Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Clostridium butyricum Attenuates Chronic Unpredictable Mild Stress-Induced Depressive-Like Behavior in Mice via the Gut-Brain Axis Jing Sun,† Fangyan Wang,‡ Xuezhen Hu,§ Changwei Yang,⊥ Hailing Xu,⊥ Ye Yao,⊥ and Jiaming Liu*,⊥
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Department of Neurology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China ‡ Department of Emergency Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China § Department of Pathophysiology, School of Basic Medicine Science, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China ⊥ Department of Preventive Medicine, School of Public Health and Management, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China ABSTRACT: Abnormal gut microbiome has been associated with depression. The mechanism of probiotics against depression remains unclear. This study aimed to determine whether Clostridium butyricum (Cb) could attenuate chronic unpredictable mild stress-induced depressive-like behavior and its possible mechanisms. Male C57BL/6 mice were subjected to chronic unpredictable mild stress (CUMS) and were treated with Cb. Depressive-like behavior was evaluated by a series of behavioral tests. The levels of cerebral 5-hydroxytryptamine (5-HT), brain derived neurotrophic factor (BDNF), glucagon-like peptide-1 (GLP-1) receptor and intestinal were measured. Cb treatment significantly improved CUMS-induced depressive-like behavior in mice. Meanwhile, Cb treatment exhibited prominent effects, increasing 5-HT and GLP-1 and upregulating BDNF expression. Furthermore, Cb-treated mice showed increased secretion of GLP-1 and upregulated GLP-1R expression. Taken together, our results demonstrate an antidepressive effect of Cb in CUMS mice partially attributed to stimulation of intestinal GLP-1 secretion and activation of cerebral GLP-1R. KEYWORDS: probiotics, depression, BDNF, glucagon-like peptide-1, gut microbiota
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INTRODUCTION Depression is a psychosis related to social, environmental, genetic, and metabolic factors.1 An increasing number of studies have recognized the potential for gut microbiota (GM) to affect the gut-brain communication in terms of health and disease.2−4 GM interacts with the host via neuroendocrine and neural pathways, which are components of the microbiota-gutbrain axis. Numerous studies have shown that GM can affect brain function and change behavior and mood.5−7 The intestinal tract is the common site of nutrient digestion, microbial colonization, and immune cell localization, which promotes their interaction.8 GM abnormalities have been strongly associated with neuropsychiatric disorders, including depression.2,4,9 GM plays a vital role in initiating signal transduction and communication between the intestinal nervous system and the central nervous system. Recent studies found that germ-free mice display exaggerated stress and anxiety-like behavior compared to specific pathogen-free (SPF) mice,10 and those anxious mice treated with Bif idobacterium infantis exhibited a complete normalization of behavior.5 Meanwhile, the loss and/or modification of the gut microflora could lead to in specified neurological diseases, such as vascular dementia11 and psychological stress.12,13 Furthermore, modulating the composition of GM using prebiotics and probiotics may produce beneficial effects on anxiety and depression. Application of probiotics regulating the composition of the gut © XXXX American Chemical Society
microbiota can have beneficial effects on anxiety and depression. The treatment with live Mycobacterium vaccae and Lactobacillus helveticus ROO52 reduces anxiety- and depressive-like behaviors and alleviates memory dysfunction.14 Probiotics have prevented or improved brain disorders by restoring normal microbial populations and bringing benefits to their hosts.4,15 However, their antidepressive mechanism remains unclear. Glucagon-like peptide-1 (GLP-1) is considered one of the chemical mediators linked the gut-brain axis.16 GLP-1 is released by intestinal L cells, which are mainly distributed in the ileum and colon. GM is directly in contact with the L cells in the intestinal tract, and the changes of gut microbes could affect the GLP-1 level.17,18 GLP-1 receptor (GLP-1R) is found in the central nervous system (CNS), and the GLP-1 signal system is closely linked to the function of CNS, including neuronutrition and neuroprotection.19 Therefore, probiotics can promote the interaction between intestinal bacteria and L cells, increase the secretion of GLP-1, and improve its beneficial effect. Received: May 9, 2018 Revised: July 11, 2018 Accepted: July 11, 2018
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DOI: 10.1021/acs.jafc.8b02462 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
