Behavioural Brain Research 291 (2015) 12–19
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Thymol produces an antidepressant-like effect in a chronic unpredictable mild stress model of depression in mice Xue-Yang Deng a , Hong-Yan Li a , Jun-Jun Chen a , Rui-Peng Li a , Rong Qu b,∗ , Qiang Fu a,∗∗ , Shi-Ping Ma a,∗ ∗ ∗ a b
Department of Pharmacology of Chinese Materia, China Pharmaceutical University, Nanjing 210009, China Department of Pharmacology of Traditional Chinese Medical Formulae, Nanjing University of Traditional Chinese Medicine, Nanjing 210029, China
h i g h l i g h t s • • • •
Thymol exhibited an antidepressant-like effect. Thymol alleviated the depression-related behaviors in mice. Thymol reversed the decrease of neurotransmitters induced by CUMS. The antidepressant-like properties of thymol are regulated via NLRP3 inflammasome mediated neuro-inflammation.
a r t i c l e
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Article history: Received 1 March 2015 Received in revised form 23 April 2015 Accepted 28 April 2015 Available online 6 May 2015 Keywords: Thymol Antidepressant Neurotransmitter Proinflammatory cytokine NLRP3
a b s t r a c t Thymol, a bioactive monoterpene isolated from Thymus vulgaris, has displayed inspiring neuroprotective properties. The present study was designed to evaluate the antidepressant-like effects of thymol on a chronic unpredictable mild stress (CUMS) model of depression in mice and explore the underlying mechanisms. It was observed that thymol treatment (15 mg/kg and 30 mg/kg) significantly reversed the decrease of sucrose consumption, the loss of body weight, the reduction of immobile time in the tail suspension tests (TST) and forced swimming tests (FST) induced by CUMS paradigm. The levels of norepinephrine (NE) and serotonin (5-HT) in the hippocampus decreased in the CUMS-treated mice. Chronic treatments with thymol significantly restored the CUMS-induced alterations of monoamine neurotransmitters in the hippocampus. Our results further demonstrated that thymol administration negatively regulated the induction of proinflammatory cytokines including interleukin (IL)-1, IL-6, and tumor necrosis factor␣ in CUMS mice. Furthermore, thymol inhibited the activation of nod-like receptor protein 3 (NLRP3) inflammasome and its adaptor, and subsequently decreased the expression of caspase-1. In sum, our findings suggested that thymol played a potential antidepressant role in CUMS mice model through up-regulating the levels of central neurotransmitters and inhibiting the expressions of proinflammatory cytokines, which might provide potential for thymol in the light of opening up new therapeutic avenues for depression. © 2015 Elsevier B.V. All rights reserved.
Abbreviations: CUMS, chronic unpredictable mild stress; FST, forced swimming tests; TST, tail suspension tests; IL-1, interleukin-1 beta; IL-6, interleukin-6; TNF-␣tumor, necrosis factor-alpha; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3; ASC, apoptosis-associated speck like protein; HPA, hypothalamic pituitary adrenal; CORT, corticosterone; FLU, fluoxetine. ∗ Corresponding author. Tel.: +86 25 85811929. ∗∗ Corresponding author at: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, China. Tel.: +86 25 83271507. ∗ ∗ ∗Corresponding author at: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, China. Tel.: +86 25 83271419. E-mail addresses: [email protected] (R. Qu), [email protected] (Q. Fu), [email protected] (S.-P. Ma). http://dx.doi.org/10.1016/j.bbr.2015.04.052 0166-4328/© 2015 Elsevier B.V. All rights reserved.
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1. Introduction Major depressive disorder is a recurring and life threatening illness, from which up to 20% of the world population suffer [1]. The World Health Organization numbered depression as the fourth leading cause of disability worldwide, which brings heavy burden to the society [2]. Although currently available antidepressants might provide 2/3 of these patients with some relief, it is far from ideal. It is still an urgent need for looking for more effective and safe drugs against depression. Inflammation has been recognized as an important mechanism of neuropsychiatric disorders, including major depression [3]. Associations between inflammatory biomarkers and depressive symptoms, such as disturbed sleep, fatigue, and cognitive impairment, have been described by numerous studies [4,5]. Patients suffered from depression exhibited increased inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-8, interferon (IFN)-␥, TNF-␣ [6]. Inflammatory cytokines access the brain and act on neurotransmitter metabolism, neuronal plasticity, and neuroendocrine function. Among these, inflammatory cytokines-induced changes in monoamine neurotransmitters have been reported to be a primary pathway to depression [7]. Inflammatory cytokines could activate brain serotonergic systems, increase brain tryptophan concentrations and have a direct influence on 5-HT and NE metabolism [8]. In addition, inflammatory cytokines have been proposed to be connected with the HPA axis abnormality, which is a potentially causative or exacerbating factor in depression, and directly induces glucocorticoids secretions from the adrenals [9]. Inflammasomes are molecular platforms activated upon detection of stress or other danger signals, among which the NLRP3 inflammasome is the most notably characterized one [10]. The NLRP3 inflammasome complex consists of the NLRP3 pattern recognition receptor, the apoptosis-associated specklike protein (ASC) adaptor, and the effector protein caspase-1 [3]. NLRP3 is activated upon pathogens exposure, cellular injury, or other environmental irritants. Once activated, NLRP3 interacts with ASC adaptor via pyrin domain hemophilic interactions, leading to the activation of caspase-1 [11], which triggers the processing of inflammatory cytokines such as IL-1 to engage the innate immune defenses [12] by cleaving its precursor pro-IL-1. Recently, NLRP3 inflammasome is increasingly suggested to play a major role in systemic diseases, such as cancer, cardiovascular diseases, diabetes and depression in particular [10,13]. Researches show that NLRP3 inflammasome is activated in depressed animal models [14] and in patients with major depression as well [15], which suggests that NLRP3 inflammasome might be a new target and offer new perspectives in the study of depression [16]. Thymol is a main bioactive monoterpene isolated from many medicinal herbs, such as Thymus vulgaris, Monarda punctate and Origanum vulgare spp. [17,18]. Thymol has been widely used as an active anti-inflammatory ingredient, which can inhibit the isoproterenol induced inflammation in myocardial infarcted rats [19], attenuate collagenase-induced osteoarthritis [20], and alleviate allergic airway inflammation in ovalbumin-induced mouse asthma [21]. Moreover, studies have reported that thymol has various bioactivities, such as anticancer [22], anti-bacterial [18], and antioxidant [23] properties. Recent studies focusing on the neuroprotective activities of thymol have shown that it attenuates amyloid  or scopolamine induced cognitive impairment in rats [24]. Thymol treatment has been reported to increase antioxidant status in rat brain [25], and has specific interactions with synaptic neural functions [26]. Further findings have reported that thymol displayed neuroprotective effects, which attributed to its potential action on GABA-mediated inhibition of synaptic transmission [27]. Considering the inspiring beneficial effects of thymol on the brain, the present study was designed to explore its antidepressant-like
