PBB-72183; No of Pages 7 Pharmacology, Biochemistry and Behavior xxx (2015) xxx–xxx
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
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Jinsong Xue, Hongyan Li, Xueyang Deng, Zhanqiang Ma, Qiang Fu ⁎, Shiping Ma ⁎⁎
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Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, PR China
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Article history: Received 7 November 2014 Received in revised form 14 April 2015 Accepted 17 April 2015 Available online xxxx
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Keywords: L-Menthone Antidepressant Unpredictable chronic mild stress (UCMS) NLRP3 inflammasome Cytokines Neurotransmitters
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(MTN) is a Chinese old remedy extracted from the genus Mentha. It has been widely used as a cooling agent and a counterirritant for pain relief, although its antidepressant-like effects have not yet been reported. The present study was designed to investigate whether MTN confers an antidepressant-like effect in mice exposed to unpredictable chronic mild stress (UCMS) and to explore its potential mechanisms. The effects of MTN on mouse behavioral changes were investigated in our study. We determined the levels of the nucleotide binding, oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, inflammatory cytokines and neurotransmitters in the hippocampus of mice. Behavioral tests, including the sucrose preference test (SPT), open field test (OFT), forced swimming test (FST) and tail suspension test (TST) revealed that MTN (15 and 30 mg/kg) treatments for 3 weeks alleviated the depression symptoms of UCMS in mice. Mice receiving MTN treatments exhibited reduced levels of NLRP3 and caspase-1. Moreover, MTN treatments reversed the UCMS-induced alterations in the concentrations of neurotransmitter norepinephrine (NE) and serotonin (5-HT) and inhibited the expression of pro-inflammatory cytokines (PIC) interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the hippocampus of mice. Taken together, our findings suggested that MTN may play a potential antidepressant-like role in the UCMS mouse model by regulating the NLRP3 inflammasome and mediating inflammatory cytokines and central neurotransmitters, which together provide insight towards the development of novel therapeutic treatments for depression. © 2015 Published by Elsevier Inc.
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confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflammatory cytokines and central neurotransmitters
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Depression is a chronic, recurring, and life-threatening mental disorder in which the underlying mechanisms remain unclear, and the effective cures are scarce (Berton and Nestler, 2006). Moreover, most of the current antidepressants used in the clinic not only have a low curative ratio, but they also may cause side effects (Zhou et al., 2011). Thus, the identification of safer and more reliable antidepressants is necessary. There are many hypotheses involved in the etiopathogenesis of depression, including the monoamine deficiency hypothesis, hyperactive HPA axis hypothesis, cytokine hypothesis, neurodegeneration hypothesis, and other hypotheses. Among these hypotheses, the emerging cytokine hypothesis has attracted interest because it can orchestrate complex responses and coordinate responses with those of other physiological
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1. Introduction
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⁎ Correspondence to: Q. Fu, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, PR China. Tel.: +86 25 3271507. ⁎⁎ Correspondence to: S.-P. Ma, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, PR China. Tel.: +86 25 3271507; fax: +86 25 3271505. E-mail addresses: [email protected] (Q. Fu), [email protected] (S. Ma).
systems in the body (Dunn et al., 2005). In the present study, we mainly focused on the modulation of the NLRP3 inflammasome on cytokines and monoamine neurotransmitters. The NLRP3 inflammasome, a new target in depression, belongs to the NLR family and consists of NLRP3, the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1, a biomarker used to evaluate the effectiveness and resistance to antidepressants (Gomez and Cordero, 2014; Schroder and Tschopp, 2010). The NLRP3 inflammasome consists of intracellular pattern-recognition molecules detecting microbial- and dangerassociated molecular patterns and has been implicated in major diseases, such as ischemic stroke, obesity and type 2 diabetes (Cassel et al., 2009; Fann et al., 2013; Shan et al., 2014; Luo et al., 2014). Importantly, microglia are a primary source of PIC in the brain and the resident macrophage in the central nervous system (CNS). Once activated, NLRP3 oligomerizes in microglia and recruits pro-caspase-1, resulting in the activation of caspase-1 and the production of IL-1β. IL1β then activates downstream pathways, such as NF-κB, and promotes the expression of IL-6 and TNF-α, which play an important role in the pathophysiology of depression (Maes et al., 2012). It has been reported that chronic stress can increase the production of IL-1β and TNF-α in the hippocampus (Munhoz et al., 2006). The effects of IL-1β on the
http://dx.doi.org/10.1016/j.pbb.2015.04.014 0091-3057/© 2015 Published by Elsevier Inc.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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2. Materials and methods
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2.1. Animals
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Male ICR mice (4 weeks, weighing 18–22 g) were purchased from the Experimental Animal Center in Jiangsu Province (Nanjing, PR China). Mice were randomly housed in cages for one week to acclimatize to their environment prior to their use in experiments. The mice had free access to food and water and were housed in a room maintained at 25 ± 1 °C with a 12/12 hour light/dark cycle. All of the animal
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2.3. Unpredictable chronic mild stress and experimental design
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Fluoxetine hydrochloride was purchased from the Changzhou Siyao Pharmaceuticals Co., Ltd (Changzhou, PR China). MTN (97%) was purchased from the Shanghai Jianglai Co., Ltd. (Shanghai, PR China). All drugs were dissolved in 0.3% sodium carboxy methyl cellulose (CMC)Na. ELISA kits (R&D, Minneapolis, MN, USA), and a bicinchoninic acid assay (BCA) kit (Beyotime Institute of Biotechnology Co., Ltd) was used in this study.
Fig. 1. Structure of L-menthone.
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The mice were randomly assigned to five groups (n = 10 for each group): control group (0.3% CMC-Na), UCMS (0.3% CMC-Na) group, UCMS + fluoxetine (FLU) (10 mg/kg) group, UCMS + MTN (15 mg/kg) group, and UCMS + MTN (30 mg/kg) group. All of the drugs were orally administered in a volume of 10 ml/kg body weight. To examine whether MTN has potential antidepressant-like effect, the UCMS model was used to induce depressive-like behavior in mice. The mice were first exposed to the UCMS procedure for 3 weeks, followed by 3 weeks of MTN or FLU, treatment during which UCMS exposure was maintained (Fig. 2). The UCMS procedure was performed as previously described (Willner et al., 1987), with minor modifications. Briefly, the mice were allowed to acclimate one week before they were subjected to the following stressors for six weeks (42 days): (1) water deprivation (24 h), (2) food deprivation (24 h), (3) overnight illumination, (4) cage tilt (45°), (5) soiled cage (200 ml of water in 100 g of sawdust bedding), (6) exposure to a foreign object, (7) light/dark perversion, (8) overhang (10 min), (9) exposure to an empty bottle, (10) 1-min tail pinch (1 cm from the beginning of the tail), (11) 5-min oscillation, and (12) white noise. All of the stressors were applied at any time of the day and night to ensure the unpredictable nature of the experiment.
