Intestinal Microbiota Mediates High-Fructose and High-Fat Diets to Induce Chronic Intestinal Inflammation

Abstract

An unhealthy diet has been linked to increased incidence of chronic diseases. To investigate the relationship between diet and intestinal inflammation, mice in two experimental groups were fed on a high-fat diet or high-fructose diet, respectively. The result showed that the defecation volume of the experimental groups was significantly reduced compared with that of the control group, and the levels of pro-inflammatory cytokines (interleukin (IL)-1β and IL-6) and IgG in serum were increased significantly. In addition, inflammatory cell infiltration was observed in intestinal tissue, indicating that a high-fructose or high-fat diet can lead to constipation and inflammation. Further analysis showed that the microbial composition of the experimental groups changed significantly, including a decrease of the Bacteroidetes/Firmicutes ratio and increased levels of Bacteroides, Akkermansia, Lactobacillus, and Ruminococcus, which might be associated with inflammation. The results of pro-inflammatory metabolites analysis showed that the levels of arachidonic acid, stearic acid, and indoxylsulfuric acid were significantly increased in the experimental groups, which were related significantly to Bacteroides, Enterococcus, and Akkermansia. Meanwhile, the content of 5-hydroxytryptamine (5-HT) was significantly decreased, which might cause constipation by reducing intestinal peristalsis. Moreover, transplantation of fecal bacteria from inflammatory mice caused constipation and inflammation in normal mice, which could be relieved by feeding a normal diet. The results of the present study indicated that changes in intestinal microbiota and microbial metabolites may underlie chronic intestinal inflammation and constipation caused by high-fructose and high-fat diets.

Keywords: fecal output; high-fat diet; high-fructose diet; intestinal inflammation; intestinal microbiota; metabolites.

Figure 1

The experimental design and animal experiment flow chart and changes in body weight and fecal volume of mice. Blue mice represent healthy mice, and pink mice represent inflamed mice. The black arrow indicates the diet-induced inflammation experiment, the red arrow indicates the fecal bacteria transplantation experiment, and the blue arrow indicates the diet recovery experiment (A). Changes in body weight of mice at 8 weeks (B). 24 h fecal volume of mice in each group (C). (****p < 0.0001).

Figure 2

Effects of high-fructose and high-fat diets on intestinal inflammation. The colon pathological sections were from the high-fructose group (A, J), the high-fat group (B, K) and the control group (C, L). The small intestine pathological sections were from the high-fructose group (D, M), the high-fat group (E, N) and the control group (F, O). The cecum pathological sections were from the high-fructose group (G, P), the high-fat group (H, Q), and the control group (I, R). In H&E staining images, the nucleus is blue and the cytoplasm is red; the red arrow indicates inflammatory cell infiltration. In the immunofluorescence staining images, the nuclei stained by DAPI are blue under UV excitation, the cells labeled for CD3 are red, and those labeled for CD4 are green. Cells expressing both CD3 and CD4 appear yellow after superimposition. The levels of IL-1 β (S), IL-2 (T), IL-6 (U), and IgG (V) were measured from the 3rd week to the 8th week. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

High-fructose and high-fat diets changes the intestinal microbiota in mice. The relative abundance of the top 10 microbes at the phyla level and genus level in each group is shown in the histogram, with the horizontal axis being the sample name and the vertical axis being the relative abundance (A). In the PCoA analysis, the abscissa represents one principal component, the ordinate represents another principal component, and the percentage represents the contribution value of principal component to the sample difference (B). Alpha diversity box chart: Observed species is the observed number of species (also known as the OTU number) (C). An evolutionary branching diagram in which the circles radiating from the inside to the outside represent the taxonomic level from phylum to genus (or species) (D). The abundance distribution box diagram of different species between groups, in which the horizontal axis is the sample grouping and the vertical axis is the relative abundance of the corresponding species (E). (* p < 0.05, **p < 0.01).

Figure 4

The effect of high-fructose and high-fat diets on metabolites. The Pearson correlation coefficient between the quality control (QC) samples was calculated based on the peak area value (A). In the PCoA analysis, the abscissa PC1 represents the first principal component score, and the scattering of different colors represent the samples of the different experimental groups. The closer the distance is, the smaller the difference between the samples is, and the ellipse is the 95% confidence interval (B). The contents of o-cresol (C), arachidonic acid (D), stearic acid (E), palmitic acid (F) and indole sulfuric acid (G) in fecal metabolites were determined. The contents of SCFAs in fecal metabolites included 3-ureopropanoic acid (H), imidazole acetic acid (I), methylvaleric acid (J), and γ-aminobutyric acid (K). The content of amino acids in fecal metabolites, including phenylalanine (L), tyrosine (M), and 5-hydroxytryptophan (N). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Fecal bacteria transplantation changes the intestinal microbiota and induces inflammation. Serum immune factors, including IL-1 β (A), IL-6 (B), TNF–α (C), and immunoglobulin IgG (D) associated with inflammation. In the PCoA analysis, the abscissa and ordinate, respectively, represent a principal component, and percentage represents the contribution value of the principal component to the sample difference (E). H&E staining showing the intestinal state of the experimental group after transplantation of fecal bacteria (F). The UPGMA cluster tree, on the left is the UPGMA cluster tree structure, and on the right is the relative abundance distribution map of species at the phylum level (G). Histogram of species distribution at the genus level (H). The histogram of LDA value distribution shows the species with significant differences in abundance among the different groups (I). (C.Fru: mice transplanted with fecal bacteria from the high-fructose group, C.Fat: mice transplanted with fecal bacteria from the high-fat group).

Figure 6

Diet changes the intestinal microbiota and then affects intestinal inflammation. Levels of proinflammatory serum immune factors, including IL-1β (A), IL-6 (B), TNF–α (C), and immunoglobulin IgG (D) associated with inflammation. H&E staining showed the intestinal state of the experimental group after transplantation of fecal bacteria (E). Based on the unweighted UniFrac distance PCoA analysis (F). In the cladogram, the circles radiating from inside to outside represent the taxonomic level from phylum to genus (or species). Each small circle at different taxonomic levels represents a taxonomic level, and the diameter of the small circle is proportional to the relative abundance (G). The histogram of LDA value distribution shows the species whose LDA score is greater than the set value (the default setting is 4); biomarkers with statistical differences between groups (H). (Fru.C, high-fructose diet changed to normal diet group; Fat.C, high-fat diet changed to normal diet group).