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effect in CUMS model in mice, which mimics several depressive syndromes observed in patients with depression, such as body weight loss, decreased responsiveness to rewarding stimuli, and insufficient self-care [28,29]. At the meantime, in order to investigate the possible mechanisms underlying the therapeutic effects of thymol, we also assessed the levels of monoamine neurotransmitters and the expressions of proinflammatory cytokines. 2. Materials and methods 2.1. Animals Male ICR mice (18–20 g) were purchased from the Experimental Animal Center of China Pharmaceutical University (Nanjing, China). Upon arrival, mice were group housed under standard housing conditions (room temperature 25 ± 2 ◦ C; a 12/12 h light/dark cycle). Water and food were provided ad libitum. Animal handling and experimental procedure were performed strictly in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation and approved by the Science and Technology Department of Jiangsu Province. 2.2. Drugs and reagents Fluoxetine (FLU) hydrochloride was purchased from Changzhou Siyao Pharmaceuticals Co., Ltd (Changzhou, China). Thymol (2isopropyl-5-methyl-phenol) was obtained from Sigma Chemical Co. (St Louis, MO, USA). Bicinchoninic acid assay (BCA) kit was produced by Beyotime Institute of Biotechnology Co., Ltd (Nanjing, China). Chloral hydrate was from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Total RNA Extraction Reagent and HiScriptTM Q RT SuperMixfor qPCR (+gDNA wiper) were obtained from Vazyme Biotech Co., Ltd (Nanjing, China). 2.3. CUMS procedure and experimental design Mice were randomly assigned into five groups (n = 12). Group I: Normal control group; Group II: CUMS model group; Group III: CUMS + thymol (15 mg/kg) group; Group IV: CUMS + thymol (30 mg/kg) group; Group V: CUMS + FLU (10 mg/kg) group. Thymol or FLU was administered intragastrically once a day for 3 weeks from the fourth week. Thymol and FLU were prepared by dd water in 0.3% carboxy methyl cellulose as an adjuvant [30]. For the normal control and CUMS model groups, mice were given an equal volume of 0.3% carboxy methyl cellulose. The levels of thymol administration chosen were similar to those used in previous trials, in which thymol protected against inflammation in isoproterenol induced myocardial infarcted rats [19], ovalbumin induced mouse asthma [21], and cisplatin induced nephrotoxic rats [31]. And these doses were considerably lower than the reported value of the median lethal dose (980 mg/kg in rats) [25]. Mice were exposed to the CUMS stimuli for 3 weeks (from 1st to 3rd week), followed by 3 weeks (from 4th to 6th week) of thymol treatment during which CUMS continued. Behavior tests were performed after the last drug administration (Fig. 1). The CUMS procedure was performed as previously described, with minor modification. Briefly, mice were group housed and allowed to acclimate one week to their environment. Then, the normal control group animals were left undisturbed in their cages in a separated room throughout the following six weeks, whereas the other four groups of mice were single housed and subjected to a variety of mild stressors for six weeks (42 days): (1) food deprivation for 24 h, (2) water deprivation for 24 h, (3) overnight illumination, (4) cage tilt (45◦ ) for 7 h, (5) soiled cage (200 mL water in 100 g sawdust bedding), (6) foreign object exposure, (7) light/dark perversion, (8) overhang (10 min), (9) physical restraint
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Fig. 1. Schematic representation of the experimental procedure for CUMS and treatments in mice. Abbreviations: TST, tail suspension tests; FST, the forced swimming tests.
for 3 h, (10) 1 min tail pinch (1 cm from the beginning of the tail), (11) 5 min oscillation, and (12) white noise. All stressors were applied randomly to ensure the unpredictability of the experiment. 2.4. Body weight and sucrose preference test Body weight and sucrose preference test were measured every week during the experiment paradigm. For sucrose preference test, mice were first deprived of water and food for 8 h before the test, then given the free choice between two bottles (one with 1% sucrose solution, and another with tap water) for 12 h. To prevent possible effects of side preference in drinking behaviors, the positions of the bottles were switched after 6 h. The consumptions of the sucrose solution and tap water were estimated by weighing the bottles. The preference for sucrose (SP) was calculated as a percentage of the consumed sucrose solution relative to the total amount of liquid drunk. SP was calculated as the formula: SP = sucrose intake/(sucrose intake + water intake) × 100. 2.5. Forced swimming test FST was performed as previously described [32]. In brief, mice were individually putted into a glass cylinder (20 cm in height, and 14 cm in diameter) with a depth of 10 cm water (25 ± 2 ◦ C) in it. The immobility time was recorded as the time when mice spent floating in the water without struggling or only making those movements necessary to keep their heads above the water. The duration of immobility was recorded at the last 4 min of the total 6 min, which indicated the depressive state. 2.6. Tail suspension test TST was conducted as previously described [33] on the next day after FST. Briefly, individual mice were acoustically and visually isolated, and suspended about 50 cm above the floor by placing adhesive tape (approximately 1 cm from the tail tip). Each mouse was suspended for total 6 min, of which the duration of immobility was recorded at the last 4 min. Mice were considered as immobile when they were passively suspended and remained completely motionless. 2.7. Serum corticosterone (CORT) level determination After behavioral tests, the blood samples were collected from the retro-orbital plexus. Referring to Arthur’s method [34], 0.2 mL serum and 2 mL of 0.1 mol/L NaOH were mixed for 30 s. 2 mL of dichloromethane was added into the mixture and scrolled for 3 min before standing for 5 min at the room temperature. After centrifugation at 2500 r/min for 20 min, sulfuric acid–alcohol (concentrated
sulfuric acid: 98% ethanol = 7:3) mixed liquor was added into 1.5 mL organic phase. Next, the above solution was whirled for 3 min, centrifuged at 2500 r/min for 20 min, then the aqueous phase was allowed to stand for 30 min. Finally, the fluorescence intensity was measured according to the following parameters: excitation wavelength at 472 nm, emission wavelength at 519 nm. 2.8. Quantitative real-time PCR detection of IL-1ˇ, IL-6 and TNF-˛ Total RNA from hippocampus samples were isolated using Trizol reagent (Vazyme Biotech Co., Ltd., Nanjing, China). The cDNA was synthesized with the Trans Script First-Strand cDNA Synthesis Super Mix (Beijing TransGen Biotech, Beijing, China). Quantitative real-time PCR was carried in 20 L reactions using SsoFastTM EvaGree Supermix (Bio-Rad, Hercules, CA, USA). Real-time PCR analysis was performed in a CFX ConnentTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The sequences of primers used in this experiment are summarized as follows: IL-1 (sense 5 -CTG TGT CTT TCC CGT GGA CC-3 ; antisense 5 -CAG CTC ATA TGG GTC CGA CA-3 ); IL-6, sense 5 -GAC CCC AAA AGA TTA AGG A-3 ; antisense 5 -CAC AAT GAG TGA CAC TGC C-3 ; TNF-␣, sense 5 -ATC CGC GAC GTG GAA CTG-3 ; antisense 5 -CAG CTC ATA TGG GTC CGA CA3 ; -actin, sense 5 -TCT GGC ACC ACA CCT TCT A-3 ; and antisense 5 -AGG CAT ACA GGG ACA GCA C-3 . 2.9. Determination of NE and 5-HT levels The levels of 5-HT and NE in hippocampus of the experimental animals were determined by HPLC coupled with MS. Briefly, A Waters ACQUITYTM triple-quadrupole tandem mass spectrometer (Waters Corp., Milford, MA, USA) was connected to the UPLC system (Waters, Milford, MA, USA) via electrospray ionization (ESI) interface. The mobile phase was composed of A (0.1% formic acid, v/v) and B (acetonitrile) with a gradient elution: 0–6 min, 90–65% A; 6–8 min, 65–0% A; 8–10 min, 0% A. The flow rate was 0.4 mL/min and the column temperatures were maintained at 30 ◦ C and 10 ◦ C. High purity nitrogen was taken as the nebulizer and auxiliary gas, and argon was taken as the collision gas. The ESI source parameters for the determination of NE and 5-HT were as follows: capillary 1.00 kV, cone 30.00 V, extractor 4.00 V. The temperatures of the source and desolvation were 120 ◦ C and 350 ◦ C. Nitrogen was used as the desolvation gas (600 L/h) and cone gas (120 L/h), and argon was used as the collision gas (0.17 mL/min) for collision-induced dissociation. 2.10. Statistical analysis All data were presented as the means ± standard error of mean (S.E.M.). Statistical evaluations of the differences among the
