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Yokukansan, a traditional Japanese herbal medicine, enhances the anxiolytic effect of fluvoxamine and reduces cortical 5-HT2A receptor expression in mice
Journal of Ethnopharmacology 216 (2018) 89–96
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Yokukansan, a traditional Japanese herbal medicine, enhances the anxiolytic effect of fluvoxamine and reduces cortical 5-HT2A receptor expression in mice
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Rintaro Ohno1, Hiroko Miyagishi1, Minoru Tsuji , Atsumi Saito, Kazuya Miyagawa, Kazuhiro Kurokawa, Hiroshi Takeda Department of Pharmacology, School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara, Tochigi 324-8501, Japan
A R T I C L E I N F O
A B S T R A C T
Chemical compounds: Fluvoxamine maleate (PubChem CID: 9560989) Ketanserin tartrate (PubChem CID: 16219944)
Ethnopharmacological relevance: Yokukansan is a traditional Japanese herbal medicine that has been approved in Japan as a remedy for neurosis, insomnia, and irritability in children. It has also been reported to improve behavioral and psychological symptoms in patients with various forms of dementia. Aim of the study: To evaluate the usefulness of co-treatment with an antidepressant and an herbal medicine in the psychiatric field, the current study examined the effect of yokukansan on the anxiolytic-like effect of fluvoxamine in mice. Materials and methods: The anxiolytic-like effect in mice was estimated by the contextual fear conditioning paradigm. Contextual fear conditioning consisted of two sessions, i.e., day 1 for the conditioning session and day 2 for the test session. The expression levels of 5-HT1A and 5-HT2A receptor in the mouse brain regions were quantified by western blot analysis. Results: A single administration of fluvoxamine (5–20 mg/kg, i.p.) before the test session dose-dependently and significantly suppressed freezing behavior in mice. In the combination study, a sub-effective dose of fluvoxamine (5 mg/kg, i.p.) significantly suppressed freezing behavior in mice that had been repeatedly pretreated with yokukansan (0.3 and 1 g/kg, p.o.) once a day for 6 days after the conditioning session. Western blot analysis revealed that the expression level of 5-HT2A receptor was specifically decreased in the prefrontal cortex of mice that had been administered yokukansan and fluvoxamine. Furthermore, microinjection of the 5-HT2A receptor antagonist ketanserin (5 nmol/mouse) into the prefrontal cortex significantly suppressed freezing behavior. Conclusion: The present findings indicate that repeated treatment with yokukansan synergistically enhances the anxiolytic-like effect of fluvoxamine in the contextual fear conditioning paradigm in mice in conjunction with a decrease in 5-HT2A receptor-mediated signaling in the prefrontal cortex. Therefore, combination therapy with fluvoxamine and yokukansan may be beneficial for the treatment of anxiety disorders.
Keywords: Yokukansan Fluvoxamine Contextual fear conditioning Anxiolytic effect 5-HT2A receptor Mouse
1. Introduction A growing body of evidence suggests that the brain serotonin (5-HT) nervous system plays a role in the mechanism of fear memory and the pathology of fear-related mental disorders (Bauer, 2015). Selective 5HT reuptake inhibitors (SSRIs), antidepressants that preferentially activate brain 5-HT neurotransmission, are clinically used for the treatment of these disorders, including obsessive-compulsive disorder (OCD) and post-traumatic stress disorder (PTSD) (Figgitt and McClellan, 2000). This clinical evidence indicates that SSRIs have not only antidepressant but also anxiolytic properties. It is well known that rodents
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that are re-exposed to the same environment where they had been previously exposed to aversive stimuli including inescapable foot-shock show freezing behavior, a response characterized by a period of crouching and complete immobility, and this behavior can be used as a model of anxiety caused by fear memory (Fanselow and Helmstetter, 1988). Furthermore, freezing behavior induced by contextual fear conditioning has been reported to typically be reduced by both acute and chronic treatment with SSRIs (Inoue et al., 2011), indicating that this animal model may be useful for evaluating the efficacy of SSRIs for the treatment of anxiety disorders. Yokukansan is a traditional Japanese herbal medicine, which has
Corresponding author. E-mail address: [email protected] (M. Tsuji). Contributed equally.
https://doi.org/10.1016/j.jep.2018.01.032 Received 14 October 2017; Received in revised form 26 January 2018; Accepted 26 January 2018 Available online 31 January 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved.
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was regulated strictly at a warehouse with low temperature (15 ℃) and low humidity (below 60%) before use. The amount of materials that is cut and mixed was based on the unit quantity of yokukansan formulation for production. Once cut, they are used immediately to prevent any deterioration. To manufacture the powdered water extract of yokukansan, approximately 500 kg of cut materials was decocted by purified 6000 L hot water. Next, a continuous centrifugal was used to separate the liquid extract from the residues of crude drugs. To concentrate the liquid extract, low-temperature concentration with a continuous film evaporator was performed. Then, the concentrated liquid that was sprayed from the top of the dryer was dried and cooled to obtain the powdered water extract. The same active ingredients derived from the herbal medicines in extract powders were also detected in standard solutions for the herbal medicines. The developed plates were either examined by spraying with a 4-dimethylaminobenzaldehyde reagent or dilute sulfuric acid, or irradiated with ultraviolet light. Upon comparison with the standard solutions for the herbal medicines, one spot among the spots from the yokukansan extract showed the same color tone and Rf value. In addition, the amounts of active ingredients such as glycyrrhizin, saikosaponin b2 and ferulic acid have been determined by high-performance liquid chromatography analysis and stable contents have been secured (see Supplementary Fig. 1). The chromatographic conditions for glycyrrhizin were column: a stainless steel column packed with octadecylsilanized silica gel for liquid chromatography, mobile phase: a mixture of H2O, CH3CN and CH3COOH, column temperature: a constant temperature of about 40 ℃, flow rate: 1.2 ml/min, detector: an ultraviolet absorption photometer (wavelength: 254 nm). The chromatographic conditions for saikosaponin b2 were column: a stainless steel column packed with octadecylsilanized silica gel for liquid chromatography, mobile phase: a mixture of H2O, MeOH and CH3CN, column temperature: a constant temperature of about 50 ℃, flow rate:1.0 ml/min, detector: an ultraviolet absorption photometer (wavelength: 254 nm). The chromatographic conditions for ferulic acid were column: a stainless steel column packed with octylsilanized silica gel for liquid chromatography, mobile phase: a mixture of H2O, CH3CN and (HCOO)2, column temperature: a constant temperature of about 25 ℃, flow rate: 1.2 ml/min, detector: an ultraviolet absorption photometer (wavelength: 320 nm). Manufacturing processes and quality are standardized based on the Good Manufacturing Practices defined by the Ministry of Health, Labor and Welfare of Japan. Fluvoxamine maleate, a selective serotonin reuptake inhibitor, was provided by Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). Ketanserin tartrate, a selective 5-HT2A receptor antagonist, was purchased from Sigma-Aldrich Co., Ltd. (MO, USA). Yokukansan and fluvoxamine were dissolved in purified water and saline, respectively. Ketanserin was prepared in a vehicle of saline to which a few drops of Tween 80 had been added. The dosage and injection route of drugs were decided based on previous reports (Miyamoto et al., 2000, 2004; Nunes-deSouza et al., 2008; Dobi et al., 2013; Tsuji et al., 2014).