until the end of the experiment. The mice in group CUMS and Con received the equivalent milk. All three groups of mice received a normal diet and were fed daily with a restricted diet (10% less than the daily ration) to reach the same amount of intake. Behavioral Test. Depression-related behaviors were assessed with OFT, TST, and FST. All behavioral experiments were monitored by a trained observer blinded to the treatments. TST. The TST was carried out as described.24 Briefly, the mice were lifted 50 cm off the ground (approximately 1 cm from the tip of each mouse’s tail). At the end of the 6 min test for 5 min of the 6 min test, the immobility time was scored for each mouse. The mouse was considered immobile only when they were passive and completely motionless. Immobility was defined as the absence of movement for 6 min and was used as evidence of hopelessness. After completion of the test, mice were returned to a holding cage. FST. The FST has been used to identify depressive-like behavior in animals. The FST was performed as previously described.24 Briefly, the mouse was individually placed into cylindrical glass container (25 cm height, 10 cm diameter) containing 20 cm water maintained at 24 ± 0.5 °C. Care was taken not to put the nose of the mouse below water level. After a 1 min habituation period, each mouse was forced to swim for 5 min, and the immobility time was recorded during these 5 min. The immobility time was defined as there is no escape behavior, i.e., when they ceased struggling and remained floating motionless, only making those movements necessary to keep the head or nose above water. After 6 min, mice were removed from the water, dried with towels, and immediately returned to their home cage. OFT. Mice were placed individually in a cardboard box (40 cm × 40 cm × 30 cm), which consisted of 25 squares (5 cm × 5 cm) at the bottom. After a 1 min habituation period, each mouse was placed in the center of the box, and the numbers of crossings (crossing the sector with all four paws) and rearings (raising the forepaws) were recorded within 5 min. The open field arena was cleaned between trials with a 5% ethanol solution. Tissue Collection. After behavioral measurements, the mice were killed and brain and colon samples were collected. Collected samples of colon placed in tubes containing EDTA and dipeptidyl peptidase-4 (DPP-4) inhibitor and centrifuged and the supernatant kept at 20 °C for further analysis. 4% paraformaldehyde solution was use to fix brain tissue, and further immunohistochemical analysis was conducted. The brain tissues were immediately frozen in liquid nitrogen until analysis. The colon was immediately frozen in PBS until analysis. A schematic representation of the intervention and assessments is shown in Figure 1. ELISA. The brain and colon tissues were frozen in PBS and kept at 80 °C until analysis. The levels of 5-HT and GLP-1 were, respectively, measured by ELISA kit (RayBiotech, Norcross, GA) and ELISA kit (Millipore, St. Louis, MO) according to the manufacturer’s instructions. Western Blot Analysis. The brain samples were homogenized in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) consisting of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X100, 1% sodium deoxycholate, 0.1% SDS, and multiple inhibitors. The samples were then analyzed by Western blot, as described in our previous research.7,26,27 The primary antibodies were BDNF and GLP-1R. The information and dilution of the antibody are as follows: BDNF (1:1000, Bioworld, Louis Park, MN), GLP-1R (1:1000, Santa Cruz Biotechnology, Dallas, TX), and β-actin (1:1000, Bioworld, Louis Park, MN). After incubation with primary antibodies; membranes were incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. β-Actin was used as the loading control. Immunohistochemistry Analysis. Consecutive 5-μm thick sections from routinely fixed and paraffin embedded tissues from the brain and colon samples were processed for immunohistochemistry. Immunohistochemistry for BDNF and GLP-1R were then carried out on the sections as previously described.11,25,28 Slices were incubated with the primary antibody against BDNF (1:200, Bioworld, Louis Park, MN), GLP-1R (1:200, Santa Cruz Biotechnology, Dallas, TX), and GLP-1 primary antibody (1:250, Abcam, Cambridge, MA)
Clostridium butyricum (Cb) is a very important intestinal probiotics, which plays an important role in regulating gut microbiota. Our previous studies showed that Cb’s treatment for vascular dementia,20 diabetes combined with cerebral ischemia,6 and traumatic brain injury is effective.7 Cb affects the metabolism of gut microbes by regulating the structure of gut microbiota.21 These effects of Cb can be primarily explained as it could increase the production of metabolic butyrate, which stimulates GLP-1 secretion.22 Although Cb has been reported to promote the release of GLP-1 in different animal models, the effect of Cb on GLP-1 secretion in depressed animal models has not yet been elucidated. Therefore, this study was designed to investigate the antidepressant effects of C. butyricum on depression induced by chronic unpredictable mild stress (CUMS) in mice. We verified the hypothesis that Cb played an antidepressive role by stimulating intestinal GLP-1 secretion and activating cerebral GLP-1R in the animal model of depression.
