Effects of alternate-day fasting, time-restricted fasting and intermittent energy restriction DSS-induced on colitis and behavioral disorders

Abstract

Intermittent fasting (IF) has been reported to have beneficial effects on improving gut function via lowering gut inflammation and altering the gut microbiome diversity. In this study, we aimed to investigate the differential effects of three different common IF treatments, alternate day fasting (ADF), time-restricted fasting (TRF), and intermittent energy restriction (IER), on a dextran sodium sulfate (DSS)-induced colitis mouse model. The results indicated that TRF and IER, but not ADF improved the survival rates of the colitis mice. TRF and IER, but not ADF, reversed the colitis pathological development by improving the gut barrier integrity and colon length. Importantly, TRF and IER suppressed the inflammatory responses and oxidative stress in colon tissues. Interestingly, TRF and IER also attenuated colitis-related anxiety-like and obsessive-compulsive disorder behavior and alleviated the neuroinflammation and oxidative stress. TRF and IER also altered the gut microbiota composition, including the decrease of the enrichments of colitis-related microbes such as Shigella and Escherichia Coli, and increase of the enrichments of anti-inflammatory-related microbes. TRF and IER also improved the short chain fatty acid formation in colitis mice. In conclusion, the TRF and IER but not ADF exhibited the protective effects against colitis and related behavioral disorders, which could be partly explained by improving the gut microbiome compositions and preventing gut leak, and consequently suppressing the inflammation and oxidative damages in both colon and brain. The current research indicates that proper IF regimens could be effective strategies for nutritional intervention for the prevention and treatment of colitis.

Keywords: Anxiety-like behavior; Colitis; Gut microbes; Inflammation; Intermittent fasting; Oxidative stress.

Graphical abstract

Fig. 1

Effects of ADF, TRF and IER on the survival rates and colitis development in DSS-treated mice. (A) Experimental schedule of animal treatments (n = 12 mice per group). (B) Timeline depicting the diet of ad libitum, ADF, TRF and IER. (C) Body weight and (D) Body weight of mice in each group on day 36. (E) Food intake of mice in each group. (F) Fluid intake of mice in each group. Data are presented as mean ± SEM and statistical significance was determined by two-way ANOVA with Newman-Keuls multiple comparisons test, n = 12 mice per group, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group). (G) DAI values in 36-day treatment. (H) Disease activity index (DAI) score on the day 36. (I) Colon length in each group of the ADF, TRF and IER regimens. (J) Survival rate of mice in each group during the 36-day treatment. Data are presented as mean ± SEM and statistical significance was determined by two-way ANOVA with Newman-Keuls multiple comparisons test, n = 12 mice per group, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group).

Fig. 2

Effects of ADF, TRF and IER on DSS-induced histopathological changes in DSS-induced colitis mice. (A) Representative images of H&E staining of colon for each group of the TRF and IER regimens. (B) Histological score based on H&E-stained sections per mouse (n = 6). (C) Representative images of Alcian blue staining of colon for each group of the TRF and IER regimens. (D) The mRNA expression of MUC-2 in colon for each group of the TRF and IER regimens. (E) Quantification of goblet cells based on Alcian blue -stained sections by ImageJ software. Data are presented as mean ± SEM and statistical significance was determined by two-way ANOVA with Newman-Keuls multiple comparisons test, n = 12 mice per group, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Effects of TRF and IER on the gut barrier integrity DSS-induced colitis mice. (A) Representative images of transmission electron microscopy of colonic ultrastructure in epithelial cells for each group of the TRF and IER regimens. (B) Representative images of immunohistochemistry of Claudin-1 in colon for each group of the TRF and IER regimens. (C) Quantitative immunohistochemical analysis of claudin-1 protein in colon: the positive region was identified by ImageJ software and the area ratio with the colon wall region was calculated. (D–F) The mRNA expression of Claudin-1, ZO-1, Occludin in colon for each group of the TRF and IER regimens. Data are presented as mean ± SEM and statistical significance was determined by two-way ANOVA with Newman-Keuls multiple comparisons test, n = 12 mice per group, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group).

Fig. 4

Effects of TRF and IER on inflammatory responses and oxidative stress in the colon of DSS-induced colitis mice. (A) LPS levels in serum. (B–C) Protein levels of inflammatory cytokines TNF-α and IL-6 in mice colon. (D) MDA level in the colon. (E–H) The mRNA expression of TNF-α, IL-1β, IL-6, IFN-γ in colon for each group of the TRF and IER regimens. Data are presented as mean ± SEM and statistical significance was determined by two-way ANOVA with Newman-Keuls multiple comparisons test, n = 12 mice per group, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group).

Fig. 5

Effects of TRF and IER on behavioral disorders, Inflammation and oxidative stress in the brain of DSS-induced colitis. (A) Diagram of an elevated plus maze (EPM). (B) The result of elevated plus maze test. (C) Diagram of a marble burying test (MBT). (D) The result of marble burying test. (E) Representative images of immunohistochemistry of IBA-1 in cortex region for each group of the TRF and IER regimens. (F–H) The mRNA expression of TNF-α, IL-1β, IL-6 in cortex for each group of the TRF and IER regimens. (I–K) The levels of MDA, GSSG and GSH in cortex. Data are presented as mean ± SEM and statistical significance was determined by two-way ANOVA with Newman-Keuls multiple comparisons test, n = 12 mice per group, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group).

Fig. 6

Effects of TRF and IER on the gut microbiome structure in DSS-induced colitis mice. (A) Partial least squares discrimination analysis (PLS-DA) of each group of the TRF and IER regimens. (B) and (C) Relative abundance of the gut microbiota in genus levels in the TRF and IER regimens. (D) and (E) Linear discriminant analysis of the TRF and IER regimens. (F) The relative abundance of Gammaproteobacteria. (G) The relative abundance of Christensenellaceae. (H) The relative abundance of Enterobacteriaceae. (I) The relative abundance of Peptostreptococcaceae. (J) The relative abundance of Shigella. (K) The relative abundance of Escherichia coli. Microbiome sequencing was performed by data of control group, Control-ADF group, Control-TRF group, Control-IER group, DSS group, DSS-ADF group, DSS -TRF group, DSS-IER group (n = 6 per group). The data of intestinal flora are presented as mean ± SEM and statistical significance was determined by Wilcox test, n = 6, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group).

Fig. 7

Effects of TRF and IER on gut microbiome and corresponding metabolites production in DSS-induced colitis mice. (A) The relative abundance of Lactobacillus. (B) The relative abundance of Rikenellaceae. (C) The relative abundance of Coprococcus. (D) The relative abundance of Ruminococcus. The data of intestinal flora are presented as mean ± SEM and statistical significance was determined by Wilcox test, n = 6, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group, ^$p < 0.05, ^$$p < 0.01, compared with the DSS-TRF group). (E) The concentration of acetate. (F) The concentration of propionate. (G) The concentration of butyrate. (H) The concentration of isobutyrate. The data of short-chain fatty acids are presented as mean ± SEM and statistical significance was determined by two-way ANOVA with Newman-Keuls multiple comparisons test, n = 6, (*p < 0.05, **p < 0.01, compared with the Control group, ^#p < 0.05, ^##p < 0.01, compared with the DSS group).