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Fig. 2. The sucrose preference test (A) and body weight (B) were measured every week during the CUMS procedure. Effects of thymol treatment on the immobility time in the FST (C) and TST (D) after the CUMS paradigm. N = 10–12. Data represent mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
groups were analyzed by one-way analysis of variance (ANOVA) with repeated measures on body weight and sucrose preference consumption, and one-way ANOVA without repeated measures followed by the post hoc Tukey test for multiple comparisons. The probability level of p < 0.05 was considered to be significant in the analyses. All statistical procedures were carried out using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Thymol treatment reversed the decreases of body weight and sucrose preference The decrease of sucrose solution consumption was widely adopted as a common measure of anhedonia-like behavior in rodents. In present experiment, sucrose preference (SP) was monitored weekly to evaluate the effectiveness of the depression model. The results of SP test during the CUMS paradigm was shown in Fig. 2A. At the beginning, SPs were similar in each group, while a repeated ANOVA with treatment as between factor and week as within factor, showed a marked effect of treatment [F(4,50) = 15.459, p < 0.001], and significant effect of week [F(6,45) = 10.017, p < 0.001]. However, no significant week × treatment interaction was observed in the experiment. Post hoc test exhibited a significant effect between the CUMS group and
15 or 30 mg/kg thymol group (p < 0.05, p < 0.05), but not 10 mg/kg FLU group. In addition, one-way ANOVA revealed a significant decrease of SP in the CUMS group from the 3rd week (p < 0.05) and sustained the following weeks (week 4: p < 0.01; week 5: p < 0.01; week 6: p < 0.001). After 3-week treatment, thymol (15, 30 mg/kg) and FLU (10 mg/kg) attenuated the decrease of SP induced by CUMS (thymol: p < 0.01, p < 0.01, respectively; FLU: p < 0.05). The body weights of the mice in each group did not have significant differences at the beginning (Fig. 2B). Over the 6week experiment, significant effect of treatment [F(4,50) = 30.515, p < 0.001] and significant effect of week [F(6,45) = 112.478, p < 0.001] were indicated by a repeated ANOVA. Furthermore, a significant week × treatment interaction [F(24,192) = 2.426, p < 0.001] was also indicated on body weight. Meanwhile, post hoc test showed a significant effect of treatment between the CUMS group and 30 mg/kg thymol (p < 0.01) group or 10 mg/kg FLU (p < 0.05) group, but not 15 mg/kg thymol. In addition, one-way ANOVA showed that the control group gained more weight than the CUMS group started from 2nd week (p < 0.05) and sustained the following weeks (week 3: p < 0.001; week 4: p < 0.001; week 5: p < 0.001; week 6: p < 0.001). After 3-week treatment, thymol (15, 30 mg/kg) and FLU (10 mg/kg) attenuated the decreases in body weight gain induced by CUMS (thymol: p < 0.05, p < 0.001, respectively; FLU: p < 0.01).
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30 mg/kg) or FLU (10 mg/kg) treatment significantly decreased IL1 transcription (thymol: p < 0.05, p < 0.01; FLU: p < 0.01) after CUMS exposure. Similarly, CUMS paradigm significantly increased IL-6 level compared with control group (p < 0.01), which were attenuated by thymol (15 mg/kg and 30 mg/kg) or FLU treatment (thymol: p < 0.05, p < 0.05, respectively; FLU: p < 0.05). Meanwhile, the abnormal level of hippocampus TNF-␣ was remarkably restored by thymol (30 mg/kg) or FLU treatment (thymol: p < 0.01; FLU: p < 0.01). 3.6. Thymol treatment recovered CUMS-induced activation of the inflammasome
Fig. 3. CUMS-induced elevation of serum CORT level was reversed by chronic thymol treatment. N = 10–12. Values represent mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
3.2. Thymol treatment shortened immobility time in TST and FST The results of tail suspension tests (TST) and forced swimming tests (FST) were exhibited in Fig. 2. CUMS paradigm induced a marked increase in immobility time during TST (Fig. 2C) and FST (Fig. 2D) as shown (p < 0.01, p < 0.05 respectively). Thymol (30 mg/kg) or FLU (10 mg/kg) treatment significantly reversed the increase of immobility time in TST (thymol: p < 0.05; FLU: p < 0.01) induced by CUMS exposure. For FST (Fig. 2D), immobility time was prolonged after CUMS challenge vs. the control group (p < 0.05). Either thymol (15, 30 mg/kg) or FLU (10 mg/kg) treatment obviously alleviated the CUMS-induced increase in immobility time (thymol: p < 0.05, p < 0.05, respectively; FLU: p < 0.05). 3.3. Effects of thymol on serum CORT To evaluate the effects of thymol on HPA axis function, blood was collected and levels of serum CORT were measured. As shown in Fig. 3, the CUMS paradigm produced a 2.6-fold elevation in serum CORT level vs. the control group (p < 0.001). Thymol (15, 30 mg/kg) or FLU (10 mg/kg) administration restored this elevation (thymol: p < 0.001, p < 0.001, respectively; FLU: p < 0.001) induced by CUMS. 3.4. Effects of thymol on neurotransmitters in the hippocampus As shown in Fig. 4A, CUMS significantly decreased the 5-HT levels in the hippocampus vs. the control group (p < 0.01). FLU (10 mg/kg) or thymol treatments (30 mg/kg) led to a significant elevation in 5-HT concentration in the hippocampus (p < 0.05, p < 0.05 respectively) compared to the CUMS group. Fig. 4B presented the effects of thymol on the alternations of NE. CUMS paradigm dramatically decreased the levels of NE in the hippocampus (p < 0.001), whereas thymol at doses of 15 mg/kg and 30 mg/kg significantly inhibited the depletion of NE levels (p < 0.05, p < 0.01). 3.5. Thymol treatment reversed CUMS-induced increases of proinflammatory cytokines The effects of thymol and FLU on the mRNA levels of IL1 (Fig. 5A), IL-6 (Fig. 5B) and TNF-␣ (Fig. 5C) in hippocampus of the CUMS treated mice were shown in Fig. 5. The levels of IL-1, IL-6, and TNF-␣ were significantly increased after CUMS exposure (p < 0.01, p < 0.01, p < 0.01, respectively). Thymol (15,