been approved in Japan as a remedy for neurosis, insomnia, and irritability and night crying in children. Increased recent clinical evidence indicates that yokukansan is also effective and well-tolerated treatment for behavioral and psychological symptoms, such as excitement, aggression, hallucinations, insomnia, anxiety, wandering and depression, in patients with Alzheimer's disease and other forms of dementia when used clinically (Matsuda et al., 2013). Interestingly, an in vitro binding study demonstrated that yokukansan binds to the 5-HT1A receptors and acts as a partial agonist (Terawaki et al., 2010). Moreover, repeated administration of yokukansan increases and decreases 5-HT1A and 5HT2A receptor function in the prefrontal cortex, respectively (Egashira et al., 2008; Ueki et al., 2015a, 2015b). Previously, both agonism of 5HT1A receptor and antagonism of 5-HT2A receptor have been shown to have anxiolytic-like effects in animal models of anxiety (Motta et al., 1992; Hashimoto et al., 1996), although paradoxical findings have also been reported regarding the latter (León et al., 2017). These reports led us to speculate that yokukansan may have a beneficial effect on anxiety disorders. Indeed, a few studies reported that yokukansan showed inhibitory effects on anxiety-like behaviors induced by both innate fear and memory-dependent fear in rat (Mizoguchi et al., 2010; Yamaguchi et al., 2012; Shoji and Mizoguchi, 2013). Clinical evidence has indicated that there are treatment-resistant populations in patients suffering anxiety disorder (Patterson and Van Ameringen, 2016). Recently, complementary and integrative medicine is attracting attention for the purpose of enhancing the efficacy and/or reducing side effects of Western medicine. Thus, we expected that yokukansan might be beneficial for augmentation therapy in treatmentresistant anxiety disorders. As a basic research to prove this hypothesis, the present study investigated the effect of yokukansan on the anxiolytic-like effect of fluvoxamine as estimated by the contextual fear conditioning paradigm in mice. Furthermore, changes in the expression of 5-HT1A and 5-HT2A receptors in the brains of mice that had been subjected to the fear conditioning paradigm were also examined. 2. Material and methods 2.1. Animals Male ICR mice (Japan SLC Inc., Shizuoka, Japan) were housed at a room temperature of 23 ± 1 ℃ with a 12 h light-dark cycle (light on 7:00 a.m. to 7:00 p.m.). Food and water were available ad libitum. All experiments were carried out during the light period. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of the International University of Health and Welfare, which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 2.2. Drugs Yokukansan is composed of seven kinds of dried medicinal herbs: 4.0 g of Atractylodis lanceae Rhizoma (Atractylodes lancea De Candolle), 4.0 g of Poria (Wolfiporia cocos Ryvarden et Gilbertson (Poria cocos Wolf)), 3.0 g of Cnidii Rhizoma (Cnidium officinale Makino), 3.0 g of Uncariae Uncis cum Ramulus (Uncaria rhynchophylla Miquel), 3.0 g of Angelicae Radix (Angelica acutiloba Kitagawa), 2.0 g of Bupleuri Radix (Bupleurum falcatum Linné), and l.5 g of Glycyrrhizae Radix (Glycyrrhiza uralensis Fischer). These herbs are registered in the Pharmacopeia of Japan ver. 17. The powdered water extract of yokukansan used in the present study was manufactured according to the formulation previously reported (Mizukami et al., 2009; Terawaki et al., 2010; Tsuji et al., 2014; Nakatani et al., 2014) and supplied by Tsumura & Co. (Tokyo, Japan). Raw materials for crude drug of yokukansan that were delivered to Shenzhen Tsumura Medicine Co., Ltd. in China or to the Ishioka Processing & Quality Control Center in Japan were stored in a low-temperature warehouse. The storage condition of these clued drugs
2.3. Apparatus and procedure for the contextual fear conditioning paradigm For the experiments, we used a plastic box (20 × 18 × 30 cm high) with a stainless steel grid floor. Intermittent inescapable electric footshocks were delivered through the grid floor by an isolated shock generator (Muromachi Kikai, Co., Ltd., Japan). The contextual conditioned fear stress procedure was performed over 2 days in accordance with our previous reports (Miyamoto et al., 2000; Takeda et al., 2002) with a minor modification; i.e., a day for the conditioning session and a day for the test session. In the conditioning session, mice were placed in the box and subjected to 36 inescapable foot-shocks (intensity 1 mA, duration 1 s) at 1–10 s intervals. After the last foot-shock, mice were immediately returned to their home cage. Twenty-four hours or a week later, mice were used in the test session. In the test session, the mice were again placed in the same box without being exposed to foot-shocks, and the duration of freezing behavior was 90
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Fig. 3. (A) Experimental schedule. (B) Effect of repeated pretreatment with yokukansan alone on freezing behavior induced by contextual fear conditioning in mice. DW: distilled water, SAL: saline, YKS: yokukansan. Values are the mean ± SEM of 8–10 mice.
(10 ml/kg) was administered orally (p.o.) once a day for 6 days from the day after the conditioning session (Figs. 2A and 3A). Ketanserin (5 nmol/0.1 μL) or vehicle was microinjected into the medial prefrontal cortex 40 min prior to the start of the test session (Fig. 6A).
Fig. 1. (A) Experimental schedule. (B) Effects of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. SAL: saline, FLV: fluvoxamine. Values are the mean ± SEM of 20–22 mice. **p < .01 (Dunnett's multiple comparison test).
2.4. Surgery and microinjection After anesthesia (pentobarbital 80 mg/kg, i.p.), mice were placed in a stereotaxic apparatus with a mouse frame adaptor (David Kopf; Tujunga, CA, USA). Mice were implanted with a unilateral guide cannula (26 gauge) in the left hemisphere, which was then fixed to the skull with dental cement. The coordinates for the medial prefrontal cortex were as follows: 2.0 mm anterior to the bregma, 0.2 mm lateral to the midsagittal line, and 2.0 mm below the dura mater. Mice were randomly assigned to 2 groups, which were injected with either ketanserin or vehicle, and the behavioral experiments were carried out 5–7 days after surgery. Ketanserin (5 nmol) or vehicle was microinjected in a volume of 0.1 μL at a rate of 0.1 μL/min via an internal cannula (33 gauge), which extended the guide cannula by 0.5 mm. At the end of the experiments, mice were sacrificed under deep anesthesia with pentobarbital (80 mg/kg, i.p.). The brains were perfused through the heart with 4% paraformaldehyde solution, before being removed and fixed in 4% paraformaldehyde. The brains were cut into 100 µm coronal sections on a vibratome. The placement of the cannula tracks and tips in slices was examined by microscopy to identify animals with exact localization. 2.5. Western blot analysis Western blot analysis was performed as described previously (Miyagishi et al., 2012, 2017). Immediately after freezing behavior was recorded, mice were sacrificed by decapitation. Their brains were rapidly removed and the prefrontal cortex, amygdala and hippocampus were dissected on ice. The brain tissues were homogenized with icecold radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 8.0), 1% Triton and 5 mM EDTA using a homogenizer (PELLET PESTLES® Cordless Motor; Techno Chemical Co., Ltd., Tokyo, Japan). The homogenates were centrifuged at 6000 × g for 15 min at 4 °C, and then supernatants were collected and stored as test samples at −70 °C for future analysis. Protein
Fig. 2. (A) Experimental schedule. (B) Influence of repeated pretreatment with yokukansan on the effect of a sub-effective dose of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. DW: distilled water, SAL: saline, YKS: yokukansan, FLV: fluvoxamine. Values are the mean ± SEM of 10–11 mice. *p < .05 (Dunnett's multiple comparison test).
