Limosilactobacillus reuteri Alleviates Anxiety-like Behavior and Intestinal Symptoms in Two Stressed Mouse Models

Abstract

Background/objectives: Limosilactobacillus (Lm.) reuteri is a widely utilized probiotic, recognized for its significant role in alleviating symptoms associated with gastrointestinal and psychiatric disorders. However, the effectiveness of Lm. reuteri is strain-specific, and its genetic diversity leads to significant differences in phenotypes among different strains. This study aims to identify potential probiotic strains by comparing the strain-specific characteristics of Lm. reuteri to better understand their efficacy and mechanisms in alleviating stress-induced anxiety-like behaviors and gastrointestinal symptoms.

Methods: We cultivated 11 strains of Lm. reuteri from healthy human samples and conducted phenotypic and genomic characterizations. Two strains, WLR01 (=GOLDGUT-LR99) and WLR06, were screened as potential probiotics and were tested for their efficacy in alleviating anxiety-like behavior and intestinal symptoms in mouse models subjected to sleep deprivation (SD) and water avoidance stress (WAS).

Results: The results showed that the selected strains effectively improved mouse behaviors, including cognitive impairment and inflammatory response, as well as improving anxiety and regulating gut microbiota composition. The improvements with WLR01 were associated with the regulation of the NLRP3 inflammasome pathway in the SD model mice and were associated with visceral hypersensitivity and intestinal integrity in the WAS model mice.

Conclusions: In summary, this study identified the Lm. reuteri strain WLR01 as having the potential to alleviate anxiety-like behavior and intestinal symptoms through the analysis of Lm. reuteri genotypes and phenotypes, as well as validation in mouse models, thereby laying the foundation for future clinical applications.

Keywords: Limosilactobacillus reuteri; anxiety-like behavior; gastrointestinal symptoms; gut microbiota; probiotics; strain-specific.

Figure 1

Comparative genome analysis and association of geno- and phenotypes of Lm. reuteri strains. (a) Phylogenetic tree constructed with the neighbor joining method based on core gene alignment (left). Genome features include genome sizes, GC%, CDs numbers, number of tRNA, and average nucleotide identity (ANI) (right). (b) Pan- and core-genome analysis showing the growing pan-gene numbers as genome numbers increase (top right); pan-genome analysis of Lm. reuteri strains. Each ring represents one strain, and each radial extension in the ring corresponds to a particular gene (red: present; blue: absent). The outer grey–green ring represents COGs functionality annotation, with grey indicating known and green indicating unknown functions. The outermost ring highlights particular gene collections: core, pink; shell genes, brown; and cloud genes, dark blue. (c) Genotypic and phenotypic analysis of carbohydrate utilization, genes related to carbohydrate activity enzymes. (d) BIOLOG test results for carbon sources assimilation. (e) Genome annotation for antibiotic resistance. (f) Experimental observation on bacterial resistance to 30 antibiotics (white = tolerant; faint yellow = intermediate; yellow = intolerant); the Lm. reuteri strain WLR08 was analyzed in parallel for reference. Abbreviations: P: penicillin; OX: oxacillin; AM: ampicillin; CB: carbenicillin; PIP: piperacillin; CA: cephalexin; CZ: cefazolin; RAD: cephradine; CXM: cefuroxime; CAZ: ceftazidime; CTR: ceftriaxone; CFP: cefoperazone; AK: amikacin; GM: gentamicin; K: kanamycin; N: neomycin; TE: tetracycline; DX: doxycycline; MI: minocycline; E: erythromycin; MD: medemcyin; NOR: norfloxacin; OFX: ofloxacin; CIP: ciprofloxacin; VA: vancomycin; PB: polymyxin B; CC: clindamycin; SXT: trimethoprim-sulfamethoxazole; FZ: furazolidone; C: chloramphenicol.

Figure 2

Biological characteristics of Lm. reuteri strains. (a) Evaluation of tolerance to acidity (pH 2.5, 3.5, and 4) and alkalinity (pH 9 and 10) (left); bile salt tolerance results (middle), following strains’ growth at different bile salt concentrations relative to the control group without bile salts (+: normal growth; −: delay of growth between the control and oxgall bile cultures); and germination rate of spores of Lm. reuteri isolates (right). (b) Production of SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and caproic acid) and other organic acids (lactic acid and succinic acid) by each of the 8 strains.