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2.4. Sucrose preference test (SPT)
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The sucrose preference test was monitored weekly throughout the experiment. Mice were deprived of water and food for 24 h, and the mice were then given the choice to drink from two bottles for 12 h: one was filled with a sucrose solution (1% w/v), and the other was filled with water. To avoid potential side-preference effects on drinking behaviors, the positions of the bottles in the cage were switched after 6 h. The consumption of water and the sucrose solution and the total intake of liquids were estimated by weighing the bottles. The preference for sucrose was measured as the percentage of consumed sucrose solution relative to the total amount of liquid intake.
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2.5. Open field test (OFT)
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To assess the MTN treatment effects on spontaneous locomotor activities, mice were subjected to the open-field paradigm as previously described (Rodrigues et al., 2002). The apparatus (40 × 60 × 50 cm) was divided into 12 equal squares and the number of crossings was recorded. The test began by placing the mouse in the center of the device so that it was acclimatized with its environment for 2 min. The number of crossings (the number of squares crossed), rearings (standing on its hind legs) and groomings (licking, cleaning and scratching its face with the forepaws) was recorded for 4 min. The apparatus was then cleaned with a 10% alcohol solution and dried after occupancy by each mouse.
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handing and experimental procedures were strictly performed in accor- 122 dance with the National Institutes of Health (NIH) Guidelines. 123
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behavioral syndrome are characterized with decreased appetite, weight loss and psychomotor retardation (Yirmiya et al., 1999). Interestingly, IL-1R-KO mice display no depressive-like symptoms (e.g., reduced sucrose preference) when exposed to unpredictable chronic stress (Goshen et al., 2008). In addition, external stressors increase IL-6 in the hippocampus, and a lack of IL-6 confers resistance to depressivelike behavior. Indeed, marked neurodegeneration and psychological distress are related to chronic IL-6 overexpression in mice (Allan and Rothwell, 2003). TNF-α is a protein that can be produced by several cell types, including lymphocytes, macrophages and glial cells in response to infection, injury or other environmental stress. Furthermore, administration of TNF-α can increase the immobility time in the TST. Similarly, treatment with a TNF-α inhibitor can also prevent an increase in the immobility time in the FST (Kaster et al., 2012). Thus, PIC may be used as a potential functional biomarker in depression. The central inflammatory cytokine network not only produces cytokines and expresses cytokine receptors but also amplifies cytokine signals, which have profound effects on neurotransmitters (Haroon et al., 2012). Specifically, overexpression of PIC in the CNS decreases serotonin production and enhances the signaling of the kynurenine pathway. Cytokines can affect the synthesis, release, and reuptake of mood relevant neurotransmitters by mediating indoleamine 2,3 dioxygenase (IDO) and mitogen-activated protein kinase (MAPK) (Miller, 2009; Andrew et al., 2009). In the present study, we focused on the expression of 5HT and NE, which directly affected inflammation and the neurotransmitters levels. As an important oxygenic terpenoid, MTN (Fig. 1), is widely used as fragrances and flavors in the cosmetic, perfume, drug, and food industries (Gong et al., 2010). Natural MTN is widespread in many plants, particularly in pennyroyal, peppermint and geranium (Hadian et al., 2008). In addition, it is a major component of the volatile oil of schizonepeta and Chaihu-jia-longgu-muli-tang (CLM). Several studies have reported that MTN can freely cross the blood–brain barrier and has been used as a cooling agent, a counterirritant for pain relief, and in the treatment of pruritus (Eccles, 1994; Hong and Shellock, 1991; Yosipovitch et al., 1996); however, its antidepressant-like effects have not yet been reported. In the present study, we evaluated the antidepressant-like effects of MTN using the UCMS mouse model of depression and explored its potential mechanisms.
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Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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Fig. 2. Schematic illustration of the experimental procedure.
2.6. Forced swimming test (FST)
2.10. Determination of NE and 5-HT neurotransmitter levels
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The determination of the 5-HT and NE levels in the hippocampus of 215 the experimental animals was performed using HPLC coupled with MS. 216
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The FST was performed as previously described (Porsolt et al., 1977), with slight modifications. Briefly, the mice were individually placed in a glass cylinder (20 cm in height, 14 cm in diameter) where water (25 ± 2 °C) was filled to 10-cm. The immobility time was defined as the amount of time spent by the mice floating in the water without struggling and making only movements that were necessary to keep their heads above the water. Each mouse was forced to swim for 6 min, and the total duration of immobility was recorded for the last 4 min.
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The TST was performed as previously described (Steru et al., 1985). Each mouse was hung 50-cm above the floor by a piece of adhesive tape placed approximately 1-cm from the tip of the tail. During the 6min test, we recorded the immobility time for the last 4 min. Mice were considered to be immobile only when they were passively suspended and remained completely motionless.
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Mice were anesthetized with 10% chloral hydrate (350 mg/kg, intraperitoneally) and then sacrificed by terminal anesthesia. After decapitation, their brains were dissected out from the skull and placed on ice packs. Hippocampi from both hemispheres were quickly and carefully removed using curved tweezers. The hippocampi were collected in cryopreservation tubes and frozen in liquid nitrogen, followed by storage at −80 °C until further assay.
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2.9. ELISA detection of the IL-1β, IL-6 and TNF-α concentrations
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Mouse hippocampus samples were homogenized in 0.9% normal saline (w:v 1:9) and then centrifuged (4 °C) at 3500 rpm for 10 min. Dilutions of protein standards and samples were added to 96-well ELISA plates, followed by the addition of a biotin-conjugated anti-cytokine antibody and avidin–horseradish peroxidase (HRP) complex according to the manufacturer's instructions. A color reaction was achieved with the addition of a substrate solution and was terminated using the stopping solution. The concentrations of IL-1β, IL-6 and TNF-α were quantified according to the optical density, as detected using a microplate reader (450 nm). A standard curve was constructed, and the experimental results (shown as picograms per milliliter) were analyzed from within the curve.
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2.10.2. Mobile phase The mobile phase consisted of A (0.1% formic acid, v/v) and B (acetonitrile). The flow rate was 0.4 ml/min, and the column temperature was maintained at 30 °C. Nitrogen was used as the desolvation gas (600 l/h) and cone gas (120 l/h).
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2.11. Western blotting analysis
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To determine the levels of NLRP3, pro-caspase-1 and caspase-1, each hippocampus was homogenized in RIPA buffer (w:v 1:5) and lysed for 30 min. The supernatant was collected after centrifugation at 12,000 rpm for 30 min. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Beyotime Institute of Biotechnology Co., Ltd.) and then heated for 10 min. After resolving the proteins by SDS-PAGE, the samples were transferred onto 0.45-μm polyvinylidene difluoride (PVDF) membranes. The membranes were then treated with blocking buffer for 2 h and incubated with NLRP3 (1:1000, Cell Signaling Technology), β-actin (1:1000, Cell Signaling Technology), pro-caspase-1 (1:1000, Abcam) and caspase-1 (1:1000, Abcam) primary antibodies for 12 h at 4 °C. The PVDF membranes were then washed with Tris-buffered saline Tween-20 (TBST) (3 × 10 min) and incubated with secondary antibodies (1:5000, Cell Signaling Technology) for 2 h at room temperature. The membranes were developed using an enhanced chemiluminescence (ECL) detection system (Dizhao, Nanjing, China) and visualized on a Molecular Imager® ChemiDoc TM XRS+ Imaging System. The protein levels were quantified by densitometry using Image-Pro Plus (IPP 6.0) software.
