Altered Transcription Of Ammonia Transporter In Rat With Fulminant Hepatic Failure

ObjectiveFulminant hepatic failure(FHF) is clinically characterized by prolonged prothrombin time and complicated with predilected and mortal hepatic encephalopathy. The study often showed that ammonia played as an crucial role in astrocytic swelling which caused a HE. It's important to study the mechanism of entrance of ammnia in to CNSand the probability of changed mechanism in FHF, which should be meanful in the protection from ammnia entrance into CNS.It found recently that ammonia tranporter was an protein which obligatedly carried ammonia. It was named AMT/MEP/RH protein and exprssed in prokaryocyte,enkaryocyte and fungus. It occluded and exprssed as RhAG,RhD,RhBG and RhCG in rat, of which the last two proteins played roles in body organs. It found that ammonia transporter were expressed in distal tubular cell and collecting cell of kidney,testis,hepatocye,duodenal cell and CNS. It remains ambiguous how it worked and expressed. The study was to ensure an effective FHF model and find out the change of transcription of ammonia transporter, including RhBG and RhCG, in rats with FHF.Materials and Methods1. Institution of animal models 28 male Wistar mice were divided into four groups. Group 1:800mg/Kg D-GaLN and 10 g/Kg LPS combined ip group,10; Group 2:400mg/Kg D-GaLN and 100 g/Kg LPScombined ip group,5; Group 3:800mg/Kg D-GaLN and 100 g/Kg LPS combined ip group,5; 4 group:400mg/Kg D-GaLN and 16 g/Kg LPS combined iv group,8。2. Serum levels of alanine transaminase (ALT) Serum levels of alanine transaminase (ALT) were tested by biochemical method to determine the biochemical changes of liver functions in fulminant hepatic failure.3. figure of survival and mortality rateRecord the mortarlity rate and time of every rat, caompare the survival figure of 4 groups.4. HE stainingHE staining was carried out in liver tissues to determine the pathological changes of liver in fulminant hepatic failure.5. ensure time plots of rats with FHF40 male Wistar mice were divided into 6 groups time plots. They were randomly divided in to 6 groups with 5 in Oh group,5 in 2h group,5 in 6h group,10 in 12h group and 10 in 24h group.6. Real-Time PCR analysisBrain RNA was extracted in brain tissues by TRIZOL method. SYBR Green I quantitative real time PCR method was used to analysis the quantitative of occludin mRNA in every group.7. Analysis of the dataAnalysis of variance was showed as:X±SE, All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 10.0 for windows). P<0.05 was considered statistically significant.Results1. ALT level and common situationAll the rats had poor appetite and activity gradually after administration with LPS/GalN exccept 800mg/Kg D-GaLN and 10μg/Kg LPS combined ip group. Compared with 800mg/Kg D-GaLN and 10μg/Kg LPS combined ip group, ALT levels in other groups were much more higher(p<0.05), of which themselves variance were no significance. The liver showed massive or submassive necrosis in 2,3 and4 group, of which the pathological changes resembled with the changes of fulminant hepatic failure in human. There is only spotted hemorrhage of liver in 1 group.2. Survival figure of ratRat died in 2,3 and 4 group, of which 2 group is 20%,3 group is 40% and 4 group arrived at 75%. Rats of 1 group survived. Tme of death of rats assembly at 9 to 13 hour.3. HE staining in liver HE stains were well.1 group were showed completely constructure, regular liver plate, an spotted necrosis and hepatocytic swelling; 2 group were showed moderately disturbed liver plate, multilocal spotted necrosis, hepacytic swelling and slight eosinophilogic change; 3,4 group were showed with obvious disturbed liver plate, dialated and congestive sinusoid, multilocal sppoted necrosis and perfusion of many red blood cells in suround liver tissue.4. Expression of occludin mRNAAfter rat with FHF with 6 time points were established, detection of target genes found RhBG,RhCG genes transcription were inclined and highest at 12h and declined afterwards. The variance between either 9h,12h or 24h group and Oh group was significant(p<0.05).Conclusion1.800mg/Kg D-GaLN and 10μg/Kg LPS combined ip group didn't show a obvious FHF model. Compared with it, ALT values of 3 other groups were much higher and death occurred, implied a severe injury liver.400mg/Kg D-GaLN and 16μg/Kg LPS combined iv group were showed with high mortality rate and obvious lver failure, which was a best choice of FHF causative drug.2. In rats with FHF group, RhBG,RhCG genes transcription were inclined andhighest at 12h and declined afterwards.