Clostridium butyricum miyairi 588 has preventive effects on chronic social defeat stress-induced depressive-like behaviour and modulates microglial activation in mice

https://doi.org/10.1016/j.bbrc.2019.06.053Get rights and content

Highlights

  • CSDS induces depression-like behaviours in mice.

  • Preventive supplementation of CBM588 ameliorates depression against stress.

  • Stress-induced inflammation could be relieved by CBM588.

  • CBM588 treatment alters intestinal microbiota for 2 weeks.

  • Altered microbiota are correlated with mouse depression-like behaviours.

Abstract

Recent studies have suggested the neuroprotective effects of Clostridium butyricum on mood disorders. However, the potential role of Clostridium butyricum in modulating the gut-brain-axis remains unknown. Here, we applied the commercial Clostridium butyricum Miyairi 588 (CBM588) strain to assess psychological behavioural alterations in mice exposed to chronic social defeat stress (CSDS). We found that preventive treatment with CBM588 for 28 days ameliorated depressive-like behaviours in CSDS mice. We showed that CSDS led to increases in cytokines (IL-1β, IL-6, and TNF-α), intestinal dysfunction and hippocampal microglial activation, while CBM588 partially relieved these alterations. By applying 16S sequencing, we found that Firmicutes was more abundant in the faeces of CBM588/CSDS mice than in the faeces of placebo/CSDS mice, and depression-like behaviours in the mice were correlated with certain strains (including Clostridium leptum, Blautia coccoides, Family_XIII_UCG-001, Candidatus Arthromitus sp-SFB-mouse-Japan and Streptococcus hyointestinalis) at the species level. Our results illustrated the preventive effect of CBM588 against stress, suggesting the beneficial role of CBM588 in regulating neuroinflammation via the gut-brain-axis. This study provides novel strategies for clinical and scientific investigations of depressive disorders.

Introduction

The abundant commensal microbiota that inhabit our bodies are important for maintaining the internal environment [1], especially in the gut. There is emerging evidence that the dysbiosis of intestinal microflora contributes to neurodevelopmental and neurological disorders, including autistic disorder [2], Alzheimer's disease [3] and psychiatric diseases [4,5] in humans and rodents. Indeed, the intestinal microbiota mediates bidirectional interactions between the central and enteric nervous systems (CNS and ENS, respectively) through the gut-brain-axis [6,7] by interacting with the host via the hypothalamus-pituitary-adrenal (HPA) axis, metabolic and immune pathway [8], and the vagus nerve [9]. Growing evidence from our group has demonstrated that faecal microbiota transplantation from MDD patients leads to a depressive-like phenotype in germ-free (GF) mice [10], suggesting an underlying mechanism involving aberrant lipid and energy metabolism in the liver [11] and upregulated glucocorticoid receptor pathway genes in the hippocampus [12].

Diets or supplements including specific bacteria (known as probiotics) are considered beneficial to mental health beyond basic nutrition in humans and animals. For example, daily treatment with Bifidobacterium longum (B.) 1714 and B. breve 1205 for 6 weeks [13] decreases immobility time and anxiety in innately anxious BALB/c mice exposed to acute stress. HFD administration in GF mice during maternal periods causes autism spectrum disorder (ASD) and synaptic impairments in offspring that can be reversed by Lactobacillus reuteri [14]. In addition to alleviating psychiatric disorders, probiotics provide a wide range of benefits for healthy subjects; healthy volunteers administered Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 for 30 days [15] achieved lower scores on the hospital anxiety and depression scale (HADs) and exhibited a decreased global severity index (GSI) of the Hopkins Symptom Checklist 90 (HSCL-90). Recently, Clostridium butyricum was reported to stimulate glucagon-like peptide-1 (GLP-1) receptor secretion [16] to attenuate depressive-like behaviours in male C57BL/6 mice. Clostridium has been suggested to increase the production of metabolic butyrate, which has been implicated in depression through HPA-axis perturbation and damage to intestinal permeability [17], by combining Free Fatty Acid Receptor 2 (FFAR2, GPR43) and FFAR3 (GPR41) [18] and regulating NFκB and PPARγ signalling [19,20]. However, the neurophysiological molecular mechanisms of Clostridium butyricum remain unclear.

