Effect Of Tumor Necrosis Factor-alpha On Expression Of Ammonia Transporters In Brain Tissue Of Mouse With Acute Hepatic Encephalopathy

Objective:Acute liver failure?ALF?is a devastating disease and associated with a high mortality.Hepatic encephalopathy?HE?is the most severe complication and major cause of death of ALF.The mechanism of HE was not completely clear while ammonia toxicity was still regarded as the leading cause.Ammonia is a weak base?pKa 9.25 at 25°C,9.15 at 37°C?.At physiologic pH value of 7.4,ammonia is protonated?NH₄+?and only less than 2%of it exists in the deprotonated form?NH3?.The free base ammonia?NH₃?is lipophilic and can passively penetrate cell membranes?Only recently,the human nonerythroid Rhesus?Rh?glycoprotein B?RhBG?and the nonerythroid Rh glycoprotein C?RhCG?as well as their non-human mammalian homologues?Rhbg/Rhcg?,members of the Amt/MEP/Rh family,have been identified as mammalian ammonium/ammonia transporters.Up to now,there is few study on the expression of ammonia transporters in brain tissues especially in acute liver failure induced hepatic encephalopathy.TNF?plays an important role on acute liver failure with various causes.In clinical and experimental studies,results have also shown that TNF?was involved in the pathogenesis of HE.Indeed,TNF?increases the ammonia diffusion in the central nervous system-derived endothelial cells,increasing ammonia concentration in the brain by this action,and consequently increasing ammonia toxicity to the central nervous system.We aim to investigate the effect of TNF?on expression of ammonia transporters in brain tissues of acute liver failure model.Materials and methodsMale C57BL/6 mice were divided into eight groups.group 1:saline control group;group 2:D-GalN 300 mg/Kg control group;group 3:LPS control group;group 4:D-GalN+LPS group;group 5:D-GalN+TNF-?group;group 6:TNF-?control group;group 7:D-GalN 600 mg/Kg control group;group 8:TNF-?IgG antibody group?anti-TNF-?+D-GalN+LPS group?.Group 1 was 0h group,group 2 to group 5 were divided into 5 time points?2h,6h,9h,12h,24h?.In Group 6 to group 8,we chose 12h as the observation time.There were 5 mice at every time point in every group.Mice were sacrificed to remain blood sample,brain tissues and liver tissues to determine relevant indexes.Serum levels of alanine transaminase?ALT?were tested by biochemical method and HE staining was carried out in liver tissue to determine the pathological and biochemical changes of liver in acute liver failure.The levels of serum TNF?were determined by ELISA method.Blood ammonia and brain ammonia concentrations were detected by ammonia assay kit.Western blot analysis was performed to determine the semiquantitative expression of ammonia transporters Rhbg/Rhcg protein.RT-PCR was performed to determine the semiquantitative expression of ammonia transporters Rhbg/Rhcg mRNA.Immunohistochemistry and immunofluorescence analysis were performed to ovserve the location of Rhcg in mice brain tissues.Results1.The establishment of ALF mice models:Mice had poor appetite and activity gradually after administration with LPS/D-GalN.The serum levels of ALT began to increase at 2h and reach a peak level at 6 hours.The liver shown massive or submassive necrosis at 9h and 12hrs.The pathological changes resembled as the changes of fulminant hepatic failure in human.The total mortality was 60%.Animals also appeared similar appearance after administration with TNF?/D-GalN.There were no death in mice pretreated with anti TNF?antibody.Serum ALT levels reduced significantly and the liver histological analysis showed significant lighten.2.Blood ammonia and brain ammonia concentrations in acute liver failureThe blood ammonia levels began to increase at 6h in D-GalN/LPS group,and reach a peak level at 9h and 12h.The brain ammonia levels began to increase at 2h in D-GalN/LPS group,and reach a peak level at 12h.Both the blood ammonia and brain ammonia levels began to increase at 2h in D-GalN/TNF?group,and reach a peak level at 9h.3.The expression of TNF?in serum and brain tissueSerum TNF-?levels were significantly increased and reached the top value at 2h in D-GalN/LPS group,declined at 6h and drop to normal level at 12h.In LPS control group,serum TNF-?levels also reached a peak level of 314.91±59.76 pg/ml?P<0.01vs NS group?and declined rapidly at 6h.The expression of TNF?in brain tissue was increased at 12h in D-GalN/LPS group compaired with D-GalN control group and LPS control group.4.The expression of ammonia transporters in brain tissues and HBMECThe protein and mRNA expression of Rhbg has no significant changes in brain tissues of D-GalN/LPS group and D-GalN/TNF-?group.Rhcg mRNA began to increase at 2h and the protein levels of Rhcg increased at 6h?D-GalN/LPS group?and2h?D-GalN/TNF?group?respectively.Both the mRNA and protein levels of Rhcg reached the highest level at 12h.Double-immunofluorescence staining of Rhcg and CD31,GFAP,and NeuN indicated that Rhcg is expressed in neurons and more robustly in BMECs and astrocytes.After administration of human TNF-?,the protein and mRNA expression of RhBG and RhCG in HBMEC were elevated at 12h and 24hrs.5.The effect of TNF-?to expression of ammonia transportersIn mice pretreated with anti-TNF?antibody,the protein and mRNA expression of Rhcg at 12h were significantly inhibited in brain tissues of D-GalN/LPS group.The protein and mRNA expression of Rhbg at 12h had no obvious changes.ConclusionThe C57BL/6 mice were injected peritoneally with D-GalN/LPS or D-GalN/TNF-?to induce ALF animal models.The blood and brain ammonia levels were significantly increased,which supported the evidence of HE.The levels of serum and brain TNF-?were increased in D-GalN/LPS group.Pretreatment by anti-TNF-?IgG antibody could prevent the occurence of acute liver failure and reduce the blood and brain ammonia levels in D-GalN/LPS group,which demonstrated that TNF-?played an important role on acute hepatic encephalopathy.Rhcg mRNA and protein levels were elevated in D-GalN/LPS group and D-GalN/TNF?group,which were consistent with the increasing time of blood and brain ammonia.It indicated that Rhcg might play an important role in ammonia transport of central nervous system.Pretreatment by anti-TNF-?IgG antibody could down-regulate the expression of Rhcg mRNA and protein in brain tissues of D-GalN/LPS group.Besides,in vitro experiment showed that TNF-?increased the expression of Rhcg in HBMEC in mRNA and protein levels.Therefore,TNF-?might promote the transport of ammonia from blood to brain tissues and exacerbate the ammonia toxicity by increasing the expression of Rhcg.