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P053. Influence of NADPH oxidase on inflammatory response in the primary intestinal epithelial cells from patients with ulcerative colitis

R. Ramonaite1, L. Jonaitis1, G. Kiudelis1, A. Tamelis2, J. Skieceviciene1, V. Borutaite3, L. Kupcinskas1, 1Lithuanian University of Health Sciences, Institute for Digestive Research, Kaunas, Lithuania, 2Lithuanian University of Health Sciences, Department of Surgery, Kaunas, Lithuania, 3Lithuanian University of Health Sciences, Institute of Neurosciences, Kaunas, Lithuania

Background

The aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells in pathogenesis of ulcerative colitis (UC).

Methods

The primary human colonic epithelial cells from 19 patients with mild to moderate inflammatory activity of UC and 14 controls were isolated using chelation method. These 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-alfa cytokine by the colonic epithelial cells was analyzed by two-site ELISA.

Results

We found that treatment of colonic epithelial cells with NADPH oxidase inhibidor apocynin and catalase increased the viability of cultured cells isolated from UC patients. In both control and UC groups, apocynin also increased the viability of cells treated with bacterial lipopolysaccharide (LPS). Addition of NADPH oxidase inhibitor diphenylene iodonium (DPI) prevented pro-inflammatory LPS effect on colonic epithelial cells by decreasing the level of ROS production in UC group and controls. Stimulation with LPS also increased concentration of TNF-alfa in cells of UC patients. The treatment of cells with apocynin prevented LPS effects and decreased the level of TNF-alfa production in cells of UC patients.

Conclusion

In conclusion, the data demonstrate that colonic epithelial cells isolated from UC patients have lower survival in cell culture conditions than cells isolated from healthy individuals due to NADPH oxidase activity generating hydrogen peroxide. The study revealed that stimulation with LPS increased the level of TNF-alfa and generation of ROS related to NADPH oxidase activity in the colonic epithelial cells of UC patients. Treatment with NADPH oxidase inhibitors apocynin/DPI had protective effect against pro-inflammatory action of LPS in these cells.