Short-Chain Naphthoquinone Protects Against Both Acute and Spontaneous Chronic Murine Colitis by Alleviating Inflammatory Responses

Abstract

Ulcerative colitis (UC) is characterised by chronic, relapsing, idiopathic, and multifactorial colon inflammation. Recent evidence suggests that mitochondrial dysfunction plays a critical role in the onset and recurrence of this disease. Previous reports highlighted the potential of short-chain quinones (SCQs) for the treatment of mitochondrial dysfunction due to their reversible redox characteristics. We hypothesised that a recently described potent mitoprotective SCQ (UTA77) could ameliorate UC symptoms and pathology. In a dextran sodium sulphate- (DSS-) induced acute colitis model in C57BL/6J mice, UTA77 substantially improved DSS-induced body weight loss, disease activity index (DAI), colon length, and histopathology. UTA77 administration also significantly increased the expression of tight junction (TJ) proteins occludin and zona-occludin 1 (ZO-1), which preserved intestinal barrier integrity. Similar responses were observed in the spontaneous Winnie model of chronic colitis, where UTA77 significantly improved DAI, colon length, and histopathology. Furthermore, UTA77 potently suppressed elevated levels of proinflammatory cytokines and chemokines in colonic explants of both DSS-treated and Winnie mice. These results strongly suggest that UTA77 or its derivatives could be a promising novel therapeutic approach for the treatment of human UC.

Keywords: cytokines; endoplasmic reticulum stress; inflammatory bowel disease; naphthoquinones; tight junction proteins.

FIGURE 1

Molecular structures of short-chain quinones (SCQs). (A) Benzoquinone idebenone and (B) novel naphthoquinone UTA77.

FIGURE 2

Effect of UTA77 on the pathology of DSS-induced experimental colitis. (A) % body weight change; (B) disease Activity Index (DAI). Statistical significance among groups evaluated by two-way ANOVA followed by Tukey’s posttest. *p < 0.05, **p < 0.01, and ****p < 0.0001 vs. DSS. (C) Colon length evaluated by one-way ANOVA followed by Tukey’s posttest. Data expressed as mean ± SEM (n = 10/group).

FIGURE 3

Effect of UTA77 on histopathology in DSS-induced colitis. (A) Histological representation of proximal colon (PC) and distal colon (DC) sections stained with H&E for healthy mice (HC), DSS-control mice (DSS), and DSS + UTA77-treated mice (DSS + UTA77) at ×20 magnification. (B) and (C) represent the histopathology scores for each animal calculated after microscopic analysis of tissue sections from the PC and DC. Statistical significance among groups was evaluated by one-way ANOVA followed by Tukey’s posttest. **p < 0.01, ****p < 0.0001, and ns: nonsignificant. Data expressed as mean ± SEM (n = 10/group). Arrows indicate goblet cells (a), crypts/regeneration of crypts (b), inflammatory cells infiltration (c), epithelium surface erosion (d), and submucosal oedema (e).

FIGURE 4

Effect of UTA77 on goblet cells. Goblet cells producing mucus stained with Alcian Blue dye for HC, DSS, and DSS + UTA77 groups in distal colon along with graphical representation of staining intensity of Alcian Blue dye for each group (n = 3/group). Statistical significance among groups evaluated by one-way ANOVA followed by Tukey’s posttest where **p < 0.01 and ***p < 0.001. Images at ×40 magnification.

FIGURE 5

Effect of UTA77 on tight junction (TJ) protein expression in DSS-induced experimental colitis. (A) Immunohistochemical analysis of occludin and ZO-1, (B) average occludin expression in distal colon, and (C) average ZO-1 expression in distal colon. Data expressed as mean ± SEM (n = 3/group) and statistical significance evaluated by one-way ANOVA followed by Tukey’s posttest where **p < 0.01 and ***p < 0.001. Images at ×40 magnification. Arrow indicates localisation of staining.

FIGURE 6

Effect of UTA77 on the level of inflammatory cytokines and chemokines in colon tissue. Tissue levels of (A) IL-1α, (B) IL-6, (C) TNF-α, (D) G-CSF, (E) GM-CSF, (F) INF-γ, (G) RANTES, (H) eotaxin, (I) IL-13, (J) IL-10, and (K) IL-3 in PC and DC were quantified by Bio-Plex assay. Data expressed as mean ± SEM (n = 3/group) and statistical significance evaluated by one-way ANOVA followed by Tukey’s posttest where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 7

Effect of UTA77 on the pathology of spontaneous chronic colitis in Winnie mice. (A) % body weight change and (B) DAI of healthy controls (HC), Winnie controls (Win), and UTA77-treated Winnie group (Win + UTA77). Statistical significance among groups evaluated by two-way ANOVA followed by Tukey’s posttest. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. Winnie. (C) Colon length evaluated by one-way ANOVA followed by Tukey’s posttest. Data expressed as mean ± SEM (n = 6/group).

FIGURE 8

Effect of UTA77 on histopathology in spontaneous chronic colitis in Winnie mice. (A) Histological representation of PC and distal colon (DC) sections stained with H&E for healthy mice (HC), Winnie control mice (Winnie), and Winnie plus UTA77-treated mice (Winnie + UTA77) at ×10 magnification. (B) and (C) represent the histopathology scores for each animal calculated after microscopic analysis of tissue sections from the PC and DC. Statistical significance among groups was evaluated by one-way ANOVA followed by Tukey’s posttest. **p < 0.01, ****p < 0.0001, and ns: nonsignificant. Data expressed as mean ± SEM (n = 6/group). Arrows indicate goblet cells loss (a), crypts loss/crypts distortion (b), inflammatory cells infiltration (c), epithelial surface erosion (d), crypt abscesses (e), and mucosal/submucosal oedema (f).

FIGURE 9

Effect of UTA77 on endoplasmic reticulum stress (ER stress) markers in spontaneous chronic colitis in Winnie mice. (A) Protein levels of GRP78 and CHOP were analysed using Western blotting in distal colon. (B, C) Densitometry of GRP78 and CHOP. Bands densities were normalised to the levels of β-actin. Data expressed as mean ± SEM (n = 3/group) using one-way ANOVA followed by Tukey’s posttest, where *p < 0.05, ***p < 0.001, and ns: nonsignificant. The controls used (HC and Win) in this figure have been published previously (Shastri et al., 2020b).

FIGURE 10

Effect of UTA77 on the level of proinflammatory cytokines and chemokines in colonic tissue explants of Winnie mice. Tissue levels of (A) IL-1α, (B) IL-1β, (C) TNF-α, (D) INF-γ, and (E) MIP-1α in PC and DC were quantified by Bio-Plex assay. Data expressed as mean ± SEM (n = 3/group) and statistical significance evaluated by one-way ANOVA followed by Tukey’s posttest where *p < 0.05 and **p < 0.01.

FIGURE 11

Effect of UTA77 on oxidative stress markers in acute and chronic colitis. (A) Malondialdehyde (MDA) levels in distal colon of acute colitic mice, (B) MDA level in distal colon of Winnie mice, (C) total SOD activity in distal colon of acute colitic mice, and (D) nitric oxide (NO) levels in distal colon of acute colitic mice. Data expressed as mean ± SEM (n = 3/group) using one-way ANOVA followed by Tukey’s posttest, where *p < 0.05, **p < 0.01, ***p < 0.001, and ns: nonsignificant.