Aryl Hydrocarbon Receptor Activation In Intestinal Obstruction Ameliorates Intestinal Barrier Dysfunction Via Suppression Of MLCK-MLC Phosphorylation Pathway

BackgroundIntestinal epithelial barrier(IEB)is of importance structural and functional barrier.It not only could participate in the intestinal structure,but also regulate the intestinal digestion and absorption.Meanwhile,IEB,which could resist the invasion of pathogenic bacteria in vitro and effective removal of pathogenic bacteria in the body,plays a crucial role in maintaining the body's homeostasis.However,various acute or chronic pathological conditions can cause its destruction,bowel dysfunction,subsequent intestinal infection,and induce bacteremia or systemic inflammatory response syndrome(SIRS).Intestinal epithelial cells(IECs)are the main components of the intestinal mucosal barrier.Along with cell membranes,tight junctions,and their associated proteins,such as transmembrane protein claudins,occludin,and cytoplasmic plaque protein zonula occludens(ZO),they make up a selectively permeable complex.This complex is not only essential to the structure of the intestinal epithelia but also acts as the key trigger of the opening of the intestinal mucosal paracellular pathway.A large number of reports have shown that phosphorylation of myosin light chain(MLC)by myosin light chain kinase(MLCK)activation can induce the breakdown of the tight junction structural proteins ZO-1 and can increase tight junction permeability under specific pathological conditions.Therefore,it seems clear that the MLCK-pMLC pathway is of profound importance to the confusion of intestinal epithelial barrier by way of tight junction dysregulation.Aryl hydrocarbon receptor(AhR),a ligand-dependent transcription factor,is widely expressed in several kinds of tissues and cells.Upon ligand binding,activated AhR is transferred from the cytosol to the nucleus,after which it dimerizes with its nuclear translocator(ARNT)to regulate AhR target genes that contain functional xenobiotic response elements(XREs).It is significant to note that exogenous AhR activation has pernicious effects on the organism,while its activation by endogenous ligands,such as 6-formylindolo(3,2-b)carbazole(FICZ),sustains basic cellular physiological processes and functions.As a key regulators of cell signaling pathways,AhR has a very intimate relationship with many of the intestine's biological processes,such as cell differentiation,apoptosis,and the inflammatory response.Accumulating evidence indicates that AhR plays a role in modulating the processes within the intestinal epithelial barrier.However,most studies have focused on AhR expression in immune cells in the intestine.In fact,AhR is abundantly expressed in intestinal epithelial cells,which have more opportunity to come into contact with AhR agonists generated from food or commensal bacterial in the intestinal lumen.In this study,we investigated the roles of AhR in a mouse model of acute intestinal obstruction(IO).This research surveyed that how AhR activation by FICZ regulate barrier function under IO by hematoxylin-eosin(HE)stain,Western blot and realtime-PCR.Methods:1.Intestine was administered by mechanical block for 24 h to simulate the model of IO in C57BL/6.Intestinal morphology and permeability were observed by HE stain,immunofluorescence and Ussing Chambers.2.In IO model,the expression and activity of AhR was detected by immunofluorescent assay and realtime-PCR.3.In murine IO,the expression and distribution of ZO-1 was evaluated by immunofluorescence and realtime-PCR.4.Immunofluorescence and realtime-PCR were used to observe the expression and distribution of MLCK and pMLC in IO model.5.In vitro,Caco-2 were exposed to hypoxia for 12 h.Western bloting,realtime-PCR,TER and immunocytochemistry were used to observe the influence of activated AhR to cell membrane barrier and MLCK-pMLC pathway.6.AhR si RNA were transfected into hypoxic Caco-2.The proteins of ZO-1,MLCK and pMLC were investigated by Western blot.Results:1.IO cause the loss and shortening of villi,distinct epithelial cell diastasis,increased intestinal permeability,while intestinal mucosa damage were alleviative after treatment with FICZ.2.In IO,the continuity of ZO-1were damaged,and the expression were lost,however FICZ ameliorate them.3.FICZ inhibits IO-induced increases of MLC phosphorylation and MLCK expression.4.AhR activation prevented hypoxia-induced morphological destruction of tight junction through the suppression of MLCK-pMLC signaling in Caco-2 monolayers.5.Loss of AhR induces hypoxia-induced upregulation of MLC phosphorylation and MLCK expression in Caco-2 monolayers.Conclusion:1.Activated AhR is capable of protecting tight junctions in intestinal epithelia from disruption and attenuatin intestinal epithelial barrier dysfunction during murine IO.2.The suppression of MLCK-pMLC pathway is the mechanism by which AhR ameliorates intestinal barrier dysfunction in IO.