Background Glucocorticoid Hormones (GC) are an effective therapeutic agent for the treatment of the intestinal inflammation of Crohn's disease. However, the precise anti-inflammatory mechanism of GC action in Crohn's disease is unclear. Recently, we have shown that the pro-inflammatory cytokine TNF-α (which is markedly increased in IBD) causes an increase in intestinal epithelial tight junction(TJ) permeability. We hypothesize that GC have a direct effect in protecting the intestinal TJ barrier from TNF-α disruption. The purpose of this study is to test this hypothesis and examine the molecular mechanisms behind GC protection of the TNF-α induced defect of the intestinal epithelial barrier.
Methods The epithelial TJ permeability of CaCo-2 intestinal cells was measured using trans-epithelial electrical resistance of filter grown monolayers. Mechanism of action was evaluated using EMSA, immunohistochemistry, Western blot and luciferase promoter assays.
Results 1) TNF-α causes a decrease in TJ epithelial permeability across CaCo-2 monolayers. 2) TNF-α causes translocation of the p65 subunit of NF-κB from the cytoplasm to the nucleus, an increase in DNA binding to an NF-κB consensus oligonucleotide and activation of an NF-κB responsive promoter. 3) TNF-α causes down-regulation of Occludin protein expression, the key component of the extra-cellular TJ barrier. 4) Prednisolone (1-10μM) and Dexamethasone (0.25μM) pre-treatment completely inhibited the TNF-α induced decrease in CaCo-2 TJ permeability. 5) GC treatment did not affect TNF-α induced NF-κB nuclear translocation, DNA binding or NF-κB responsive promoter activation. 6) GC inhibited the TNF-α induced down-regulation of Occludin protein expression.
Conclusion Prednisolone inhibits the increase in CaCo-2 TJ permeability caused by TNF-α. The mechanism of prednisolone action involved inhibition of the TNF-α induced decrease in TJ protein expression without altering NF-κB activation. Future studies will elucidate the mechanism of GC action on TNF-α responsive tight junction proteins.