Convergent evidence has illustrated that exposure to acute or chronic psychosocial stress decreases the relative abundance of Bacteroides spp. and Clostridium spp. in the caecum [21]. This mental stress increases interleukin-6 (IL-6) and chemokine CCL2 (also known as MCP1) levels in the periphery, assembles reactive oxygen species (ROS) in the colon [22] of murine models, and increases toll-like receptor-3 (TLR3) and toll-like receptor-4 (TLR4) expression [23] in the prefrontal cortex (PFC) of depressed suicidal (DS) patients, indicating that stress causes general immune activation in vivo. Microglia are resident macrophage cells that contribute to brain inflammatory activation [24] and synaptic terminal modification [25] in addition to removing dead cells. Despite this, the relationships between cerebral microglial activity and probiotics in the depression process are poorly understood.

In the present study, we applied a commercial Clostridium butyricum agent, Miyairi 588, to investigate whether oral Miyairi 588 pretreatment can ameliorate stress-induced behavioural deficits, specifically deficits in sociability and depressive- and anxiety-like behaviours in mice. We explored intestinal and neuronal inflammation. In parallel, we assayed faecal microbiota transplantation and intestinal flora changes in the colon between CSDS mice fed different diets. This research lays a foundation for understanding the neuroinflammatory response of Clostridium butyricum in the adaptation of the gut-microbiota-brain axis to chronic stress.

Section snippets

Animals

Male C57BL/6 mice (8–12 weeks of age, 20–25 g) and male CD-1 (ICR) mice (16–20 weeks of age, 35–40 g) were obtained from the laboratory animal centre of Chongqing Medical University (Chongqing, China). The mice were individually caged with sawdust under a 12/12-h light/dark cycle (lights on at 8:00 a.m.) with controlled temperature (22 ± 2 °C) and humidity of 55 ± 5% and were fed autoclaved chow and sterile water ad libitum.

Probiotic treatment

A group of SPF mice (8–12 w, 20–25 g, n = 32) were provided sterile

CBM588 pre-feeding attenuates social avoidance and depressive-like behaviours in CSDS mice

Mice exposed to 10 consecutive days of defeat showed significant social avoidance due to both physical and psychological stress (p < 0.001). Among these mice, the CSDS group of mice pretreated with CBM588 (also known as the CBM588/stress group) (n = 25) remained in the interaction zone longer than the CSDS mice fed sterile water (also known as the placebo/stress group) (n = 25) (Fig. 2A, p = 0.064).

We next determined whether probiotic pretreatment can impact mouse phenotypes such as depressive-

Discussion

In this novel study, we fed mice with CBM588 for 4 weeks before exposure to CSDS to examine its effect on psychological phenotypes after stress. To our surprise, CSDS mice that were pre-treated with CBM588 were less susceptible then the CSDS mice fed sterile water to depression-like behaviours. CBM588 may preventively ameliorate inflammation both in the colon and hippocampus, by decreasing cytokines (including IL-1β, IL-6, and TNF-α) and inflammatory microglia activation. Evidence suggests that

Ethical statements

All experiments were approved by the Ethics Committee of Chongqing Medical University, and all animal treatment procedures were in accordance with the National Institutes of Health Guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals, NIH Publication No. 80–23, revised 1996).

Conflicts of interest

All authors have agreed to the submission of this manuscript, and there is no conflict of interest.

Author contributions

Conceived and designed the study: P.X, T.T.

Performed the experimental procedures: T.T., B.X., Y.H.Q., L.F. and M.G.B.

Analysed the data: J.J.C., P.Z., J.L. H.Y.W., J.C.P. and X.G.

Drafted the manuscript: T.T.

Acknowledgments

This study was supported by the National Key R&D Program of China (No. 2017YFA0505700), National Key Program International Cooperation Project (No. 81820108015) and Chongqing Science and Technology Project (No. 20170113).

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    These authors contributed equally to this work.

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