| Objective: Fulminant hepatic failure is a syndrome with the characteristic of severe liver function damage caused by a great quantity of liver cell's necrosis resulted from many reasons. It has the characteristics of acute onset, progress quickly, many complications, high motality and poor prognosis. It is currently the hot and tough issue on liver disease. Its pathogenesis is complex and has so far not been fully clarified.In China, the main cause of FHF is viral hepatitis, next, are drugs and poisons. The current studies suggest that the activation of cytokines play an important role in the pathogenesis of FHF. It has been confirmed that nuclear factor-κB has an regulatory role in inflammatory response in fulminant hepatic failure. NF-κB is an important transcriptional regulation factor, it has been considered one of the key factors in regulating expression of pro-inflammatory cytokines gene. It can induce the expression of inflammatory cytokines (TNF-α, IL-1, IL-6, etc.), chemokines, adhesion molecules (ICAM-1, VCAM-1, etc.), inflammatory enzymes (iNOS, COX-2, etc.) and be highly activated in inflammatory diseases. It has been confirmed that the pathophysiology of hepaic inflammatory, fibrosis, apoptosis and regeneration. Ischemia-reperfusion injury is associated with NF-κB activation.TNF-α, iNOS are important inflammatory mediums. TNF-αis an intensive sensitizer of NF-κB. And NF-κB activation can promote the activation and release of TNF-α, creating a vicious circle. Both TNF-αand NF-κB can induce iNOS, which led to the release of NO and form a cascading effect. This can promote the development of liver injury and lead to substantial necrosis of liver cells. PDTC is an antioxidant and can specially inhibit the activation of NF-κB. However, there are little research of the effect of the inhibition of NF-κB in fulminant hepatic failure. Based on the above, we detected the expression of NF-κB, TNF-αand iNOS in lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced fulminant hepatic failure in rats and both in the PDTC group, then analyze the relationship between NF-κB and liver injury, TNF-α, iNOS expression , eventually discuss the effect and molecular mechanism of the inhibition of NF-κB by PDTC in rat fulminant hepatic failure inflammatory injury, to further confirm the role of NF-κB in inflammatory injury of fulminant hepatic failure from the perspective of molecular biology, and provide a theory basis for clinical application of NF-κB inhibitor in fulminant hepatic failure.Methods:1 study objects: 60 masculinity depuratory Wistar rat weighted 180-200g were divided randomly to model group and PDTC group, which has 30 rats respectively. Fulminant hepatic failure model was established by peritoneal injection of D-galactosamine 800 mg/kg, afterward intradermally injection of LPS 10μg/kg in the model group. PDTC group were given the same treatment besides a prtreatment of PDTC 100mg/kg intraperitoneally.2 Sample collection and conservation: The rats were executed by femoral vein exanguinate at 2, 4, 8, 12 and 24 hours. 6 rats were executed in model group and PDTC group respectively. The serum was kept in -20℃. Adequate liver tissue was respectively fixed by 10% neutral formaldehyde solution; Some of the liver tissue was freezed in liquid nitrogen immediately and then put it into the -80℃refrigerator.3 Routine HE staining of hepatic tissue: Liver histopathology change was observed under the light microscope.4 Immunohistochemistry staining of hepatic tissue: The NF-κB, TNF-αand iNOS expression were detected by immunohistochemistry s-p method.5 The levels of serum ALT and TB were detected by using automatic biochemical analyzer.6 The expression of serum TNF-αwere detected by using ELISA method.7 The changes of serum NO were detected by using nitrate reductase assay.8 The expression of NF-κB,TNF-αand iNOS mRNA were detected by RT-PCR in the two groups.9 Statistics: All data are given as mean±SE,using rank sum test for analysis. P <0.05 were considered significant.Results:1 Observation of rat general state: The general state aggravated gradually with the prolongation of administration time in model group. Rat had appeared hydroposia decrease, horripilation, downcast, reaction dullness and drowsiness with the passing of time. The situation of PDTC group has improved compared with model group at Corresponding different time points, not appearance drowsiness.2 Liver tissues morphology general observation: (1)Liver general specimen observation: the model group had shown hyperemia, bleeding, hyperemia, ecchymosis and to attain bulk of necrosis. The PDTC group has improved compared with model group at corresponding different time points and has not underwent