Activation Of Aryl Hydrocarbon Receptor Maintained The Intestinal Epithelial Barrier Function Through Notch1 Dependent Signaling Pathway

Background:Intestinal ischemia/reperfusion(I/R)is the common cause of the intestinal barrier dysfunction.It can lead to the shedding of the epithelium,high mucosal permeability,bacterial translocation and enterogenic infection,initiating the systemic inflammatory response syndrome(SIRS)and finally leading to the multiple organ dysfunction syndrome(MODS)with high mortality rate in hospital.Tight junctions(TJs),which is composed of a cluster of transmembrane proteins(including ZOs,Occluin and Claudins)and firmly connected between the adjacent epithelial cells,regulates the permeability of ions,water and nutrients via the paracellular pathway,establishes the cell polarity and functions as a primary determinant of intestinal epithelial barrier(IEB)function.Previous studies have demonstrated Aryl hydrocarbon receptor(AhR)plays a vital role in maintaining the intestinal Immunologic barrier function.AhR activation could ameliorate the mucosal injury in several animal models of experimental colitis by up-regulation of the suppression of inflammatory factors such as IL-10 and IL-22,and down-regulation of the pro-inflammatory factors like IFN-??TNF-?,etc.However,the precise mechanism of AhR in regulating the intestinal epithelial barrier function still remains unclear.Notch1 signaling is proved to be a downstream signaling of AhR and also has been reported to play a key role in the development of tight junctions(TJs)and maintenance of the intestinal homeostasis.Since both AhR and Notch1 expressed in the intestinal epithelium,Therefore,we hypothesized that AhR activation could attenuate the intestinal barrier dysfunction via the regulation of the TJ proteins through Notch1 dependent signaling pathway.In the present study,we investigated the role of AhR activation by Ficz in modulation of intestinal barrier function in a mouse model of intestinal I/R and in hypoxic condition in Caco-2 cell line.This study might be conducive to provide a new targets and theoretical basis for the prevention and cure of the intestinal barrier dysfunction induced by I/R.Methods:1.Adult C57BL/6J mice were sacrificed after intestinal I/R and the intestines were harvested for histological examination by H & E stain and Chiu's score,and mucosal permeability investigation by Ussing Chambers.2.In a mouse model of I/R,the expression and distribution of ZO-1 and Occludin were detected by Immunohistochemical staining and Western blot assay.3.In murine I/R,the expression and activity of AhR-Notch1 were examine by Immunohistochemical staining,RT-qPCR and Western blot assay.4.In a hypoxic Caco-2 cell culture model,the role of AhR activation by Ficz in regulation of the expression and activity of Notch1 signaling components was investigated by Western blot assay and Immunofluorescence staining.5.In hypoxic Caco-2 cell line,the effect of AhR activated by Ficz on modulation of the expression and distribution of TJ proteins(ZO-1 and Occludin)was assessed by Immunofluorescence staining and Western blot assay.Epithelial permeability was detected by trans-epithelium electric resistance(TER).6.In hypoxic Caco-2 monolayers with or without Ficz or DAPT,the expression and activity of AhR-Notch1 signaling components and TJ proteins(ZO-1 and Occludin)were detected by Western blot assay.Results:1.In Murine I/R,the normal structure of the intestinal epithelial cell was collapsed,including the villi lodging,breaking,edema and shedding.The mucosal TER decreased while the permeability increased.However,Ficz significantly attenuated the intestinal tissue damage and obtunded the decrease in TER.2.In Murine I/R,the continuous distribution and the expression of TJs(ZO-1 and Occludin)were disorganized while Ficz markedly alleviated the disrupted distribution of TJs(ZO-1 and Occludin),increased the protein expression of ZO-1 and Occludin.3.Murine I/R inhibited the AhR-Notch1 signaling pathway while Ficz significantly activated the AhR-Notch1 signaling after I/R.4.In a hypoxic Caco-2 cell culture model,the expression of the component of AhR-Notch1 signaling decreased and the general architecture of the TJs(ZO-1 and Occludin)were collapsed.Ficz activated the epithelial Notch1 signaling pathway,maintained the distribution and expression of TJs,and ameliorated the epithelial barrier dysfunction caused by hypoxia.5.In hypoxic condition,blocking of Notch1 signaling with DAPT has no remarkably changes on the expression and activity of AhR signaling with or without Ficz,it counteracted the development of TJs(ZO-1 and Occludin)induced by Ficz.Conclusion:1.Activation of AhR by Ficz could maintain the intestinal histomorphology and the osmotic balance of the intestinal epithelium through the regulation of the distribution and expression of the TJs,thereby maintained the IEB function.2.Activation of Notch1 signaling is the underlying mechanism by which AhR activation regulation of the expression of TJs.