| Objective: Fulminant hepatic failure caused by a great quantity of liver cell's necrosis resulted from many reasons. Previous results show that :apoptosis,release of ROS,activation of cytokine network and of thrombin chain reaction play a important role in hepatocyte inflammation and necrosis which caused fulminant hepatic failure. Its exact pathogenesis has not been fully clarified,which make the cure of FHF difficult and has a high mortality. The current studies suggest that the apoptosis of hepatocyte play an important role in the pathogenesis of FHF besides cell necrosis. Therefore,a deep study of the pathgenesis is the crucial thing to prevent and cure the FHF. It has been confirmed that FADD (Fas associated death domain protein) participated in many death signal pathways induced by death receptor and has an connection role in apoptosis signal pathway. FADD is an adaptive protein, it has been considered of the indispensable protein of TNF receptor superfamilies member such as Fas,TNFR1,DR3,DR4,DR5 and p75NTR inducing cells apoptosis. TNF-α, TNF-R1, Fas, caspase-8,TRAIL are important proapoptotic protein. FADD is considered of the common transmission factor of death receptor mediated signal pathway, eg Fas, TNFR1 and TRAIL .When FADD binding with receptors ,it could recruitment and activated procasepase-8. Casepase-8 released as a activated caspases from the complex usually referred to as DISC(death inducing signalling complex),and activated cascade reaction inducing cell apoptosis by cleave the downstream capase (caspase-3,caspase-6 and caspase-7). It has been confirmed that the pathophysiology of fulminant hepatic failure, viral hepatitis, alcoholic hepatitis,non-alcoholic steatohepatitis ,hepatic cirrhosis and hepatocellular carcinoma are associated with apoptosis inducing by FADD .However, the effect and influencing factors of FADD on apoptosis signal pathway in hapetocyte of experimental fulminant hepatic failure still been known poorly.The dynamic expression of hepatocyte apoptosis ,FADD and other factors associated with apoptosis signal pathway in lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced fulminant hepatic failure in rats was detected, the role of apoptosis, FADD and correlation factors in fulminant hepatic failure was discuss. We analysed the relationship of FADD and apoptosis factors (TNF-α,TNFR-1,caspase8)expression,apoptosis index, eventually discussed the effect and molecular mechanism of the FADD in rat fulminant hepatic failure injury, to further confirm the role of FADD in apoptosis of fulminant hepatic failure from the perspective of molecular biology, and provide a theory basis for clinical application of apotosis inhibitor in fulminant hepatic failure.Methods:1 Study objects: 60 masculinity depuratory Wistar rat weighted 180-200g were divided randomly to model group and control group, which has 30 rats respectively. The fulminant hepatic failure model was established by peritoneal injection of D-galactosamine 800 mg/kg, afterward intradermally injection of LPS 10μg/kg. Control group were only given the physiological saline 2ml 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 control 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 FADD ,TNF-α,TNFR-1 and caspase8 expression were detected by immunohistochemistry pv method.5 The expression of FADD ,TNF-α,TNFR-1 and caspase8 mRNA were detected by RT-PCR in the two groups. 6 The hepatocyte apoptosis rate were detected by FCM in two groups.7 Statistics: All data are given as mean±SE,using rank sum test for analysis. P <0.05 were considered significant.Results:1 The rat general state observation: The general state 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. The situation of control group keep normal all the time.2 Liver tissues morphology general observation: (1)Liver general specimen observation: the model group had shown hyperemia, bleeding, , ecchymosis and to attain bulk of necrosis. The contol group has not appear the above situation. (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 control group were only observe a little inflammation and necrosis.3 FADD protein and mRNA expression of hepatic tissue in the two group: (1) The expression of FADD protein: The positive staining cell was mainly hepatic cell in model group, and most cytoplasm staining, increased with time, and was strongly positive at 12 hour. The positive cells decreased at 24 hour with the necrosis. At 4, 8, 12 ,24 hour, the expression of FADD protein in control group was distinct lower than model group at corresponding different time. And the difference had statistical significance (P<0.05). (2)The expression of FADD mRNA: It increased after administration and peaked at 12 hour, then gradually declined.?The difference was statistically significant (χ2=15.845,P=0.003). The expression of FADD mRNA in control group was lower than model group at corresponding different time. And the difference had statistical significance (P<0.05). 4 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=18.427,P=0.001). The expression of TNF-αprotein in control 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=15.910, P=0.003). The expression of TNF-αmRNA in control group was lower than that of model group at corresponding different time. The difference had statistical significance(P<0.05).5 TNF-R1protein and mRNA expression of hepatic tissue in the two group: (1) The expression of TNF-R1protein: The positive staining cell was mainly hepatocyte in model group, and most cytoplasm staining, increased with time. And it strongly positive expressed at 12 hour, then gradually declined. And the difference had statistical significance (χ2=23.182, p=0.000). At control group was lower than model group at corresponding different time. And the difference had statistical significance (P<0.05). (2) The expression of TNF-R1 mRNA: It increased after administration and peaked at 12 hour, then gradually declined in model group. And the difference had statistical significanc(eχ2=23.659,P=0.000). At control group was lower than that of model group at corresponding different time. The difference had statistical significance(P<0.05).6 Caspase-8 protein and mRNA expression of hepatic tissue in the two group: (1) The expression of caspase-8 protein: The positive staining cell was mainly hepatocyte in model group, and most cytoplasm staining, increased with time. And it strongly positive expressed at 12 hour, then gradually declined. And the difference had statistical significance (χ2 =23.621, p=0.000). At control group was lower than model group at corresponding different time. And the difference had statistical significance (P<0.05). (2) The expression of caspase-8 mRNA: It increased after administration and peaked at 12 hour, then gradually declined in model group. And the difference had statistical significanc(eχ2=23.709,P=0.000). At control group was lower than that of model group at corresponding different time. The difference had statistical significance(P<0.05).7 Changing of hepatocyte apoptosis: Both the apoptotic rate detected by FCM exhibited an upward trend of apoptotic hypatocytes with the passing of time in model group. In control group hepatic tissue showed less apoptotic hypatocytes (p<0.05).8 The relationship of apoptotic hepatocyte and FADD,TNF-αin experimental fulminant hepatic failure: There was a positive correlation between apoptotic rate and FADD,TNF-α(r=0.775,0.736,p=0.000,0.000).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 The expression of FADD,TNF-α,TNFR-1,caspase8 were increased and has a positive correlation with the expression of hepatocyte necrosis and apoptosis in hepetic tissue of fulminant hepatic failure.3 There was a close relationship between FADD and TNF-α, TNFR-1, caspase8 , hepatocyte apoptosis in rate hepetic tissue of fulminant hepatic failure. It was confirmed that FADD and the related apoptosis signal pathway play a important role in hepatic apoptosis of experimental fulminant hepatic failure on the perspective of cytolog,protein and gene. Scientifically regulation of the expression FADD may play an important role in prevention and treatment of FHF. |
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