Effect Of Tumor Necrosis Factor-α On Permeability And Expression Of Tight Junctional Protein Occludin In Blood-brain Barrier Of Mouse With Fulminant Hepatic Failure

AIMFulminant hepatic failure (FHF) is a devastating disease and associated with a high mortality. Cerebral edema leading to cerebral herniation is a prevalent clinical problem and a major cause of death. The mechanism responsible for development of cerebral edema includes vasogenic edema and cytotoxic edema. Increased permeability of the blood brain barrier ( BBB) is the cause of vasogenic edema. Up to now, there is few study on the changes of structure and function of BBB in FHF. And there is little known about the mechanisms of vasogenic e-dema in serious hepatic injury.TNFa plays an important role on hepatic injury. In clinical studies, results have also shown that the TNFα levels of serum increased in patients with serious liver failure. Many studies have shown that TNFa may lead to increase of the permeability of the BBB in many disease such as focal cerebral ischemia, brain trauma, malaria and multiple sclerosis. We studied the morphological change of BBB and the effect of TNFa on permeability of BBB in FHF animal model.The major structure of BBB is tight junction (TJ) , which is believed to be correlated with disruption of BBB in many patients with central nervous system disease. Occludin is the major functional protein and regulatory protein in TJ complexes. Previous studies have gained insight into the molecular structure of TJ. Reports of the regulation of TJ - associated protein under physiological and pathophysiological conditions is less. We explored the molecule bases of change in TJ structure through studied expression of occludin protein and occludin mR-NA. And illuminated the effect of TNFa on brain edema in fulminant hepaticfailure.Materials and methodsMale Balb/c mice were divided into eight groups, group 1: saline control group, group 2: LPS control group, group 3: GalN control group, group 4: LPS hepatic failure group ( LPS + GalN) , group 5: anti - TNFct group ( anti - TNFa - IgG - antibody + LPS + GalN ) , group 6: anti - TNFa - R1 - group ( anti -TNFa - Rl - antibody + LPS + GalN ) , group 7: TNFa hepatic failure group (TNFa + GalN) , group 8: TNFa control group. There were 16 mice at every time point in every group. Eight mice were detected the permeability of BBB with EB, and other eight mice were remained blood sample, brain tissues and liver tissues to determined other objects.Serum levels of alanine transaminase ( ALT) were tested by biochemical method and HE staining was carried out in liver tissue to determine the pathological and biochemical changes of liver in fulminant hepatic failure. The levels of serum TNFa were determined by ELISA method.Mice were injected intravenously 2% EB 2 hours before sacrificed. Then brain tissues were placed in formamide solution. The optical density ( OD) of the EB formamide solution was determined at 632 nm. A standard curve of EB in blank formamide was used to convert EB content in brain tissues.The pathological changes of brain tissues were observed by optics microscope, and the ultrastructure were observed by transmission electromicroscope.TUNEL method was used to detect apoptic cells in brain tissues.Immunohistochemistry method was used to test the distribution and expression of occludin in brain tissues. The immunoreactive signal were seen in cytoplasm of endothelial cells in vessels.Western blot analysis was performed to determined the semiquantitative of occludin protein expression.Brain RNA was extracted in brain tissues by guanidine isothiocyanate -phenol - chloroform method. SYBR Green I quantitative real time PCR method was used to analysis the quantitative of occludin mRNA in every group.Results1. The effect of TNFa in FHF animal model;Mice had poor appetite and activity gradually after administration with LPS/ GalN. The serum levels of ALT began to increase from 6 hours. The liver shown massive or submassive necrosis. The pathological changes resembled with the changes of fulminant hepatic failure in human. The total mortality was 66.7%. Animals also appeared similar appearance after administration with TNFa/GalN. The levels of serum TNFa appeared two peaks at 2 hours point and 9 hours point respectively after injection of LPS/GalN, and there was statistical difference compared with other control groups ( P <0.01). But there were no death in mice which pretreated with anti - TNFa - IgG - antibody and anti - TNFa - Rl -antibody. Serum ALT levels reduced significantly ( P <0. 01). The liver histological analysis showed significant lighten.2. Examination of permeability of BBB;The EB concentration of brain tissue began to increase at 2 hours. There were no significant difference among every time point in group 4 and 7. But there were significant difference compared with other control groups at every time point (P < 0. 05). The concentration of EB decreased significantly after pre-treatment by anti - TNFa - IgG - antibody and anti - TNFa - R1 - antibody (P<0.05).3. Histopathologic analysis and apoptosis detection in brain tissues;There were no positive apoptosis signals including endothelial cells, astrocytes and neurons in brain tissues sections at every time point in every group. And there were no evidence changes in histopathologic analysis of brain tissues in every group.4. Ultrastructual examination of the BBB;The ultrastructure of the BBB changed significantly from 2 hours in group 4 and 7. The basement membranes were thickening. Perivascular astroglia foot appeared to be markedly swollen with an evident reduced electron density. The endothelial cells were swollen and shrank. Some microvilli were projected to thelumen. And the number of vesicles and vacuoles were increased in endothelial cells. Some of the intracellular tight junctions between endothelial cells were rupture. But in antibodies groups, perivascular astroglia foot appeared to be no markedly swollen, and the pathological changes were significant lighten.5. Expression of occludin protein :In FHF groups and TNFα control group , the immunoreactive signal showed moderate or trace staining and even no positive signal in brain tissues of mice. Western blot analysis confirmed that low expression levels of occludin especially at 6 hours and 9 hours point. But after pretreatment by anti - TNFa - IgG -antibody and anti - TNFa - R1 - antibody , positive immunoreactive signal showed significant increased. These findings were consistent with the results of the western blot analysis.6. Expression of occludin mRNA :In FHF groups, expression of occludin mRNA decreased significantly at 2 hours point and 6 hours point after injection( P <0. 05). But the levels of mRNA approached to normal at 9 hours point. And there were no obvious change of expression of mRNA in antibodies groups and other control groups.ConclusionThe levels of serum TNFα increased significantly in FHF animal which were injection with LPS and GalN. Pretreatment by anti - TNFα - IgG - antibody and anti - TNFα - R1 - antibody could prevent the fulminant hepatic failure. Results demonstrated one step further that TNFa played an important role on fulminant hepatic failure.The phenomenon that the concentration of EB in brain tissues significandy increased indicated that the permeability of BBB increased in FHF animal model. And the increase of the permeability were correlated with the increase levels of TNFα.There were no positive apoptosis of vascular endothelial cells in FHF animal model. That indicated apoptosis mechanism was not involved in the disruption of the BBB.