Joshua A. Englert

Bile modulates intestinal epithelial barrier function via an extracellular signal related kinase 1/2 dependent mechanism.

ObjectiveObstructive jaundice is frequently complicated by infections and has been associated with increased bacterial translocation and gut mucosal hyperpermeability in animal models. Proper expression of the tight junction (TJ) proteins ZO-1 and occludin is important for normal gut barrier function. We tested whether bile modulates intestinal epithelial ZO-1 and occludin expression.Animals(a) Male C57BL/6 mice; (b) male Sprague-Dawley rats.Interventions(a) Mice were subjected to common bile duct ligation (CBDL) or a sham procedure, and 96xa0h later all surviving animals were killed for measurement of ileal mucosal permeability to FITC-labeled dextran (everted gut sac technique), bacterial translocation to mesenteric lymph nodes, and ileal epithelial ZO-1 and occludin expression (western blots). (b) Rat IEC-6 enterocytic monolayers were incubated in the presence or absence of graded concentrations of rat bile and/or U0126, an inhibitor of extracellular signal related kinase (ERK) 1/2 activation.Results(a) Compared to sham-treated controls, CBDL significantly increased gut mucosal permeability and bacterial translocation and markedly decreased ileal epithelial expression of ZO-1 and occludin. In a follow-up in vivo experiment, gavaging mice with fresh rat bile twice daily significantly ameliorated the deleterious effects of CBDL on gut barrier function. (b) Addition of 1% (v/v) bile to media enhanced phosphorylation of ERK1/2, increased the expression of ZO-1 and occludin and decreased permeability to FITC-dextran. All of these bile-mediated effects were blocked by 10xa0µM U0126.ConclusionsThese data support the view that the presence of bile in the intestinal lumen is essential for normal gut barrier function, possibly because compounds present in bile initiate ERK1/2-dependent signaling that is essential for normal expression of key TJ proteins.

Ethacrynic acid inhibits multiple steps in the NF-κB signaling pathway

Ethacrynic acid has been used as a safe and effective diuretic for more than 30 years. In this study, we tested the hypothesis that ethacrynic acid is also an anti-inflammatory agent that inhibits signaling by the proinflammatory transcription factor NF-κB. We showed that ethacrynic acid inhibited luciferase expression in lipopolysaccharide-stimulated macrophage-like RAW 264.7 cells transfected with an NF-κB-dependent luciferase reporter vector and also inhibited NF-κB DNA binding in lipopolysaccharide-stimulated RAW 264.7 cells (electrophoretic mobility shift assay). Ethacrynic acid inhibited degradation of IκBα and IκBβ in lipopolysaccharide-stimulated RAW 264.7 cells. Ethacrynic acid impaired DNA binding of wild-type p65 subunits of NF-κB in cells. However, DNA binding of a Cys38 → Ser p65 mutant was not inhibited by ethacrynic acid, suggesting that ethacrynic acid inhibits DNA binding by alkylating p65 at Cys38. In a cell-free system, binding of p50 homodimers to an NF-κB consensus sequence was inhibited by ethacrynic acid at concentrations from 10 to 100 μM, indicating that ethacrynic acid probably also covalently modifies the p50 subunit. These data indicate that ethacrynic acid inhibits activation of the NF-κB pathway at multiple points and suggest that this well-studied drug warrants further investigation as a potential therapeutic for various conditions that are associated with excessive inflammation.