Splenectomy attenuates severe thermal trauma-induced intestinal barrier breakdown in rats / 华中科技大学学报（医学）（英德文版）

The severe local thermal trauma activates a number of systemic inflammatory mediators, such as TNF-α, NF-κB, resulting in a disruption of gut barrier. The gastrointestinal tight junction (TJ) is highly regulated by membrane-associated proteins including zonula occludens protein-1 (ZO-1) and occludin, which can be modulated by inflammatory cytokines. As splenectomy has been shown to reduce secretion of cytokines, we hypothesized that (1) severe scald injury up-regulates TNF-α and NF-κB, meanwhile down-regulates expression of ZO-1 and occludin, leading to the increased intestinal permeability, and (2) splenectomy can prevent the burn-induced decrease in ZO-1 and occludin expression, resulting in improved intestinal barrier. Wistar rats undergoing a 30% total body surface area (TBSA) thermal trauma were randomized to receive an accessorial splenectomy meanwhile or not. Intestinal injury was assessed by histological morphological analysis, and serum endotoxin levels, TNF-α, NF-κB, ZO-1 and occludin levels were detected by Western blotting in the terminal ileum mucosal tissue. 30% TBSA burn caused a significant increase in serum endotoxin levels, but NF-κB, and TNF-α, and the average intestinal villus height and mucosal thickness were decreased significantly. Burn injury could also markedly decrease the levels of ZO-1 and occludin in terminal ileum mucosal tissue (all P<0.01). Splenectomy at 7th day after burn significantly reversed the burn-induced breakdown of ZO-1 and occludin (all P<0.01). The results of this study suggest that severe thermal injury damages the intestinal mucosal barrier. Splenectomy may provide a therapeutic benefit in restoring burn-induced intestinal barrier by decreasing the release of inflammatory cytokines and recovering TJ proteins.

THE EXPRESSION AND ASSOCIATION OF CONNEXIN 36 WITH ZONULA OCCLUDENS PROTEIN 1 IN HeLa CELLS / 解剖学报

Objective To investigate the expression and association of Cx36 with ZO-1 in Cx36 transfected HeLa cells. Methods RT-PCR,PCR products cloning into PCR2.1TOPO vector,Cx36-pcDNA3 expression vector construction,cell culture,transient transfection using lipofectamine 2000,stable clone screening with G418,Western blotting,double immunofluorescence,and immunoprecipitation(IP) were used. Results Cx36-pcDNA3 expression vector was constructed and transfected into HeLa cells.Using homogenates from Cx36 transfected HeLa cells,Western blotting showed Cx36 band.By immunofluorescence microscopy,Cx36 transfected HeLa cells displayed punctate immunolabeling for Cx36 between cells.Irmmunolabeling for ZO-1 in these cells exhibited similar distribution to Cx36.By laser scanning confocal microscopy after double labeling with Cx36 and ZO-1 antibody revealed a high degree of Cx36 and ZO-1 colocalization at sites of cell-cell contact.Cx36 transfected HeLa cells were taken for IP with ZO-1 antibody,blots of IP protein probed with Cx36 antibody showed detectin of Cx36.Blots showed detection of ZO-1 after IP with Cx36 antibody from Cx36 transfected HeLa cells.Conclusion Cx36 was expressed in Cx36 transfected HeLa cells,Cx36 colocalizated and associated with ZO-1 in Cx36 transfected HeLa cells.