Fructo-oligosaccharide intensifies visceral hypersensitivity and intestinal inflammation in a stress-induced irritable bowel syndrome mouse model

Figure 1 Effect of water avoidance stress on rate of weight gain and visceral sensitivity. A: Rate of weight gain (g) was lower in the water avoidance stress (WAS) group (n = 13) compared to the sham-WAS (n = 14) group. Values represent mean weight gain ± SD, repeated analysis of variance (ANOVA); B: Abdominal withdrawal reflex (AWR) scores in response to colorectal distension were increased in the WAS group compared to the sham-WAS group. Lines within the box represent the median value, ends of the box represent 25^th and 75^th percentiles, and the error bars represent 5^th and 95^th percentiles. Wilcoxon two-sample test; C: Pain threshold was decreased in the WAS group compared to the sham-WAS group. Values represent mean ± SD, Student’s t-test; D: Volume threshold was decreased in the WAS group compared to the sham-WAS group. Values represent means values ± SD, Student’s t-test. ^aP < 0.05, Sham vs WAS.

Figure 2 Effect of oral gavage of fructo-oligosaccharide on visceral sensitivity. A: Fructo-oligosaccharide (FOS) increased abdominal withdrawal reflex (AWR) scores in response to colorectal distension compared to saline administration following water avoidance stress (WAS). Values represent median, 25^th, 75^th, 5^th, and 95^th percentiles. Sham + saline (n = 7), sham + FOS (n = 7), WAS + saline (n = 6), WAS + FOS (n = 7). Kruskal-Wallis one-way ANOVA; B: Pain threshold decreased in FOS-administered compared to saline-administered mice following WAS. Values represent mean ± SD, one-way ANOVA; C: Volume threshold decreased in FOS compared to saline-administered mice following WAS. Values represent mean ± SD, one-way ANOVA. ^aP < 0.05, sham + saline vs WAS + saline; ^cP < 0.05, WAS + saline vs WAS + FOS.

Figure 3 Effect of oral gavage of fructo-oligosaccharide on intestinal histological scores. A: Ileal tissues stained with hematoxylin-eosin (HE) for evaluation of inflammation score (magnification, 200×); B: No difference in structural histology among the four groups; C: Colonic tissues stained with H&E for evaluation of inflammation score (magnification, 200×); D: No difference in structural histology among the four groups. Values represent median with 5^th and 95^th percentiles; sham + saline (n = 7), sham + FOS (n = 7), WAS + saline (n = 6), WAS + FOS (n = 7); Kruskal-Wallis one-way ANOVA. FOS: Fructo-oligosaccharide; WAS: Water avoidance stress.

Figure 4 Effect of oral gavage of fructo-oligosaccharide on short chain fatty acid concentrations. The average concentrations of total SCFA, acetic, propionic, and butyric acids increased in FOS-administered mice compared to saline-administered mice following WAS intervention. No difference was observed in total SCFA, acetic, propionic, and butyric acid levels with FOS or saline administration in mice following sham-WAS. Values represent mean ± SD; sham + saline (n = 7), sham + FOS (n = 7), WAS + saline (n = 6), WAS + FOS (n = 7); one-way ANOVA. ^aP < 0.05, WAS + saline vs WAS + FOS. SCFA: Short chain fatty acids; FOS: Fructo-oligosaccharide; WAS: Water avoidance stress.

Figure 5 Effect of oral gavage of fructo-oligosaccharide on intestinal cytokine expression. A: Among saline-administered mice, IL-6 and TNF-α expressions increased in the ileum in the WAS group compared to the sham-WAS group. IL-23 expression increased in FOS compared to saline-administered mice following WAS; B: In saline-administered mice, colonic IL-6 and IL-1β expression increased in the WAS group compared to the sham-WAS group. IL-1β expression increased in FOS compared to saline-administered mice in the WAS group. Values represent mean ± SD; sham + saline (n = 7), sham + FOS (n = 7), WAS + saline (n = 6), WAS + FOS (n = 7); one-way ANOVA. ^aP < 0.05, sham + saline vs WAS + saline; ^cP < 0.05, WAS + saline vs WAS + FOS. FOS: Fructo-oligosaccharide; WAS: Water avoidance stress; IL: Interleukin; TNF: Tumor necrosis factor.

Figure 6 Effect of oral gavage of fructo-oligosaccharide on the number of mucosal mast cells (arrows). A: Ileal tissues stained with mast cell tryptase (magnification, 400 ×); B: In saline administered mice, mast cell counts increased in WAS compared to the sham-WAS group. Mast cell counts increased in FOS compared to saline administered mice following WAS; C: Colon stained with mast cell tryptase (magnification, 400 ×); D: In saline-administered mice, mast cell count increased in the WAS group compared to the sham-WAS group. Mast cell count increased in FOS compared to saline-administered mice following WAS. Values represent mean ± SD; sham + saline (n = 7), sham + FOS (n = 7), WAS + saline (n = 6), WAS + FOS (n = 7); one-way ANOVA. ^aP < 0.05, sham + saline vs WAS + saline; ^cP < 0.05, WAS + saline vs WAS + FOS. FOS: Fructo-oligosaccharide; WAS: Water avoidance stress.