Further, we measured the expression of hippocampus NLRP3 inflammasome complex. As shown in Fig. 6, the protein levels of NLRP3 and its adaptor ASC (Fig. 6B and C) were markedly elevated (p < 0.01, p < 0.05) in CUMS mice. Similarly, CUMS paradigm significantly activated procaspase-1 into cleaved caspase-1 (P10, p < 0.01) in CUMS mice vs. control group (Fig. 6D). Thymol (15 mg/kg and 30 mg/kg) or FLU administration remarkably inhibited the activation of NLRP3 inflammasome (thymol: p < 0.05, p < 0.01, respectively; FLU: p < 0.01), and caspase-1 cleavage (thymol: p < 0.05, p < 0.01, respectively; FLU: p < 0.01). Meanwhile, thymol (30 mg/kg) showed a significant effect on ASC adaptor (p < 0.05). 4. Discussion 4.1. Effects of thymol on depression-related behaviors The sucrose preference tests were explored to reflect the anhedonia-like behavior which was defined as a core symptom of depression in depressive rodent models [28]. In the present study, we measured the sucrose consumption weekly to monitor the effectiveness of CUMS paradigm, and found that six weeks’ stimuli exposure caused anhedonia-like behaviors in mice (21% reduction of sucrose solution intake). The FST and TST tests were performed to reflect the behavioral despair [35]. Here, we observed that the immobility time was markedly prolonged both in FST and TST, while thymol or FLU administration restored these alterations induced by CUMS. Moreover, CUMS significantly decreased the body weight gain as compared with control group. Thymol or FLU treatment reversed the reduction of body weight gain of CUMSexposed mice. This was different from some studies, which had reported that the body weight was not affected by CUMS protocol [36]. This discrepancy may be explicated as different parameters in the experimental design, such as stressors and its duration time, etc. [37]. 4.2. Effects of thymol on neurotransmitters Depression is connected with the disturbance of brain neurotransmitters. Several researches have reported that CUMS paradigm caused a significant reduction of monoamine neurotransmitters, which have been proposed as a principal pathogenic factor in depression [38]. In our study, neurotransmitter 5-HT and NE decreased markedly after the CUMS exposure, which were consistent with those previous studies. Thymol administration effectively reversed the reduction of 5-HT and NE levels in CUMS-induced depressive mice. It demonstrated that the antidepressant-like efficacy of thymol was possibly achieved through the enhancement of the monoaminergic responses. 4.3. Effects of thymol on inflammation Neuroinflammatory molecules affect the synthesis, release, and reuptake of neurotransmitters including 5-HT and NE [39]. Data
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Fig. 4. Effects of thymol treatment on 5-HT (A) and NE (B) levels in the hippocampus of CUMS mice. N = 5. Data represent mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
from clinical and preclinical studies suggested that inflammatory cytokines increased in depressed patients and in animal models of depression, and the inhibition of the inflammatory processes might ameliorate their depressive symptoms [40]. Thymol exhibited strong anti-inflammatory activities in the models of osteoarthritis [20], asthma [21], skin inflammation [41], ear edema [42], and peritonitis [43] etc. It has been reported to attenuate inflammation through downregulating the expressions of proinflammatory cytokines, such as TNF-␣ [19,30,44], IL-1 [19], and IL-6 [19,44]. Furthermore, thymol could inhibit the activation of NF-B signaling in LPS treated macrophages [45], and down-regulate the MAPK pathway in mammary epithelial cells [46]. In the present study, we observed that the levels of IL-1, IL-6 and TNF-␣ in hippocampus were markedly increased after CUMS challenge, while treatment with thymol effectively attenuated the elevation of these proinflammatory cytokines induced by CUMS, indicating that the antidepressant action of thymol might be contributed to its anti-inflammatory properties. The inflammasome pathway links psychological stress, depression and comorbid systemic illnesses, and it has been proposed as a novel strategy for treating depression [10]. We examined the levels of NLRP3 inflammasome constituents, NLRP3, ASC and caspase-1, which had significant elevations in the CUMS group compared with the control. Thymol treatment (15, 30 mg/kg) obviously reversed these alterations. Thus, thymol inhibited the formation of NLRP3 inflammasome and subsequent
cleaved caspase-1, leading to the reduction of bioactive IL-1 transformation and release.
4.4. Effects of thymol on serum CORT In the present study, we showed that chronic stress engendered HPA dysfunction, as reflected by the significantly elevated serum CORT levels, and chronic administration of thymol reversed this alteration. Inflammatory cytokines are important mediators in the regulation of HPA axis function, specifically during a CUMS exposure [47]. Moreover, basal inflammatory molecules are required for the continuous CORT responses to chronic stresses. Thymol conferred an antidepressant-like effect partially through attenuating the hyperactivity of the HPA axis. In conclusion, the present research demonstrated that thymol conferred an antidepressant-like effect, at least in CUMS model of depression in mice. Thymol treatment restored the decrease of monoamine neurotransmitters induced by CUMS, and adjusted the dysfunction of the HPA axis. Furthermore, chronic administration of thymol inhibited the activation of proinflammatory cytokines, possibly through the regulation of NLRP3/caspase-1 pathway. Our finding may provide an insight into the potential of thymol in therapeutic implications for depression.
Fig. 5. Effects of thymol on the IL-1 (A), IL-6 (B) and TNF-␣ (C) mRNA in the hippocampus of CUMS mice. N = 4. Values represent mean ± S.E.M. # p < 0.05, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
##
p < 0.01,
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Fig. 6. (A) Chronic thymol treatment restored NLRP3 inflammasome activation and caspase-1 cleavage induced by CUMS. (B) Relative NLRP3 protein level. (C) Relative ASC protein level. (D) Relative cleaved caspase-1 protein level (P10). N = 4. Data represent mean ± S.E.M. # p < 0.05, ## p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. CUMS group.
Acknowledgements This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Fundamental Research Funds for the Central Universities (ZJ15030) and Research Innovation Program Project for Graduate Students in Jiangsu Province (KYLX 0639). We also thank Ding L. for assisting in the preparation of this manuscript. References [1] Gao S, Cui YL, Yu CQ, Wang QS, Zhang Y. Tetrandrine exerts antidepressantlike effects in animal models: role of brain-derived neurotrophic factor. Behav Brain Res 2013;238:79–85. [2] Bromet E, Andrade LH, Hwang I, Sampson NA, Alonso J, de Girolamo G, et al. Cross-national epidemiology of DSM-IV major depressive episode. BMC Med 2011;9:90. [3] Singhal G, Jaehne EJ, Corrigan F, Toben C, Baune BT. Inflammasomes in neuroinflammation and changes in brain function: a focused review. Front Neurosci 2014;8:315. [4] Motivala SJ, Sarfatti A, Olmos L, Irwin MR. Inflammatory markers and sleep disturbance in major depression. Psychosom Med 2005;67:187–94. [5] Meyers CA, Albitar M, Estey E. Cognitive impairment, fatigue, and cytokine levels in patients with acute myelogenous leukemia or myelodysplastic syndrome. Cancer 2005;104:788–93. [6] Dowlati Y, Herrmann N, Swardfager W, Liu H, Sham L, Reim EK, et al. A metaanalysis of cytokines in major depression. Biol Psychiatry 2010;67:446–57. [7] Miller AH. Mechanisms of cytokine-induced behavioral changes: psychoneuroimmunology at the translational interface. Brain Behav Immun 2009;23:149–58. [8] Dunn AJ, Swiergiel AH, de Beaurepaire R. Cytokines as mediators of depression: what can we learn from animal studies. Neurosci Biobehav Rev 2005;29:891–909. [9] Gadek-Michalska A, Tadeusz J, Rachwalska P, Bugajski J. Cytokines, prostaglandins and nitric oxide in the regulation of stress-response systems. Pharmacol Rep 2013;65:1655–62. [10] Iwata M, Ota KT, Duman RS. The inflammasome: pathways linking psychological stress, depression, and systemic illnesses. Brain Behav Immun 2013;31:105–14. [11] Davis BK, Wen H, Ting JP-Y. The inflammasome NLRs in immunity, inflammation, and associated diseases. Annu Rev Immunol 2011;29:707–35.
