Idebenone Protects against Acute Murine Colitis via Antioxidant and Anti-Inflammatory Mechanisms

Abstract

Oxidative stress is a key player of the inflammatory cascade responsible for the initiation of ulcerative colitis (UC). Although the short chain quinone idebenone is considered a potent antioxidant and a mitochondrial electron donor, emerging evidence suggests that idebenone also displays anti-inflammatory activity. This study evaluated the impact of idebenone in the widely used dextran sodium sulphate (DSS)-induced mouse model of acute colitis. Acute colitis was induced in C57BL/6J mice via continuous exposure to 2.5% DSS over 7 days. Idebenone was co-administered orally at a dose of 200 mg/kg body weight. Idebenone significantly prevented body weight loss and improved the disease activity index (DAI), colon length, and histopathological score. Consistent with its reported antioxidant function, idebenone significantly reduced the colonic levels of malondialdehyde (MDA) and nitric oxide (NO), and increased the expression of the redox factor NAD(P)H (nicotinamide adenine dinucleotide phosphate) dehydrogenase quinone-1 (NQO-1) in DSS-exposed mice. Immunohistochemistry revealed a significantly increased expression of tight junction proteins, which protect and maintain paracellular intestinal permeability. In support of an anti-inflammatory activity, idebenone significantly attenuated the elevated levels of pro-inflammatory cytokines in colon tissue. These results suggest that idebenone could represent a promising therapeutic strategy to interfere with disease pathology in UC by simultaneously inducing antioxidative and anti-inflammatory pathways.

Keywords: cytokines; idebenone; inflammatory bowel disease; lipid peroxidation; superoxide dismutase; tight junction proteins and ulcerative colitis.

Figure 1

Effect of idebenone on the pathology of dextran sodium sulphate (DSS)-induced experimental colitis. (A) Experimental design for the administration of DSS and idebenone in C57BL/6J mice, (B) % body weight change, (C) disease activity index (DAI) of healthy controls (HC), DSS and DSS-plus-idebenone-treated mice (DSS + I). Statistical significance among the groups was evaluated using two-way ANOVA followed by Tukey’s post-test, where * p < 0.05 and **** p < 0.0001 versus DSS. Data are expressed as a mean ± SEM (n = 10/group). (D) Colon length and (E) macroscopic appearance of colon as a mean ± SEM (n = 10/group), evaluated using one-way ANOVA followed by Tukey’s post-test.

Figure 2

Effect of idebenone on histopathology in DSS-induced colitis. (A) Histological representation of proximal colon (PC) and distal colon (DC) sections stained with haematoxylin and eosin (H&E) for healthy controls (HC), DSS-treated mice (DSS) and DSS-plus-idebenone-treated mice (DSS + I) at 20× magnification. (B,C) Histopathology scores for each animal calculated after microscopic analysis of tissue sections from the PC and DC. Statistical significance among groups was evaluated using one-way ANOVA followed by Tukey’s post-test, where ns denotes non-significance, ** p < 0.01 and **** p < 0.0001. Data are expressed as a mean ± SEM (n = 10/group). Arrows indicate crypts/regeneration of crypts (red), goblet cells (blue), epithelium surface erosion (black), inflammatory cells infiltration (white) and submucosal oedema (yellow).

Figure 3

Effect of idebenone on tight junction protein expression in DSS-induced experimental colitis. (A) Immunohistochemical analysis of occludin and zona-occludin 1 (ZO-1), (B) average occludin expression in the distal colon and (C) average ZO-1 expression in the distal colon. Data are expressed as a mean ± SEM (n = 3/group) and statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-test, where ** p < 0.01, ***p < 0.001 and **** p < 0.0001. Images are at 40× magnification. Arrow indicates the localization of staining.

Figure 4

Effect of idebenone on goblet cell loss in colitis. Goblet cells producing mucus stained with alcian blue dye for HC, DSS and DSS + I groups in the distal colon, along with a graphical representation of the staining intensity of alcian blue dye for each group (n = 3/group). Statistical significance among groups was evaluated using one-way ANOVA followed by Tukey’s post-test, where * p < 0.05 and *** p < 0.001. Images are at 40× magnification.

Figure 5

Effect of idebenone on oxidative stress in DSS-induced colitis. (A) Malondialdehyde (MDA) levels in the distal colon, (B) total superoxide dismutase (SOD) activity in the distal colon expressed as a percentage inhibition rate of reduction of xanthine oxidase activity and (C) nitric oxide (NO) concentration (μM)/gram in distal colon tissue. Data are expressed as a mean ± SEM (n = 3/group). Statistical significance among groups was evaluated using one-way ANOVA followed by Tukey’s post-test, where * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Effect of idebenone on NAD(P)H dehydrogenase quinone-1 (NQO-1) expression in colon tissue. (A) Protein levels of NQO-1 analysed using western blotting. (B) Immunohistochemical analysis of NQO-1 for all the groups, along with their graphical representation of the percentage expression in the distal colon. Data are expressed as a mean ± SEM (n = 3/group) and statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-test, where ns denotes non-significance, * p < 0.05.Images captured using a microscope at 40×. Arrow indicates localisation of the staining.

Figure 7

Effect of idebenone on the level of pro-inflammatory cytokines and chemokines in colon tissue. (A) Tissue levels of interleukin (IL)-1α, IL-6, tumour necrosis factor alpha (TNF-α), macrophage inflammatory protein 1 alpha (MIP-1α), MIP-1β, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage CSF (GM-CSF), RANTES, eotaxin, IL-10, IL-17 and IL-3 in the distal colon and (B) the levels of IL-6, TNF-α, GM-CSF, eotaxin and IL-17 in the proximal colon were quantified using a Bio-Plex assay. Data are expressed as a mean ± SEM (n = 3/group) and statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-test, where * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 8

Schematic illustration of the proposed mode of action of idebenone in a mouse model of DSS-induced acute colitis. Induction of DSS disrupts tight junctions (ZO-1 and occludin) and the mucus film covering epithelial cells, resulting in the increased infiltration of harmful microbes and toxins into the lamina propria. This uptake activates macrophages, neutrophils and lymphocytes, causing dissemination of pro-inflammatory cytokines (IL-6, IL-1a, TNF-a, IL-17, IL-3, GM-CSF and G-CSF), chemokines and generates oxidative and nitrosative stress (free radicals and NO). In colitis, NO is released by immune cells, as well as by IECs, which further damages tight junctions. All these factors contribute to the inflammation of the colon. Increased levels of oxidative stress via altered redox levels between oxidative molecules and anti-oxidative enzymes (NQO-1 and SOD) damages tissue and cells through oxidative damage to macromolecules, including lipids. Supplementation with idebenone maintains the barrier integrity by protecting tight junctions and the mucin layer. Idebenone also supresses the pro-inflammatory cytokines, chemokines, NO production and LPO. In addition, by increasing the levels of detoxifying enzyme NQO-1 and SOD, idebenone is thought to prevent colonic inflammation by simultaneously protecting against oxidative stress and inflammation. IECs—intestinal epithelial cells, G-CSF—granulocyte colony stimulating factor, GM-CSF—granulocyte macrophage colony stimulating factor, IL—interleukin, LPO—lipid peroxidation, NO—nitric oxide, NQO-1—NAD(P)H dehydrogenase quinone 1, SOD—superoxide dismutase, TNF-α—tumor necrosis factor alpha and ZO-1—zona occludin 1.