recorded for 360 s. The duration of freezing behavior was recorded by an activity-monitoring system (SUPER-MEX, Muromachi Kikai). Fluvoxamine (5, 10 and 20 mg/kg) or saline (10 ml/kg) was administered intraperitoneally (i.p.) 30 min prior to the start of the test session (Figs. 1A, 2A and 3A). Yokukansan (0.3 and 1 g/kg) or distilled water 91
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Quantitative results were expressed as the ratio of the band intensity of the protein of interest to the band intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Protein extracts were loaded on SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride paper (Millipore, Billerica, MA, USA). The membranes were blocked in blocking buffer containing 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.05% Tween 20%, and 5% skim milk for 1 h at room temperature and incubated with anti5-HT2A antibody (Santa Cruz Biotechnology, CA, USA; diluted 1:500) or anti-5-HT1A antibody (Abcam, UK; diluted 1:1000) overnight at 4 °C. The membranes were washed repeatedly in Tris-buffered saline (20 mM Tris–HCl (pH 7.6), 137 mM NaCl) containing 0.05% Tween 20, and then horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immunoresearch Laboratories, PA, USA) was added for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence (Santa Cruz Biotechnology), and scanned, optimized and analyzed by Chemi Doc XRS (Bio-Rad Laboratories, Hercules, CA, USA).
2.6. Statistical analysis All data are expressed as the mean ± S.E.M. Statistical significance was assessed by Student's t-test or one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's multiple comparison tests. Probability values of less than 0.05 were considered to indicate statistical significance.
Fig. 4. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT2A receptor in the prefrontal cortex (A), amygdala (B) and hippocampus (C). Photographs show representative western blots of 5-HT2A receptor. Graphs show the relative densities of bands. Quantitative data were normalized and expressed as a percentage of the expression of 5-HT2A receptor in the DW plus SAL group. DW: distilled water, SAL: saline, YKS: yokukansan, FLV: fluvoxamine. Values are the mean ± SEM of 9–11 mice. *p < .05 (Dunnett's multiple comparison test).
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3. Results 3.1. Effects of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice Fig. 1B shows the effects of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. A single administration of fluvoxamine 30 min prior to the test session dose-dependently suppressed freezing behavior, and significant effects were observed at 10 and 20 mg/kg. 3.2. Influence of repeated pretreatment with yokukansan on the effect of a sub-effective dose of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice Fig. 2B shows the influence of repeated pretreatment with yokukansan on the effect of a sub-effective dose of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. A single administration of fluvoxamine 30 min prior to the test session at a dose of 5 mg/kg tended to suppress freezing behavior, but this effect was not statistically significant. In contrast, this sub-effective dose of fluvoxamine significantly suppressed freezing behavior in mice that had been repeatedly pretreated with yokukansan at a dose of either 0.3 g/kg or 1 g/kg once a day for 6 days after the conditioning session. Fig. 5. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5HT1A receptor in the prefrontal cortex in mic. Photographs show representative western blots of 5-HT1A receptor. Graph shows the relative densities of bands. Quantitative data were normalized and expressed as a percentage of the expression of 5-HT1A receptor in the DW plus SAL group. DW: distilled water, SAL: saline, YKS: yokukansan, FLV: fluvoxamine. Values are the mean ± SEM of 9–11 mice.
3.3. Effect of repeated pretreatment with yokukansan alone on freezing behavior induced by contextual fear conditioning in mice Fig. 3B shows the effect of repeated pretreatment with yokukansan alone on freezing behavior induced by contextual fear conditioning in mice. Repeated administration of yokukansan at a dose of 1 g/kg once a day for 6 days after the conditioning session did not affect the freezing behavior induced by contextual fear conditioning.
4. Discussion In this study, we demonstrated that treatment with the selective 5HT reuptake inhibitor fluvoxamine before the test session in the contextual fear conditioning paradigm dose-dependently suppressed freezing behavior in mice (Fig. 1). This result was consistent with previous reports (Miyamoto et al., 2000, 2004; Nishikawa et al., 2007), suggesting that enhancing the availability of 5-HT in the brain by inhibiting the reuptake of 5-HT is beneficial for reducing anxiety. This supposition may be supported by clinical evidence that SSRIs are clinically used for the treatment of not only depressive but also anxiety disorders (Figgitt and McClellan, 2000). The neural circuitry of contextual fear conditioning primarily includes the dorsal hippocampus, medial prefrontal cortex and the amygdaloid complex (Bauer, 2015), and all of these structures contain dense concentrations of 5-HT1A and 5-HT2A receptors (Cornea-Hébert et al., 1999; Parsey et al., 2002). In general, 5-HT can have differential effects on behavior depending on the receptor subtypes present. The 5HT1A and 5-HT2A receptors may play an opposite role on contextual fear conditioning, since 5-HT1A and 5-HT2A receptor agonist have reported to decreases and increases the freezing behavior, respectively (Inoue et al., 2011; Zhang et al., 2013). Considering these reports, it is suggested that the anxiolytic-like effect of fluvoxamine may express through, at least in part, the activation of 5-HT1A receptors. In contrast, 5-HT2A receptors may act rather to attenuate the anxiolytic-like effect of fluvoxamine. In the combination study, yokukansan and fluvoxamine had synergistic effects on anxiety; i.e., a sub-effective dose of fluvoxamine significantly suppressed freezing behavior in mice that had been repeatedly pretreated with yokukansan once a day for 6 days after the conditioning session (Fig. 2). It has been reported that repeated treatment with yokukansan once a day for 14 days significantly suppressed the freezing behavior (Yamaguchi et al., 2012). In order to investigate
3.4. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT2A receptor in mouse brain regions Fig. 4 shows the effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT2A receptor in the prefrontal cortex, amygdala and hippocampus in mice. The brain regions were dissected from whole brains of mice upon completion of the fear conditioning experiments. Western blot analysis revealed that the expression level of 5-HT2A receptor was significantly decreased in the prefrontal cortex of mice that had been administered yokukansan and fluvoxamine. However, no changes in the expression of 5-HT2A receptor were observed in the amygdala or hippocampus. 3.5. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT1A receptor in the prefrontal cortex in mice Fig. 5 shows the effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT1A receptors in the prefrontal cortex in mice. There were no significant differences in the expression level of 5-HT1A receptors in the prefrontal cortex among any of the groups. 3.6. Effects of microinjection of ketanserin into the medial prefrontal cortex on freezing behavior induced by contextual fear conditioning in mice Fig. 6C shows the effects of microinjection of ketanserin, a 5-HT2A receptor antagonist, into the medial prefrontal cortex on freezing behavior induced by contextual fear conditioning in mice. A single administration of ketanserin at a dose of 5 nmol 40 min prior to the test session significantly suppressed freezing behavior. 93
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Fig. 6. (A) Experimental schedule. (B) Schematic diagram of microinjection sites within the prefrontal cortex of mice used in the present study. The injection sites are indicated by asterisks. (C) Effects of microinjection of ketanserin into the medial prefrontal cortex on freezing behavior induced by contextual fear conditioning in mice. V: vehicle, KTS: ketanserin. Values are the mean ± SEM of 7 mice. *p < .05 (Student's t-test).
