Fig. 5: Fructose produced from raffinose promotes ISC proliferation.

a Summary of significantly changed pathways involved in carbohydrate metabolism based on metagenomic data of fecal samples from control and stressed mice fed with chow diet. Pathways with p ≤ 0.05 by Wilcoxon rank-sum test are reported. b, c Box plot showing differential enrichment of indicated KOs involved in raffinose transport (b) and metabolism (c) by KEGG analysis. Box limits are the 10th and 90th percentiles, center lines are median, and the whiskers are the minimal and maximal values. Statistical analysis was performed by Wilcoxon rank-sum test for n = 4, *p < 0.05. d Relative level of fructose in the ileum of mice fed with PD or RD. Data are represented as mean ± SEM for the PD group (n = 4) and RD group (n = 7). e Representative images of intestinal organoids treated with blank culture (d-hanks), raffinose (1 mM), galactose (1 mM), glucose (1 mM), or fructose (1 mM). Scale bar: 200 µm. Organoids per crypt and crypt domains per crypt were represented as mean ± SEM for n = 3. f Schematic of the experimental design to explore the effect of fructose supplementation on ISC renewal. g Representative Olfm4 staining images of intestinal crypts and quantitation of Olfm4-positive cells per crypt. Scale bar: 100 µm. Data are represented as an average number of positive staining per crypt ± SEM for n = 4 (p = 0.0051). h Organoids derived from the intestinal crypts of PD-fed mice with or without fructose supplementation during stress. Scale bar: 200 µm. Data are expressed as Mean ± SEM for organoid per crypt (n = 4, p = 0.0015) or crypt domain per organoid (n = 6), respectively. i Schematic of the experimental design to understand the effect of L. reuteri on raffinose metabolism. j Disease activity index (DAI) score and colon length of mice with colitis. Data are represented as mean ± SEM for CD-GF/DSS group (n = 6) and CD-L. reuteri/DSS group (n = 5). p = 0.017 for colon length; p < 0.0001 for DAI. k Representative H&E staining of distal colon section of GF and GF-L. reuteri mice with colitis. Scale bar: 100 µm. Data are represented as mean ± SEM for n = 4 biologically independent samples. l Representative Lgr5 staining images and quantification of Lgr5-positive cells in the crypt of colonic tissue. Scale bar: 100 µm. Data are represented as mean ± SEM for n = 4 biologically independent samples. m Relative raffinose and fructose level in the colonic tissue of GF and L. reuteri colonized mice. Data are represented as mean ± SEM for n = 5 (p = 0.0003 for raffinose; p = 0.0071 for fructose). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (d, e) or unpaired two-tailed Student’s t-test (g, h, j–m). ns no significance. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.