Influence of NADPH oxidase on inflammatory response in primary intestinal epithelial cells in patients with ulcerative colitis

Rima Ramonaite,Laimas Jonaitis,Limas Kupcinskas,Gediminas Kiudelis,Vilmante Borutaite,Jurgita Skieceviciene,Paulius Cizas,Algimantas Tamelis

doi:10.1186/1471-230x-13-159

Rima Ramonaite, Laimas Jonaitis + Show 6 more

Open Access

https://doi.org/10.1186/1471-230x-13-159

Copy DOI

Abstract

BackgroundThe aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells during the active phase of UC.MethodsThe primary human colonic epithelial cells were isolated from 19 patients with mild to moderate inflammatory activity of UC and 14 controls using chelation method. The cells were cultivated under the effect of mediators. Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorimetrically using Amplex Red. Production of TNF-α cytokine by the colonic epithelial cells was analysed by ELISA.ResultsThe results of our study showed that unstimulated cells of UC patients had a decreased viability, increased ROS production, but similar TNF-α level when compared to the controls. Stimulation with LPS increased hydrogen peroxide and TNF-α level in the UC group. Treatment of colonic epithelial cells with NADPH oxidase inhibitor increased cell viability decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from UC patients.ConclusionsOur study showed that bacterial endotoxins induced NADPH oxidase activation in the colonic epithelial cells. Moreover, we revealed that treatment with NADPH oxidase inhibitors had a protective effect against pro-inflammatory action of LPS in human colonic epithelium cells during inflammation.

Highlights

The aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells during the active phase of Ulcerative colitis (UC)
Assessment of viability of human colonic epithelial cells Firstly, we investigated whether there was any difference in survival of colonic epithelial cells isolated from UC patients and control individuals in cell culture
Stimulation of colonic epithelial cells with LPS significantly reduced the number of live cells from 72% to 57% and increased necrotic cell death from 18% to 37% in the control group; whereas the viability of cells in UC group remained unchanged upon LPS treatment (Figure 1B)

Summary

Introduction

The aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells during the active phase of UC. One of the major factors in the onset of UC is inappropriate mucosal immune response towards the intestinal microbiota leading to mucosal tissue damage and chronic inflammation [1,2,3]. Increased production of reactive oxygen species (ROS) and oxidant-induced protein or lipid alterations have been implicated in tissue damage observed in chronic inflammatory disorders, such as IBD [4]. Previous studies have shown that epithelial NADPH oxidase mediating formation of ROS might be involved in host defence system and inflammatory responses at mucosal surfaces [6,7,8]. The variations have been found in genes responsible for localization of the NADPH oxidase complex (including p47phox and p67phox and RAC2) to the membrane [10,11]

Objectives

Methods

Results

Conclusion