Effect of dysbiosis in Irritable Bowel Syndrome (IBS) via the modulation of the intestinal epithelial barrier : regulation of ROS production

Abstract

Microbial imbalance, known as dysbiosis, is considered one of the leading pathophysiological mechanisms during the onset of irritable bowel syndrome (IBS). Escherichia coli O157:H7, commensal Escherichia coli and Lactobacillus rhamnosus GG (commensal bacterial strains) are among the main bacteria dysregulated during IBS. Intestinal flora modulates the function of intestinal epithelial cells. In response to bacterial infection, excessive production of reactive oxygen species (ROS) produced by intestinal epithelial and immune cells leads to oxidative stress that disrupts mucosal barrier function through mechanisms that destroy tight junctions’ proteins (TJs) such as oxidation, nitration, disassembly and instability of actin cytoskeleton in addition to the production of zonulin, which is a protein affecting tight junctions’ proteins. In this context studying the effect of Escherichia coli O157:H7, commensal Escherichia coli and Lactobacillus rhamnosus GG on intestinal epithelial cells function, may open new perspectives in the treatment of IBS. Our aim is to elucidate the effect of these bacterial strains on the function of human intestinal epithelial cells. First, ROS production by human colorectal carcinoma cells HCT116 has been assessed using nitroblue tetrazolium (NBT) reduction assay in coincubation systems. In parallel, mRNA has been extracted and the gene expression of ROS- producing enzymes NOX1 and NOX1 regulatory component NOXO1 (NADPH oxidase organizer 1) has been quantified by qRT-PCR. Finally, as increased zonulin levels have been shown to promote tight junctions’ disruption and to affect epithelial barrier integrity during IBS, expression of zonulin gene has been determined as well. Commensal E. coli was found to be, for the first time, a potent inducer of ROS in HCT116 cells. While E. coli O157:H7 was found to be a good inducer of ROS, L. rhamnosus induced moderate ROS production in HCT116 cells. On the other hand, we couldn’t determine a gene expression for NOX1, NOXO1 an Zonulin in HCT116 cells after 24 hours of coincubation with the three types of bacteria.