Latilactobacillus sakei Furu2019 and stachyose as probiotics, prebiotics, and synbiotics alleviate constipation in mice

Abstract

Introduction: Slow transit constipation (STC) is a common disorder in the digestive system. This study aimed to evaluate the effects of stachyose (ST) and Latilactobacillus sakei Furu 2019 (L. sakei) alone or combined on diphenoxylate-induced constipation and explore the underlying mechanisms using a mouse model.

Methods: ICR mice were randomly divided into five groups. The normal and constipation model groups were intragastrically administrated with PBS. The ST, L. sakei, and synbiotic groups were intragastrically administrated with ST (1.5 g/kg body weight), alive L. sakei (3 × 10⁹ CFU/mouse), or ST + L. sakei (1.5 g/kg plus 3 × 10⁹ CFU/mouse), respectively. After 21 days of intervention, all mice except the normal mice were intragastrically administrated with diphenoxylate (10 mg/kg body weight). Defecation indexes, constipation-related intestinal factors, serum neurotransmitters, hormone levels, short-chain fatty acids (SCFAs), and intestinal microbiota were measured.

Results: Our results showed that three interventions with ST, L. sakei, and synbiotic combination (ST + L. sakei) all alleviated constipation, and synbiotic intervention was superior to ST or L. sakei alone in some defecation indicators. The RT-PCR and immunohistochemical experiment showed that all three interventions relieved constipation by affecting aquaporins (AQP4 and AQP8), interstitial cells of Cajal (SCF and c-Kit), glial cell-derived neurotrophic factor (GDNF), and Nitric Oxide Synthase (NOS). The three interventions exhibited a different ability to increase the serum excitatory neurotransmitters and hormones (5-hydroxytryptamine, substance P, motilin), and reduce the serum inhibitory neurotransmitters (vasoactive intestinal peptide, endothelin). The result of 16S rDNA sequencing of feces showed that synbiotic intervention significantly increased the relative abundance of beneficial bacteria such as Akkermansia, and regulated the gut microbes of STC mice. In conclusion, oral administration of ST or L. sakei alone or combined are all effective to relieve constipation and the symbiotic use may have a promising preventive effect on STC.

Keywords: Latilactobacillus sakei; intestinal flora; slow transit constipation; stachyose; synbiotics.

Figure 1

Effects of ST or L. sakei alone or combined on defecation parameters and gastrointestinal transit in mice. (A) Times to first black stool defecation; (B) Fecal number; (C) Fecal weight; (D) Fecal water contents; (E) Gastrointestinal transit. Data represent the mean ± SD (n = 6). Statistical analysis was conducted using One-way ANOVA followed by Tukey's multiple comparisons test for each group. The bars bearing different letters indicate a significant difference, P < 0.05.

Figure 2

The effect of ST or L. sakei alone or combined on the mRNA expression levels of constipation-related intestinal factors in mice. (A) Changes in mRNA levels of AQP4; (B) Changes in mRNA levels of AQP8; (C) Changes in mRNA levels of c-Kit; (D) Changes in mRNA levels of SCF; (E) Changes in mRNA levels of NOS; (F) Changes in mRNA levels of GDNF. Data represent the mean ± SD (n = 6). Statistical analysis was conducted using One-way ANOVA followed by Tukey's multiple comparisons test for each group. Bars bearing different letters indicate a significant difference, P < 0.05.

Figure 3

The effect of ST or L. sakei alone or combined on the protein levels of constipation-related intestinal factors in mice. (A) Protein expressions of AQP4, AQP8, c-kit, and SCF in the colons of mice were detected by immunohistochemistry and were observed under a 200×00 microscope. The mean optical density (MOD) of AQP4 (B), AQP8 (C), c-kit (D), and SCF (E) was calculated and evaluated by an experienced person who was blind to the samples in terms of treatment. Data represent the mean ± SD (n = 6). Statistical analysis was conducted using One-way ANOVA followed by Tukey's multiple comparisons test for each group using GraphPad Prism5. Bars bearing different letters indicate a significant difference, P < 0.05.

Figure 4

Effects of ST or L. sakei alone or combined on intestinal hormone levels in mice. (A) 5-HT level; (B) SP level; (C) MTL level; (D) VIP level; (E) ET level. 5-HT, 5-hydroxytryptamine; SP, substance P; MTL, motilin; VIP, vasoactive intestinal peptide; ET, endothelin. Statistical analysis was conducted using One-way ANOVA followed by Tukey's multiple comparisons test for each group. Bars bearing different letters indicate a significant difference, P < 0.05.

Figure 5

The effect of ST or L. sakei alone or combined on short-chain fatty acid levels of constipation-related intestinal factors in mice. (A) Acetic acid; (B) Propionic acid; (C) Butyric acid; (D) Isovaleric acid; (E) Isobutyric acid; (F) Valeric acid. Data represent the mean ± SD (n = 6). Statistical analysis was conducted using one-way ANOVA followed by Tukey's multiple comparisons test for each group. Bars bearing different letters indicate a significant difference, P < 0.05.

Figure 6

The effect of ST or L. sakei alone or combined on the alpha diversity analysis and beta diversity analysis in mice microbiota. (A) Ace index; (B) Chao index; (C) Shannon index; (D) PCA score plot at the phylum level; (E) NMDS plot at the genus level. Data represent the mean ± SD (n = 6–8). Wilcoxon signed-rank test of non-parameter statistics was used for constipation group vs. normal group, ***p < 0.001. Wilcoxon signed-rank test of non-parameter statistics was used for ST, L. sakei, and ST + L. sakei groups, vs. constipation group *p < 0.05, **p < 0.01.

Figure 7

The effect of ST or L. sakei alone or combined on the phylum- and genus-level structures of the gut microbiota. (A) Relative abundance bar plot at the phylum level; (B) Relative abundance at the genus level; (C) LEfSe analysis of gut microbiota. Data represent the mean ± SD (n = 6–8). Wilcoxon signed-rank test of non-parameter statistics was used for constipation group vs. normal group, ***p < 0.001. Wilcoxon signed-rank test of non-parameter statistics was used for ST, L. sakei, and ST + L. sakei groups, vs. constipation group *p < 0.05.