PPI ADMINISTRATION CAUSES COLONIC TIGHT JUNCTION BARRIER DYSFUNCTION VIA INCREASE INENETROCOCCUS FECALIS

Abstract BACKGROUND Proton pump inhibitors (PPIs) are highly effective antagonists of gastric acid secretion and are widely used to treat a number of gastroesophageal disorders, including peptic ulcer disease, GERD, Barrett’s esophagus. PPI-induced elevation in intra-gastric pH and subsequent alterations of gastrointestinal physiology are known to cause undesired effects on the entire GI tract. Gastric acid suppression can change the composition of the intestinal microbiota causing intestinal dysbiosis and increased risk for intestinal inflammation. The intrinsic intestinal barrier is composed of a single layer of columnar epithelial cells and inter-epithelial tight junctions (TJ) residing at the apical-most region of the intercellular space. Defective intestinal TJ barrier is an important pathogenic factor for intestinal inflammation. The role of PPI on Intestinal TJ barrier is not known. AIM The aim of the present study was to study the effect of PPI on intestinal dysbiosis and mouse colonic TJ permeability. METHODS 8 to 12-week-old male C57BL/6N mice were divided into two groups (Controls and PPI treatments (Omeprazole 20mg/kg B.W.) and were fed a nutritionally adequate feed for 30 days. Fecal samples were collected from both treated and untreated mice after 30 days. Fecal DNA was isolated using the Qiagen kit according to the manufactures Instructions. 16s amplicon DNA was sequenced by Genewiz (South Plainfield, NJ). Degenerate primers that amplify the V3-V4 region of bacterial rDNA genes were used (F341-806R pair).The amplicon pool sequenced on Illumina MiSeq generated paired-end 301 bp reads was multiplexed using Illumina’s fastq. At the end of the experiments, the colonic permeability was measured on Ussing chambers. Western blot (WB) and Immunofluorescence (IF) was used to study myosin light chain kinase (MLCK, a key regulator of TJ barrier) protein expression in mice colon. RESULTS The microbiome changes identified by 16S rRNA sequencing showed a significant increased proportion of Firmucutis in the fecal samples of PPI treated mice compared with untreated mice. Also, PPI group showed prominent increase in enterococcus spp which was identified as Enterococcus fecalis (EF). In Ussing chambers studies, PPI administration caused a decrease in transepithelial resistance (TER), and increase in urea and inulin flux when compared to untreated colonic tissues. Immunofluorescence and Western blot analysis showed that PPI administration also caused an increase in MLCK and phospho- MLC protein expression in mice colonic tissues. Also, PPI treatment caused increased focal sloughing of colonic surface epithelium with focal to diffuse lymphocytic infiltration in the colonic lamina propria in mice colon. CONCLUSION Our results suggest that the PPI causes overgrowth of a pathobiont EF associated increase in mouse colonic TJ barrier permeability.