Effect Of Tumor Necrosis Factor-α On Expression Of Tight Junctional Protein ZO-1 In Blood-brain Barrier With Fulminant Hepatic Failure

AbstractThe prognosis of severe hepatitis is serious, and its case - fatality is high. The complication of hepatic encephalopathy is severe, and the brain edema can directly lead to death, but the pathogenesis of brain edema is not clear. The permeability increase of blood - brain barrier is one of the main manifestation, and its mechanism need further researches.Subject and MethodThe severe hepatitis group is as follow . 29 patients is confirmed as severe hepatitis by hepatic tissue examination after clinical death. The control group is as follow. 20 patients dead of transportation accident is confirmed that there are not serious hepatic tissue inflammation.The condition of experimental animal is as follow. 60 Balb/c mouse is 6 — 8 weeks old, and their body weights are 18 -22 g. All mouse are provided by the China Medical University animal experimental central.MethodThe brain tissue is dyeing by HE, and the pathomorphology changes of the brain tissue and the grades of the brain tissue are observed under light microscope.The ultramicroconstruction features are observed under electron micro-scope.TJ protein in the brain tissue of severe hepatitis patients are histochemical stained.TJ protein in the brain tissue of severe hepatitis experimental Balb/c mouse are histochemical stained, and the relative amounts of ZO - 1 in the brain tissues are examined by WESTERN BLOT and ZO -1 mRNA is examined by REAL TIME QRT - PCR.The same work is done after TNFa intervention.Results1. Brain edema was seen in the brain tissue of patients with severe hepatitis. It is maily vasogenic edema was seen. Endothelial cell shrinkage in the BBB of the brain tissue by transmission electron microscope,villus tubercle projected to the lumina,cellular membrane obscure,intranuclear heterochromatin collected sidely and conglomeration, basal membrane defect,chondrosome of the endothelial cell express vacuolar degeneration and many vescicles around it. TJ which lies inter endothelial cells loosen and broken.2. The immunihistory analyse of ZO - 1 protain in the brain tissue of patients with severe type hepatitis". Strong positive straining was seen in the intra-cytoplasmic or along the EC endomembrane of the blood vessel. Obvious weaken of the positive staining in the vessels of the FHF group and brown staining cant be seen in the whole slice.The immunihistory analyse of ZO - 1 protain in Balb/c mouse is the same as that of patients with severe type hepatitis.The semiquantitive analysis of ZO -1 protein;The semiquantitive analysis of the protein was done by Western Blot. There is an evid strap at 225KDa in the control group wheras the expression of ZO -1 protein weakened in the hepatic failure group.The expression of ZO -1 mRNA: The expression of ZO -1 mRNA of the hepatic failure mouse lowered and had significance difference with the control group. P <0.01.3. Intervention of TNFaThe results of ZO -1 immunohistochemistry staining in the brain tissue associated with TJ protein in hepatic necrosis model induced by TNFa, the relative amount of ZO -1 in the brain tissue by Western Blot and expression of ZO -1 mRNA by real time qrt - per were resemble to the above mentioned.Conclusions1. It can see different degree of brain edema in the brain tissue of severe hepatitis patients, which mainly present in vasogenic edema type.2. The ultrastructural characteristic of blood brain barrier ( BBB) in the brain tissue of severe hepatitis patients are: endothelial cell shrinkage, basal membrane defect, the tight junction ( TJ ) between cells were exist, loose, and disrupt.3. As the degree of edema is more aggravation in severe hepatitis patients, the ultrastructural characteristic of BBB presents more and more severe of destroy in endothelial cell membrane and basal membrane.4. The expression of protein ZO - 1 associated with tight junction (TJ) is significantly down regulation in the brain tissue of severe hepatitis patients, combining with the significantly down regulation of ZO -1 in animal model, it suggest that ZO -1 is the fundament of form and structure changing in TJ.5. The expression of ZO -1 protein is regulated by the mRNA transcription of ZO - 1, and it may be the direct evidence of separation and ruptured in TJ.6. In the animal model of FHF induced by TNFot/GalN, the TNFa participated in the regulation of ZO -1 and was the main reason of decreased expression of ZO -1.7. The results of intervention by TNFa suggests that TNFa participates in the signal conduction regulation of TJ by regulating to the ZO -1.