regulating peroxisome proliferator-receptor alpha function in previous studies.But the exactly regulating function of ERS with TXNIP-NLRP3 inflammasome pathway has not yet been fully understood.Part One NLRP3 inflammasome activation and TXNIP expression in HBV related acute on chronic liver failure patientsObjection:In the presented study,we collected plasma samples from chronic hepatitis B(CHB)patients and early stage HBV related acute on chronic liver failure(HBV-ACLF)patients and peripheral blood mononuclear cells(PBMCs)to evaluate the biochemical markers,NLRP3inflammasome associated molecules and the expression of TXNIP.Correlation analysis were performed between NLRP3 inflammasome related molucules,TXNIP and liver injury markers.Methods:1 Evaluation of NLRP3 Inflammasome activation and TXNIP Expression in HBV-ACLF patientsThirty patients,including 10 healthy patients,10 chronic hepatitis B patients and 10 early-stage HBV-ACLF patients,were enrolled in this study.The diagnosis of chronic hepatitis B and HBV-ACLF are according to the"Guidelines for the Prevention and Treatment of Chronic Hepatitis B(2015Update)"and the"Guidelines for the Diagnosis and Treatment of Liver Failure(2012 Edition)".2 Biochemical markers were analyzed by a automatic chemical analyzer.3 The peripheral blood mononuclear cells(PBMCs)of all subjects were isolateded to evaluate the expression of NLRP3 inflammasome related molecules and TXNIP.Results:1.Population characteristics and biochemical characteristics of the all enrolled subjectsALT,AST,TB,and DB in the HBV-ACLF group were significantly higher than those in the CHB group and the Control group,and the TB levels in the CHB group were significantly higher than those in the Control group(P<0.05 or 0.01).2.NLRP3 inflammasome activation and expression of TXNIP were elevated in HBV-ACLF patientsNLRP3 inflammasome related ASC mRNA,NLRP3 mRNA,Caspase-1mRNA,IL-18 mRNA and IL-1βin HBV-ACLF group were significantly inceased compared with Control group and CHB group,P all<0.01.The serum concentration of IL-18 and IL-1βin HBV-ACLF group was significantly higher than those in the Control group and CHB group,P all<0.01.The expression of TXNIP mRNA in the PBMC of HBV-ACLF patients was significantly higher than that of the Control group and the CHB group,P all<0.01.3.The correlation analysis showed that the expression of TXNIP mRNA in PBMCs was positively correlated with ALT,AST,TB,and DB levels(r=0.8161,P<0.01;r=0.7597,P<0.01;r=0.8171,P<0.01;r=0.7972,P<0.01).There was a positive correlation between IL-1βconcentration in the peripheral blood and ALT levels(r=0.78,P<0.01).Summary:NLRP3 inflammasome activation and elevated expression of TXNIP were detected in HBV-ACLF patients,which were related with the liver injury.Part two Role of TXNIP-NLRP3 inflammasome pathway in acute liver failure miceObjection:A mouse model of acute liver failure was constructed and transduced with the adeno-associated virus vector to knock down the expression of TNXIP with TXNIP sh RNA in some mice to investigate the role of the TXNIP-NLRP3 inflammasome pathway in the pathogenesis of acute liver failure in mice.Methods:1 Transfection of mice with AAV-TXNIP shRNA60 healthy male BALB/c mice aged 8 weeks and weighing 20-25 g were randomly divided into normal control group(NC,n=20),TXNIP shRNA group(transfected with TXNIP shRNA to knockdown TXNIP expression,n=20)and Control shRNA group(transfected with control shRNA,n=20).Three mice in each group were sacrificed after anesthesia to evaluate the knockdown efficiency of TXNIP shRNA.The remaining mice were treated with D-GalN/LPS to evaluate the effect of TXNIP knockdown on the liver failure induced by D-GalN/LPS.2 Establishment of D-Gal N/LPS induced acute liver failure modelTwenty-seven male BALB/c mice aged 12 weeks and weighing 20-25 g were randomly divided into healthy control group(NC,n=10)and acute liver failure model group(D-GalN/LPS,n=17).Corresponding drug treatment was given together with mice in TXNIP shRNA group and the Control shRNA group described above:mice in the NC group were given intraperitoneal injections of PBS;mice in the D-GalN/LPS group,TXNIP shRNA group,and Control sh RNA group were given intraperitoneal injections of D-GalN/LPS.Ten mice in each group were used to observe the general state and 24-hour survival rate.The remaining mice were sacrificed 6 hours after the D-Gal N/LPS injection.Plasma and tissues were obtained for HE,TUNEL staining,qPCR or Western Blot tests.Results:1.Transfection of TXNIP sh RNA significantly inhibits TXNIP expression in miceUsing AAV as a vector to transfect mice with TXNIP shRNA,the expression of TXNIP were significantly lower than those in normal control mice.Among them,the suppression in liver was the most obvious,P<0.01.2.Knockdown of TXNIP protected mice form ALF induced by D-GalN/LPSThe 24-hour survival rate of mice in TXNIP shRNA group was 60%,significantly higher than that in D-GalN/LPS