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2.10.1. Equipment Waters ACQUITY™ triple-quadrupole tandem mass spectrometer (Waters Corp., Milford, MA, USA), Waters ACQUITY UPLC system (Waters, Milford, MA, USA), and ACQUITY UPLC™ BEH C18 (2.1 × 100 mm I.D., 1.7 μm, Waters, Milford, USA) column were used in this study.
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The data in the figures are presented as the mean ± standard error of 249 the mean (S.E.M.). Statistical evaluations of the differences among the 250
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
3.1. MTN improves depression-related behaviors in the UCMS mouse model
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All data were recorded by a trained observer who was blind to the experimental group. Mice were deprived of water and food 1 h after the last treatment (drug administration and UCMS), and the SPT was performed 24 h later, followed by the OFT, FST and TST.
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3.3. Effects of MTN on IL-1β, IL-6 and TNF-α production in the hippocampus 302 The concentration of the hippocampus cytokines IL-1β, IL-6 and TNF-α was determined using ELISA. As shown in Fig. 5, the levels of IL-1β [F (4, 25) = 8.887, P b 0.001], IL-6 [F (4, 25) = 4.238, P b 0.01] and TNF-α [F (4, 25) = 6.179, P b 0.01] in the hippocampus were markedly increased in UCMS-treated mice compared with the control group.
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3.1.2. Effects of MTN on locomotor activity in the OFT Spontaneous locomotor activities were measured in the open field test. As shown in Fig. 3B, mice exposed to stressors showed a significant decrease in the number of crossings [F (4, 45) = 4.217, P b 0.01] and rearings [F (4, 45) = 4.033, P b 0.01], while no significant difference was found in the number of groomings [F (4, 45) = 0.935, P = 0.452]. We observed that mice treated with FLU (10 mg/kg) or MTN (15, 30 mg/kg) demonstrated a reversal of the number of crossings
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As shown in Fig. 4A and B, UCMS significantly decreased the levels of 5-HT [F (4, 25) = 6.482, P b 0.01] and NE [F (4, 25) = 5.915, p b 0.01] in the hippocampus versus the control group. Furthermore, FLU (10 mg/kg) or MTN treatments (15, 30 mg/kg) resulted in a significant elevation in the 5-HT (P b 0.05, P b 0.05, P b 0.05, respectively) and NE concentrations (P b 0.01, P b 0.05, P b 0.01, respectively) in the hippocampus.
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3.2. Effects of MTN on neurotransmitters levels in the hippocampus
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3.1.1. Effects of MTN on sucrose consumption in the SPT Anhedonia is a core symptom of depression. The reduced consumption of sucrose solution is a well-established measure of anhedonia. The SPT is shown in Fig. 3A. At the beginning of the test, there were no significant differences among the five groups. After 3 weeks of the experiment, the UCMS procedure significantly decreased the sucrose preference of mice compared with non-UCMS mice [F (4, 45) = 8.450, P b 0.001]. Long-term treatment with FLU (10 mg/kg) and MTN (15, 30 mg/kg) for 3 weeks significantly ameliorated the UCMS procedureinduced sucrose preference decreases at the end of the 6-week UCMS treatment (Fig. 3A, P b 0.001, P b 0.001, P b 0.001, respectively).
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3.1.3. Effects of MTN on immobility time in the TST and FST Fig. 3C [F (4, 45) = 5.544, P b 0.01] showed the effects of MTN on the immobility time in the TST. Mice exposed to UCMS exhibited a significant increase in the immobility time compared with the control group (P b 0.001). FLU (10 mg/kg), and MTN (15, 30 mg/kg) markedly ameliorated the helpless behavior exhibited by the mice versus the UCMS treatment group (P b 0.01, P b 0.05, P b 0.01, respectively). As indicated in Fig. 3D, the immobility time in the FST was recorded to measure the depressive-like behavior. Mice in the UCMS group displayed a significant increase in immobility time during the FST [F (4, 45) = 8.099, P b 0.001]. Furthermore, treatment with FLU (10 mg/kg) (P b 0.01) or MTN (15, 30 mg/kg) (P b 0.01, P b 0.01, respectively) attenuated these behavioral changes.
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(P b 0.05, P b 0.05, P b 0.05, respectively) and rearings (P b 0.01, 279 P b 0.05, P b 0.05, respectively) induced by UCMS. 280
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groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's test. A P-value less than 0.05 was considered statistically significant.
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Fig. 3. Behavioral changes of UCMS mice and treatments. (A) The sucrose preference test was performed every week during the UCMS procedure. (B) The open field test (the number of rearings, squares crossed and groomings) was evaluated after the UCMS procedure. (C) The TST was performed after the UCMS procedure. (D) The FST was performed after the UCMS procedure. N = 10, values represent the mean ± S.E.M. ##P b 0.01, ###P b 0.001 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs. UCMS group.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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3.4. UCMS-induced activation of the NLRP3 inflammasome was reversed by chronic MTN treatment
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For the first time, the present study demonstrated that MTN exerted antidepressant-like effects in the UCMS mouse model of depression. Stress is an important risk factor in depression, and UCMS has been widely adopted by researchers as a standardized behavioral model to induce a depressive-like state for evaluating drug activity evaluation and is performed as a consecutive exposure of a series of various types of unpredictable mild stressors in animals to simulate a series of life stress events (Willner, 1997; Lin et al., 2014). Indeed, the UCMS-
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As shown in Fig. 6, compared with the control group, UCMS significantly increased the levels of NLRP3 [F (4, 10) = 6.572, P b 0.01] and caspase-1 [F (4, 10) = 17.975, P b 0.001] in the hippocampus. Our results showed that FLU (10 mg/kg) (P b 0.05) and MTN (15, 30 mg/kg) (P b 0.05, P b 0.01, respectively) dramatically decreased the levels of NLRP3 compared with the UCMS group. The hippocampal caspase-1 levels were also decreased after treatment with FLU (10 mg/kg) (P b 0.01) and MTN (15, 30 mg/kg) (P b 0.01, P b 0.01, respectively). However, there was no significant difference in the pro-caspase-1 levels [F (4, 10) = 0.265, P = 0.894] among the five groups.
