[The impact of tumor necrosis factor alpha on expression of intestinal epithelial tight junction protein occludin in fulminant hepatic failure mice model].
OBJECTIVE
We reported recently that intestinal epithelial tight junction protein occludin was reduced in mice with fulminant hepatic failure (FHF), when there was accompanying high levels of serum TNFalpha. This study was to explore the role of TNFalpha in the changes of intestinal epithelial tight junction protein occludin in mice with FHF.


METHODS
Male BALB/c mice were divided into 6 groups. In one group, the mice were intraperitoneally injected with lipopolysaccharide (LPS, 10 microg/kg) and D-galactosamine (GalN, 800 mg/kg) to induce acute liver necrosis. Anti-TNFalpha-IgG (100 microg/mouse) was injected through vena caudalis 30 minutes before GalN/LPS administration. The remaining groups were intraperitoneally injected with GalN, LPS, TNFalpha (10 microg/kg) or saline respectively to serve as controls. Mice were executed 6 and 9 hours after administration and intestinal specimens were obtained. The location and expression of intestinal epithelial tight junction protein occludin were detected with immunohistochemistry and Western blot. Real time quantitative PCR was used to detect the mRNA level of occludin.


RESULTS
Tight junction protein occludin was localized on the apical region of lateral plasma membrane representing the region of tight junctions in the surface and crypt epithelial cells. In the mucosal tissue from mice with FHF and with TNFalpha injection for 9 hours, occludin-staining was globally down-regulated. Results from Western blot demonstrated consistent and significant reduction of occludin expression in the mice with FHF (0.36 +/- 0.05 vs 0.71 +/- 0.09, P < 0.05) and with TNFalpha injection for 9 hours (0.48 +/- 0.02 vs 0.71 +/- 0.09, P < 0.05). Anti-TNFalpha-IgG can prevent the changes of occludin as examined both with immunohistochemistry and with Western blot (0.74 +/- 0.03 vs 0.71 +/- 0.09, P > 0.05). Furthermore we evaluated occludin mRNA expression by real time quantitative PCR and observed marked down-regulation of occludin mRNA in the mice with FHF and TNFalpha injection for 6 hours as compared with the controls (0.72 +/- 0.04, 0.81 +/- 0.03 vs 1.00 +/- 0.05, P < 0.05), Anti-TNF-IgG restored the state of occludin mRNA expression in the mice with FHF to that in controls (1.01 +/- 0.10 vs 1.00 +/- 0.05, P > 0.05).


CONCLUSIONS
The reduction of tight junction protein occludin in intestinal epithelial cells may be a result of the production of TNFalpha in mice with fulminant hepatic failure.
Department of Digestive Diseases, The First Affiliated Hospital of China Medical University, Shenyang 110001, China.
Wei
W
Cui
Ying
Y
Wang
Li
L
Ma
Ying
Y
Wen
Pei
P
Liu
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D015153:Blotting, Western; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D007274:Injections, Intraperitoneal; D007420:Intestine, Large / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017114:Liver Failure, Acute / Q000503:physiopathology*; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D062793:Occludin; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000008:administration & dosage / Q000276:immunology / Q000494:pharmacology*
D000906:Antibodies; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha