Nicotinamide Ameliorates Dextran Sulfate Sodium-Induced Chronic Colitis in Mice through Its Anti-Inflammatory Properties and Modulates the Gut Microbiota

Abstract

Vitamin B (nicotinamide (NAM)), one of the most important nutritional components for humans, exerts anti-inflammatory activity. This study was aimed at investigating the effect of NAM on the gut microbiota and short-chain fatty acids (SCFAs) in mice with chronic colitis. Colitis was induced in C57BL/6 male mice by administration of 1.5% dextran sulfate sodium (DSS), and the mice were intraperitoneally injected with normal saline (NS) or NAM. NAM treatment ameliorated weight loss and changes in colon length, disease activity index (DAI) score, and histologic scores. Moreover, enzyme-linked immunosorbent assay (ELISA) analysis of LPL cells revealed that the level of interleukin- (IL-) 6, IL-12p70, IL-1β, tumor necrosis factor- (TNF-) α, interferon- (IFN-) γ, IL-21, and IL-17A was increased, while IL-10 was reduced, in the chronic colitis group compared to the control group, but the levels of all these factors were restored after NAM treatment. Then, 16S rRNA sequencing of the large intestinal content was performed, and analysis of alpha diversity and beta diversity showed that the richness of the gut microbiota was decreased in the DSS group compared to the control group and restored after NAM treatment. In addition, NAM modulated specific bacteria, including Odoribacter, Flexispira, and Bifidobacterium, in the NAM+chronic colitis group. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated that NAM treatment restored disruptions in the functions of the gut microbiota (replication and repair, cell motility) in mice with DSS-induced colitis. Furthermore, NAM also restored the reduction in valeric acid in mice with DSS-induced chronic colitis. Our results suggest that NAM treatment could alleviate DSS-induced chronic colitis in mice by inhibiting inflammation and regulating the composition and function of gut microbiota.

Figure 1

Chemical structure of NAM.

Figure 2

Flow chart of the experiment: (a) control group; (b) chronic colitis group; (c) NAM group; (d) NAM+chronic colitis group.

Figure 3

NAM ameliorated DSS-induced chronic colitis: (a) survival rate (t (d): time displayed as days); (b) changes in body weight (%); (c) disease activity index (DAI); (d) colon length (the data are presented as the mean ± SD; the body weight and DAI score were analyzed by two-way ANOVA; when the P value was less than 0.05, the difference was considered significant. ^∗P < 0.05) (n = 8).

Figure 4

(a) Large intestinal pathology (×40 and ×200): intestinal tissue revealed the infiltration of inflammatory cells and disruption of crypts (the arrow) in the chronic colitis group. (b) Pathological damage scores for the large intestines (the data are presented as the mean ± SD; the pathological damage scores were analyzed by t-test; ^∗P < 0.05) (n = 8).

Figure 5

(a) Cytokine production by unstimulated LPL cells was analyzed by ELISA. (b) LPL cells with or without anti-CD3 and anti-CD28 mAb stimulation (the data are presented as the mean ± SD, the cytokine was analyzed by one-way ANOVA followed by LSD post hoc test, and previously, a normality test had been applied; when the P value was less than 0.05, the difference was considered significant. ^∗P < 0.05) (n = 6).

Figure 6

NAM intervention counteracted DSS-induced shifts in the gut microbiota. (a) Species accumulation curves: the species accumulation boxplots reached stable values, indicating that the sequencing covered most phylotypes. (b) Venn diagram of the overlap of operational taxonomic units and differences in operational taxonomic units among groups: 1914 of all OTUs accounting for total richness were universal and found in all samples in the control, chronic colitis, and NAM+chronic colitis groups; 399 OTUs in the NAM+chronic colitis group were not found in the control or chronic colitis group. (c) The alpha diversity index observed_species. (d) The alpha diversity index Shannon index; the alpha diversity index indicated that bacterial species richness was decreased in the chronic colitis group but restored by NAM treatment. (e) The plots shown were generated using weighted UniFrac NMDS. (f) The relative abundance of bacteria at the phylum level in the four groups. (A: control group, n = 12; B: chronic colitis group, n = 12; C:NAM group, n = 10; D: NAM+chronic colitis group, n = 15).

Figure 7

Taxonomic difference in the gut microbiota between the chronic colitis group and control group: (a) a LEFSE cladogram representing different taxa in a tree-like structure; (b) taxa in the control (green) group and chronic colitis (red) group were analyzed by linear discriminant analysis (LDA) effect size. Significantly differentially abundant taxa are listed in the bar plot based on effect size (LDA score (log 10) > 2) and P value < 0.05.

Figure 8

Taxonomic difference in the gut microbiota between the chronic colitis group and NAM+chronic colitis group. (a) A LEFSE cladogram representing different taxa in a tree-like structure. (b) Taxa in the chronic colitis (green) group and NAM+chronic colitis (red) group were analyzed by linear discriminant analysis (LDA) effect size. Significantly differentially abundant taxa are listed in the bar plot based on effect size (LDA score (log 10) > 2) and P value < 0.05. (c–e) Significance analysis of differential gut microbial communities in the large intestinal contents of mice in the chronic colitis group and NAM+chronic colitis group. The cladogram and results of LDA are shown. Relative abundances of Odoribacter, Flexispira, and Bifidobacterium in the chronic colitis group and NAM+chronic colitis group are shown. The average and median values for the relative abundance of the taxon in each group are shown by solid lines and dashed lines, respectively, to directly reflect differences between groups. (B: chronic colitis group; D: NAM+chronic colitis group).

Figure 9

PICRUSt analysis of KEGG pathways. Functional predictions of the gut microbiota in the different groups: (a, b) functional differences in cellular processes and genetic information processing between the control and chronic colitis groups; (c, d) Functional differences in cellular processes and genetic information processing between the chronic colitis and NAM+chronic colitis groups.

Figure 10

OPLS-DA score plots for the control, chronic colitis, and NAM+chronic colitis groups. Colors indicate the different experimental groups. From the results of OPLS-DA shown as a score chart, we can see very significant distinction among the three groups of samples, and data for all the samples are in the 95% confidence interval.

Figure 11

Relative quantitative values of acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and caproic acid concentrations in the large intestinal contents in the control, DSS (chronic colitis), and NAM+chronic colitis groups (n = 5 per group). The data are expressed as the mean. Differences were assessed by Student's t-test. Significant differences were established at an adjusted P value < 0.05 (^∗P < 0.05) with a fold change < 0.5 or >2 (ns: no significant difference).

Figure 12

Schematic representation of the proposed anti-IBD effects of NAM on DSS-induced colitis in mice. After the onset of DSS-induced colitis, Th cells are activated, which can increase the production of inflammatory cytokines such as IL-6 and IL-1β and decrease production of the anti-inflammatory cytokine IL-10. Moreover, colitis also changes the composition and function of the gut microbiota, which manifests as an increase in harmful bacteria, disrupts the function of the gut microbiota, and disrupts the production of SCFAs. However, the changes of valeric acid can be restored by NAM. On the one hand, NAM can directly protect the gut barrier by regulating the production of inflammatory cytokines, increasing the proportion of the beneficial microbiota, restoring the function of the gut microbiota, and increasing the production of SCFAs. On the other hand, beneficial microbiota and SCFAs can also protect the gut barrier by regulating the production of inflammatory cytokines.