S1354 Modification of the Tight Junctional Barrier in Human Duodenal Mucosal by Oral Zinc Administration in a Patient-Based Study: RNA-Seq and Western Immunoblot Analyses

Abstract

Introduction: Mucosal barrier function is targeted in a wide variety of GI disease. Celiac disease, IBD, GI malignancies, sepsis and a wide variety of GI microbial infectious diseases all compromise the GI barrier. The tight junctional (TJ) complex is pivotal in this regard. The ability to safely modify the TJ complex and reduce its leakiness could be prophylactically and/or therapeutically useful. Methods: Patients 18-80 years of age presenting for endoscopic medical surveillance of gastroesophageal disorders but free of current small bowel disease were recruited for study. Patients were instructed to administer orally either a zinc-gluconate medication (12 patients) (26 mg elemental zinc) or a sodium gluconate placebo (11 patients), BID, for 14 days immediately prior to their EGD. During their EGD, standard duodenal bulb biopsies were taken for Western immunoblot analysis and for mRNA analysis. Biopsies were immediately flash frozen on dry ice in the procedures room and stored at -195oC for subsequent extraction. For protein studies, biopsies were homogenized in lysis buffer, sonicated and then analyzed by PAGE and Western immunoblot using a variety of antibodies to TJ proteins. Analysis of the biopsy transcriptome was performed by high-throughput RNA-seq. Results: Transcriptome analysis of over 20,000 genes revealed upregulation of many known zinc-regulated genes such as metallothioneins A, B, F, G, H, L, and M. The TJ barrier gene, occludin (OCLN), was also upregulated. Enrichment analysis using GSEA showed zinc upregulating several gene ontology (GO) biological processes as well as cellular components and pathways (Reactome). These included Zinc Ion Homeostasis (GO), Response to Zinc Ion (GO), Cell Junction Organization (Reactome), Cell-Cell Junction Assembly (GO), and Tight Junction (GO). Western immunoblot analyses showed statistically significant upregulation (two-tailed t-test) of the TJ proteins, claudin-2 and tricellulin, in mucosal biopsies of the zinc-treated group. Claudins -3 and -5 showed significant upregulations, and claudin-7, a significant down regulation, in a one-tailed t-test. Claudins -1, -4 and -18 showed no trend regarding a zinc-induced change. Conclusion: This is the first evidence of intended modification of TJ complexes in humans in situ. The results follow on a large body of published literature showing zinc-induced TJ changes and improved barrier function in a variety of human epithelial cell culture and animal models.