Effects Of PDTC On Expression Of NF-κB And Hepatocellular Apoptosis In Experimental Fulminant Hpatic Failure

Objective: Fulminant hepatic failure (FHF) is a clinical syndrome with a high morbidity. Currently liver transplantation is the only treatment measure to cure FHF. However it's restricted in clinical implementation because of the shortage of hepatic source, high cost of operating and long-term application of immunosuppressive agents after liver transplantation. Therefore, novel and effective clinical medical treatment based on pathogenesis is necessary. In recent years, there are many achievements on FHF, but the precise mechanism is still vague.It's showed that activated inflammatory factors play an important role in FHF. Since nuclear factor-κB (NF-κB) regulates in?ammatory cytokine production, including inducible nitric oxide synthesis (iNOS), tumor necrosis factor (TNF), the inhibition of NF-κB activation would offer a new treatment for liver failures. It has been suggested that hepatocellular apoptosis is an important factor contributing to FHF besides hepatocyte necrosis recently. There's a close relationship between necrosis and apoptosis. Many proinflammatory factors regulated by NF-κB play an important role in cell apoptosis. NF-κB also regulates a large array of pro-apoptotic and anti-apoptotic genes, including caspases, death receptors, IAPs, members of Bcl-2 and some oncogenes. NF-κB can act both as pro-apoptotic and anti-apoptotic factor in different pathways depending on different stimulations, actived target genes. So far there was little data on the roles of NF-κB in hepatocellular apoptosis in FHF. Thus, the actions of NF-κB in hepatocytes apoptosis of FHF were not well understood.In this research, Wistar rats were administered with D-GalN and LPS to manufacture FHF animal model. There was also a PDTC (Pyrrolidine dithiocarbamate) group established which pretreated with PDTC and then accepted the same administration with model group. Hematoxylin-eosin (HE) staining, flow cytometry (FCM), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods were used to evaluate hepatocellular apoptosis in both the two groups. Immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) methods were used to detect the expressions of NF-κB, c-Myc, survivin in the two groups. Results of two groups were analized and compared to investigate how does NF-κB affect hepatocellular apoptosis and provide new treatment measures of antioxidant in FHF.Methods: 1 Animals: 60 masculinity depuratory Wistar rats weighted 180-200g were randomly divided into model group 30 and PDTC group 30. The fulminant hepatic failure model was manufactured by peritoneal injection of D-galactosamine 800mg/kg, afterward intradermally injection of LPS 10 ug/kg. PDTC group were given the same treatment besides a prtreatment of PDTC 100mg/kg intraperitoneally. 2 Sample collection and conservation: 6 rats in model group and PDTC group respectively were sacrificed at 2, 4, 8, 12, 24 hours after injection by femoral vein exanguinate. Adequate liver tissue was respectively fixed by 10% neutral formaldehyde solution, 70% ethanol. Some of the liver tissue was freezed in liquid nitrogen immediately and then put it into the -80℃refrigerator. 3 Liver histopathology changes under the light microscope were observed. 4 The serum alanine aminotransferase (ALT) and total bilirubin (TB) levels were detected by Olympus AU2700 auto-biochemical analysor. 5 Immunohistochemistry staining of hepatic tissue specimen: NF-κB, Survivin and c-Myc were detected by routine immunohistochemistry S-P method and evaluated with Fromowitz's comprehend regulation.The final result is demonstrated as negative, weakly, positive and strongly positive. 6 NF-κB, survivin and c-myc mRNA of hepatic tissue semiquantitativly were analysed by RT-PCR. 7 The apoptotic rate was detected by flow cytometry method. 8 The apoptotic index was detected by TUNEL. 9 Statistically analysis: SPSS 11.5 statistical software was used for statistical analysis, data were analyzed by grade and rank and Spearman's rank correlation test. P<0.05, a significant difference.Results:1 Observation of rat general state of health: General state of health aggravated gradually with the prolongation of administration time in model group. Rats had appeared hydroposia decrease, horripilation, downcast, reaction dullness and drowsiness with the passing of time. General state of health in PDTC group seemed better than model group. The rats in PDTC group only appeared horripilation and hydroposia decrease untill 24th hour.2 Liver tissues morphology general observation: (1) Liver tissues had showed hyperaemia, bleeding point, partly congestion and necrosis in model group. The fragility of hepatic tissue has increased and there is massive necrosis in the model group with the passing of time. Liver tissue in PDTC group showed less bleeding point and congestion than model group at the same time spot. (2) Hepatic tissue HE staining: The pathological change aggravated gradually with the passing of time in model group. Hepatocyte edema and acidophilia, apoptotic bodies formed, inflammatory cell infiltration, liver tissue hemorrhage, hepatocyte massive necrosis and hepatic lobules disorganization were discovered in liver tissue under light microscope In PDTC group hepatic tissue showed less apoptotic and necrotic hepatocytes than model group.3 Biochemical analysis: ALT and TB levels rose moderately in the early period and sharply arise in the late hours. Compared with model group, PDTC group showed lower ALT and TB levels (p<0.05).4 Changing of hepatocyte apoptosis: Both the apoptotic rate detected by FCM and apoptotic index by TUNEL exhibited an upward trend of apoptotic hypatocytes with the passing of time in model group. In PDTC group hepatic tissue showed less apoptotic hypatocytes than model group (p<0.05).5 The expression of NF-κB: Both the p65 protein expressed in nucleus and NF-κB mRNA gradually increased in model group. In PDTC group hepatic tissue showed less NF-κB protein and mRNA expression compared with model group (p<0.05).6 The expression of c-Myc,Survivin: In model group there were two peaks of hepatic c-Myc expression, respectively at 4th and 24th hour. It didn't show a decrease in PDTC group (p>0.05). In model group survivin protein expression gradually increased, with a two mRNA expression peak respectively at 4th and 24th hour. It also showed no dcrease in PDTC group (p>0.05).7 The relationship of apoptotic hepatocyte and survivin, c-myc in experimental fulminant hepatic failure: There was a negative correlation between apoptotic rate and surviving, c-myc (r=-0.491, -0.414, p=0.000, 0.004).Conclusions:1 The rat model induced by D-GalN+LPS can reflect the pathological changes of fulminant hepatic failure. Hepatocellular apoptosis was observed morphologically, cytologically and from gene level. It's confirmed that hepatocyte apoptosis plays an important role in the progression of FHF. 2 NF-κΒexpression was dynamically observed from protein, cyto, gene levels, it suggested that NF-κΒplays a pro-apoptotic role in FHF model induced by D-GalN+LPS except its proinflammatory function.3 PDTC treatment attenuated hepatic mRNA and protein expression levels of NF-kappaB in FHF model induced by D-GalN+LPS.4 Hepatocellular apoptosis was attenuated by PDTC in FHF rat model induced by D-GalN+LPS through a NF-κΒ-dependent way. Thus it offered a clinical application with NF-κB inhibitor to treat FHF.5 The anti-apoptotic roles of c-Myc,survivin were observed in the study. But c-Myc,survivin were not definitely regulated by NF-κΒ. It's suggested that NF-κΒregulate hepatocellular apoptosis through complex pathways which need further investigation.