Figure 3

Chronic stresses induce extensive behavioral and inflammatory factors responses in mice (n = 6/group). (a) Schematic representation of the experimental design. (b) Percentage of weight gain during SD on the last day. (c) Distance of motion in open arms. (d) The time spent in open arms in SD mice. (e) Percentage of weight gain during WAS on the last day. (f) Distance of motion in open arms. (g) The time spent in open arms in WAS mice. (h) Distance of motion in novel arm. (i) The time spent in novel arms. (j) Spontaneous alternation rate. ELISA analyses of (k) TNF-α, (l) IL-6, (m) MDA, and (n) IL-1β in SD mice. (o) Abdominal withdrawal reflex scores in response to CRD. (p) The numbers of fecal pellets found in containers during WAS on the last day. ELISA analyses of (q) TNF-α, (r) IL-6, (s) IL-1β, and (t) MDA in WAS mice. Statistics: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Chronic stresses induce physiological responses (NLRP3 inflammasome, HPA axis, and intestinal mucosal barrier) and gut microbiota dysbiosis in mice (n = 6/group). RT-qPCR of hippocampal caspase-1 mRNA normalized to expression of (a) NLRP3, (b) Caspase-1, and (c) ASC. The hypothalamic–pituitary–adrenal axis index of (d) CRH, (e) CORT, (f) colonic ZO-1 mRNA, and (g) occludin, normalized to expression of GAPDH. (h) Immunohistochemistry of intestinal tissue based on ZO-1 and occludin protein. (i,j) Chao1 index and Shannon index of fecal microbiota in SD mice. (k) Microbial taxa at the phylum level in SD mice. (l) The top 15 microbial taxonomic groups at the genus level in SD mice. (m) Distribution histogram based on LDA (LDA > 2) in SD mice. (n,o) Chao1 index and Shannon index of fecal microbiota in WAS mice. (p) Microbial taxa at the phylum level in WAS mice. (q) The top 15 microbial taxonomic groups at the genus level in WAS mice. (r) Distribution histogram based on LDA (LDA > 2) in WAS mice. Statistics: ns: not significant (p > 0.05), * p < 0.05, ** p < 0.01.

Figure 5

Lm. reuteri alleviates extensive behavioral and inflammatory factor responses in chronic stressed mice (n = 6/group). (a) Percentage of weight gain on the last day in SD mice. (b) Distance of motion in open arms. (c) The time spent in open arms. (d) Percentage of weight gain on the last day in WAS mice. (e) Distance of motion in open arms. (f) The time spent in open arms. (g) Distance of motion in novel arm. (h) The time spent in novel arms. (i) Spontaneous alternation rate. ELISA analyses of (j) TNF-α, (k) IL-6, (l) IL-1β, and (m) MDA in SD mice. (n) Abdominal withdrawal reflex scores in response to CRD. (o) The numbers of fecal pellet found in containers during WAS on the last day. ELISA analyses of (p) TNF-α, (q) IL-6, (r) IL-1β, and (s) MDA in WAS mice. Statistics: * p < 0.05, ** p < 0.01.

Figure 6

Lm. reuteri improved physiological responses (NLRP3 inflammasome, HPA axis, and intestinal mucosal barrier) and gut microbiota dysbiosis in chronically stressed mice (n = 6/group). RT-qPCR of hippocampal (a) NLRP3, (b) Caspase-1, and (c) ASC mRNA normalized to expression of GAPDH. The hypothalamic–pituitary–adrenal axis index of (d) CRH and (e) CORT. RT-qPCR of (f) colonic ZO-1 mRNA and (g) occludin, normalized to expression of GAPDH. (h) Immunohistochemistry of intestinal tissue based on ZO-1 and occludin protein in WAS mice. (i,j) Chao1 index and Shannon index of fecal microbiota in SD mice. (k) Microbial taxa at the phylum level in SD mice. (l) The top 15 microbial taxonomic groups at the genus level in SD mice. (m) Distribution histogram based on LDA (LDA > 2) in SD mice. (n,o) Chao1 index and Shannon index of fecal microbiota in WAS mice. (p) Microbial taxa at the phylum level in WAS mice. (q) The top 15 microbial taxonomic groups at the genus level in WAS mice. (r) Distribution histogram based on LDA (LDA > 2) in WAS mice. Statistics: ns: not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001.