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Research report
Thymol produces an antidepressant-like effect in a chronic unpredictable mild stress model of depression in mice Xue-Yang Deng a , Hong-Yan Li a , Jun-Jun Chen a , Rui-Peng Li a , Rong Qu b,∗ , Qiang Fu a,∗∗ , Shi-Ping Ma a,∗ ∗ ∗ a b
Department of Pharmacology of Chinese Materia, China Pharmaceutical University, Nanjing 210009, China Department of Pharmacology of Traditional Chinese Medical Formulae, Nanjing University of Traditional Chinese Medicine, Nanjing 210029, China
h i g h l i g h t s • • • •
Thymol exhibited an antidepressant-like effect. Thymol alleviated the depression-related behaviors in mice. Thymol reversed the decrease of neurotransmitters induced by CUMS. The antidepressant-like properties of thymol are regulated via NLRP3 inflammasome mediated neuro-inflammation.
a r t i c l e
i n f o
Article history: Received 1 March 2015 Received in revised form 23 April 2015 Accepted 28 April 2015 Available online 6 May 2015 Keywords: Thymol Antidepressant Neurotransmitter Proinflammatory cytokine NLRP3
a b s t r a c t Thymol, a bioactive monoterpene isolated from Thymus vulgaris, has displayed inspiring neuroprotective properties. The present study was designed to evaluate the antidepressant-like effects of thymol on a chronic unpredictable mild stress (CUMS) model of depression in mice and explore the underlying mechanisms. It was observed that thymol treatment (15 mg/kg and 30 mg/kg) significantly reversed the decrease of sucrose consumption, the loss of body weight, the reduction of immobile time in the tail suspension tests (TST) and forced swimming tests (FST) induced by CUMS paradigm. The levels of norepinephrine (NE) and serotonin (5-HT) in the hippocampus decreased in the CUMS-treated mice. Chronic treatments with thymol significantly restored the CUMS-induced alterations of monoamine neurotransmitters in the hippocampus. Our results further demonstrated that thymol administration negatively regulated the induction of proinflammatory cytokines including interleukin (IL)-1, IL-6, and tumor necrosis factor␣ in CUMS mice. Furthermore, thymol inhibited the activation of nod-like receptor protein 3 (NLRP3) inflammasome and its adaptor, and subsequently decreased the expression of caspase-1. In sum, our findings suggested that thymol played a potential antidepressant role in CUMS mice model through up-regulating the levels of central neurotransmitters and inhibiting the expressions of proinflammatory cytokines, which might provide potential for thymol in the light of opening up new therapeutic avenues for depression. © 2015 Elsevier B.V. All rights reserved.
Abbreviations: CUMS, chronic unpredictable mild stress; FST, forced swimming tests; TST, tail suspension tests; IL-1, interleukin-1 beta; IL-6, interleukin-6; TNF-␣tumor, necrosis factor-alpha; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3; ASC, apoptosis-associated speck like protein; HPA, hypothalamic pituitary adrenal; CORT, corticosterone; FLU, fluoxetine. ∗ Corresponding author. Tel.: +86 25 85811929. ∗∗ Corresponding author at: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, China. Tel.: +86 25 83271507. ∗ ∗ ∗Corresponding author at: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, China. Tel.: +86 25 83271419. E-mail addresses: [email protected] (R. Qu), [email protected] (Q. Fu), [email protected] (S.-P. Ma). http://dx.doi.org/10.1016/j.bbr.2015.04.052 0166-4328/© 2015 Elsevier B.V. All rights reserved.
X.-Y. Deng et al. / Behavioural Brain Research 291 (2015) 12–19
1. Introduction Major depressive disorder is a recurring and life threatening illness, from which up to 20% of the world population suffer [1]. The World Health Organization numbered depression as the fourth leading cause of disability worldwide, which brings heavy burden to the society [2]. Although currently available antidepressants might provide 2/3 of these patients with some relief, it is far from ideal. It is still an urgent need for looking for more effective and safe drugs against depression. Inflammation has been recognized as an important mechanism of neuropsychiatric disorders, including major depression [3]. Associations between inflammatory biomarkers and depressive symptoms, such as disturbed sleep, fatigue, and cognitive impairment, have been described by numerous studies [4,5]. Patients suffered from depression exhibited increased inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-8, interferon (IFN)-␥, TNF-␣ [6]. Inflammatory cytokines access the brain and act on neurotransmitter metabolism, neuronal plasticity, and neuroendocrine function. Among these, inflammatory cytokines-induced changes in monoamine neurotransmitters have been reported to be a primary pathway to depression [7]. Inflammatory cytokines could activate brain serotonergic systems, increase brain tryptophan concentrations and have a direct influence on 5-HT and NE metabolism [8]. In addition, inflammatory cytokines have been proposed to be connected with the HPA axis abnormality, which is a potentially causative or exacerbating factor in depression, and directly induces glucocorticoids secretions from the adrenals [9]. Inflammasomes are molecular platforms activated upon detection of stress or other danger signals, among which the NLRP3 inflammasome is the most notably characterized one [10]. The NLRP3 inflammasome complex consists of the NLRP3 pattern recognition receptor, the apoptosis-associated specklike protein (ASC) adaptor, and the effector protein caspase-1 [3]. NLRP3 is activated upon pathogens exposure, cellular injury, or other environmental irritants. Once activated, NLRP3 interacts with ASC adaptor via pyrin domain hemophilic interactions, leading to the activation of caspase-1 [11], which triggers the processing of inflammatory cytokines such as IL-1 to engage the innate immune defenses [12] by cleaving its precursor pro-IL-1. Recently, NLRP3 inflammasome is increasingly suggested to play a major role in systemic diseases, such as cancer, cardiovascular diseases, diabetes and depression in particular [10,13]. Researches show that NLRP3 inflammasome is activated in depressed animal models [14] and in patients with major depression as well [15], which suggests that NLRP3 inflammasome might be a new target and offer new perspectives in the study of depression [16]. Thymol is a main bioactive monoterpene isolated from many medicinal herbs, such as Thymus