Dynamic Changes in Gut Microbiome of Ulcerative Colitis: Initial Study from Animal Model

Abstract

Background: An animal model of DSS-induced UC has been widely used in basic research, and the dysbiosis of gut microbiome is one of the important pathogenetic mechanisms of DSS-induced UC, but its dynamic changes and correlation with inflammatory factors are not clear yet.

Methods: Clinical signs and tissue damage degree of C57BL/6 ulcerative colitis mice model induced by different concentrations of DSS were compared with that of normal mice, and finally the optimal concentration of DSS was determined. Then we analyzed the sequencing results of gut microbiome and inflammatory factors to determine the dynamic patterns of gut microbiome and their correlation with the inflammatory factors.

Results: DSS at 2.5% and 3.0% concentration could cause intestinal injury and induce colitis. However, 3.0% DSS resulted in higher mortality. In addition, there were dynamic changes of gut microbiome in DSS-induced UC model: the relative abundance of intestinal flora increased first and then decreased in Bacteroides, Parabacteroides, Romboutsia, Clostridium_sensu_stricto_1, Lachnospiraceae_NK4A136_group, norank_f_norank_o_Clostridia_UCG-014, Parasutterella, and decreased first and then increased in Lactobacillus, Muribaculum, norank_f_Muribaculaceae, in addition, Bifidobacterium, Coriobacteriaceae_UCG-002 and Enterorhabdus did not change in the first 14 days but increased significantly on day 21. Moreover, inflammatory cytokines were closely associated with the imbalance of the intestinal microbiota in mice with UC: most pathogenic bacteria in the intestinal tract of the UC animal model were positively correlated with pro-inflammatory factors and negatively correlated with anti-inflammatory factors, while beneficial bacteria were the opposite.

Conclusion: Intestinal microecology plays an important role in DSS-induced UC model, and the relative abundance of gut microbiome changes dynamically in the occurrence and development of ulcerative colitis.

Keywords: 16S rDNA; DSS; dynamic changes; gut microbiome; ulcerative colitis.

Figure 1

Experimental protocol of DSS-induced ulcerative colitis mice.

Figure 2

General condition of DSS-induced ulcerative colitis mice. (A) Mice survival was monitored daily. (B) The body weight of mice was measured once daily. (C) The DAI score was calculated by measuring the clinical score of body weight change, stool, and fecal occult blood. The graphs are represented as mean±SD. *,^#p<0.05, **,^##p<0.01. *Control and model 2.5. ^#Control and model 3.0.

Figure 3

Colons tissue of the proximal and distal colon sections were stained with H&E, and the histological images were obtained through an optical microscope. Histopathological scores of inflammation cell infiltration, depth of lesions, destruction of crypts, width of lesions, and crypt damage were measured. The graphs are represented as mean ±SD. *p<0.05, ^#p<0.01. *Control and model 2.5. ^#Control and model 3.0.

Figure 4

Levels of inflammatory cytokines was measured by ELISA in DSS-induced ulcerative colitis. (A) The level of inflammatory cytokines such as IL-2, IL-4, IL-6, IL-10, IL-22, or 5-HT from the serum of the mice. (B) The level of inflammatory cytokines such as IL-2, IL-4, IL-6, IL-10, IL-22, TNF-α, IFN-γ, or 5-HT from the colon of the mice. The graphs are represented as mean ±SD. *p<0.05, **p<0.01 and ***p<0.001.

Figure 5

DSS changed the structure of gut microbiome in UC model mice. (A) Rarefaction/Shannon curve indicates the amount of sequencing data is large enough to reflect the true level of bacterial diversity. (B) Pan/core analysis shows the amount of sequencing data is reasonable. (C) Venn diagram (OTU and genus) analysis notes DSS could reduce the richness of bacterial community. (D) Beta-diversity analyses illustrates the gut microbiome composition of different groups differed at different time points.

Figure 6

Differential gut microbiome results. (A and B) The difference genera of intestinal flora in mice at phylum (A) and genus (B) levels at different time points was analyzed. The graphs are represented as mean±SD. *,^#p<0.05, **,^##p<0.01. *Control and model 2.5. ^#Control and model 3.0.

Figure 7

Heatmap of correlation analysis between gut microbiome and inflammatory cytokines in UC mice. Spearman’s rank correlation coefficient is indicated using a color gradient: red indicates positive correlation; green indicates negative correlation,*p<0.05, **p<0.01.