induced depressive animal model has good validity and reliable predictability in screening new antidepressant drugs using a series of behavioral tests (Willner, 1997). We selected the sucrose preference test, a core symptom of depression, to represent the behavioral change. Mice demonstrated decreased sucrose intake when exposed to UCMS, while FLU and MTN treatment reversed the reduction in sucrose consumption in the model group. In addition, to assess the effects of MTN on locomotor activity and exploratory behaviors, we recorded the number of crossings, rearings and groomings. Mice in the UCMS group exhibited a decreased number of crossing and rearing times compared with the control group in the OFT. Long-term administration of FLU and MTN could also reverse these alterations. Taken together, these data provide insight into the potential of MTN in therapeutic implications for depression. Anhedonia-like behavior is associated with the latency to float and the duration of immobility in the FST (Robert and Franco, 2006). In our study, the immobility time during the FST and TST was recorded as behavioral alterations. The FST, a behavioral despair test with good reliability and predictive validity, has been widely used to screen new antidepressant drugs (Petit-Demouliere et al., 2005). Previous studies have demonstrated that chronic stress dramatically increases the immobility time in the FST in each mouse (Zhou et al., 2007; Mao et al., 2009). The TST is also widely used to assess the antidepressant-like properties of new drugs and represents a failure of persistence in the escape-directed behavior (Cryan et al., 2005). Consistent with the previous literature, we showed that UCMS increased the immobility time in the FST and TST. Moreover, FLU treatments have a lower immobility time in contrast to the model group. In addition, 3-week MTN treatments counteracted the UCMS-induced elevation in immobility time.
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However, treatment with FLU (10 mg/kg) (P b 0.001, P b 0.01, P b 0.05, respectively), MTN (15 mg/kg) (P b 0.01, P b 0.01,P b 0.05, respectively) and MTN (30 mg/kg) (P b 0.001, P b 0.01, P b 0.05, respectively) markedly inhibited UCMS-induced increases in IL-1β, IL-6 and TNF-α levels.
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Fig. 4. Effects of MTN treatment on 5-HT (A) and NE (B) levels in the hippocampus of UCMS mice. N = 6. Values represent the mean ± S.E.M. ##P b 0.01, ###P b 0.001 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. UCMS group.
Fig. 5. (A) Effects of MTN on the IL-1β levels in the hippocampus of UCMS mice. (B) Effects of MTN on the IL-6 levels in the hippocampus of UCMS mice. (C) Effects of MTN on the TNF-α levels in the hippocampus of UCMS mice. N = 6. Values represent the mean ± S.E.M. ##P b 0.01, ###P b 0.001 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs. UCMS group.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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We also observed behavioral changes from different aspects using the SPT (i.e., anhedonia-like behavioral test), OFT (i.e., locomotor/exploratory behavioral test), FST and TST (i.e., desperate behavioral test). On the basis of these results, we can conclude that MTN confers antidepressant-like effects in the UCMS model of depression. The NLRP3 inflammasome has been implicated in a series of diseases, including sepsis, gout, diabetes mellitus and atherosclerosis (Strowing et al., 2012). In addition, the inflammasome is a novel target in depression, and inhibition of cytokine production can result in a reduction of inflammation-induced depressive-like behavior. Moreover, psychological and physical stressors can contribute to the development of depression via the inflammasome (Iwata et al., 2013). Formation of the NLRP3 inflammasome can convert precursor caspase-1 into cleaved caspase-1. Cleaved caspase-1 can then transform IL-1β into biologically active mature pro-inflammatory cytokines and release them into the extracellular environment (Martinon et al., 2002). The findings obtained from preclinical and clinical studies suggest that inflammation stimulation induces depressive-like symptoms, thereby supporting the view that inflammatory cytokines are causally involved in depression (Evans et al., 2012). The hippocampus plays a major role in emotional behavior and neuroendocrine regulations. We showed that the IL-1β, IL-6 and TNF-α levels in the hippocampus were markedly increased after the UCMS procedure, while treatment with MTN reduced depressive-like symptoms and effectively attenuated the elevation of UCMS-induced central IL-1β, IL-6 and TNF-α, indicating that the antidepressant-like activities of MTN might be related to its anti-inflammatory effects. The NLRP3 inflammasome has emerged as an unexpected sensor for the pathogenesis of auto-inflammation, metabolic danger and stress. It has been reported that FLU could significantly reduce the levels of NLRP3 and caspase-1 in CUMS rats (Pan et al., 2014). Consistent with a previous study, our results showed that FLU and MTN significantly decreased the levels of NLRP3 and caspase-1 compared with the UCMS group. Thus, our present study illuminated the antidepressant-like mechanism
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Fig. 6. (A) Western blotting analyses of NLRP3, pro-caspase-1, caspase-1 protein levels in the hippocampus of UCMS-induced mice. (B, C and D) Quantification of NLRP3, pro-caspase-1, caspase-1 protein bands. N = 3. Values represent the mean ± S.E.M. #P b 0.05, ##P b 0.01 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. UCMS group.
of MTN on regulation of the NLRP3 inflammasome and associated inflammatory processes. In addition, we further demonstrated that MTN altered the levels of central neurotransmitters. It is well known that the metabolism of 5-HT and NE plays an important role in the modulation of depression. It has been reported that the 5-HT pathway in the hippocampus increases resistance or tolerance to stress, and modification of 5-HT neurotransmission interferes with cognitive function (Frederico et al., 1996). A body of evidence has shown that monoamine neurotransmitters are decreased by UCMS and are reversed by antidepressants (Brkris et al., 2005; Savegnago et al., 2007; Yi et al., 2008). Indeed, most treatments for depression act by increasing the levels of monoamine neurotransmitters (O' Leary et al., 2007; Southwick et al., 2005). Monoamine neurotransmitters (5-HT and NE) in the central nervous system (CNS) have been reported as the main biochemical cause of depression (Savegnago et al., 2007; Wong et al., 1995). Furthermore, alterations in the metabolism of 5-HT and NE have been associated with potent effects of inflammatory cytokines on pathways involved in the pathophysiology of depression (Dunn et al., 1999; Capuron and Miller, 2011). IDO activated by pro-inflammatory cytokines break down tryptophan into kynurenine rather than serotonin, which has been proposed to contribute to decreased serotonin and depressive-like behavior (Schwarcz and Pellicciari, 2002). In the present study, we found that MTN administration could effectively reverse the alterations in the concentrations of 5-HT and NE in UCMS-induced depressive mice. These data provide support to the hypothesis that the antidepressant-like effects of MTN are partly mediated by an alteration of monoaminergic responses. According to the cytokine hypothesis of depression (Maes, 2008), we can infer that the decrease in 5-HT and NE levels in the hippocampus are caused by an activation of the NLRP3 inflammasome, which promote inflammatory processes, and the anti-inflammatory effects of MTN might have a direct effect on neurotransmitter metabolism, resulting in antidepressant-like effects.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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In conclusion, we report, for the first time, that MTN confers antidepressant-like effects in the UCMS mouse model. Behavioral changes in the SPT, OFT, FST and TST were ameliorated by MTN treatment. In addition, the present results are the first piece of evidence that MTN can modulate UCMS-induced NLRP3 inflammasome levels, with a corresponding downregulation of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Thus, MTN may inhibit the activities and expression of pro-inflammatory cytokines and may restore the 5-HT and NE levels. Taken together, our findings may provide further insight into the potential of MTN as a therapeutic treatment for depression.