vulgaris, Monarda punctate and Origanum vulgare spp. [17,18]. Thymol has been widely used as an active anti-inflammatory ingredient, which can inhibit the isoproterenol induced inflammation in myocardial infarcted rats [19], attenuate collagenase-induced osteoarthritis [20], and alleviate allergic airway inflammation in ovalbumin-induced mouse asthma [21]. Moreover, studies have reported that thymol has various bioactivities, such as anticancer [22], anti-bacterial [18], and antioxidant [23] properties. Recent studies focusing on the neuroprotective activities of thymol have shown that it attenuates amyloid  or scopolamine induced cognitive impairment in rats [24]. Thymol treatment has been reported to increase antioxidant status in rat brain [25], and has specific interactions with synaptic neural functions [26]. Further findings have reported that thymol displayed neuroprotective effects, which attributed to its potential action on GABA-mediated inhibition of synaptic transmission [27]. Considering the inspiring beneficial effects of thymol on the brain, the present study was designed to explore its antidepressant-like
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effect in CUMS model in mice, which mimics several depressive syndromes observed in patients with depression, such as body weight loss, decreased responsiveness to rewarding stimuli, and insufficient self-care [28,29]. At the meantime, in order to investigate the possible mechanisms underlying the therapeutic effects of thymol, we also assessed the levels of monoamine neurotransmitters and the expressions of proinflammatory cytokines. 2. Materials and methods 2.1. Animals Male ICR mice (18–20 g) were purchased from the Experimental Animal Center of China Pharmaceutical University (Nanjing, China). Upon arrival, mice were group housed under standard housing conditions (room temperature 25 ± 2 ◦ C; a 12/12 h light/dark cycle). Water and food were provided ad libitum. Animal handling and experimental procedure were performed strictly in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation and approved by the Science and Technology Department of Jiangsu Province. 2.2. Drugs and reagents Fluoxetine (FLU) hydrochloride was purchased from Changzhou Siyao Pharmaceuticals Co., Ltd (Changzhou, China). Thymol (2isopropyl-5-methyl-phenol) was obtained from Sigma Chemical Co. (St Louis, MO, USA). Bicinchoninic acid assay (BCA) kit was produced by Beyotime Institute of Biotechnology Co., Ltd (Nanjing, China). Chloral hydrate was from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Total RNA Extraction Reagent and HiScriptTM Q RT SuperMixfor qPCR (+gDNA wiper) were obtained from Vazyme Biotech Co., Ltd (Nanjing, China). 2.3. CUMS procedure and experimental design Mice were randomly assigned into five groups (n = 12). Group I: Normal control group; Group II: CUMS model group; Group III: CUMS + thymol (15 mg/kg) group; Group IV: CUMS + thymol (30 mg/kg) group; Group V: CUMS + FLU (10 mg/kg) group. Thymol or FLU was administered intragastrically once a day for 3 weeks from the fourth week. Thymol and FLU were prepared by dd water in 0.3% carboxy methyl cellulose as an adjuvant [30]. For the normal control and CUMS model groups, mice were given an equal volume of 0.3% carboxy methyl cellulose. The levels of thymol administration chosen were similar to those used in previous trials, in which thymol protected against inflammation in isoproterenol induced myocardial infarcted rats [19], ovalbumin induced mouse asthma [21], and cisplatin induced nephrotoxic rats [31]. And these doses were considerably lower than the reported value of the median lethal dose (980 mg/kg in rats) [25]. Mice were exposed to the CUMS stimuli for 3 weeks (from 1st to 3rd week), followed by 3 weeks (from 4th to 6th week) of thymol treatment during which CUMS continued. Behavior tests were performed after the last drug administration (Fig. 1). The CUMS procedure was performed as previously described, with minor modification. Briefly, mice were group housed and allowed to acclimate one week to their environment. Then, the normal control group animals were left undisturbed in their cages in a separated room throughout the following six weeks, whereas the other four groups of mice were single housed and subjected to a variety of mild stressors for six weeks (42 days): (1) food deprivation for 24 h, (2) water deprivation for 24 h, (3) overnight illumination, (4) cage tilt (45◦ ) for 7 h, (5) soiled cage (200 mL water in 100 g sawdust bedding), (6) foreign object exposure, (7) light/dark perversion, (8) overhang (10 min), (9) physical restraint
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Fig. 1. Schematic representation of the experimental procedure for CUMS and treatments in mice. Abbreviations: TST, tail suspension tests; FST, the forced swimming tests.
for 3 h, (10) 1 min tail pinch (1 cm from the beginning of the tail), (11) 5 min oscillation, and (12) white noise. All stressors were applied randomly to ensure the unpredictability of the experiment. 2.4. Body weight and sucrose preference test Body weight and sucrose preference test were measured every week during the experiment paradigm. For sucrose preference test, mice were first deprived of water and food for 8 h before the test, then given the free choice between two bottles (one with 1% sucrose solution, and another with tap water) for 12 h. To prevent possible effects of side preference in drinking behaviors, the positions of the bottles were switched after 6 h. The consumptions of the sucrose solution and tap water were estimated by weighing the bottles. The preference for sucrose (SP) was calculated as a percentage of the consumed sucrose solution relative to the total amount of liquid drunk. SP was calculated as the formula: SP = sucrose intake/(sucrose intake + water intake) × 100. 2.5. Forced swimming test FST was performed as previously described [32]. In brief, mice were individually putted into a glass cylinder (20 cm in height, and 14 cm in diameter) with a depth of 10 cm water (25 ± 2 ◦ C) in it. The immobility time was recorded as the time when mice spent floating in the water without struggling or only making those movements necessary to keep their heads above the water. The duration of immobility was recorded at the last 4 min of the total 6 min, which indicated the depressive state. 2.6. Tail suspension test TST was conducted as previously described [33] on the next day after FST. Briefly, individual mice were acoustically and visually isolated, and suspended about 50 cm above the floor by placing adhesive tape (approximately 1 cm from the tail tip). Each mouse was suspended for total 6 min, of which the duration of immobility was recorded at the last 4 min. Mice were considered as immobile when they were passively suspended and remained completely motionless. 2.7. Serum corticosterone (CORT) level determination After behavioral tests, the blood samples were collected from the retro-orbital plexus. Referring to Arthur’s method [34], 0.2 mL serum and 2 mL of 0.1 mol/L NaOH were mixed for 30 s. 2 mL of dichloromethane was added into the mixture and scrolled for 3 min before standing for 5 min at the room temperature. After centrifugation at 2500 r/min for 20 min, sulfuric acid–alcohol (concentrated