the synergistic effect of fluvoxamine and yokukansan, the present study set treatment period under which yokukansan alone will not develop a significant anxiolytic-like effect (Fig. 3). A growing body of evidence has shown that yokukansan improves behavioral and psychological symptoms, such as excitement, aggression, hallucinations, insomnia, anxiety, wandering and depression, in patients with Alzheimer's disease and other forms of dementia, without severe adverse effects (Matsuda et al., 2013). Recently, it has also been reported that yokukansan alleviates preoperative anxiety without undesirable sedation, compared to diazepam (Arai et al., 2014; Wada et al., 2017). These clinical effects are supported by the finding in preclinical studies that yokukansan reduces anxiety in animal models (Mizoguchi et al., 2010; Yamaguchi et al., 2012; Shoji and Mizoguchi, 2013). In addition to these previous reports, the present findings provide the new insight that yokukansan may be effective for enhancing the effect of fluvoxamine in the treatment of anxiety disorders. There are now believed to be seven kinds of 5-HT receptor families, 5-HT1–7, that are comprised of 14 structurally and pharmacologically distinct 5-HT receptor subtypes (Hoyer et al., 2002). Among them, the 5-HT1 and 5-HT2 receptor subtypes have received particular attention due to their implication in emotional modulation and in several psychiatric disorders. Indeed, 5-HT1A receptor agonist as well as 5-HT2 receptor antagonist are known to be effective in the treatment of anxiety and depression (Jones and Blackburn, 2002). Therefore, the
present study tried to clarify the changes in the expression levels of 5HT1A and 5-HT2 receptors in brain regions of mice that had been subjected to the fear conditioning paradigm. The results showed that the expression level of 5-HT2A receptor is specifically decreased in the prefrontal cortex of mice that had been administered yokukansan and fluvoxamine (Fig. 4). This finding is in good agreement with a previous report (Egashira et al., 2008). Another report indicated that isolation stress significantly increased 5-HT2A receptor density in the prefrontal cortex, and repeated treatment with yokukansan significantly downregulated this increase (Ueki et al., 2015b). Thus, yokukansan may attenuate the function of cortical 5-HT2A receptor. These findings may explain the mechanism of the effectiveness of yokukansan for psychiatric disorders, since it has been reported that cortical 5-HT2A receptor may play a critical role in the expression of anxiety (Aznar and Klein, 2013). For example, cortical 5-HT2A receptors have been shown to play a role in modulating the anxiety response in a transgenic mouse model, where selective restoration of 5-HT2A receptor signaling in the cortex normalized conflict anxiety-like behavior in 5-HT2A receptor knockout (htr2a-/-) mice (Weisstaub et al., 2006). In contrast, a recent behavioral pharmacological study demonstrated that microinjection of ketanserin, a selective 5-HT2A receptor antagonist, into the infralimbic and prelimbic cortices had anxiolytic-like effects in the elevated plusmaze test and a contextual fear conditioning paradigm (León et al., 2017). The present study confirmed this report in that the freezing 94
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Author contributions
behavior of mice induced by contextual fear conditioning was suppressed by the microinjection of ketanserin into the medial prefrontal cortex before the test session (Fig. 6). The blockade of 5-HT2A receptor may make it difficult for 5-HT to bind to the 5-HT2A receptor, and may also create an environment amenable to other receptor subtypes that are important for the expression of an anxiolytic effect such as 5-HT1A receptor. Taken together, the present findings, along with previous reports, suggest that reduction of cortical 5-HT2A receptor-mediated signaling may be involved, at least in part, in the enhancement of the anxiolytic-like effect of fluvoxamine by repeated pretreatment with yokukansan. Additionally, it has been reported that the downregulation of 5-HT2A receptors is suggested to be induced by a synergistic effect of Bupleuri Radix, Uncariae Uncis cum Ramulus, Angelicae Radix and Glycyrrhizae Radix among the seven constituent components of yokukansan (Ueki et al., 2015b). Thus, these multiple herbal medicine containing a large number of ingredients may be responsible for the synergistic effects of yokukansan and fluvoxamine on anxiety. In contrast to 5-HT2A receptor, no significant changes in the expression of cortical 5-HT1A receptor were observed in the present study in mice that had been repeatedly treated with yokukansan (Fig. 5). However, it has been reported that repeated treatment of yokukansan did not change protein or mRNA levels of 5-HT1A receptor in the prefrontal cortex of mice, but significantly increased the density of receptor as detected by a receptor binding assay (Ueki et al., 2015a). Furthermore, enhancement of the 8-OH-DPAT-induced decrease in rearing behavior of mice, a behavior that is considered to reflect the function of postsynaptic 5-HT1A receptor in the cerebral cortex (Bert et al., 2006), was also observed under repeated treatment with yokukansan, suggesting enhancement of the function of cortical 5-HT1A receptor (Ueki et al., 2015a). Otherwise, an in vitro binding assay revealed that yokukansan had a partial agonistic effect on 5-HT1A receptor (Terawaki et al., 2010), and geissoschizine methyl ether, an indole alkaloid derived from Uncariae Uncis cum Ramulus, has been identified as the active ingredient responsible for the partial agonism of 5-HT1A receptors by yokukansan (Nishi et al., 2012). Additionally, there is a report suggesting that feurlic acid, an active ingredient contained in Cnidii Rhizoma, also possesses agonist activity to 5-HT1A receptors (Zeni et al., 2012). It is well known that activation of 5-HT1A receptor consistently reduces freezing behavior of mice subjected to a fear conditioning paradigm (Inoue et al., 2011). Therefore, it is also possible that yokukansan may positively affect cortical 5-HT1A receptor-mediated signaling and enhance the anxiolytic-like effect of fluvoxamine. Further detailed studies will be needed to evaluate this hypothesis.