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This work was supported by grants obtained from the Priority
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Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
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Jinsong Xue, Hongyan Li, Xueyang Deng, Zhanqiang Ma, Qiang Fu ⁎, Shiping Ma ⁎⁎
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Article history: Received 7 November 2014 Received in revised form 14 April 2015 Accepted 17 April 2015 Available online xxxx
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Keywords: L-Menthone Antidepressant Unpredictable chronic mild stress (UCMS) NLRP3 inflammasome Cytokines Neurotransmitters
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(MTN) is a Chinese old remedy extracted from the genus Mentha. It has been widely used as a cooling agent and a counterirritant for pain relief, although its antidepressant-like effects have not yet been reported. The present study was designed to investigate whether MTN confers an antidepressant-like effect in mice exposed to unpredictable chronic mild stress (UCMS) and to explore its potential mechanisms. The effects of MTN on mouse behavioral changes were investigated in our study. We determined the levels of the nucleotide binding, oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, inflammatory cytokines and neurotransmitters in the hippocampus of mice. Behavioral tests, including the sucrose preference test (SPT), open field test (OFT), forced swimming test (FST) and tail suspension test (TST) revealed that MTN (15 and 30 mg/kg) treatments for 3 weeks alleviated the depression symptoms of UCMS in mice. Mice receiving MTN treatments exhibited reduced levels of NLRP3 and caspase-1. Moreover, MTN treatments reversed the UCMS-induced alterations in the concentrations of neurotransmitter norepinephrine (NE) and serotonin (5-HT) and inhibited the expression of pro-inflammatory cytokines (PIC) interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the hippocampus of mice. Taken together, our findings suggested that MTN may play a potential antidepressant-like role in the UCMS mouse model by regulating the NLRP3 inflammasome and mediating inflammatory cytokines and central neurotransmitters, which together provide insight towards the development of novel therapeutic treatments for depression. © 2015 Published by Elsevier Inc.
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Depression is a chronic, recurring, and life-threatening mental disorder in which the underlying mechanisms remain unclear, and the effective cures are scarce (Berton and Nestler, 2006). Moreover, most of the current antidepressants used in the clinic not only have a low curative ratio, but they also may cause side effects (Zhou et al., 2011). Thus, the identification of safer and more reliable antidepressants is necessary. There are many hypotheses involved in the etiopathogenesis of depression, including the monoamine deficiency hypothesis, hyperactive HPA axis hypothesis, cytokine hypothesis, neurodegeneration hypothesis, and other hypotheses. Among these hypotheses, the emerging cytokine hypothesis has attracted interest because it can orchestrate complex responses and coordinate responses with those of other physiological
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⁎ Correspondence to: Q. Fu, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, PR China. Tel.: +86 25 3271507. ⁎⁎ Correspondence to: S.-P. Ma, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, PR China. Tel.: +86 25 3271507; fax: +86 25 3271505. E-mail addresses: [email protected] (Q. Fu), [email protected] (S. Ma).
systems in the body (Dunn et al., 2005). In the present study, we mainly focused on the modulation of the NLRP3 inflammasome on cytokines and monoamine neurotransmitters. The NLRP3 inflammasome, a new target in depression, belongs to the NLR family and consists of NLRP3, the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1, a biomarker used to evaluate the effectiveness and resistance to antidepressants (Gomez and Cordero, 2014; Schroder and Tschopp, 2010). The NLRP3 inflammasome consists of intracellular pattern-recognition molecules detecting microbial- and dangerassociated molecular patterns and has been implicated in major diseases, such as ischemic stroke, obesity and type 2 diabetes (Cassel et al., 2009; Fann et al., 2013; Shan et al., 2014; Luo et al., 2014). Importantly, microglia are a primary source of PIC in the brain and the resident macrophage in the central nervous system (CNS). Once activated, NLRP3 oligomerizes in microglia and recruits pro-caspase-1, resulting in the activation of caspase-1 and the production of IL-1β. IL1β then activates downstream pathways, such as NF-κB, and promotes the expression of IL-6 and TNF-α, which play an important role in the pathophysiology of depression (Maes et al., 2012). It has been reported that chronic stress can increase the production of IL-1β and TNF-α in the hippocampus (Munhoz et al., 2006). The effects of IL-1β on the
http://dx.doi.org/10.1016/j.pbb.2015.04.014 0091-3057/© 2015 Published by Elsevier Inc.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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Male ICR mice (4 weeks, weighing 18–22 g) were purchased from the Experimental Animal Center in Jiangsu Province (Nanjing, PR China). Mice were randomly housed in cages for one week to acclimatize to their environment prior to their use in experiments. The mice had free access to food and water and were housed in a room maintained at 25 ± 1 °C with a 12/12 hour light/dark cycle. All of the animal
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Fluoxetine hydrochloride was purchased from the Changzhou Siyao Pharmaceuticals Co., Ltd (Changzhou, PR China). MTN (97%) was purchased from the Shanghai Jianglai Co., Ltd. (Shanghai, PR China). All drugs were dissolved in 0.3% sodium carboxy methyl cellulose (CMC)Na. ELISA kits (R&D, Minneapolis, MN, USA), and a bicinchoninic acid assay (BCA) kit (Beyotime Institute of Biotechnology Co., Ltd) was used in this study.
Fig. 1. Structure of L-menthone.
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The mice were randomly assigned to five groups (n = 10 for each group): control group (0.3% CMC-Na), UCMS (0.3% CMC-Na) group, UCMS + fluoxetine (FLU) (10 mg/kg) group, UCMS + MTN (15 mg/kg) group, and UCMS + MTN (30 mg/kg) group. All of the drugs were orally administered in a volume of 10 ml/kg body weight. To examine whether MTN has potential antidepressant-like effect, the UCMS model was used to induce depressive-like behavior in mice. The mice were first exposed to the UCMS procedure for 3 weeks, followed by 3 weeks of MTN or FLU, treatment during which UCMS exposure was maintained (Fig. 2). The UCMS procedure was performed as previously described (Willner et al., 1987), with minor modifications. Briefly, the mice were allowed to acclimate one week before they were subjected to the following stressors for six weeks (42 days): (1) water deprivation (24 h), (2) food deprivation (24 h), (3) overnight illumination, (4) cage tilt (45°), (5) soiled cage (200 ml of water in 100 g of sawdust bedding), (6) exposure to a foreign object, (7) light/dark perversion, (8) overhang (10 min), (9) exposure to an empty bottle, (10) 1-min tail pinch (1 cm from the beginning of the tail), (11) 5-min oscillation, and (12) white noise. All of the stressors were applied at any time of the day and night to ensure the unpredictable nature of the experiment.