sulfuric acid: 98% ethanol = 7:3) mixed liquor was added into 1.5 mL organic phase. Next, the above solution was whirled for 3 min, centrifuged at 2500 r/min for 20 min, then the aqueous phase was allowed to stand for 30 min. Finally, the fluorescence intensity was measured according to the following parameters: excitation wavelength at 472 nm, emission wavelength at 519 nm. 2.8. Quantitative real-time PCR detection of IL-1ˇ, IL-6 and TNF-˛ Total RNA from hippocampus samples were isolated using Trizol reagent (Vazyme Biotech Co., Ltd., Nanjing, China). The cDNA was synthesized with the Trans Script First-Strand cDNA Synthesis Super Mix (Beijing TransGen Biotech, Beijing, China). Quantitative real-time PCR was carried in 20 L reactions using SsoFastTM EvaGree Supermix (Bio-Rad, Hercules, CA, USA). Real-time PCR analysis was performed in a CFX ConnentTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The sequences of primers used in this experiment are summarized as follows: IL-1 (sense 5 -CTG TGT CTT TCC CGT GGA CC-3 ; antisense 5 -CAG CTC ATA TGG GTC CGA CA-3 ); IL-6, sense 5 -GAC CCC AAA AGA TTA AGG A-3 ; antisense 5 -CAC AAT GAG TGA CAC TGC C-3 ; TNF-␣, sense 5 -ATC CGC GAC GTG GAA CTG-3 ; antisense 5 -CAG CTC ATA TGG GTC CGA CA3 ; -actin, sense 5 -TCT GGC ACC ACA CCT TCT A-3 ; and antisense 5 -AGG CAT ACA GGG ACA GCA C-3 . 2.9. Determination of NE and 5-HT levels The levels of 5-HT and NE in hippocampus of the experimental animals were determined by HPLC coupled with MS. Briefly, A Waters ACQUITYTM triple-quadrupole tandem mass spectrometer (Waters Corp., Milford, MA, USA) was connected to the UPLC system (Waters, Milford, MA, USA) via electrospray ionization (ESI) interface. The mobile phase was composed of A (0.1% formic acid, v/v) and B (acetonitrile) with a gradient elution: 0–6 min, 90–65% A; 6–8 min, 65–0% A; 8–10 min, 0% A. The flow rate was 0.4 mL/min and the column temperatures were maintained at 30 ◦ C and 10 ◦ C. High purity nitrogen was taken as the nebulizer and auxiliary gas, and argon was taken as the collision gas. The ESI source parameters for the determination of NE and 5-HT were as follows: capillary 1.00 kV, cone 30.00 V, extractor 4.00 V. The temperatures of the source and desolvation were 120 ◦ C and 350 ◦ C. Nitrogen was used as the desolvation gas (600 L/h) and cone gas (120 L/h), and argon was used as the collision gas (0.17 mL/min) for collision-induced dissociation. 2.10. Statistical analysis All data were presented as the means ± standard error of mean (S.E.M.). Statistical evaluations of the differences among the
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Fig. 2. The sucrose preference test (A) and body weight (B) were measured every week during the CUMS procedure. Effects of thymol treatment on the immobility time in the FST (C) and TST (D) after the CUMS paradigm. N = 10–12. Data represent mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
groups were analyzed by one-way analysis of variance (ANOVA) with repeated measures on body weight and sucrose preference consumption, and one-way ANOVA without repeated measures followed by the post hoc Tukey test for multiple comparisons. The probability level of p < 0.05 was considered to be significant in the analyses. All statistical procedures were carried out using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Thymol treatment reversed the decreases of body weight and sucrose preference The decrease of sucrose solution consumption was widely adopted as a common measure of anhedonia-like behavior in rodents. In present experiment, sucrose preference (SP) was monitored weekly to evaluate the effectiveness of the depression model. The results of SP test during the CUMS paradigm was shown in Fig. 2A. At the beginning, SPs were similar in each group, while a repeated ANOVA with treatment as between factor and week as within factor, showed a marked effect of treatment [F(4,50) = 15.459, p < 0.001], and significant effect of week [F(6,45) = 10.017, p < 0.001]. However, no significant week × treatment interaction was observed in the experiment. Post hoc test exhibited a significant effect between the CUMS group and
15 or 30 mg/kg thymol group (p < 0.05, p < 0.05), but not 10 mg/kg FLU group. In addition, one-way ANOVA revealed a significant decrease of SP in the CUMS group from the 3rd week (p < 0.05) and sustained the following weeks (week 4: p < 0.01; week 5: p < 0.01; week 6: p < 0.001). After 3-week treatment, thymol (15, 30 mg/kg) and FLU (10 mg/kg) attenuated the decrease of SP induced by CUMS (thymol: p < 0.01, p < 0.01, respectively; FLU: p < 0.05). The body weights of the mice in each group did not have significant differences at the beginning (Fig. 2B). Over the 6week experiment, significant effect of treatment [F(4,50) = 30.515, p < 0.001] and significant effect of week [F(6,45) = 112.478, p < 0.001] were indicated by a repeated ANOVA. Furthermore, a significant week × treatment interaction [F(24,192) = 2.426, p < 0.001] was also indicated on body weight. Meanwhile, post hoc test showed a significant effect of treatment between the CUMS group and 30 mg/kg thymol (p < 0.01) group or 10 mg/kg FLU (p < 0.05) group, but not 15 mg/kg thymol. In addition, one-way ANOVA showed that the control group gained more weight than the CUMS group started from 2nd week (p < 0.05) and sustained the following weeks (week 3: p < 0.001; week 4: p < 0.001; week 5: p < 0.001; week 6: p < 0.001). After 3-week treatment, thymol (15, 30 mg/kg) and FLU (10 mg/kg) attenuated the decreases in body weight gain induced by CUMS (thymol: p < 0.05, p < 0.001, respectively; FLU: p < 0.01).
