Effect Of The Tumour Necrosis Factor On Intestinal Mucosa Permeability In Mice With Fulminant Hepatic Failure

ObjectiveHepatic failure caused by a variety of factors, serious damage to liver cells, leading to their synthesis, detoxification and biotransformation functions seriously impaired. Multi-organ failure, hepatic encephalopathy, brain edema, secondary infection, coagulation disorder, hemodynamic and metabolic disorders and a variety of renal complications may occur frequently after hepatic failure. A large number of studies have shown that cytotoxic cytokines such as tumor necrosis factor(TNF-a),y-Interferon (IFN-y), interleukin(IL) significantly increased after hepatic failure, which can directly cause tissue edema and damage. At the same time cytotoxic cytokines can also damage the tight junctions between cells and cause the intestinal mucosal injury, result in. increased intestinal mucosal permeability.Increase of intestinal mucosal permeability is closely related with endotoxemia, spontaneous bacterial peritonitis (SBP) and hepatic encephalopathy. At present, most studies suggested that abnormal increase of cytokines after hepatic failure affected or damaged the intestinal mucosal epithelial cell-cell junctional complex, resulting in increased intestinal mucosal permeability through a certain mechanism. Some bacterial toxins, bacteria, macromolecular substances can pass through the intestinal mucosal barrier into the blood or abdominal cavity when intestinal mucosal permeability significantly increased. These substances passed through the intestinal mucosal barrier are closely related with endotoxemia, spontaneous bacterial peritonitis and hepatic encephalopathy.These complications can aggravate the pathogenetic condition of the patients with hepatic failure. Therefore it is very necessary to study the mechanism of increased intestinal mucosal permeability. Inhibition of the increase of intestinal mucosal permeability is important to the treatment and prevention of complications of hepatic failure that has important clinical significance.Materials and Methods一.Reagent:Tumor necrosis factor alpha (TNF-α),LPS,GalN,Occludin antibody,Claudin-1 antibody,FITC marker goat-anti-rabbit IgG,anti-TNFa-IgG,D-Lactate,D-lactic acid dehydrogenase二.Animal:Six-week-old male BALB/c mice, body weight was from 18 to 22 gram三.Methods:1.animal models:Mice with FHF were injected intraperitioncally Galactosamine (GalN) (800mg/kg body weight), followed by lipopolysaccharide(LPS)(100μg/kg body weight). For preparation of mice with GalN/TNF-a-treated induced mice model with FHF were given an intraperitoncal injection of GalN(800mg/kg body weight), followed by TNFa(10μg/kg body weight).In control groups, mice were given an intraperitoincal injection of GalN (800mg/kg body weight), LPS(100μg/kg body weihgt), NS(equal volum GalN and LPS, body weihgt), respectively.Anti-TNF-a monoclonal antibody (100μg/mouse) was injected intravenously into GalN/LPS-treated mice 30 minutes before GalN/LPS administration.2.Serum levels of D-Lactate:Serum levels of D-Lactate were tested by spectrophotography to evaluate the changes of intestinal mucosa permeability in fulminant hepatic failure.3.Ultrastructures of intestinal mucosa were observed under transmission electron microscope.(H-600; HITACHI, Japan) 4.Real-Time PCR analysis:Intestine 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 mRNAand Claudin-1 mRNAin every group.5.Western blot analysis:Western blot analysis was performed to determined the quantitative of Occludin protein expression and Claudin-1 protein expression.6.Immunohistochemistry staining:Immunohistochemistry method was used to detect the distribution and expression of Occludin and Claudin-1 in frozen tissue sections.7. Analysis of the data:Analysis of variance was showed as:X±SE, All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS13.0 for windows). P <0.05 was considered statistically significant.Results1.Serum levels of D-LactateMice had poor appetite and activity gradually after administration with LPS/GalN. The serum levels of D-Lactate increase obviously after injected 6 hours and have significantly difference with control groups(P<0.01). Prophylactic treatment with anti-TNFα-IgG antibody prevented the increase of D-Lactate concentration with mice in FHF.2.Transmission Electron MicroscopeWhen these TJs in FHF mice were examined by TEM, there were significant differences in the appearances of TJ strands at 9h after GalN/LPS administration and control groups.The appearance of TJ strands in anti-TNFa-IgG antibody treated group were not change comparing with normal control group.The morphological abnormalities at 9h in FHF group included that the integrity of tight junctions was disrupted,and microvilli shorted, broken.sheded.3. Expression of Occludin mRNA and Claudin-1 mRNAThere were obvious change of expression of mRNA between 9h group and Oh control group. The expression of Occludin mRNA and Claudin-1 mRNA in FHF group decreased obviously compared with anti-TNFa-IgG antibody group, this showed that the decrease of expression of Occludin mRNA and Claudin-1 mRNA maybe the mechanism of increase of intestinal mucosa permeability in FHF.4. Expression of Occludin protein and Claudin-1 proteinWestern Blot analysis confirmed that low expression levels of Occludin and Claudin-1 especially at 9 hours. But pretreatment by anti-TNFa-IgG antibody prevented low expression levels of Occludin and Claudin-1. These findings were consistent with the results of the Real-Time PCR analysis.5.ImmunohistochemistryLocal high intesity of Occludin protein and Claudin-1 protein were seen in the apical region of the later membrane representing TJs of intestinal epithelial cells. There were significant differences in the appearance of Occludin protein and Claudin-1 protein among FHF group and control group. The results under light microscopy revealed that Occludin protein and Claudin-1 protein were exclusively concentrated on the intestinal epithelial cells.In experiment group, the distribution of Occludin protein and Claudin-1 protein at 2h after GalN/LPS administration were almost unchangeed comparing with NS, anti-TNFa-IgG treated groups.Decreased intensity of Occludin protein and Claudin-1 protein staining in epithelial cells was observed at 6h and 9h after GalN/LPS administration and at 6h after GalN/TNF-a administration.Conclusion1. The D-Lactate concentration in FHF group increased significantly compared with control group. But prophylactic treatment with anti-TNFa-IgG antibody prevented the increase of D-Lactate concentration with mice in FHF.This proved that TNF-a can induce the increase of intestinal mucosa permeability2. Anti-TNFα-IgG antibody can pervent the disruption of intestinal epithelial intercellular tight junction in mice model with FHF and the low expression of Occludin Portein and Claudin-1 Portein in intestinal epithelial cells of mice model with FHF. TNF-αwas one of main factor of the increase of intestinal mucosa permeability, that decreased the expression of Occludin and Claudin-1 in intestional epithelial cells of mice model with FHF.