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The sucrose preference test was monitored weekly throughout the experiment. Mice were deprived of water and food for 24 h, and the mice were then given the choice to drink from two bottles for 12 h: one was filled with a sucrose solution (1% w/v), and the other was filled with water. To avoid potential side-preference effects on drinking behaviors, the positions of the bottles in the cage were switched after 6 h. The consumption of water and the sucrose solution and the total intake of liquids were estimated by weighing the bottles. The preference for sucrose was measured as the percentage of consumed sucrose solution relative to the total amount of liquid intake.
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To assess the MTN treatment effects on spontaneous locomotor activities, mice were subjected to the open-field paradigm as previously described (Rodrigues et al., 2002). The apparatus (40 × 60 × 50 cm) was divided into 12 equal squares and the number of crossings was recorded. The test began by placing the mouse in the center of the device so that it was acclimatized with its environment for 2 min. The number of crossings (the number of squares crossed), rearings (standing on its hind legs) and groomings (licking, cleaning and scratching its face with the forepaws) was recorded for 4 min. The apparatus was then cleaned with a 10% alcohol solution and dried after occupancy by each mouse.
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behavioral syndrome are characterized with decreased appetite, weight loss and psychomotor retardation (Yirmiya et al., 1999). Interestingly, IL-1R-KO mice display no depressive-like symptoms (e.g., reduced sucrose preference) when exposed to unpredictable chronic stress (Goshen et al., 2008). In addition, external stressors increase IL-6 in the hippocampus, and a lack of IL-6 confers resistance to depressivelike behavior. Indeed, marked neurodegeneration and psychological distress are related to chronic IL-6 overexpression in mice (Allan and Rothwell, 2003). TNF-α is a protein that can be produced by several cell types, including lymphocytes, macrophages and glial cells in response to infection, injury or other environmental stress. Furthermore, administration of TNF-α can increase the immobility time in the TST. Similarly, treatment with a TNF-α inhibitor can also prevent an increase in the immobility time in the FST (Kaster et al., 2012). Thus, PIC may be used as a potential functional biomarker in depression. The central inflammatory cytokine network not only produces cytokines and expresses cytokine receptors but also amplifies cytokine signals, which have profound effects on neurotransmitters (Haroon et al., 2012). Specifically, overexpression of PIC in the CNS decreases serotonin production and enhances the signaling of the kynurenine pathway. Cytokines can affect the synthesis, release, and reuptake of mood relevant neurotransmitters by mediating indoleamine 2,3 dioxygenase (IDO) and mitogen-activated protein kinase (MAPK) (Miller, 2009; Andrew et al., 2009). In the present study, we focused on the expression of 5HT and NE, which directly affected inflammation and the neurotransmitters levels. As an important oxygenic terpenoid, MTN (Fig. 1), is widely used as fragrances and flavors in the cosmetic, perfume, drug, and food industries (Gong et al., 2010). Natural MTN is widespread in many plants, particularly in pennyroyal, peppermint and geranium (Hadian et al., 2008). In addition, it is a major component of the volatile oil of schizonepeta and Chaihu-jia-longgu-muli-tang (CLM). Several studies have reported that MTN can freely cross the blood–brain barrier and has been used as a cooling agent, a counterirritant for pain relief, and in the treatment of pruritus (Eccles, 1994; Hong and Shellock, 1991; Yosipovitch et al., 1996); however, its antidepressant-like effects have not yet been reported. In the present study, we evaluated the antidepressant-like effects of MTN using the UCMS mouse model of depression and explored its potential mechanisms.
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Fig. 2. Schematic illustration of the experimental procedure.
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The determination of the 5-HT and NE levels in the hippocampus of 215 the experimental animals was performed using HPLC coupled with MS. 216
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The FST was performed as previously described (Porsolt et al., 1977), with slight modifications. Briefly, the mice were individually placed in a glass cylinder (20 cm in height, 14 cm in diameter) where water (25 ± 2 °C) was filled to 10-cm. The immobility time was defined as the amount of time spent by the mice floating in the water without struggling and making only movements that were necessary to keep their heads above the water. Each mouse was forced to swim for 6 min, and the total duration of immobility was recorded for the last 4 min.
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The TST was performed as previously described (Steru et al., 1985). Each mouse was hung 50-cm above the floor by a piece of adhesive tape placed approximately 1-cm from the tip of the tail. During the 6min test, we recorded the immobility time for the last 4 min. Mice were considered to be immobile only when they were passively suspended and remained completely motionless.
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Mice were anesthetized with 10% chloral hydrate (350 mg/kg, intraperitoneally) and then sacrificed by terminal anesthesia. After decapitation, their brains were dissected out from the skull and placed on ice packs. Hippocampi from both hemispheres were quickly and carefully removed using curved tweezers. The hippocampi were collected in cryopreservation tubes and frozen in liquid nitrogen, followed by storage at −80 °C until further assay.
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Mouse hippocampus samples were homogenized in 0.9% normal saline (w:v 1:9) and then centrifuged (4 °C) at 3500 rpm for 10 min. Dilutions of protein standards and samples were added to 96-well ELISA plates, followed by the addition of a biotin-conjugated anti-cytokine antibody and avidin–horseradish peroxidase (HRP) complex according to the manufacturer's instructions. A color reaction was achieved with the addition of a substrate solution and was terminated using the stopping solution. The concentrations of IL-1β, IL-6 and TNF-α were quantified according to the optical density, as detected using a microplate reader (450 nm). A standard curve was constructed, and the experimental results (shown as picograms per milliliter) were analyzed from within the curve.
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2.10.2. Mobile phase The mobile phase consisted of A (0.1% formic acid, v/v) and B (acetonitrile). The flow rate was 0.4 ml/min, and the column temperature was maintained at 30 °C. Nitrogen was used as the desolvation gas (600 l/h) and cone gas (120 l/h).
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To determine the levels of NLRP3, pro-caspase-1 and caspase-1, each hippocampus was homogenized in RIPA buffer (w:v 1:5) and lysed for 30 min. The supernatant was collected after centrifugation at 12,000 rpm for 30 min. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Beyotime Institute of Biotechnology Co., Ltd.) and then heated for 10 min. After resolving the proteins by SDS-PAGE, the samples were transferred onto 0.45-μm polyvinylidene difluoride (PVDF) membranes. The membranes were then treated with blocking buffer for 2 h and incubated with NLRP3 (1:1000, Cell Signaling Technology), β-actin (1:1000, Cell Signaling Technology), pro-caspase-1 (1:1000, Abcam) and caspase-1 (1:1000, Abcam) primary antibodies for 12 h at 4 °C. The PVDF membranes were then washed with Tris-buffered saline Tween-20 (TBST) (3 × 10 min) and incubated with secondary antibodies (1:5000, Cell Signaling Technology) for 2 h at room temperature. The membranes were developed using an enhanced chemiluminescence (ECL) detection system (Dizhao, Nanjing, China) and visualized on a Molecular Imager® ChemiDoc TM XRS+ Imaging System. The protein levels were quantified by densitometry using Image-Pro Plus (IPP 6.0) software.
