Polysaccharides from Chrysanthemum morifolium Ramat ameliorate colitis rats by modulating the intestinal microbiota community

Abstract

The gut microflora dysbiosis has been closely related with the inflammatory bowel disease (IBD). In this study, the effect of polysaccharides from Chrysanthemum morifolium Ramat on the gut microbiota was evaluated by ulcerative colitis (UC) rat model. Physiological and pathological analyses suggested that Chrysanthemum polysaccharides possessed notably protective effects on UC in vivo. Based on the Illumina MiSeq platform, 16S rRNA sequencing of the rat colonic contents indicated that the intestinal flora structure remarkably changed in the model rats and the tendency was alleviated to a certain degree by treatment with different dosages of Chrysanthemum polysaccharides. In normal groups, there were more Firmicutes than Bacteroidetes, but this change lost at the pathological state. Following Chrysanthemum polysaccharides, rising Firmicutes/Bacteroidetes ratio was validated. Besides the microbial diversity and the community richness of the UC rats were improved by Chrysanthemum polysaccharides, the composition of intestinal microflora in the model group were also restored after oral administration of Chrysanthemum polysaccharides. The abundance of opportunistic pathogens was decreased (Escherichia, Enterococcus and Prevotella), while the levels of protective bacteria such as Butyricicoccus and Clostridium (butyrate-producing bacteria), Lactobacillus and Bifidobacterium (probiotics), Lachnospiraceae and Rikenellaceae elevated in various degrees. Correlation analysis between intestinal flora and biochemical factors suggested that the relative abundance of protective bacteria was positively correlated with the levels of anti-inflammatory cytokines such as IL-4, IL-10 and IL-11, while aggressive bacteria were positively correlated with proinflammatory cytokine such as IL-23、IL-6、 IF-17、TNF-α、IL-1β and IFN-γ. The above results showed that the intestinal flora were closely related to the secretion and expression of cytokines in the body, and they interacted with each other to regulate immune function. Thus, Chrysanthemum polysaccharides could ameliorate ulcerative colitis by fostering beneficial intestinal flora growth, modulating the balance of intestinal microecology and restoring the immune system.

Keywords: 16S rRNA; chrysanthemum polysaccharides; microbial diversity; short chain fatty acids; ulcerative colitis.

Figure 1. Chrysanthemum polysaccharides ameliorated TNBS-induced colitis in SD rats

(A) Change of body weight during the disease process. (B) DAI based on weight loss, hematochezia, and diarrhea. (C) Statistics of colon length of each group. (D) Histopathological changes of colons. Significant differences (P < 0.05) between treatments were indicated by the letters a, b, or c. The results were presented as the mean ± SD; n = 6 for each treatment. N, normal group; M, TNBS-induced group; PP, SASP 0.5g/kg; HP, 200 mg/kg; MP, 100 mg/kg; LP, 50 mg/kg.

Figure 2. Chrysanthemum polysaccharides regulated cytokine profiles in colons mucosa of SD rats with TNBS-induced colitis

Cytokine levels in homogenated colonic proteins were assessed by ELISA. Significant differences (P < 0.05) between treatments were indicated by the letters a, b, or c. The results were presented as the mean ± SD; n = 6 for each treatment. N, normal group; M, TNBS-induced group; PP, SASP 0.5g/kg; HP, 200 mg/kg; MP, 100 mg/kg; LP, 50 mg/kg.

Figure 3. OTU network

OTU network analysis of bacterial communities from different treatment groups (N, M, PP, HP, MP, LP) for the V3-V4 16S rRNA region.

Figure 4. PCA (left) and PCoA (right) analysis of variation between the bacterial communities present in all biopsy samples

Each data point represents an individual sample.

Figure 5

(A) Relative abundance of the main phyla and genes in the intestinal microbiota. Left: Phyla; Right: Genes. (B) Average relative abundance of seven protective bacteria in intestinal contents of different treatment groups. (C) Average relative abundance of five aggressive bacteria in intestinal contents of different treatment groups. Significant differences (P < 0.05) between treatments were indicated by the letters a, b, or c. The results were presented as the mean ± SD; n = 5 for each treatment. N, normal group; M, TNBS-induced group; PP, SASP 0.5g/kg; HP, 200 mg/kg; MP, 100 mg/kg; LP, 50 mg/kg.

Figure 6

(A) The relationship between inflammatory factors/cytokines in colon epithelial tissue samples and intestinal flora based on CCA. (B) Heatmap correlation analysis of intestinal microflora and biochemical factors.