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EInhibitory effect of yokukansan on the decrease in the hippocampal excitatory amino acid transporter EAAT2 in stress-maladaptive mice. J. Tradit. Complement. Med. 2017 (7), 371–374. Miyamoto, J., Tsuji, M., Takeda, H., Nawa, H., Matsumiya, T., 2000. Pretreatment with diazepam suppresses the reduction in defensive freezing behavior induced by fluvoxamine in the conditioned fear stress paradigm in mice. Eur. J. Pharmacol. 409, 81–84. Miyamoto, J., Tsuji, M., Takeda, H., Ohzeki, M., Nawa, H., Matsumiya, T., 2004. Characterization of the anxiolytic-like effects of fluvoxamine, milnacipran and risperidone in mice using the conditioned fear stress paradigm. Eur. J. Pharmacol. 504, 97–103. Mizoguchi, K., Tanaka, Y., Tabira, T., 2010. Anxiolytic effect of a herbal medicine, yokukansan, in aged rats: involvement of serotonergic and dopaminergic transmissions in the prefrontal cortex. J. Ethnopharmacol. 127, 70–76. Mizukami, K., Asada, T., Kinoshita, T., Tanaka, K., Sonohara, K., Nakai, R., Yamaguchi, K., Hanyu, H., Kanaya, K., Takao, T., Okada, M., Kudo, S., Kotoku, H., Iwakiri, M., Kurita, H., Miyamura, T., Kawasaki, Y., Omori, K., Shiozaki, K., Odawara, T., Suzuki, T., Yamada, S., Nakamura, Y., Toba, K., 2009. A randomized cross-over study of a traditional Japanese medicine (kampo), yokukansan, in the treatment of the behavioural and psychological symptoms of dementia. Int. J. Neuropsychopharmacol. 12, 191–199. Motta, V., Maisonnette, S., Morato, S., Castrechini, P., Brandão, M.L., 1992. Effects of
5. Conclusion The present study found that repeated treatment with yokukansan synergistically enhances the anxiolytic-like effect of fluvoxamine in the contextual fear conditioning paradigm in mice in conjunction with a decrease in 5-HT2A receptor-mediated signaling in the prefrontal cortex. Therefore, the combination of yokukansan and fluvoxamine may prove useful as a complementary and integrative medicine for augmentation therapy in treatment-resistant anxiety disorders.
Acknowledgements We would like to thank Tsumura & Co. (Tokyo, Japan) for kindly supply the extract of yokukansan and its component analysis data.
Conflict of interest The authors declare that they have no conflicts of interest to disclose. 95
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Tsuji, M., Takeuchi, T., Miyagawa, K., Ishii, D., Imai, T., Takeda, K., Kitajima, M., Takeda, H., 2014. Yokukansan, a traditional Japanese herbal medicine, alleviates the emotional abnormality induced by maladaptation to stress in mice. Phytomedicine 21, 363–371. Ueki, T., Mizoguchi, K., Yamaguchi, T., Nishi, A., Ikarashi, Y., Hattori, T., Kase, Y., 2015a. Yokukansan increases 5-HT1A receptors in the prefrontal cortex and enhances 5-HT1A receptor agonist-induced behavioral responses in socially isolated mice. Evid. Based Complement. Altern. Med. 2015, 726471. Ueki, T., Mizoguchi, K., Yamaguchi, T., Nishi, A., Sekiguchi, K., Ikarashi, Y., Kase, Y., 2015b. Yokukansan, a traditional Japanese medicine, decreases head-twitch behaviors and serotonin 2A receptors in the prefrontal cortex of isolation-stressed mice. J. Ethnopharmacol. 166, 23–30. Yamaguchi, T., Tsujimatsu, A., Kumamoto, H., Izumi, T., Ohmura, Y., Yoshida, T., Yoshioka, M., 2012. Anxiolytic effects of yokukansan, a traditional Japanese medicine, via serotonin 5-HT1A receptors on anxiety-related behaviors in rats experienced aversive stress. J. Ethnopharmacol. 143, 533–539. Wada, S., Inoguchi, H., Hirayama, T., Matsuoka, Y.J., Uchitomi, Y., Ochiai, H., Tsukamoto, S., Shida, D., Kanemitsu, Y., Shimizu, K., 2017. Yokukansan for the treatment of preoperative anxiety and postoperative delirium in colorectal cancer patients: a retrospective study. Jpn. J. Clin. Oncol. 47, 844–848. Weisstaub, N.V., Zhou, M., Lira, A., Lambe, E., González-Maeso, J., Hornung, J.P., Sibille, E., Underwood, M., Itohara, S., Dauer, W.T., Ansorge, M.S., Morelli, E., Mann, J.J., Toth, M., Aghajanian, G., Sealfon, S.C., Hen, R., Gingrich, J.A., Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice. Science 313, 536–540, 2006. Zeni, A.L., Zomkowski, A.D., Maraschin, M., Rodrigues, A.L., Tasca, C.I., 2012. Ferulic acid exerts antidepressant-like effect in the tail suspension test in mice: evidence for the involvement of the serotonergic system. Eur. J. Pharmacol. 679, 68–74. Zhang, G., Ásgeirsdóttir, H.N., Cohen, S.J., Munchow, A.H., Barrera, M.P., Stackman Jr., R.W., 2013. Stimulation of serotonin 2A receptors facilitates consolidation and extinction of fear memory in C57BL/6J mice. Neuropharmacology 64 (403-113).
blockade of 5-HT2 receptors and activation of 5-HT1A receptors on the exploratory activity of rats in the elevated plus-maze. Psychopharmacology 107, 135–139. Nakatani, Y., Tsuji, M., Amano, T., Miyagawa, K., Miyagishi, H., Saito, A., Imai, T., Takeda, K., Ishii, D., Takeda, H., 2014. Neuroprotective effect of yokukansan against cytotoxicity induced bycorticosterone on mouse hippocampal neurons. Phytomedicine 21, 1458–1465. Nishi, A., Yamaguchi, T., Sekiguchi, K., Imamura, S., Tabuchi, M., Kanno, H., Nakai, Y., Hashimoto, K., Ikarashi, Y., Kase, Y., 2012. Geissoschizine methyl ether, an alkaloid in Uncaria hook, is a potent serotonin1A receptor agonist and candidate for amelioration of aggressiveness and sociality by yokukansan. Neuroscience 207, 124–136. Nishikawa, H., Inoue, T., Izumi, T., Koyama, T., 2007. Synergistic effects of tandospirone and selective serotonin reuptake inhibitors on the contextual conditioned fear stress response in rats. Eur. Neuropsychopharmacol. 17, 643–650. Nunes-de-Souza, V., Nunes-de-Souza, R.L., Rodgers, R.J., Canto-de-Souza, A., 2008. 5HT2 receptor activation in the midbrain periaqueductal grey (PAG) reduces anxietylike behaviour in mice. Behav. Brain Res. 187, 72–79. Parsey, R.V., Oquendo, M.A., Simpson, N.R., Ogden, R.T., Van Heertum, R., Arango, V., Mann, J.J., 2002. Effects of sex, age, and aggressive traits in man on brain serotonin 5-HT1A receptor binding potential measured by PET using [C-11]WAY-100635. Brain Res. 954, 173–182. Patterson, B., Van Ameringen, M., 2016. Augmentation strategies for treatment-resistant anxiety disorders: a systematic review and meta-analysis. Depress. Anxiety 33 (728376). Shoji, H., Mizoguchi, K., 2013. Brain region-specific reduction in c-Fos expression associated with an anxiolytic effect of yokukansan in rats. J. Ethnopharmacol. 149, 93–102. Takeda, H., Tsuji, M., Miyamoto, J., Matsumiya, T., 2002. Rosmarinic acid and caffeic acid reduce the defensive freezing behavior of mice exposed to conditioned fear stress. Psychopharmacology 164, 233–235. Terawaki, K., Ikarashi, Y., Sekiguchi, K., Nakai, Y., Kase, Y., 2010. Partial agonistic effect of yokukansan on human recombinant serotonin 1A receptors expressed in the membranes of Chinese hamster ovary cells. J. Ethnopharmacol. 127, 306–312.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Yokukansan, a traditional Japanese herbal medicine, enhances the anxiolytic effect of fluvoxamine and reduces cortical 5-HT2A receptor expression in mice
T
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Rintaro Ohno1, Hiroko Miyagishi1, Minoru Tsuji , Atsumi Saito, Kazuya Miyagawa, Kazuhiro Kurokawa, Hiroshi Takeda Department of Pharmacology, School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara, Tochigi 324-8501, Japan
A R T I C L E I N F O
