Exploring the Dose-Effect Relationship of Bifidobacterium longum in Relieving Loperamide Hydrochloride-Induced Constipation in Rats through Colon-Released Capsules

Abstract

Constipation is a common disease affecting humans. Bifidobacterium longum is reportedly effective in relieving constipation. Current studies generally focus on the dose-response relationship of oral doses; however, the dose-effect relationship of B. longum in the colon, which is the primary site where B. longum exerts constipation-relieving effects, to treat constipation has not been studied. Herein, three strains of B. longum (FGSZY6M4, FJSWXJ10M2, and FSDJN6M3) were packaged in colon-released capsules to explore the dose-effect relationship in the colon. For each strain, three groups of capsules (10⁴, 10⁶, and 10⁸ CFU/capsule, respectively) and one group of free probiotics (10⁸ CFU/mL) were used to explore the colonic dose effect of B. longum. The results showed that the three strains of B. longum improved fecal water content and promoted intestinal motility by regulating gastrointestinal peptide (MTL, GAS, and VIP), aquaporin-3, and 5-hydroxytryptamine levels while promoting gastrointestinal motility and relieving constipation by regulating the intestinal flora composition of constipated rats and changing their metabolite content (short-chain fatty acids). Among the three free bacterial solution groups (10⁸ CFU/mL), FGSZY6M4 was the most effective in relieving constipation caused by loperamide hydrochloride in rats. The optimal effective dose of each strain was 6M4 (10⁴ CFU/day), 10M2 (10⁶ CFU/day), and S3 (10⁸ CFU/day) of the colon-released capsules. Therefore, for some effective strains, the dose of oral probiotics can be reduced by colon-released capsules, and constipation can be relieved without administering a great number of bacterial solutions. Therefore, investigating the most effective dose of B. longum at the colon site can help to improve the efficiency of relieving constipation.

Keywords: Bifidobacterium longum; colon-released capsules; constipation; dose–effect relationship.

Figure 1

Intestinal fluorescence of rats indicating capsule transport. (a–e) The gastrointestinal fluorescent images of rats at different time points. The red color indicates a higher fluorescence signal, and the blue color indicates a lower fluorescence signal.

Figure 2

Apparent indicators of constipation: (a) fecal water content (%); (b) first black stool defecation time (min); (c) small intestine propulsion rate (%). Data are shown as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the model group.

Figure 3

Serum gastrointestinal active peptide levels (a–c), colonic aquaporin-3, and serotonin levels. (a) Serum gastrin (GAS); (b) serum motilin (MTL); (c) serum vasoactive intestinal peptide (VIP); (d) colonic aquaporin-3 (AQP3); (e) colonic serotonin (5-HT). Data are shown as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the model group.

Figure 4

The short-chain fatty acid level of fecal samples by GC-MS. (a) Acetic acid (μmol/g); (b) propionic acid (μmol/g); (c) butyric acid (μmol/g); (d) isobutyric acid (μmol/g); (e) isovaleric acid (μmol/g); (f) valeric acid (μmol/g). Data are shown as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, compared with the model group. # p < 0.05, compared with the control group. ns, not significant.

Figure 5

The composition of intestinal flora was changed after being given different doses of B. longum. (a) Shannon index of α-diversity; (b) Chao1 index of α-diversity; (c) principal coordinate analysis (PCoA) of the microbial group indicating β-diversity; (d) relative abundance of phyla; (e–h) relative abundance of Firmicutes, Bacteroidetes, Firmicutes/Bacteroidetes, and Actinomyces. Data are shown as means ± standard deviations. * p < 0.05, ** p < 0.01, compared with the model group.

Figure 6

The linear discriminant analysis effect size (LEfSe) analysis of fecal flora. (a) LDA score of characteristic bacteria (LDA > 2); (b) phylogenetic tree illustrating the taxonomic hierarchy of bacteria, which varies in terms of relative abundance between the various groupings. The relative abundance of each taxon is proportional to the size of the circle.

Figure 7

Animal experiment design schematic.