Role Of NLRP3 Inflammasome On The Pathogenesis Of HBV Related Acute-on-chronic Liver Failure

Objective: Hepatitis B virus(HBV)related acute-on-chronic liver failure(ACLF)is a life threatening syndrome,with poor prognosis and high mortality,which is characterized by damaged hepatic function and multiple organ dysfunction,many scholars believed that immune injury play a important role on the development of liver failure.Immune dysfunction may occur in the peripheral blood,however,there is no clear indicators to quantify the state of immune.NLRP3 inflammasome are intracellular multiprotein complexes which is the part of innate immunity.The function of promoting inflammation had been reported about acute liver failure,but the relationship between HBV related ACLF and NLRP3 remains unclear.Hence,our study aims to explore the impact of NLRP3 in the Hepatitis B virus related acute-on-chronic liver failure by investigating the levels of NLRP3 and NLRP3 related components in peripheral blood mononuclear cell and liver tissues among the different research objects.Method: Total 75 subjects were enrolled in our study and divided into 3groups including 30 HBV related acute on chronic liver failure patients,30 chronic hepatitis B patients and 15 healthy controls.At the same period,8liver tissues were obtained from HBV related ACLF patients,8 CHB patients and 5 liver donors by liver transplantations and liver biopsy.Liver function parameters,coagulation function parameters,serum virology and serum HBV DNA loads were determined.The expression of NLRP3 were analyzed by flow cytometry.In addition,the NLRP3,Caspase-1,IL-1β,IL-18 mRNA expression in PBMC(include the whole PBMC,CD14+monocyte and CD14-PBMC)and liver tissues were analyzed by RT-qPCR.Further,immunohistochemical staining was performed to illustrate the distributions and amounts of NLRP3 in different groups and the co-expression of NLRP3 and CD68.Finally,we made correlation analysis between NLRP3 and MELD score.Results:1 General informationThe demographic data of the subjects were comparable for age and sex.The ACLF patients displayed obviously higher levels of ALT,AST,TB,DB and INR,but a lower ALB levels and HBV DNA loads(P<0.01).2 HBV related ACLF patients had lower levels of NLRP3 in PBMC.The levels of NLRP3 were measured by flow cytometry,It was suggested that the levels of NLRP3 in HBV related ACLF patients were lower than CHB patients and healthy controls,and compared with healthy controls,the NLRP3 levels of CHB patients were also lower(P<0.01).Then,compared with early stage of ACLF patients,middle and late stage of ACLF patients had lower expression of NLRP3(P<0.01).In PBMC,the expression of NLRP3 mRNA in HBV related ACLF patients were obvious lower than CHB patients and healthy controls(P<0.05),compared with healthy controls,the NLRP3 levels of CHB patients were also lower(P<0.01).3 NLRP3 were mainly expressed in CD14+CD16+monocytes.Subgroup analysis:on CD14++CD16-monocytes CD16++CD14-monocytes,groups of data in each group of between comparisons,not obvious difference,non-statistics significance(P>0.05).HBV related ACLF patients and CHB patients had lower expression of NLRP3 on CD14+CD16+monocytes than healthy controls(P<0.01),especially in the middle and late ACLF patients.On CD14+monocytes,The levels of NLRP3 mRNA in HBV related ACLF patients and CHB patients were lower than healthy controls(P<0.05),groups of data in CD14-PBMC of between comparisons,not obvious difference,non-statistics significance(P>0.05).4 NLRP3 were expressed higher in liver tissues.By the immunohistochemistry,the higher expression of HBV related ACLF patients were observed compared with NLRP3 in CHB patients(P<0.01).There was no expression of NLRP3 in healthy controls.The expression of NLRP3 were gathered in interlobular portal areas and hepatic lobule,especially in hepatic lobule.NLRP3 were showed mainly in kuffer cell cytoplasm by co-expression of CD68.By RT-qPCR,The higher expression of NLRP3 mRNA in HBV related ACLF patients and CHB patients were observed compared with healthy controls(P<0.01),particular in HBV related ACLF patients(P<0.01).5 HBV related ACLF patients had lower levels of NLRP3 related gene in PBMC.The expression of Caspase-1 mRNA and IL-1β mRNA in PBMC of HBV related ACLF patients were lower than CHB patients(P<0.05).there was no difference in IL-18 and there had no statistically significant between HBV related ACLF patients and healthy controls(P>0.05).What is more,the expression of Caspase-1 mRNA,IL-1β mRNA and IL-18 mRNA in CD14+monocytes of HBV related ACLF patients were lower than CHB patients and healthy controls(P<0.05),the expression of Caspase-1mRNA,IL-1β mRNA and IL-18 mRNA in CD14-PBMC as well(P < 0.05).There had no statistically significant between HBV related ACLF patients and healthy controls(P>0.05).6 HBV related ACLF patients had higher levels of NLRP3 related gene in liver tissues.In liver tissues,the expression of IL-1β mRNA and IL-18 mRNA in HBV related ACLF patients and CHB patients were obvious higher than healthy controls(P<0.01),particular in HBV-related ACLF patients,But the expression of Caspase-1 mRNA in HBV related ACLF patients had no statistically significant with CHB patients(P>0.05).7 The relationship between NLRP3 and MELD scoreThe expression of NLRP3 in ACLF patients CD14+CD16+ monocytes were negatively correlated with the MELD score(P<0.01).Conclusions:1 NLRP3 express lower in the peripheral blood of HBV related ACLFand may be correlated with immunosuppression.2 NLRP3 were mainly expressed in CD14+CD16+monocytes,they may be gathered to liver to damage hepatocyte.