The Effect of Formula-based Nutritional Treatment on Colitis in a Murine Model

Abstract

Background: Exclusive enteral nutrition (EEN) induces remission in pediatric Crohn's disease (CD). The exact mechanism of EEN therapy in CD is unknown, but alteration of the intestinal microflora after EEN is thought to affect mucosal healing. To determine the link between EEN therapy and therapeutic efficacy in CD, we established a murine model of dextran sulfate sodium (DSS)-induced colitis and applied EEN therapy.

Methods: Eight-week-old C57BL/6 mice were administered DSS for 4 days to induce colitis, and either normal chow (NC) or EEN was administered for the following 4 days. The mice were grouped according to the feeding pattern after DSS administration: DSS/NC and DSS/EEN groups. The clinical course was confirmed via daily observation of the weight and stool. Fecal samples were collected and 16sRNA sequencing was used. The mice were sacrificed to confirm colonic histopathology.

Results: Weight reduction and increase in disease activity were observed as the day progressed for 4 days after DSS administration. There was significant weight recovery and improvement in disease activity in the EEN group compared to that in the NC group. Verrucomicrobia and Proteobacteria abundances tended to increase and Bacteroidetes abundance decreased in the EEN group. In the EEN group, significant changes in the β-diversity of the microbiota were observed. In the analysis of microbiome species, abundances of Akkermansia muciniphila, Clostridium cocleatum, mucin-degrading bacteria, Flintibacter butyricus, and Parabacteroides goldsteinii, which are beneficial microbiota, were significantly increased in the EEN group compared to those in the NC group. More abundant mucins were confirmed in the colonic histopathology of the EEN group. These microbial and histopathological differences suggested that EEN might improve colitis symptoms in a murine colitis model by promoting mucin recycling and subsequently inducing the healing effect of the gut barrier.

Conclusion: EEN showed clinical efficacy in a murine model of colitis. Based on the increase in mucin-degrading bacteria and the pathological increase in mucin production after EEN administration, it can be observed that mucin plays an important role in the therapeutic effect of EEN.

Keywords: Exclusive Enteral Nutrition; Microbiome; Mucin; Murine Model.

Fig. 1. Changes of body weight and disease activity by days. (A) The change in weight is represented by the ratio. The change in body weight showed statistical difference between DSS/ NC group and DSS/EEN group on day 8 (3.30% in DSS/EEN group vs. −7.21% in DSS/NC group, P < 0.05). (B) Disease activity index is represented. Disease activity index is composed of weight change, hematochezia, and diarrhea. There was statistical difference between DSS/NC and DSS/EEN groups on day 8 (2.8% in DSS/EEN group vs. 5.0% in DSS/NC group, P < 0.05).

DSS = dextran sulfate sodium, NC = normal chow, EEN = exclusive enteral nutrition.

Fig. 2. Alpha diversity by group. (A) The number of observed ASVs of control, DSS/NC, and DSS/EEN groups. The number of observed ASVs in the control group was significantly higher than that in DSS/NC and DSS/EEN groups (P < 0.01). (B) Dominance D values of control, DSS/NC, and DSS/EEN groups. Dominance D in the control group was significantly higher than that in DSS/NC and DSS/EEN groups (P < 0.001). (C) Shannon H value of control, DSS/NC, and DSS/EEN groups. Shannon H value of the DSS/EEN group was significantly higher than that in the control group (P < 0.05). Significance among the groups was statistically tested using one-way analysis of variance, followed by Tukey’s multiple comparison test. Data are expressed as the mean ± standard deviation of the mean.

ASV = ampilcon sequence variant, DSS = dextran sulfate sodium, NC = normal chow, EEN = exclusive enteral nutrition. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Fig. 3. Relative abundance of microbiomes by groups. (A) Relative abundance of control, DSS/NC, and DSS/EEN group at the phylum level. (B) Relative abundance comparison in eight bacterial phyla: Actinobacteria, Bacteroidetes, Deferribacteres, Firmicutes, Proteobacteria, Tenericutes, Verrucomicrobia and their corresponding others. Statistical analysis was performed using one-way analysis of variance followed by Newman–Keuls multiple comparison test.

DSS = dextran sulfate sodium, NC = normal chow, EEN = exclusive enteral nutrition. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Fig. 4. β-diversity of control, DSS/NC, and DSS/EEN groups. Similarity among the groups was tested statistically using analysis of similarity.

DSS = dextran sulfate sodium, NC = normal chow, EEN = exclusive enteral nutrition, NMDS = non-metric multi-dimensional scaling.

Fig. 5. LDA score of relative abundance at the species level. LEfSe and LDA score ≥ 3.5 are shown. (A) LDA scores showing significantly different abundance between DSS/NC and control groups. (B) Cladogram of LDA scores showing significantly different abundance between DSS/NC and control groups. (C) LDA scores showing significantly different abundance between DSS/EEN and control groups. (D) Cladogram of LDA scores showing significantly different abundances between DSS/EEN and control groups.

LDA = linear discriminant analysis, LEfSe = linear discriminant analysis effect size, DSS = dextran sulfate sodium, NC = normal chow, EEN = exclusive enteral nutrition.

Fig. 6. Colon histology of DSS/NC and DSS/EEN groups. Colon tissues were collected on day 8 and the specimens were stained with hematoxylin and eosin. These samples were analyzed using mouse colitis histology index.

DSS = dextran sulfate sodium, NC = normal chow, EEN = exclusive enteral nutrition.