bulk of ecchymosis. (2)Hepatic tissue HE staining: the pathological changes aggravated gradually with the passing of time in model group, followed by degeneration of liver cells, balloon-like change, inflammatory cell infiltration, eosinophilic change, until a large hemorrhage, hepatocellular necrosis fusion, nuclear dissolution, fragmentation, fiber network structure collapse. The PDTC group were obviously ameliorated compared with model group at corresponding different time. The inflammation and necrosis were reduced.3 The changes of serum ALT,TB,TNF-αand NO: In model group, ALT and TB levels increased slowly at 2, 4, 8 hour after administration and dramatically increased at 12 and 24 hours. They were significantly lower in PDTC group. Except for 8 hour, the ALT levels were lower than the model group of the remaining time points, the difference had statistical significance (P<0.05). The TB level were lower than the model group at 4, 12, 24 hour, and the difference had statistical significance (P<0.05).In model group, the serum TNF-αsignificantly increased at 2 hour after administration, and gradually declined with the time passed. They were lower at corresponding different time in PDTC group, and the difference had statistical significance (P<0.05).In model group, the serum NO significantly increased at 2 hour after administration, at 12 hour increased most, and decrease gradually. They were lower than PDTC group at 2, 8, 12 hour, and the difference had statistical significance (P<0.05).4 NF-κB protein and mRNA expression of hepatic tissue in the two group: (1) The expression of NF-κB protein: The positive staining cell was mainly hepatic cell in model group, and most Nuclear staining, increased with time, and was strongly positive at 12 hour. The positive cells decreased at 24 hour with the necrosis. At 2, 8, 12 hour, the expression of NF-κB protein in PDTC group was lower than model group at corresponding different time. And the difference had statistical significance (P<0.05). (2)The expression of NF-κB mRNA: It increased after administration and peaked at 8 hour, then gradually declined. The difference was statistically significant (χ2 = 26.307, P = 0.000). At 4, 8 hour, the expression of NF-κB mRNA in PDTC group was lower than model group at corresponding different time. And the difference had statistical significance (P<0.05).5 TNF-αprotein and mRNA expression of hepatic tissue in the two group: (1) The expression of TNF-αprotein: The positive staining cell is mainly hepatocyte in model group, and most cytoplasm staining, increased with time. And the difference had statistical significance (χ2=19.361,P=0.000). At 4, 8, 12 hour, the expression of TNF-αprotein in PDTC group was lower than model group at corresponding different time. And the difference had statistical significance (P<0.05). (2) The expression of TNF-αmRNA: It increased after administration and peaked at 12 hour, then gradually decline in model group. And the difference had statistical significance (χ2=25.385,P=0.000). Except for 2 hour, the expression of TNF-αmRNA in PDTC group was lower than that of model group at corresponding different time. The difference had statistical significance(P<0.05).6 iNOS protein and mRNA expression of hepatic tissue in the two group: (1) The expression of iNOS protein: The positive staining cell was mainly hepatocyte in model group, and most cytoplasm staining, increased with time. And it strongly positive expressed at 8 hour, then gradually declined. And the difference had statistical significance(χ2=22.895, P=0.000). At 4,8 hour, the expression of iNOS protein in PDTC group was lower than model group at corresponding different time. And the difference had statistical significance (P<0.05).(2) The expression of iNOS mRNA: It increased after administration and peaked at 8 hour, then gradually declined in model group. And the difference had statistical significance(χ2=25.281,P=0.000). At 4 and 8 hour, the expression of iNOS mRNA in PDTC group was lower than that of model group at corresponding different time. The difference had statistical significance(P<0.05).Conclusions:1 TNF-α, iNOS and NO play an important role in the hepatic inflammation of D-GalN+LPS-induced fulminant hepatic failure.2 The NF-κB of liver tissue is correlated with iNOS, TNF-αin D-GalN+LPS-induced fulminant hepatic failure. It further confirmed that NF-κB plays an important role in hepatocelluar inflammation and necrosis in FHF.3 The expression of hepatic NF-κΒcan be inhibited by PDTC in D-GalN+LPS-induced FHF.4 PDTC can attenuate hepatic inflammation and necrosis in D-GalN+LPS-induced FHF through the inhibition of NF-κΒ.
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