A B S T R A C T
Chemical compounds: Fluvoxamine maleate (PubChem CID: 9560989) Ketanserin tartrate (PubChem CID: 16219944)
Ethnopharmacological relevance: Yokukansan is a traditional Japanese herbal medicine that has been approved in Japan as a remedy for neurosis, insomnia, and irritability in children. It has also been reported to improve behavioral and psychological symptoms in patients with various forms of dementia. Aim of the study: To evaluate the usefulness of co-treatment with an antidepressant and an herbal medicine in the psychiatric field, the current study examined the effect of yokukansan on the anxiolytic-like effect of fluvoxamine in mice. Materials and methods: The anxiolytic-like effect in mice was estimated by the contextual fear conditioning paradigm. Contextual fear conditioning consisted of two sessions, i.e., day 1 for the conditioning session and day 2 for the test session. The expression levels of 5-HT1A and 5-HT2A receptor in the mouse brain regions were quantified by western blot analysis. Results: A single administration of fluvoxamine (5–20 mg/kg, i.p.) before the test session dose-dependently and significantly suppressed freezing behavior in mice. In the combination study, a sub-effective dose of fluvoxamine (5 mg/kg, i.p.) significantly suppressed freezing behavior in mice that had been repeatedly pretreated with yokukansan (0.3 and 1 g/kg, p.o.) once a day for 6 days after the conditioning session. Western blot analysis revealed that the expression level of 5-HT2A receptor was specifically decreased in the prefrontal cortex of mice that had been administered yokukansan and fluvoxamine. Furthermore, microinjection of the 5-HT2A receptor antagonist ketanserin (5 nmol/mouse) into the prefrontal cortex significantly suppressed freezing behavior. Conclusion: The present findings indicate that repeated treatment with yokukansan synergistically enhances the anxiolytic-like effect of fluvoxamine in the contextual fear conditioning paradigm in mice in conjunction with a decrease in 5-HT2A receptor-mediated signaling in the prefrontal cortex. Therefore, combination therapy with fluvoxamine and yokukansan may be beneficial for the treatment of anxiety disorders.
Keywords: Yokukansan Fluvoxamine Contextual fear conditioning Anxiolytic effect 5-HT2A receptor Mouse
1. Introduction A growing body of evidence suggests that the brain serotonin (5-HT) nervous system plays a role in the mechanism of fear memory and the pathology of fear-related mental disorders (Bauer, 2015). Selective 5HT reuptake inhibitors (SSRIs), antidepressants that preferentially activate brain 5-HT neurotransmission, are clinically used for the treatment of these disorders, including obsessive-compulsive disorder (OCD) and post-traumatic stress disorder (PTSD) (Figgitt and McClellan, 2000). This clinical evidence indicates that SSRIs have not only antidepressant but also anxiolytic properties. It is well known that rodents
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that are re-exposed to the same environment where they had been previously exposed to aversive stimuli including inescapable foot-shock show freezing behavior, a response characterized by a period of crouching and complete immobility, and this behavior can be used as a model of anxiety caused by fear memory (Fanselow and Helmstetter, 1988). Furthermore, freezing behavior induced by contextual fear conditioning has been reported to typically be reduced by both acute and chronic treatment with SSRIs (Inoue et al., 2011), indicating that this animal model may be useful for evaluating the efficacy of SSRIs for the treatment of anxiety disorders. Yokukansan is a traditional Japanese herbal medicine, which has
Corresponding author. E-mail address: [email protected] (M. Tsuji). Contributed equally.
https://doi.org/10.1016/j.jep.2018.01.032 Received 14 October 2017; Received in revised form 26 January 2018; Accepted 26 January 2018 Available online 31 January 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved.
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was regulated strictly at a warehouse with low temperature (15 ℃) and low humidity (below 60%) before use. The amount of materials that is cut and mixed was based on the unit quantity of yokukansan formulation for production. Once cut, they are used immediately to prevent any deterioration. To manufacture the powdered water extract of yokukansan, approximately 500 kg of cut materials was decocted by purified 6000 L hot water. Next, a continuous centrifugal was used to separate the liquid extract from the residues of crude drugs. To concentrate the liquid extract, low-temperature concentration with a continuous film evaporator was performed. Then, the concentrated liquid that was sprayed from the top of the dryer was dried and cooled to obtain the powdered water extract. The same active ingredients derived from the herbal medicines in extract powders were also detected in standard solutions for the herbal medicines. The developed plates were either examined by spraying with a 4-dimethylaminobenzaldehyde reagent or dilute sulfuric acid, or irradiated with ultraviolet light. Upon comparison with the standard solutions for the herbal medicines, one spot among the spots from the yokukansan extract showed the same color tone and Rf value. In addition, the amounts of active ingredients such as glycyrrhizin, saikosaponin b2 and ferulic acid have been determined by high-performance liquid chromatography analysis and stable contents have been secured (see Supplementary Fig. 1). The chromatographic conditions for glycyrrhizin were column: a stainless steel column packed with octadecylsilanized silica gel for liquid chromatography, mobile phase: a mixture of H2O, CH3CN and CH3COOH, column temperature: a constant temperature of about 40 ℃, flow rate: 1.2 ml/min, detector: an ultraviolet absorption photometer (wavelength: 254 nm). The chromatographic conditions for saikosaponin b2 were column: a stainless steel column packed with octadecylsilanized silica gel for liquid chromatography, mobile phase: a mixture of H2O, MeOH and CH3CN, column temperature: a constant temperature of about 50 ℃, flow rate:1.0 ml/min, detector: an ultraviolet absorption photometer (wavelength: 254 nm). The chromatographic conditions for ferulic acid were column: a stainless steel column packed with octylsilanized silica gel for liquid chromatography, mobile phase: a mixture of H2O, CH3CN and (HCOO)2, column temperature: a constant temperature of about 25 ℃, flow rate: 1.2 ml/min, detector: an ultraviolet absorption photometer (wavelength: 320 nm). Manufacturing processes and quality are standardized based on the Good Manufacturing Practices defined by the Ministry of Health, Labor and Welfare of Japan. Fluvoxamine maleate, a selective serotonin reuptake inhibitor, was provided by Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). Ketanserin tartrate, a selective 5-HT2A receptor antagonist, was purchased from Sigma-Aldrich Co., Ltd. (MO, USA). Yokukansan and fluvoxamine were dissolved in purified water and saline, respectively. Ketanserin was prepared in a vehicle of saline to which a few drops of Tween 80 had been added. The dosage and injection route of drugs were decided based on previous reports (Miyamoto et al., 2000, 2004; Nunes-deSouza et al., 2008; Dobi et al., 2013; Tsuji et al., 2014).
