Original Article
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 21, 2014; 20(35): 12533-12541
Published online Sep 21, 2014. doi: 10.3748/wjg.v20.i35.12533
Protective action of NADPH oxidase inhibitors and role of NADPH oxidase in pathogenesis of colon inflammation in mice
Rima Ramonaite, Jurgita Skieceviciene, Simonas Juzenas, Violeta Salteniene, Juozas Kupcinskas, Paulius Matusevicius, Vilmante Borutaite, Limas Kupcinskas
Rima Ramonaite, Jurgita Skieceviciene, Simonas Juzenas, Violeta Salteniene, Juozas Kupcinskas, Limas Kupcinskas, Institute for Digestive Research, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas LT 44307, Lithuania
Juozas Kupcinskas, Limas Kupcinskas, Department of Gastroenterology, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas LT 50161, Lithuania
Paulius Matusevicius, Department of Animal Nutrition, Academy of Veterinary, Lithuanian University of Health Sciences, Kaunas LT 47181, Lithuania
Vilmante Borutaite, Institute for Neurosciences, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas LT 44307, Lithuania
Author contributions: Ramonaite R and Skieceviciene J performed the experiments, analysed the data, interpreted the results, prepared the figures, and drafted the manuscript; Juzenas S and Salteniene V performed the experiments and analysed the data; Matusevicius P and Kupcinskas J interpreted the results, analysed the data, and drafted the manuscript; Borutaite V and Kupcinskas L contributed to the conception and design of the research, interpreted the results, and approved the final version of the manuscript.
Correspondence to: Limas Kupcinskas, MD, Professor, Institute for Digestive Research, Academy of Medicine, Lithuanian University of Health Sciences, A. Mickeviciaus str. 9, Kaunas LT 44307, Lithuania. kupcinskas@gmail.com
Telephone: +37-37-326508 Fax: + 37-37-326508
Received: December 17, 2013
Revised: February 11, 2014
Accepted: June 12, 2014
Published online: September 21, 2014
Processing time: 276 Days and 17.1 Hours
Abstract

AIM: To investigate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation in a mouse model of dextran sulphate sodium (DSS)-induced colitis.

METHODS: Balb/c mice were divided into three groups: 8 mice with acute DSS-induced colitis (3.5% DSS solution; 7 d), 8 mice with chronic DSS-induced colitis (3.5% DSS solution for 5 d + water for 6 d; 4 cycles; total: 44 d) and 12 mice without DSS supplementation as a control group. Primary colonic epithelial cells were isolated using chelation method. The cells were cultivated in the presence of mediators (lipopolysaccharide (LPS), apocynin or diphenyleneiodonium). Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorometrically using Amplex Red. Production of tumour necrosis factor-alpha (TNF-α) by the colonic epithelial cells was analysed by ELISA. Nox1 gene expression was assessed by real-time PCR.

RESULTS: Our study showed that TNF-α level was increased in unstimulated primary colonic cells both in the acute and chronic colitis groups, whereas decreased viability, increased ROS production, and expression of Nox1 was characteristic only for chronic DSS colitis mice when compared to the controls. The stimulation by LPS increased ROS generation via NADPH oxidase and decreased cell viability in mice with acute colitis. Treatment with NADPH oxidase inhibitors increased cell viability and decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from mice of both acute and chronic colitis groups.

CONCLUSION: Our study revealed the importance of NADPH oxidase in the pathogenesis of both acute and chronic inflammation of the colon.

Keywords: Apocynin; Diphenyleneiodonium; Dextran sulphate sodium-induced colitis; Inflammatory bowel disease, Nicotinamide adenine dinucleotide phosphate oxidase; Reactive oxygen species; Tumour necrosis factor-α

Core tip: Reactive oxygen species (ROS)-induced oxidative stress is one of the most important etiological factors involved in the inflammation. The key producers of ROS in cells are nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes. Several studies have shown that epithelial NADPH oxidase might be responsible for normal immune response to antigens in the gut. On the other hand, little is known about the molecular pathways controlling ROS production via NADPH oxidase in primary intestinal epithelial cells during inflammation. The aim of this study was to investigate the role of NADPH oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation using a mouse model of dextran sulphate sodium-induced colitis. The results of our study revealed the importance of NADPH oxidase in the pathogenesis of colon inflammation.