Btk Inhibitors Alleviate Acute Liver Failure In Mice And Its Mechanism Investigation

Background and aimsLiver failure(LF)is a clinical syndrome of severe liver dysfunction due to death of massive hepatocytes,posing a serious hazard to human health around the world.Until now,there is no effective drug treatment for LF in the clinic.In recent years,a new insight into the LF mechanisms is the emerging role of liver injury caused by immune response.Both innate and adaptive immunity are involved in the progression of LF.As a member of Tec family,Bruton's tyrosine kinase(Btk)is generally expressed in all hematopoietic lineages except for T cell and plasma cell.A large number of studies have shown that Btk plays an important role in regulating immune responses and the expressions of inflammatory factors.This study focuses on the potential protective effects of Btk inhibitors on acute liver failure(ALF).Based on clarifying the protective effects of Btk inhibitors on two different mice models of-ALF,we further explore its possible involved mechanisms.The aim of this research is to verify that Btk functions as a promising target for the therapy of ALF,providing a new approach for the prevention and treatment of LF.MethodsPart 1:After two difierent Btk inhibitors(Ibrutinib and Acalabrutinib)were administered at different doses,models of ALF in mice were established by using Lipopolysaccharides(LPS)combined with D-galactosamine(D-GalN),and Concanavalin A(ConA).The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in mouse serum were detected by automatic chemical analyzer,and liver pathological characters were analyzed by hematoxylin-eosin staining.The expressions of cytokines in mouse livers,RAW264.7 cells and human primary CD 14+monocytes were measured by real-time PCR and enzyme-linked immune sorbent assay(ELISA).Part 2:The expression of Btk was determined by immunohistochemistry analysis of human liver tissue and Western Blot analysis of Romas cells,THP-1 cells and L02 cells.The expression of p-Btk under inflammatory stimuli was confirmed by Western Blot and flow cytometry.The mRNA levels of various cytokines in RAW264.7 cells stimulated by LPS were measured by real-time PCR.The expression of NOD-Like Receptor Protein 3(NLRP3)and nuclear factor-KB(NF-?B)was detected by Western Blot,and the expression level of components in NLRP3 inflammasome was verified by real-time PCR.CD69 expressed on T cell subsets,CD80 and CD86 on B cells and dendritic cells(DCs)were analysed by flow cytometry.ResultsPart 1:Btk inhibitors reduced the elevation of liver enzymes and alleviated hepatocyte necrosis and inflammatory infiltration in both LPS/D-GalN-and ConA-induced mice models.Btk inhibitors downregulated the expression levels of TNF-? IL-1?,IL-6 and IL-10.Part 2:Btk was not expressed in hepatocytes.Inflammatory stimuli caused dramatic changes in Btk phosphorylation.In addition to TNF-?,IL-1(3,IL-6 and IL-10,Btk inhibitors also downregulated the expressions of IL-2,IL-4,IFN-? and IL-12.Btk inhibitors reduced the expression of IL-1? via suppressing the activation of NLRP3 inflammasome.Btk inhibitors attenuated T cell activities by reducing the expression of CD86 on B lymphocytes and DCs.Conclusions1)Btk inhibitors were first demonstrated to play protective roles on ALF in mice,and Btk is a feasible and effective drug target for the treatment of ALF.2)Btk inhibitors reduce the expression of IL-1? by suppressing the activation of NLRP3 inflammasome,and alleviate cytokine storm,playing protective roles on death of massive hepatocytes.3)Btk inhibitors attenuate T cell activities by inhibiting antigen presentation function,alleviating T cell-induced liver injury in mice.