Mesalamine-Mediated Amelioration of Experimental Colitis in Piglets Involves Gut Microbiota Modulation and Intestinal Immune Cell Infiltration

Abstract

Mesalamine (MES), also known as 5-aminosalicylic acid, is effective in treating mild to moderate ulcerative colitis (UC). The mechanisms of its actions are not fully elucidated. The aim of this study was to investigate the effects of MES treatment on intestinal microbiota and immune system in an dextran sulfate sodium (DSS)-induced UC model in postweaning piglets. Eighteen weaned piglets were assigned randomly to the following treatments: control group (CON, distilled water), DSS group (DSS, 3% DSS), and MES group (MES, 3% DSS + 2 g/day MES). Our results showed that MES treatment alleviates DSS-induced colitis in piglets, as evidenced by a reduced diarrhea index score and increased average daily gain (P < 0.05). This is accompanied by decreased diamine oxidase activity, D-lactate level (P < 0.05), and attenuated mucosal damage. MES treatment also decreased the abundance of Methanogens and reduced colon CD11b⁺ macrophage and CD3⁺ T-cell infiltrations in piglets with DSS-induced colitis (P < 0.05). Collectively, these data indicate that MES treatment-mediated colitis protection may involve microbiota and immune cell alterations.

Keywords: colitis; gut microbiota; immune cells; mesalamine; piglet.

Figure 1

MES alleviates DSS-induced colitis in piglets. (A) Schematic diagram of the colitis model of weaned piglets used in this study; (B) average daily feed intake, (C) average daily gain, and (D) feed conversion rate during DSS treatment; (E) diarrhea index; (F) serum levels of D-lactic acid and (G) serum diamine oxidase; (H) H&E-stained colon sections. Scale bar = 50 µm. Data were presented as mean ± SEM (n = 6 per group). Statistical significance was determined using one-way ANOVA, followed by Tukey test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

MES suppresses immune responses in piglets with DSS-induced colitis. (A) Volcano plot of DEGs between DSS and MES groups. The X-axis represents the fold change in the expression of DEGs, and the Y-axis represents the statistical significance of the fold change. Each point represents a DEG. (B) Cluster heatmap of DEGs; top 20 KEGG annotation of (C) downregulated and (F) upregulated DEGs. The numbers of DEGs in each pathway are counted, and the KEGG gene ratio and adjusted P-values are acquired by hypergeometric distribution test. Heatmap of DEGs enriched in cAMP signaling pathway-related KEGG pathways (D), cytokine–cytokine receptor interaction (E, G) Relative mRNA abundances of IL-1α, IL-1β, CXCL11, CXCL9, CCL21, FOSB, and TNFSF8 validated by RT-qPCR. Values were means ± SEM (n = 6 per group). *P < 0.05, **P < 0.01.

Figure 3

MES reduces the recruitment of immune cells in piglets with DSS-induced colitis. (A) Colonic mucosa mRNA levels of IL-6, IL-17A, IL-22, and IFN-γ; (B, D) intestinal frequencies of CD11b⁺ macrophages and CD3⁺ T cells; (C) serum levels of MPO, IL-1β, IL-22, and IL-12 p70; (E, F) representative images of intestinal infiltrations of CD11b⁺ myeloid cells and CD3⁺ T cells. Scale bar = 50 µm. Data were presented as mean ± SEM (n = 6 per group). Statistical significance was determined using one-way ANOVA, followed by Tukey test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

MES modulates gut microbiota in piglets with DSS-induced colitis. (A) Rank abundance curves of beta diversity in the CON, DSS, and MES groups; (B) PCoA plots upon MES therapy assessed by PERMANOVA; (C) heatmaps of species abundance at the genus level; (D) LEFSe analysis revealed the dominant bacteria in the CON, DSS, and MES groups; relative abundance of top 10 (E) phyla, (F) families, (G) genera in the CON, DSS, and MES groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

MES-mediated mitigative effects in piglets with DSS-induced colitis are correlated with gut microbiota alteration and colonic inflammation: heatmap of the Spearman correlation analysis between the gut microbiota and colonic proinflammatory factors.