been approved in Japan as a remedy for neurosis, insomnia, and irritability and night crying in children. Increased recent clinical evidence indicates that yokukansan is also effective and well-tolerated treatment for behavioral and psychological symptoms, such as excitement, aggression, hallucinations, insomnia, anxiety, wandering and depression, in patients with Alzheimer's disease and other forms of dementia when used clinically (Matsuda et al., 2013). Interestingly, an in vitro binding study demonstrated that yokukansan binds to the 5-HT1A receptors and acts as a partial agonist (Terawaki et al., 2010). Moreover, repeated administration of yokukansan increases and decreases 5-HT1A and 5HT2A receptor function in the prefrontal cortex, respectively (Egashira et al., 2008; Ueki et al., 2015a, 2015b). Previously, both agonism of 5HT1A receptor and antagonism of 5-HT2A receptor have been shown to have anxiolytic-like effects in animal models of anxiety (Motta et al., 1992; Hashimoto et al., 1996), although paradoxical findings have also been reported regarding the latter (León et al., 2017). These reports led us to speculate that yokukansan may have a beneficial effect on anxiety disorders. Indeed, a few studies reported that yokukansan showed inhibitory effects on anxiety-like behaviors induced by both innate fear and memory-dependent fear in rat (Mizoguchi et al., 2010; Yamaguchi et al., 2012; Shoji and Mizoguchi, 2013). Clinical evidence has indicated that there are treatment-resistant populations in patients suffering anxiety disorder (Patterson and Van Ameringen, 2016). Recently, complementary and integrative medicine is attracting attention for the purpose of enhancing the efficacy and/or reducing side effects of Western medicine. Thus, we expected that yokukansan might be beneficial for augmentation therapy in treatmentresistant anxiety disorders. As a basic research to prove this hypothesis, the present study investigated the effect of yokukansan on the anxiolytic-like effect of fluvoxamine as estimated by the contextual fear conditioning paradigm in mice. Furthermore, changes in the expression of 5-HT1A and 5-HT2A receptors in the brains of mice that had been subjected to the fear conditioning paradigm were also examined. 2. Material and methods 2.1. Animals Male ICR mice (Japan SLC Inc., Shizuoka, Japan) were housed at a room temperature of 23 ± 1 ℃ with a 12 h light-dark cycle (light on 7:00 a.m. to 7:00 p.m.). Food and water were available ad libitum. All experiments were carried out during the light period. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of the International University of Health and Welfare, which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 2.2. Drugs Yokukansan is composed of seven kinds of dried medicinal herbs: 4.0 g of Atractylodis lanceae Rhizoma (Atractylodes lancea De Candolle), 4.0 g of Poria (Wolfiporia cocos Ryvarden et Gilbertson (Poria cocos Wolf)), 3.0 g of Cnidii Rhizoma (Cnidium officinale Makino), 3.0 g of Uncariae Uncis cum Ramulus (Uncaria rhynchophylla Miquel), 3.0 g of Angelicae Radix (Angelica acutiloba Kitagawa), 2.0 g of Bupleuri Radix (Bupleurum falcatum Linné), and l.5 g of Glycyrrhizae Radix (Glycyrrhiza uralensis Fischer). These herbs are registered in the Pharmacopeia of Japan ver. 17. The powdered water extract of yokukansan used in the present study was manufactured according to the formulation previously reported (Mizukami et al., 2009; Terawaki et al., 2010; Tsuji et al., 2014; Nakatani et al., 2014) and supplied by Tsumura & Co. (Tokyo, Japan). Raw materials for crude drug of yokukansan that were delivered to Shenzhen Tsumura Medicine Co., Ltd. in China or to the Ishioka Processing & Quality Control Center in Japan were stored in a low-temperature warehouse. The storage condition of these clued drugs
2.3. Apparatus and procedure for the contextual fear conditioning paradigm For the experiments, we used a plastic box (20 × 18 × 30 cm high) with a stainless steel grid floor. Intermittent inescapable electric footshocks were delivered through the grid floor by an isolated shock generator (Muromachi Kikai, Co., Ltd., Japan). The contextual conditioned fear stress procedure was performed over 2 days in accordance with our previous reports (Miyamoto et al., 2000; Takeda et al., 2002) with a minor modification; i.e., a day for the conditioning session and a day for the test session. In the conditioning session, mice were placed in the box and subjected to 36 inescapable foot-shocks (intensity 1 mA, duration 1 s) at 1–10 s intervals. After the last foot-shock, mice were immediately returned to their home cage. Twenty-four hours or a week later, mice were used in the test session. In the test session, the mice were again placed in the same box without being exposed to foot-shocks, and the duration of freezing behavior was 90
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Fig. 3. (A) Experimental schedule. (B) Effect of repeated pretreatment with yokukansan alone on freezing behavior induced by contextual fear conditioning in mice. DW: distilled water, SAL: saline, YKS: yokukansan. Values are the mean ± SEM of 8–10 mice.
(10 ml/kg) was administered orally (p.o.) once a day for 6 days from the day after the conditioning session (Figs. 2A and 3A). Ketanserin (5 nmol/0.1 μL) or vehicle was microinjected into the medial prefrontal cortex 40 min prior to the start of the test session (Fig. 6A).
Fig. 1. (A) Experimental schedule. (B) Effects of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. SAL: saline, FLV: fluvoxamine. Values are the mean ± SEM of 20–22 mice. **p < .01 (Dunnett's multiple comparison test).
2.4. Surgery and microinjection After anesthesia (pentobarbital 80 mg/kg, i.p.), mice were placed in a stereotaxic apparatus with a mouse frame adaptor (David Kopf; Tujunga, CA, USA). Mice were implanted with a unilateral guide cannula (26 gauge) in the left hemisphere, which was then fixed to the skull with dental cement. The coordinates for the medial prefrontal cortex were as follows: 2.0 mm anterior to the bregma, 0.2 mm lateral to the midsagittal line, and 2.0 mm below the dura mater. Mice were randomly assigned to 2 groups, which were injected with either ketanserin or vehicle, and the behavioral experiments were carried out 5–7 days after surgery. Ketanserin (5 nmol) or vehicle was microinjected in a volume of 0.1 μL at a rate of 0.1 μL/min via an internal cannula (33 gauge), which extended the guide cannula by 0.5 mm. At the end of the experiments, mice were sacrificed under deep anesthesia with pentobarbital (80 mg/kg, i.p.). The brains were perfused through the heart with 4% paraformaldehyde solution, before being removed and fixed in 4% paraformaldehyde. The brains were cut into 100 µm coronal sections on a vibratome. The placement of the cannula tracks and tips in slices was examined by microscopy to identify animals with exact localization. 2.5. Western blot analysis Western blot analysis was performed as described previously (Miyagishi et al., 2012, 2017). Immediately after freezing behavior was recorded, mice were sacrificed by decapitation. Their brains were rapidly removed and the prefrontal cortex, amygdala and hippocampus were dissected on ice. The brain tissues were homogenized with icecold radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 8.0), 1% Triton and 5 mM EDTA using a homogenizer (PELLET PESTLES® Cordless Motor; Techno Chemical Co., Ltd., Tokyo, Japan). The homogenates were centrifuged at 6000 × g for 15 min at 4 °C, and then supernatants were collected and stored as test samples at −70 °C for future analysis. Protein
Fig. 2. (A) Experimental schedule. (B) Influence of repeated pretreatment with yokukansan on the effect of a sub-effective dose of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. DW: distilled water, SAL: saline, YKS: yokukansan, FLV: fluvoxamine. Values are the mean ± SEM of 10–11 mice. *p < .05 (Dunnett's multiple comparison test).
