The Role Of Bruton’s Tyrosine Kinase In Acetaminophen-Induced Acute Liver Injury In Mice And Its Possible Mechanism

Objective(1)To observe the dynamic changes of Bruton’s tyrosine kinase(Btk),p-Btk,NF-κB,NLRP3,IL-1β,TNF-α in acute liver injury induced by Acetaminophen(APAP)in mice,and preliminarily explore the role of Btk in acute liver injury induced by APAP.(2)To investigate the effect of Btk inhibitor(LFM-A13)on acute liver injury induced by acetaminophen(APAP)in mice and its possible mechanism.Methods(1)54 SPF C57BL/6J male mice were randomly divided into nine groups,including normal control group,APAP 0.5h group,APAP1 h group,APAP3 h group,APAP6 h group,APAP12 h group,APAP24 h group,APAP36 h group and APAP48 h group.After fasting for 12 h,mice in the corresponding groups were sacrificed after a single intraperitoneal injection of APAP(300mg/kg)for 0.5h,1h,3h,6h,12 h,24h,36 h and48h.Serum ALT and AST were measuredby chemical method.Liver tissue damage was observed by HE staining.IL-1β and TNF-α in liver tissue homogenate were measured by ELSIA.The expressionof Btk,p-Btk,NF-κB and NLRP3 in liver was determined by western blot.(2)32 SPF C57BL/6J male mice were randomly divided into four groups: normal control group,model group(APAP group),LFM-A13 low-dose group(LFM-A13(3mg/kg)+APAP),and LFM-A13 high-dose group(LFM-A13(30mg/kg)+APAP),with8 mice in each group.After 12 h of fasting,mice in the normal group were intraperitoneally injected with 0.9% normal saline,mice in the model group were givensingle intraperitoneal injection of APAP(300mg/kg),mice in the LFM-A13 low-dose group and in the LFM-A13 high-dose group were intraperitoneally injected with LFM-A13 3mg/kg and LFM-A13(30mg/kg)1h before single intraperitoneal injection of APAP,respectively.Mice in each group were sacrificed after single intraperitoneal injection of APAP for 24 h.serum ALT and AST in each group were measured.Pathological changes of liver in each group were observed,HAI scores were calculated for each group.IL-1β and TNF-α-in liver tissue homogenate were measured by ELSIA.The expressions of Btk,p-Btk,NF-κB and NLRP3 in liver were determined by Western blot.Results(1)Compared with the normal control group,serum ALT(F=100.968,P=0.000),AST(F=73.539,P=0.000),liver necrosis degree(F=29.845,P=0.000),liver Btk(F=441.280,P=0.000),p-Btk(F=1323.022,P=0.000),NF-кB p65(F=133.125,P=0.000),NLRP3levels(F=820.095,P=0.000)and The contents of IL-1β(F=8.643,P=0.000),TNF-α(F=20.545,P=0.000)were all changed.Among them,the various kinds index of 24 h was respectively the highest.(2)Compared with the normal control group,serum ALT and AST were significantly increased in APAP group(P<0.01).Compared with the APAP group,the serum ALT and AST of mice in the LFM-A13 intervention group decreased significantly,and the decrease was the most significant in the LFM-A13 low-dose group(P<0.01).In the APAP group,hepatic sinus congestion was evident,with extensive necrosis,mainly manifested as lobular central necrosis.Hepatic sinus congestion and necrosis area were significantly reduced in all LFM-A13 intervention groups,histological activity index score in the APAP group was significantly higher than that in the normal group(P<0.01),and HAI score in the LFM-A13 intervention group was significantly lower than that in the APAP group(P<0.01).The levels of IL-1β and TNF-α in APAP groupwere significantly higher than those in normal group and LFM-A13 intervention group(P<0.01).Western blot indicated that the p-Btk/Btk expression gray ratio,NF-кB p65 and NLRP3 in APAP group were higher than those in normal group(p<0.01).,the corresponding indexes of each LFM-A13 intervention group were significantly lower than that of the APAP group(P<0.05).Conclusion(1)Liver injury induced by single intraperitoneal injection of APAP showed dynamic changes;(2)Increased Btk expression and its phosphorylation,NLRP3 inflammasome formation,NF-κB activation,and IL-1β and TNF-α levels were observed in APAP-induced acute liver injury.(3)LFM-A13 can alleviate acute liver injury induced by APAP in mice,and its mechanism may be related to the inhibition of Btk expression and its phosphorylation,reduction of NLRP3 inflammasome formation,NF-κB activation,and elevation of IL-1β and TNF-α.