| Background: Inflammatory bowel disease(IBD),which is comprised of Ulcerative colitis(UC)and Crohn's disease(CD),is a chronic nonspecific intestinal inflammatory disease.IBD is common in developed countries.In China,Singapore and other Asian countries,the incidence of IBD is increased rapidly recent decades.Although the etiology of IBD is not yet clear,a combination of genetic susceptibility,intestinal flora,immune response,environmental exposure,etc.are involved in the development of IBD.The nicotinamide adenine dinucleotide phosphate oxidase(NADPH oxidase,NOX)enzyme family includes NADPH oxidases 1 to 5(NOX1-NOX5),dual oxidases 1 and dual oxidases 2(DUOX1-DUOX2).Most of the reactive oxygen species(ROS)in colon are from NADPH oxidases,95% of 5-Hydroxytryptamine(serotonin or 5-HT)is synthesized by enterochromaffin cells(ECs),and mainly located in the intestines.5-HT plays an important role in the regulation of intestinal functions.Both NADPH oxidases and 5-HT are implicated in the pathogenesis of IBD.Objective: To investigate the impact of 5-HT on the expression of NADPH oxidases and in dextran sulfate sodium(DSS)-induced mouse colitis.Methods: Six to eight-week-old C57BL/6J(B6)male mice were randomly divided into 4 groups(n=8 each group): mice in control group drank water freely,in 5-HT group were treated with 1.0 mg/kg of 5-HT every 3 days by enema,in DSS group were exposed to 1.0% DSS,in 5-HT+DSS group were concurrently treated with 1.0mg/kg of 5-HT and 1.0% DSS.At the end of 6th day of experiment,mice were scarified to collect colon tissue.The severity of colitis was analyzed by disease activity index(DAI),colon length and tissue pathology.Infiltrating leukocytes were counted by counting number of myeloperoxidase(MPO)positive cells.Pro-inflammatory cytokine interleukin(IL)-1?,IL-6,IL-8,tumor necrosis factor-?(TNF-?)and Nox1,Nox2,Nox4 and Duox2 m RNAs in colon tissue were measured by quantitative realtime PCR(q RT-PCR).The protein of Nox1,Nox2,Nox4 and Duox2 were evaluated by immunohistochemistry(IHC)and Western blot.Results:(1)Mice in control group and 5-HT group had no signs of inflammation.Mild colitis was found in mice treated with 1.0% DSS.Mice receiving both 5-HT and DSShad severe colitis.(2)The expression of IL-1?,IL-6,IL-8 and TNF-? mRNA were increased corresponding to the severity of colitis.With 5-HT or DSS treatment there were more MPO positive cells in mice colon,when mice cotreated with 5-HT and DSS,MPO positive cells were increased furthurly.(3)The expression of Nox1,2,4 and Duox2 mRNA in 5-HT group were increased 3.4-,6.5-,3.8-and 2.5-fold compared to control group(both P < 0.05).In mice treated with DSS,Nox2 and Nox4 increased 7.0-and 2.1-fold,respectively(both P < 0.05),Duox2 increased 1.8-fold,Nox1 decreased 3.3-fold(P < 0.05).Furthermore,in 5-HT+DSS group,Nox2 and Nox4 increased 11.9 and 3.4-fold(both P < 0.05),Duox2 decreased 1.7-fold,Nox1 mRNA was undetectable(P < 0.001).IHC results showed that Nox2 and Nox4 were mainly located in infiltrating leukocytes and elevated in the colons of mice treated with 5-HT,which consistent with the gene expression.Nox1 protein were mainly expressed at the brush border membrane of colonic epithelium cells and goblet cells,Duox2 were also expressed at the brush border membrane of colonic epithelium cells as well as in the cytoplasm,the expression of Nox1 and Duox2 protein were also consistent with the gene expression.In addition,we determined the expression of NADPH oxidases by Western blot,the results showed similar results to IHC semi-qualification.Conclusions:(1)Mice received 5-HT alone did not show signs of colitis,however,5-HT could aggravated DSS-induced mouse colitis by recruiting leukocytes in mice colon.(2)5-HT alone increased the expression of Nox1,Nox2,Nox4 and Duox2 in mice colon.That the increased NADPH oxidase expression by 5-HT might be one of the mechanisms in onset of exacerbated DSS-induced colitis.The disparity expression of NADPH oxidase in mice colitis indicating that it may play different roles in IBD. |
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