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2.10.1. Equipment Waters ACQUITY™ triple-quadrupole tandem mass spectrometer (Waters Corp., Milford, MA, USA), Waters ACQUITY UPLC system (Waters, Milford, MA, USA), and ACQUITY UPLC™ BEH C18 (2.1 × 100 mm I.D., 1.7 μm, Waters, Milford, USA) column were used in this study.
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The data in the figures are presented as the mean ± standard error of 249 the mean (S.E.M.). Statistical evaluations of the differences among the 250
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
3.1. MTN improves depression-related behaviors in the UCMS mouse model
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All data were recorded by a trained observer who was blind to the experimental group. Mice were deprived of water and food 1 h after the last treatment (drug administration and UCMS), and the SPT was performed 24 h later, followed by the OFT, FST and TST.
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3.3. Effects of MTN on IL-1β, IL-6 and TNF-α production in the hippocampus 302 The concentration of the hippocampus cytokines IL-1β, IL-6 and TNF-α was determined using ELISA. As shown in Fig. 5, the levels of IL-1β [F (4, 25) = 8.887, P b 0.001], IL-6 [F (4, 25) = 4.238, P b 0.01] and TNF-α [F (4, 25) = 6.179, P b 0.01] in the hippocampus were markedly increased in UCMS-treated mice compared with the control group.
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3.1.2. Effects of MTN on locomotor activity in the OFT Spontaneous locomotor activities were measured in the open field test. As shown in Fig. 3B, mice exposed to stressors showed a significant decrease in the number of crossings [F (4, 45) = 4.217, P b 0.01] and rearings [F (4, 45) = 4.033, P b 0.01], while no significant difference was found in the number of groomings [F (4, 45) = 0.935, P = 0.452]. We observed that mice treated with FLU (10 mg/kg) or MTN (15, 30 mg/kg) demonstrated a reversal of the number of crossings
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As shown in Fig. 4A and B, UCMS significantly decreased the levels of 5-HT [F (4, 25) = 6.482, P b 0.01] and NE [F (4, 25) = 5.915, p b 0.01] in the hippocampus versus the control group. Furthermore, FLU (10 mg/kg) or MTN treatments (15, 30 mg/kg) resulted in a significant elevation in the 5-HT (P b 0.05, P b 0.05, P b 0.05, respectively) and NE concentrations (P b 0.01, P b 0.05, P b 0.01, respectively) in the hippocampus.
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3.1.1. Effects of MTN on sucrose consumption in the SPT Anhedonia is a core symptom of depression. The reduced consumption of sucrose solution is a well-established measure of anhedonia. The SPT is shown in Fig. 3A. At the beginning of the test, there were no significant differences among the five groups. After 3 weeks of the experiment, the UCMS procedure significantly decreased the sucrose preference of mice compared with non-UCMS mice [F (4, 45) = 8.450, P b 0.001]. Long-term treatment with FLU (10 mg/kg) and MTN (15, 30 mg/kg) for 3 weeks significantly ameliorated the UCMS procedureinduced sucrose preference decreases at the end of the 6-week UCMS treatment (Fig. 3A, P b 0.001, P b 0.001, P b 0.001, respectively).
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3.1.3. Effects of MTN on immobility time in the TST and FST Fig. 3C [F (4, 45) = 5.544, P b 0.01] showed the effects of MTN on the immobility time in the TST. Mice exposed to UCMS exhibited a significant increase in the immobility time compared with the control group (P b 0.001). FLU (10 mg/kg), and MTN (15, 30 mg/kg) markedly ameliorated the helpless behavior exhibited by the mice versus the UCMS treatment group (P b 0.01, P b 0.05, P b 0.01, respectively). As indicated in Fig. 3D, the immobility time in the FST was recorded to measure the depressive-like behavior. Mice in the UCMS group displayed a significant increase in immobility time during the FST [F (4, 45) = 8.099, P b 0.001]. Furthermore, treatment with FLU (10 mg/kg) (P b 0.01) or MTN (15, 30 mg/kg) (P b 0.01, P b 0.01, respectively) attenuated these behavioral changes.
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groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's test. A P-value less than 0.05 was considered statistically significant.
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Fig. 3. Behavioral changes of UCMS mice and treatments. (A) The sucrose preference test was performed every week during the UCMS procedure. (B) The open field test (the number of rearings, squares crossed and groomings) was evaluated after the UCMS procedure. (C) The TST was performed after the UCMS procedure. (D) The FST was performed after the UCMS procedure. N = 10, values represent the mean ± S.E.M. ##P b 0.01, ###P b 0.001 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs. UCMS group.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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3.4. UCMS-induced activation of the NLRP3 inflammasome was reversed by chronic MTN treatment
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For the first time, the present study demonstrated that MTN exerted antidepressant-like effects in the UCMS mouse model of depression. Stress is an important risk factor in depression, and UCMS has been widely adopted by researchers as a standardized behavioral model to induce a depressive-like state for evaluating drug activity evaluation and is performed as a consecutive exposure of a series of various types of unpredictable mild stressors in animals to simulate a series of life stress events (Willner, 1997; Lin et al., 2014). Indeed, the UCMS-
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As shown in Fig. 6, compared with the control group, UCMS significantly increased the levels of NLRP3 [F (4, 10) = 6.572, P b 0.01] and caspase-1 [F (4, 10) = 17.975, P b 0.001] in the hippocampus. Our results showed that FLU (10 mg/kg) (P b 0.05) and MTN (15, 30 mg/kg) (P b 0.05, P b 0.01, respectively) dramatically decreased the levels of NLRP3 compared with the UCMS group. The hippocampal caspase-1 levels were also decreased after treatment with FLU (10 mg/kg) (P b 0.01) and MTN (15, 30 mg/kg) (P b 0.01, P b 0.01, respectively). However, there was no significant difference in the pro-caspase-1 levels [F (4, 10) = 0.265, P = 0.894] among the five groups.
induced depressive animal model has good validity and reliable predictability in screening new antidepressant drugs using a series of behavioral tests (Willner, 1997). We selected the sucrose preference test, a core symptom of depression, to represent the behavioral change. Mice demonstrated decreased sucrose intake when exposed to UCMS, while FLU and MTN treatment reversed the reduction in sucrose consumption in the model group. In addition, to assess the effects of MTN on locomotor activity and exploratory behaviors, we recorded the number of crossings, rearings and groomings. Mice in the UCMS group exhibited a decreased number of crossing and rearing times compared with the control group in the OFT. Long-term administration of FLU and MTN could also reverse these alterations. Taken together, these data provide insight into the potential of MTN in therapeutic implications for depression. Anhedonia-like behavior is associated with the latency to float and the duration of immobility in the FST (Robert and Franco, 2006). In our study, the immobility time during the FST and TST was recorded as behavioral alterations. The FST, a behavioral despair test with good reliability and predictive validity, has been widely used to screen new antidepressant drugs (Petit-Demouliere et al., 2005). Previous studies have demonstrated that chronic stress dramatically increases the immobility time in the FST in each mouse (Zhou et al., 2007; Mao et al., 2009). The TST is also widely used to assess the antidepressant-like properties of new drugs and represents a failure of persistence in the escape-directed behavior (Cryan et al., 2005). Consistent with the previous literature, we showed that UCMS increased the immobility time in the FST and TST. Moreover, FLU treatments have a lower immobility time in contrast to the model group. In addition, 3-week MTN treatments counteracted the UCMS-induced elevation in immobility time.
