Effect And Mechanism Of NF-κB In The Hepatic Cells' Inflammation Damage Of Experimental Fulminant Hepatic Failure

Objective: Fulminant hepatic failure (FHF) is a syndrome with the characteristic of severe liver function damage which is caused by a great quantity of liver cells'necrosis because of many reasons. It has the characteristics of acute onset, bad prognosis and high case fatality and there is no utility treatment till now. Its case fatality rate is 40-70% [1] oversears. Case fatality rate is 50% in China after continuous research [2]. Reducing its fatality rate has become a problem crying for settlement. The pathogenesis of FHF has not been completely elucidated though we had achieved an obvious progress in its pathogenesis, clinical diagnosis and treatment. The clarification of its pathogenesis will certainly improve the level of prevention and cure.Presently, we emphasis the endotoxin induced trigger action in the research of FHF pathogenesis domestically and abroad. Present research had indicated that the toxic action of endotoxin is mainly indirect. It depends on the releasing of many inflammation mediators. It also emphasizes the function of cytokine, among which TNF-αplayed a crucial role [3]. We had observed the increase of TNF-αin the FHF induced liver functional failure patients clinically and basically and elucidated the function of TNF-αin its pathogenesis [4]. It is generally believed that endotoxin entered liver and activated monocaryon/macrophage system; thus, caused the release of many inflammation mediators which was centered by TNF-αmediated inflammation reaction and resulted in the liver cell death.NF-κB is a multiple stage transcription factor which has an intimate correlation with organism immunization and inflammation process by coding virogene and gene of inflammatory cell factor, chemotatic factor, interferon, MHC protein, growth factor and cell adhesion factor [5]. Cell factors involved in the synthesis regulation of inflammatory reaction related protein mainly contains: pro-inflammatory cell factor, such as TNF-α, IL-2, IL-1β, IL-6, GM-CSF and G-CSF; chemotatic factors, such as IL-8, phagocyte inflammatory protein-1, phagocyte inducing protein-1; inflammatory reaction enzymes; adhesion molecule, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin; Receptors, such as IL-2 receptor, T cell receptor (βchain). They are mainly involved in the organism inflammatory reaction. They can promote the inflammatory cell's accumulation, activation or the release of inflammation mediators and aggravate inflammatory reaction.LPS and TNF-αare the intense sensitizers of NF-κB [6]. NF-κB is the nucleus link to mediate excess inflammation reaction. The research about NF-κB is mainly focused on tumor and ischemic/reperfusion aspect domestically and abroad. Yet, there is little research about its effect and mechanism in liver cell inflammation damage in FHF. Based on the above theory, we use immunohistochemistry method to detect NF-κB's expression variation in the FHF patho-process to confirm NF-κB's function in the FHF and thus, to clarify FHF's pathogenesis.Methods: 1. Study objects: 45 masculinity depuratory Wistar rat which is weighted 180-200g. Divided rat randomly to model group and control group, which has rats 30 and 15 respectively. Produce fulminant hepatic failure model by peritoneal injection of D-galactosamine 800mg/kg, afterward intradermally inject LPS 10μg/kg in the model group. Inject equal volume of physiological saline in the peritoneal of the control group. 2. Sample collection and conservation: execute rat by femoral vein exanguinate at 2, 4, 8, 12 and 24 hours. Execute 6 and 3 rats in model group and control group respectively. Keep blood serum and obtain liver by cutting the belly open immediately after execution. Retain quantity sufficient liver tissue by putting it into 10% neutral formalin; quick freeze the rest in the liquid nitrogen and put them in the -80℃refrigerator. 3. Routine HE staining of hepatic tissue specimen: Observe liver histopathology change under the light microscope. Observe the formation and development of inflammation and necrosis. 4. Immunohistochemistry staining of hepatic tissue specimen: Use routine immunohistochemistry S-P method to detect NF-κB, iNOS and TNF-αexpression. Refer to Fromowitz's [7] comprehend regulation to evaluate the expression of NF-κBp65 and TNF-α. Refer to Shimizu's [8] assessment standard and use semi-quantitative method to evaluate iNOS expression. Score it by the distribution of staining cell and staining extent. The final result is demonstrated as negative, weakly positive, positive and strongly positive. 