Tu1374 1,25-Dihydroxyvitamin D3 Preserves Intestinal Epithelial Barrier Function From TNF-α Induced Injury via Suppression of MLCK-Mlc Phosphorylation Signaling Pathway

Abstract

Intestinal barrier dysfunction occurs inmultiple enteropathies. Some known proinflammatory cytokines, such as TNF-a, INF-γ, play a pivotal role as mediator between various pathogens and gut epithelium. In our previous work, TNF-α injured Caco-2 monolayers were used to screen potential barrier protective reagents. 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) turned out to be a very effective protector. This is consistent with literatures which report that 1,25(OH)2D3 has anti-inflammation effect in bowel diseases. To elucidate the underlying mechanism, we performed this work. Given the fact that tight junction (TJ) is the important intercellular structure related with intestinal epithelial barrier function, and that the key role of myosin light chain (MLC) phosphorylation in regulating TJ. The TJ status and MLC phosphorylation was studied.Methods Experiments were conducted on Caco-2 monolayers. Barrier dysfunction was generated by 10ng/ml TNF-α treatment for up to 48h. Barrier function was represented by trans-epithelial electrical resistance (TEER), and macro molecule tracer, FITC-Dextran 40,000 Da (FD-40) trans-membrane flux. Morphological changes of intercellular TJ were visualized by immunofluorescence staining of ZO-1 and Occludin. The expression level of phosphorylated MLC (P-MLC), and its upstream MLC kinase (MLCK) were determined by immunoblotting. The nuclear translocation and activation of NF-kB p65 was analyzed by Electrophoretic Mobility Shift Assay (EMSA) and immunofluorescent staining. Results TNF-α decreased TEER by ~60% after 48h treatment, 10-7M 1,25(OH)2D3 reversed TEER to ~80% of original level. Consistently, TNF-α increased FD-40 flux through monolay by nearly 5 folds, 1,25(OH)2D3 repressed the number to about2 folds. TJ structure altered significantly following TNF-α treatment. 1,25(OH)2D3 ameliorated TNF-α-induced TJ damage. The MLC phosphorylation, as well as MLCK protein expression were induced by TNF-α. These increases were significantly inhibited by 1,25(OH)2D3 treatment. Additionally, 1,25(OH)2D3 suppressed the nuclear translocation and activation of NF-kB p65.Conclusion 1,25(OH)2D3 attenuates TNF-α-induced intestinal epithelial barrier dysfunction by inhibiting the signaling pathway ofMLCK-dependentMLCphosphorylation via NF-kB p65 pathway.