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30 mg/kg) or FLU (10 mg/kg) treatment significantly decreased IL1 transcription (thymol: p < 0.05, p < 0.01; FLU: p < 0.01) after CUMS exposure. Similarly, CUMS paradigm significantly increased IL-6 level compared with control group (p < 0.01), which were attenuated by thymol (15 mg/kg and 30 mg/kg) or FLU treatment (thymol: p < 0.05, p < 0.05, respectively; FLU: p < 0.05). Meanwhile, the abnormal level of hippocampus TNF-␣ was remarkably restored by thymol (30 mg/kg) or FLU treatment (thymol: p < 0.01; FLU: p < 0.01). 3.6. Thymol treatment recovered CUMS-induced activation of the inflammasome
Fig. 3. CUMS-induced elevation of serum CORT level was reversed by chronic thymol treatment. N = 10–12. Values represent mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
3.2. Thymol treatment shortened immobility time in TST and FST The results of tail suspension tests (TST) and forced swimming tests (FST) were exhibited in Fig. 2. CUMS paradigm induced a marked increase in immobility time during TST (Fig. 2C) and FST (Fig. 2D) as shown (p < 0.01, p < 0.05 respectively). Thymol (30 mg/kg) or FLU (10 mg/kg) treatment significantly reversed the increase of immobility time in TST (thymol: p < 0.05; FLU: p < 0.01) induced by CUMS exposure. For FST (Fig. 2D), immobility time was prolonged after CUMS challenge vs. the control group (p < 0.05). Either thymol (15, 30 mg/kg) or FLU (10 mg/kg) treatment obviously alleviated the CUMS-induced increase in immobility time (thymol: p < 0.05, p < 0.05, respectively; FLU: p < 0.05). 3.3. Effects of thymol on serum CORT To evaluate the effects of thymol on HPA axis function, blood was collected and levels of serum CORT were measured. As shown in Fig. 3, the CUMS paradigm produced a 2.6-fold elevation in serum CORT level vs. the control group (p < 0.001). Thymol (15, 30 mg/kg) or FLU (10 mg/kg) administration restored this elevation (thymol: p < 0.001, p < 0.001, respectively; FLU: p < 0.001) induced by CUMS. 3.4. Effects of thymol on neurotransmitters in the hippocampus As shown in Fig. 4A, CUMS significantly decreased the 5-HT levels in the hippocampus vs. the control group (p < 0.01). FLU (10 mg/kg) or thymol treatments (30 mg/kg) led to a significant elevation in 5-HT concentration in the hippocampus (p < 0.05, p < 0.05 respectively) compared to the CUMS group. Fig. 4B presented the effects of thymol on the alternations of NE. CUMS paradigm dramatically decreased the levels of NE in the hippocampus (p < 0.001), whereas thymol at doses of 15 mg/kg and 30 mg/kg significantly inhibited the depletion of NE levels (p < 0.05, p < 0.01). 3.5. Thymol treatment reversed CUMS-induced increases of proinflammatory cytokines The effects of thymol and FLU on the mRNA levels of IL1 (Fig. 5A), IL-6 (Fig. 5B) and TNF-␣ (Fig. 5C) in hippocampus of the CUMS treated mice were shown in Fig. 5. The levels of IL-1, IL-6, and TNF-␣ were significantly increased after CUMS exposure (p < 0.01, p < 0.01, p < 0.01, respectively). Thymol (15,
Further, we measured the expression of hippocampus NLRP3 inflammasome complex. As shown in Fig. 6, the protein levels of NLRP3 and its adaptor ASC (Fig. 6B and C) were markedly elevated (p < 0.01, p < 0.05) in CUMS mice. Similarly, CUMS paradigm significantly activated procaspase-1 into cleaved caspase-1 (P10, p < 0.01) in CUMS mice vs. control group (Fig. 6D). Thymol (15 mg/kg and 30 mg/kg) or FLU administration remarkably inhibited the activation of NLRP3 inflammasome (thymol: p < 0.05, p < 0.01, respectively; FLU: p < 0.01), and caspase-1 cleavage (thymol: p < 0.05, p < 0.01, respectively; FLU: p < 0.01). Meanwhile, thymol (30 mg/kg) showed a significant effect on ASC adaptor (p < 0.05). 4. Discussion 4.1. Effects of thymol on depression-related behaviors The sucrose preference tests were explored to reflect the anhedonia-like behavior which was defined as a core symptom of depression in depressive rodent models [28]. In the present study, we measured the sucrose consumption weekly to monitor the effectiveness of CUMS paradigm, and found that six weeks’ stimuli exposure caused anhedonia-like behaviors in mice (21% reduction of sucrose solution intake). The FST and TST tests were performed to reflect the behavioral despair [35]. Here, we observed that the immobility time was markedly prolonged both in FST and TST, while thymol or FLU administration restored these alterations induced by CUMS. Moreover, CUMS significantly decreased the body weight gain as compared with control group. Thymol or FLU treatment reversed the reduction of body weight gain of CUMSexposed mice. This was different from some studies, which had reported that the body weight was not affected by CUMS protocol [36]. This discrepancy may be explicated as different parameters in the experimental design, such as stressors and its duration time, etc. [37]. 4.2. Effects of thymol on neurotransmitters Depression is connected with the disturbance of brain neurotransmitters. Several researches have reported that CUMS paradigm caused a significant reduction of monoamine neurotransmitters, which have been proposed as a principal pathogenic factor in depression [38]. In our study, neurotransmitter 5-HT and NE decreased markedly after the CUMS exposure, which were consistent with those previous studies. Thymol administration effectively reversed the reduction of 5-HT and NE levels in CUMS-induced depressive mice. It demonstrated that the antidepressant-like efficacy of thymol was possibly achieved through the enhancement of the monoaminergic responses. 4.3. Effects of thymol on inflammation Neuroinflammatory molecules affect the synthesis, release, and reuptake of neurotransmitters including 5-HT and NE [39]. Data
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Fig. 4. Effects of thymol treatment on 5-HT (A) and NE (B) levels in the hippocampus of CUMS mice. N = 5. Data represent mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
from clinical and preclinical studies suggested that inflammatory cytokines increased in depressed patients and in animal models of depression, and the inhibition of the inflammatory processes might ameliorate their depressive symptoms [40]. Thymol exhibited strong anti-inflammatory activities in the models of osteoarthritis [20], asthma [21], skin inflammation [41], ear edema [42], and peritonitis [43] etc. It has been reported to attenuate inflammation through downregulating the expressions of proinflammatory cytokines, such as TNF-␣ [19,30,44], IL-1 [19], and IL-6 [19,44]. Furthermore, thymol could inhibit the activation of NF-B signaling in LPS treated macrophages [45], and down-regulate the MAPK pathway in mammary epithelial cells [46]. In the present study, we observed that the levels of IL-1, IL-6 and TNF-␣ in hippocampus were markedly increased after CUMS challenge, while treatment with thymol effectively attenuated the elevation of these proinflammatory cytokines induced by CUMS, indicating that the antidepressant action of thymol might be contributed to its anti-inflammatory properties. The inflammasome pathway links psychological stress, depression and comorbid systemic illnesses, and it has been proposed as a novel strategy for treating depression [10]. We examined the levels of NLRP3 inflammasome constituents, NLRP3, ASC and caspase-1, which had significant elevations in the CUMS group compared with the control. Thymol treatment (15, 30 mg/kg) obviously reversed these alterations. Thus, thymol inhibited the formation of NLRP3 inflammasome and subsequent
cleaved caspase-1, leading to the reduction of bioactive IL-1 transformation and release.
4.4. Effects of thymol on serum CORT In the present study, we showed that chronic stress engendered HPA dysfunction, as reflected by the significantly elevated serum CORT levels, and chronic administration of thymol reversed this alteration. Inflammatory cytokines are important mediators in the regulation of HPA axis function, specifically during a CUMS exposure [47]. Moreover, basal inflammatory molecules are required for the continuous CORT responses to chronic stresses. Thymol conferred an antidepressant-like effect partially through attenuating the hyperactivity of the HPA axis. In conclusion, the present research demonstrated that thymol conferred an antidepressant-like effect, at least in CUMS model of depression in mice. Thymol treatment restored the decrease of monoamine neurotransmitters induced by CUMS, and adjusted the dysfunction of the HPA axis. Furthermore, chronic administration of thymol inhibited the activation of proinflammatory cytokines, possibly through the regulation of NLRP3/caspase-1 pathway. Our finding may provide an insight into the potential of thymol in therapeutic implications for depression.
Fig. 5. Effects of thymol on the IL-1 (A), IL-6 (B) and TNF-␣ (C) mRNA in the hippocampus of CUMS mice. N = 4. Values represent mean ± S.E.M. # p < 0.05, ### p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. CUMS group.
##
p < 0.01,
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Fig. 6. (A) Chronic thymol treatment restored NLRP3 inflammasome activation and caspase-1 cleavage induced by CUMS. (B) Relative NLRP3 protein level. (C) Relative ASC protein level. (D) Relative cleaved caspase-1 protein level (P10). N = 4. Data represent mean ± S.E.M. # p < 0.05, ## p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. CUMS group.
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