group,P=0.044;The levels of ALT and AST in mice in TXNIP shRNA group were significantly lower than those in D-GalN/LPS group(P<0.01).HE staining showed that the liver injury of mice in TXNIP shRNA group was significantly relieved.3.Knockdown of TXNIP significantly reduced the hepatocyte apoptosis in ALF miceApoptosis of hepatocytes was detected by TUNEL staining.The results showed that the ratio of TUNEL positive cells in the liver of TXNIP shRNA group was significantly lower than that in D-GalN/LPS group,P<0.01.4.Knockdown of TXNIP inhibited NLRP3 inflammasome activationThe mRNA and protein expressions of NLRP3 inflammasome associated molecules in the liver of mice in TXNIP shRNA group was significantly lower than that in D-GalN/LPS group,and the interaction between NRLP3inflammasome related molecule ASC and NLRP3 was interfered.The plasma concertrations of IL-18 and IL-1β,markers of NLRP3 inflammasome activation,in TXNIP shRNA gourp was significantly lower than those in D-GalN/LPS group,P all<0.01.Summary:1.Intraperitoneal injection of D-GalN/LPS successfully constructed mouse model of acute liver failure.2.NLRP3 inflammasome was activated in ALF mice.3.Transfection of TXNIP shRNA with AAV significantly inhibited the expression of TXNIP in mice,which was mostly suppressed in liver.4.Knockdown of TXNIP expression significantly reduced NLRP3inflammasome activation in mice,reduced hepatocyte injury,and improved24-hour survival in ALF mice,suggesting that the TXNIP-NLRP3inflammasome pathway acts as a regulatory role in the pathogenesis of ALF.Part three Effects of endoplasmic reticulum stress on the TXNIP-NLRP3 inflammasome pathway in acute liver failure miceObjection:The effect of endoplasmic reticulum stress(ERS)on the TXNIP-NLRP3 inflammasome pathway was observed using a cell co-culture model.Methods:Firstly,qPCR and Western blot were performed to detect ERS in acute hepatic failure model mice.Then MACS was used to sort mice primary splenic macrophages(F4/80~+cells were defined as macrophages)in ALF mice.Transwell was used to constructa co-culture system of primary splenic macrophages and mouse hepatocyte cell line AML-12 cells.Tunicamycin(TM),an ERS inducer,or DMSO was used to induce ERS,observing the effect of ERS to TXNIP-NLRP3 inflammasome pathway.The specific cell grouping were:DMSO+AML-12+NC Mφgroup;TM+AML-12+NC Mφgroup;DMSO+AML-12+TXNIP shRNA Mφgroup;TM+AML-12+TXNIP shRNA Mφgroup.Results:1.Expression of GRP78 and CHOP were significantly elevated in ALF mice;after the knockdown of TXNIP expression,ERS was obviously relieved.2.Application of TM could induce ERS in AML-12 cells.The expressions of GRP78,CHOP mRNA and protein in AML-12 cells were significantly higher than those in the control group,P<0.01.3.Activate ERS to reverse the protective effect of TXNIPThe results of CCK8 showed that there was no significant difference in the survival rate of AML-12 cells in AML-12+TXNIP shRNA Mφco-culture system and AML-12+NC Mφco-culture system after application of TM to induce ERS in AML-12 cells,and TUNEL staining revealed no significant difference in apoptosis between the two groups.4.Activation of ERS reversed the suppression of inhibition effect on NLRP3 inflammasome induced by TXNIP knockdownAfter treatment with TM,there was no difference in mRNA and protein expression levels of NLRP3 inflammasome associated molecules between AML-12+TXNIP shRNA Mφand AML-12+NC Mφco-culture system,and supernatant IL-18,IL-1βconcentration showed no difference.Summary:1.ERS in liver tissue of ALF mice was significantly enhanced,and knockdown of TXNIP significantly relieved the ERS and decreased the aptosis of hepatocytes,suggesting that inhibition of TNXIP can reduce hepatocyte apoptosis by alleviating hepatocyte ERS.2.Application of TM,activating the cellular ERS,reversed inhibition effect on NLRP3 inflammasome activation induced by TXNIP knockdown.3.Application of TM to activate cellular ERS could reverse the protective effect induced by TXNIP knockdown against ALF.4.ERS may play a role in the pathogenesis of acute hepatic failure,and may be related to the regulation of TXNIP-NLRP3 inflammasome pathway.Conclusions:1.NLRP3 inflammasome was activated in HBV-ACLF patients along with elevated expression of TXNIP,which were related with liver injury.2.The activation of NLRP3 inflammasome in ALF mice was significant.TXNIP-knockdown in mice by TXNIP sh RNA significantly inhibited the activation of NLRP3 inflammasome in ALF mice,reducing NLRP3inflammasome-associated liver injury,and furtherly improving survival.3.Typical ERS response was observed in ALF mice.Knockdown of TXNIP in mice can significantly relieve ERS in hepatocytes and decrease the apoptosis,which makes the inhibition of TXNIP-mediated NLRP3inflammasome activation and the related protective effect disappear.4.TXNIP-NLRP3 inflammasome pathway was involved in the pathogenesis of liver failure and its function could be regulated by ERS.