Oral Probiotics Ameliorate the Behavioral Deficits Induced by Chronic Mild Stress in Mice via the Gut Microbiota-Inflammation Axis

Abstract

In recent years, a burgeoning body of research has revealed links between depression and the gut microbiota, leading to the therapeutic use of probiotics for stress-related disorders. In this study, we explored the potential antidepressant efficacy of a multi-strain probiotics treatment (Lactobacillus helveticus R0052, Lactobacillus plantarum R1012, and Bifidobacterium longum R0175) in a chronic mild stress (CMS) mouse model of depression and determined its probable mechanism of action. Our findings revealed that mice subjected to CMS exhibited anxiety- and depressive-like behaviors in the sucrose preference test, elevated plus maze, and forced swim test, along with increased interferon-γ, tumor necrosis factor-α, and indoleamine 2,3-dioxygenase-1 levels in the hippocampus. Moreover, the microbiota distinctly changed from the non-stress group and was characterized by highly diverse bacterial communities associated with significant reductions in Lactobacillus species. Probiotics attenuated CMS-induced anxiety- and depressive-like behaviors, significantly increased Lactobacillus abundance, and reversed the CMS-induced immune changes in the hippocampus. Thus, the possible mechanism involved in the antidepressant-like activity of probiotics is correlated with Lactobacillus species via the gut microbiota-inflammation-brain axis.

Keywords: 3-dioxygenase; Bifidobacteria; Lactobacillus; chronic mild stress; indoleamine 2; inflammation; microbiota; probiotics.

FIGURE 1

Experimental design. Representation of the study design. C57BL/6 mice were fed for a total of 4 weeks with a mixture of three probiotics (Lactobacillus helveticus R0052, L. plantarum R1012, and Bifidobacterium longum R0175), the antidepressant fluoxetine, or saline. After 4-week treatment, animals underwent a battery of testing relevant to anxiety. All groups were sacrificed on the same day. Cecal contents and the entire hippocampus tissue were harvested for further analysis. SPT, sucrose preference test; EPM, elevated plus maze; FST, forced swim test; CMS + NS, subjected to the CMS procedure and received saline; CMS + F, subjected to the CMS procedure and received fluoxetine; CMS + P, subjected to the CMS procedure and received probiotics; CON + NS, not subjected to any stress and received saline; CON + F, not subjected to any stress and received fluoxetine; and CON + P, not subjected to any stress and received probiotics.

FIGURE 2

Behavioral assessments in mice. (A) There were no differences among six groups in sucrose preference at baseline before exposure to the CUS protocol. (B) SPT results showed no significant differences in total liquid consumption. (C) Sucrose preference was significantly decreased after 4 weeks of CMS, and this effect of stress was reversed by fluoxetine, but not by probiotics. (D) EPM test results showed that the time spent in the open arms was significantly reduced in the CMS group, and this effect of stress was reversed by fluoxetine and probiotics. (E) Results of FST showed that the immobility time was significantly increased in the CMS group; the change of immobility time induced by CMS was reversed by fluoxetine administration or probiotics administration. The comparision of three groups without CMS revealed that fluoxetine administration led a significantly decreased in the immobility time compared with probiotics administration. Data are presented as mean ± SEM; n = 8 per group; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. the CMS + NS group, ^#P < 0.05 vs. the CON + P group. SPT, sucrose preference test; EPM, elevated plus maze; FST, forced swim test; CMS + NS, subjected to the CMS procedure and received saline; CMS + F, subjected to the CMS procedure and received fluoxetine; CMS + P, subjected to the CMS procedure and received probiotics; CON + NS, not subjected to any stress and received saline; CON + F, not subjected to any stress and received fluoxetine; and CON + P, not subjected to any stress and received probiotics.

FIGURE 3

Alterations of gut microbiota. (A) Unweighted UniFrac Anosim was significantly different among groups. (B) Weighted UniFrac Anosim was significantly different among groups. (C) Two-dimensional principal coordinate analysis (PCoA) of unweighted UniFrac distances showed a clear difference in the microbial community compositions between the stress and non-stress groups. (D) The α-diversity index, Chao1 revealed differences in species richness; stress-exposed samples presented significantly higher diversity. (E) The α-diversity index, Simpson revealed differences in species evenness; stress-exposed samples presented significantly higher species evenness. (F) Relative abundance of the genus Lactobacillus in the six groups, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. the CMS + NS group. (G) The most abundant taxa at the genus level in all samples; n = 8 per group.

FIGURE 4

Cytokine concentrations in the hippocampus. (A) There was no significant difference in hippocampal IL-6 level among six groups. (B) CMS significantly increased IFN-γ expression in the hippocampus, which could be reversed by supplementation with probiotics or fluoxetine. (C) CMS significantly increased TNF-α level in the hippocampus, which could be reversed by supplementation with probiotics or fluoxetine. (D) There was no significant difference in hippocampal IL-10 level among six groups. Data are presented as mean ± SEM; n = 8 per group; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. the CMS + NS group.

FIGURE 5

IDO1 protein levels in the hippocampus. Above: western blots for IDO1. Below: ImageJ analysis of IDO1 protein intensity normalized to β-actin; n = 4 per group; ^∗P < 0.05; ^∗∗P < 0.01.