5. Conduct statistics analysis by using SPSS 11.0: Use grade to demonstrate numeration data. Use Wilcoxon rank sum test and rank correlation analysis to analyze every target at different time spot. The dependability of every two targets is analyzed by using rank correlation analysis. P<0.01 indicates that there is significant difference.Results: 1. Observation of rat general state of health: General state of health is good in control group and there is no death. 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. One rat had died at 11 hour. 2. Liver tissues morphology general observation: (1) Liver general specimen observation: There is little difference in liver tissue morphology at different time spot in control group. In control group, rat liver is bright red; Luster is good; texture is soft and flexible; the edge is sharp. 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. (2) Hepatic tissue HE staining: There is little difference in hepatic tissue morphology at different time spot in control group. The construction of hepatic lobules is concinnous and the line of hepatic plate is in order. There is no degeneration and necrosis in control group. The pathological change aggravated gradually with the passing of time in model group. Light microscope liver tissue pathology had indicated liver cellular edema, sinus hepaticus congestion, inflammatory cell infiltration, hepatic tissue bleeding, hepatic cell massive necrosis and hepatic lobules disorganization. The main style of hepatic cell damage is inflammation and necrosis. A few hepatic cells have shown the apoptosis morphology change. 3. Immunohistochemistry change in each hepatic tissue: (1) NF-κBp65 protein expression: Some nonparenchymal cell (sinus hepaticus endotheliocyte, Kupffer cell) had shown positive staining in the control group. Cytoplasm had shown weakly positive expression and nuclear positive expression is rare in control group. The positive staining cell is mainly hepatic cell and Kupffer cell in model group. It is mainly cytoplasm staining of nonparenchymal cell in 2 hour group. It is mainly liver cell nucleus staining in 4, 8, 12 and 24 hour group. The number of nucleus positive staining cells increased obviously in 4, 8, 12 hour group and is much higher than control goup. (2) INOS protein expression: Some nonparenchymal cell (Kupffer cell, sinus hepaticus endotheliocyte) had shown positive staining in the control group. The ratio of positive staining increased and staining extent deepened with the development of liver inflammatory degree in the model group. (3) TNF-αprotein expression: Some nonparenchymal cell (Kupffer cell) had shown positive staining in the control group. The ratio of positive staining increased and staining extent deepened with the development of liver inflammatory degree in the model group. 4. The relationship of NF-κB, iNOS and TNF-αin the experimental fulminant hepatic failure: the research result had indicated that the degree of rat liver degeneration, necrosis and inflammation is severe in model group and the degree of necrosis and inflammation increased with the passing of time. There are positive correlation between NF-κB and iNOS (r=0.801, P<0.01); NF-κB and TNF-αalso have positive correlation (r=0.852, P<0.01).Conclusions:1 The D-GalN+LPS model can reflect the pathological change process of fulminant hepatic failure. We can observe the liver tissue pathological change in FHF. Thus, it is suitable for the pathogenetic research of FHF.2 Observe the activation process of NF-κB from cytoplasm to nucleus in fulminant hepatic failure from protein level. It proves that NF-κB's activation takes part in the FHF pathological lesion process and is an important pathogenesy of FHF.3 The high expression of NF-κB in liver cells in FHF had indicated that in fulminant hepatic failure pathological process, liver cell is not only the target cell of causative agent attack, it can also play a vital role in its onset process.4 INOS, TNF-αand NF-κB have intimate correlation in FHF. The expression of iNOS and TNF-αincreased obviously with the activation of NF-κB. The research had proved that NF-κB played an important role in liver tissue inflammation and necrosis of FHF. It also indicated that NF-κB is a critical element of FHF onset process. This had provided original interfere target for the clinical treatment of FHF.