Gastroesophageal Reflux Attenuates The Barrier Function Of Mouse Esophageal Epithelium Through NFκB-mediated Inflammation

Background and Aim:The barrier function of esophageal epithelium is a major defense against gastroesophageal reflux. Previous studies have shown that reflux damage is reflected by increased permeability of esophageal mucosa for ions and macromolecules and dilated intercellular space in the esophageal epithelium. In order to develop novel therapies, it is critical to understand how contact with a refluxates attenuates esophageal barrier function. Barrett metaplasia usually occurs as a consequence of gastroesophageal reflux disease, but the exact molecular mechanism of gastroesophageal reflux progressing into Barrett esophagus remains poorly understood. This study aims1) investigating the role of NFκB-mediated inflammation in attenuated esophageal barrier function due to gastroesophageal reflux;2) investigating the molecular mechanism of gastroesophageal reflux progressing into Barrett esophagus.Methods:In this study, gastric, duodenal and mixed reflux models were developed in mice through surgical performance. Transepithelial electrical resistance (TEER) was measured by mini-Ussing chamber for different reflux models, while dilated intercellular space (DIS) observed with electrical microscope. Mouse esophageal epithelium is analyzed by gene microarray and statistical strategies (i.e.,Gene Set Enrichment Analysis and Significance Analysis of Microarray), which screened out activated transcription factors, canonical signaling pathways and gene ontology, as well as up-or down-regulated genes in mouse genome. Genes screened were confirmed by gene expression assays (i.e.,Real-time PCR, Western Blotting, immunohistochemical staining and ELISA). Distribution of inflammatory cells in esophageal mucosa of different reflux models was shown by specific histochemical staining, while semi-quantification of inflammatory cells was caculated. Upon mice treated with NFκB inhibitor Bay11-7085(20mg/kg/day, i.p.), TEER and cytokines were measured in esophageal mucosa of duodenal and mixed reflux models.Results:A decrease of TEER and DIS was observed in esophageal mucosa of duodenal and mixed reflux models as compared with control, but not in that of gastric reflux model. GSEA showed the activation of inflammation-related gene sets and canonical signaling pathways in esophageal epithelium of different reflux models, and activation of NFκB due to duodenal and mixed reflux. SAM revealed up or down-regulation of NFκB target genes, Barrett-related genes and tight junction genes. Real-time PCR confirmed down-regulation of Cldnl, Cldn4, Cldn10and Cldn23and up-regulation of Cldn7. Immunohistochemical staining showed up-regulation of NFκB p50/p65, NFκB target genes MMP3/MMP9and Cdx2in esophageal epithelium of mixed and duodenal reflux models. An increase of cytokines (i.e. IL1β, IL6and IL8) and cell counts (i.e. neutrophils, eosinophils and mast cells) was observed in esophageal mucosa of duodenal and mixed reflux models as compared with control. Treatment with an NFκB inhibitor, Bay11-7085counteracted the effects of duodenal and mixed reflux on TEER and cytokines.Conclusion:Our results suggested1) gastroesophageal reflux attenuates the barrier function of mouse esophageal epithelium through NFκB-mediated inflammation;2) Gastroesophageal reflux induces Barrett’s metaplasia in esophageal epithelium through activation of Barrett’s genes.Nineteen pictures,5tables and85references are included in this manuscript.