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However, treatment with FLU (10 mg/kg) (P b 0.001, P b 0.01, P b 0.05, respectively), MTN (15 mg/kg) (P b 0.01, P b 0.01,P b 0.05, respectively) and MTN (30 mg/kg) (P b 0.001, P b 0.01, P b 0.05, respectively) markedly inhibited UCMS-induced increases in IL-1β, IL-6 and TNF-α levels.
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Fig. 4. Effects of MTN treatment on 5-HT (A) and NE (B) levels in the hippocampus of UCMS mice. N = 6. Values represent the mean ± S.E.M. ##P b 0.01, ###P b 0.001 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. UCMS group.
Fig. 5. (A) Effects of MTN on the IL-1β levels in the hippocampus of UCMS mice. (B) Effects of MTN on the IL-6 levels in the hippocampus of UCMS mice. (C) Effects of MTN on the TNF-α levels in the hippocampus of UCMS mice. N = 6. Values represent the mean ± S.E.M. ##P b 0.01, ###P b 0.001 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs. UCMS group.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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We also observed behavioral changes from different aspects using the SPT (i.e., anhedonia-like behavioral test), OFT (i.e., locomotor/exploratory behavioral test), FST and TST (i.e., desperate behavioral test). On the basis of these results, we can conclude that MTN confers antidepressant-like effects in the UCMS model of depression. The NLRP3 inflammasome has been implicated in a series of diseases, including sepsis, gout, diabetes mellitus and atherosclerosis (Strowing et al., 2012). In addition, the inflammasome is a novel target in depression, and inhibition of cytokine production can result in a reduction of inflammation-induced depressive-like behavior. Moreover, psychological and physical stressors can contribute to the development of depression via the inflammasome (Iwata et al., 2013). Formation of the NLRP3 inflammasome can convert precursor caspase-1 into cleaved caspase-1. Cleaved caspase-1 can then transform IL-1β into biologically active mature pro-inflammatory cytokines and release them into the extracellular environment (Martinon et al., 2002). The findings obtained from preclinical and clinical studies suggest that inflammation stimulation induces depressive-like symptoms, thereby supporting the view that inflammatory cytokines are causally involved in depression (Evans et al., 2012). The hippocampus plays a major role in emotional behavior and neuroendocrine regulations. We showed that the IL-1β, IL-6 and TNF-α levels in the hippocampus were markedly increased after the UCMS procedure, while treatment with MTN reduced depressive-like symptoms and effectively attenuated the elevation of UCMS-induced central IL-1β, IL-6 and TNF-α, indicating that the antidepressant-like activities of MTN might be related to its anti-inflammatory effects. The NLRP3 inflammasome has emerged as an unexpected sensor for the pathogenesis of auto-inflammation, metabolic danger and stress. It has been reported that FLU could significantly reduce the levels of NLRP3 and caspase-1 in CUMS rats (Pan et al., 2014). Consistent with a previous study, our results showed that FLU and MTN significantly decreased the levels of NLRP3 and caspase-1 compared with the UCMS group. Thus, our present study illuminated the antidepressant-like mechanism
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Fig. 6. (A) Western blotting analyses of NLRP3, pro-caspase-1, caspase-1 protein levels in the hippocampus of UCMS-induced mice. (B, C and D) Quantification of NLRP3, pro-caspase-1, caspase-1 protein bands. N = 3. Values represent the mean ± S.E.M. #P b 0.05, ##P b 0.01 vs. control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. UCMS group.
of MTN on regulation of the NLRP3 inflammasome and associated inflammatory processes. In addition, we further demonstrated that MTN altered the levels of central neurotransmitters. It is well known that the metabolism of 5-HT and NE plays an important role in the modulation of depression. It has been reported that the 5-HT pathway in the hippocampus increases resistance or tolerance to stress, and modification of 5-HT neurotransmission interferes with cognitive function (Frederico et al., 1996). A body of evidence has shown that monoamine neurotransmitters are decreased by UCMS and are reversed by antidepressants (Brkris et al., 2005; Savegnago et al., 2007; Yi et al., 2008). Indeed, most treatments for depression act by increasing the levels of monoamine neurotransmitters (O' Leary et al., 2007; Southwick et al., 2005). Monoamine neurotransmitters (5-HT and NE) in the central nervous system (CNS) have been reported as the main biochemical cause of depression (Savegnago et al., 2007; Wong et al., 1995). Furthermore, alterations in the metabolism of 5-HT and NE have been associated with potent effects of inflammatory cytokines on pathways involved in the pathophysiology of depression (Dunn et al., 1999; Capuron and Miller, 2011). IDO activated by pro-inflammatory cytokines break down tryptophan into kynurenine rather than serotonin, which has been proposed to contribute to decreased serotonin and depressive-like behavior (Schwarcz and Pellicciari, 2002). In the present study, we found that MTN administration could effectively reverse the alterations in the concentrations of 5-HT and NE in UCMS-induced depressive mice. These data provide support to the hypothesis that the antidepressant-like effects of MTN are partly mediated by an alteration of monoaminergic responses. According to the cytokine hypothesis of depression (Maes, 2008), we can infer that the decrease in 5-HT and NE levels in the hippocampus are caused by an activation of the NLRP3 inflammasome, which promote inflammatory processes, and the anti-inflammatory effects of MTN might have a direct effect on neurotransmitter metabolism, resulting in antidepressant-like effects.
Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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In conclusion, we report, for the first time, that MTN confers antidepressant-like effects in the UCMS mouse model. Behavioral changes in the SPT, OFT, FST and TST were ameliorated by MTN treatment. In addition, the present results are the first piece of evidence that MTN can modulate UCMS-induced NLRP3 inflammasome levels, with a corresponding downregulation of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Thus, MTN may inhibit the activities and expression of pro-inflammatory cytokines and may restore the 5-HT and NE levels. Taken together, our findings may provide further insight into the potential of MTN as a therapeutic treatment for depression.
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This work was supported by grants obtained from the Priority
444 Academic Program Development of Jiangsu Higher Education 445 Q10 Institutions and the Natural Science Foundation of Jiangsu Province of
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Please cite this article as: Xue J, et al, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflamma..., Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.014
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