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MATERIALS AND METHODS
Animals. Adult (age 6−8 weeks, 18−22 g) male C57BL/6J mice were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. Mice were housed standard environmental conditions (22 ± 1 °C; humidity 55 ± 5%; and 12-h light/dark cycle. Mice were acclimated for a week before the formal experiment. All experiments were carried out in accordance with the Guide for Animal Experimentation of Wenzhou Medical University. Induction of Depressive Mouse Model. The procedure of CUMS was performed as described previously23 and as performed in our previous study,24 with slight modification. In short, the CUMS protocol contained a variety of mild stressors: 24-h shortages of food and water, tightness of the tail, tilt of the cage, cold swimming, physical restraint, reversed cycle of light-dark, soiled bedding, foot shooting, heat stimulation, and suspension. One of the stressors (random order) within 4 weeks was given every day. The procedural sequence was performed as follows (Figure 1): (1) stress procedure
Figure 1. Experimental schematic diagram. Mice were treated for a total of 4 weeks with Cb or vehicle. After 4 weeks of treatment, animals underwent a battery of tests relevant to depression. All groups were sacrificed on the same day. Tissues were harvested for further analysis. CUMS, chronic unpredictable mild stress; OFT, open field test; TST, tail suspension test; FST, forced swim test. during weeks 1−4, (2) open-field test (OFT) on day 29, and (3) tail suspension test (TST) and forced swimming test (FST) on day 30. In the behavior of the listed above tests, the mice were transferred to the experimental room for adaptation for at least 1 h prior to the test. After each test, mice were sent back to their home cages. Mice in the control group were placed in a separate room and were not in contact with the stress mice. Bacteria Preparation and Groups. C. butyricum WZMC1018 (CGMCC 9830) was provided by the General Microbiological Culture Collection Center China. The bacterial solution was prepared every day in sterile milk and treated orally at a concentration of 5 × 108 CFU/0.5 mL/day/mice for 28 consecutive days. The dose of C. butyricum used in the present experiment was chosen based on previous studies.6,11,25 Mice were randomly divided into three groups (n = 10 per group): the control (Con), CUMS (CUMS), and C. butyricum (Cb) groups. Every morning, the Cb group was treated intragastrically at a dose of 0.5 mL of Cb once daily during the stress B
DOI: 10.1021/acs.jafc.8b02462 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. Effects of Cb on depressive-like behavior. (A) The tail suspension test, (B) the forced swimming test, and (C) open field test. Error bars indicate the SEM; **P < 0.01 vs Con, #P < 0.05 vs CUMS, and ##P < 0.01 vs CUMS, n = 6−8 per group. overnight at 4 °C. Then, the sections were incubated with secondary antibody (1:400) and were visualized using diaminobenzidine (DAB) as the chromogen. Cells with brown granules were considered immunoreactions-positive cells. The slides were observed under light microscopy and photographed. Data Analysis. All data were expressed as the mean ± standard error of mean (SEM). The data were analyzed by ANOVA followed by Bonferroni’s test to determine the significant difference between each group. A value of P < 0.05 was considered significant.
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RESULTS Effect of Cb on Depressive-Like Behavior. The effect of Cb on depressive-like behavior in CUMS mice was assessed by FST and TST. The mice exposed to CUMS exhibited a significantly longer immobility time in the TST than the mice in the Con group (P < 0.01, Figure 2A), and Cb treatment reversed this phenomenon (P < 0.05, Figure 2A). Similar results were found in the FST. Compared with the Con group, the CUMS-exposed mice had significantly increased immobility time in the FST (P < 0.01, Figure 2B). However, treatment with Cb significantly abolished the adverse effect of CUMS (P < 0.01, Figure 2B). To eliminate the excitatory or inhibitory effects of CUMS and Cb on behavior, the locomotion counts were analyzed before each depression or anxiety behavior test. The locomotion counts did not show significantly different crossings or rearings in the OFT (P > 0.05, Figure 2C), suggesting that Cb did not affect the locomotor activity. Effects of Cb on Cerebral 5-HT Level. As shown in Figure 3, CUMS-exposed mice had significantly reduced 5-HT levels in the brain compared to those in the Con group (P < 0.05), while those given Cb treatment had significantly higher 5-HT levels than those in the CUMS group (P < 0.05). Effects of Cb on Cerebral BDNF Level. To investigate the effect of Cb on the level of BDNF in the brain, we measured BDNF by Western blot and immunohistochemistry. As shown in Figure 4, BDNF in the CUMS group was markedly decreased compared to that in the Con group (P