recorded for 360 s. The duration of freezing behavior was recorded by an activity-monitoring system (SUPER-MEX, Muromachi Kikai). Fluvoxamine (5, 10 and 20 mg/kg) or saline (10 ml/kg) was administered intraperitoneally (i.p.) 30 min prior to the start of the test session (Figs. 1A, 2A and 3A). Yokukansan (0.3 and 1 g/kg) or distilled water 91
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Quantitative results were expressed as the ratio of the band intensity of the protein of interest to the band intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Protein extracts were loaded on SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride paper (Millipore, Billerica, MA, USA). The membranes were blocked in blocking buffer containing 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.05% Tween 20%, and 5% skim milk for 1 h at room temperature and incubated with anti5-HT2A antibody (Santa Cruz Biotechnology, CA, USA; diluted 1:500) or anti-5-HT1A antibody (Abcam, UK; diluted 1:1000) overnight at 4 °C. The membranes were washed repeatedly in Tris-buffered saline (20 mM Tris–HCl (pH 7.6), 137 mM NaCl) containing 0.05% Tween 20, and then horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immunoresearch Laboratories, PA, USA) was added for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence (Santa Cruz Biotechnology), and scanned, optimized and analyzed by Chemi Doc XRS (Bio-Rad Laboratories, Hercules, CA, USA).
2.6. Statistical analysis All data are expressed as the mean ± S.E.M. Statistical significance was assessed by Student's t-test or one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's multiple comparison tests. Probability values of less than 0.05 were considered to indicate statistical significance.
Fig. 4. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT2A receptor in the prefrontal cortex (A), amygdala (B) and hippocampus (C). Photographs show representative western blots of 5-HT2A receptor. Graphs show the relative densities of bands. Quantitative data were normalized and expressed as a percentage of the expression of 5-HT2A receptor in the DW plus SAL group. DW: distilled water, SAL: saline, YKS: yokukansan, FLV: fluvoxamine. Values are the mean ± SEM of 9–11 mice. *p < .05 (Dunnett's multiple comparison test).
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3. Results 3.1. Effects of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice Fig. 1B shows the effects of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. A single administration of fluvoxamine 30 min prior to the test session dose-dependently suppressed freezing behavior, and significant effects were observed at 10 and 20 mg/kg. 3.2. Influence of repeated pretreatment with yokukansan on the effect of a sub-effective dose of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice Fig. 2B shows the influence of repeated pretreatment with yokukansan on the effect of a sub-effective dose of fluvoxamine on freezing behavior induced by contextual fear conditioning in mice. A single administration of fluvoxamine 30 min prior to the test session at a dose of 5 mg/kg tended to suppress freezing behavior, but this effect was not statistically significant. In contrast, this sub-effective dose of fluvoxamine significantly suppressed freezing behavior in mice that had been repeatedly pretreated with yokukansan at a dose of either 0.3 g/kg or 1 g/kg once a day for 6 days after the conditioning session. Fig. 5. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5HT1A receptor in the prefrontal cortex in mic. Photographs show representative western blots of 5-HT1A receptor. Graph shows the relative densities of bands. Quantitative data were normalized and expressed as a percentage of the expression of 5-HT1A receptor in the DW plus SAL group. DW: distilled water, SAL: saline, YKS: yokukansan, FLV: fluvoxamine. Values are the mean ± SEM of 9–11 mice.
3.3. Effect of repeated pretreatment with yokukansan alone on freezing behavior induced by contextual fear conditioning in mice Fig. 3B shows the effect of repeated pretreatment with yokukansan alone on freezing behavior induced by contextual fear conditioning in mice. Repeated administration of yokukansan at a dose of 1 g/kg once a day for 6 days after the conditioning session did not affect the freezing behavior induced by contextual fear conditioning.
4. Discussion In this study, we demonstrated that treatment with the selective 5HT reuptake inhibitor fluvoxamine before the test session in the contextual fear conditioning paradigm dose-dependently suppressed freezing behavior in mice (Fig. 1). This result was consistent with previous reports (Miyamoto et al., 2000, 2004; Nishikawa et al., 2007), suggesting that enhancing the availability of 5-HT in the brain by inhibiting the reuptake of 5-HT is beneficial for reducing anxiety. This supposition may be supported by clinical evidence that SSRIs are clinically used for the treatment of not only depressive but also anxiety disorders (Figgitt and McClellan, 2000). The neural circuitry of contextual fear conditioning primarily includes the dorsal hippocampus, medial prefrontal cortex and the amygdaloid complex (Bauer, 2015), and all of these structures contain dense concentrations of 5-HT1A and 5-HT2A receptors (Cornea-Hébert et al., 1999; Parsey et al., 2002). In general, 5-HT can have differential effects on behavior depending on the receptor subtypes present. The 5HT1A and 5-HT2A receptors may play an opposite role on contextual fear conditioning, since 5-HT1A and 5-HT2A receptor agonist have reported to decreases and increases the freezing behavior, respectively (Inoue et al., 2011; Zhang et al., 2013). Considering these reports, it is suggested that the anxiolytic-like effect of fluvoxamine may express through, at least in part, the activation of 5-HT1A receptors. In contrast, 5-HT2A receptors may act rather to attenuate the anxiolytic-like effect of fluvoxamine. In the combination study, yokukansan and fluvoxamine had synergistic effects on anxiety; i.e., a sub-effective dose of fluvoxamine significantly suppressed freezing behavior in mice that had been repeatedly pretreated with yokukansan once a day for 6 days after the conditioning session (Fig. 2). It has been reported that repeated treatment with yokukansan once a day for 14 days significantly suppressed the freezing behavior (Yamaguchi et al., 2012). In order to investigate
3.4. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT2A receptor in mouse brain regions Fig. 4 shows the effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT2A receptor in the prefrontal cortex, amygdala and hippocampus in mice. The brain regions were dissected from whole brains of mice upon completion of the fear conditioning experiments. Western blot analysis revealed that the expression level of 5-HT2A receptor was significantly decreased in the prefrontal cortex of mice that had been administered yokukansan and fluvoxamine. However, no changes in the expression of 5-HT2A receptor were observed in the amygdala or hippocampus. 3.5. Effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT1A receptor in the prefrontal cortex in mice Fig. 5 shows the effects of co-treatment with yokukansan and fluvoxamine on the expression of 5-HT1A receptors in the prefrontal cortex in mice. There were no significant differences in the expression level of 5-HT1A receptors in the prefrontal cortex among any of the groups. 3.6. Effects of microinjection of ketanserin into the medial prefrontal cortex on freezing behavior induced by contextual fear conditioning in mice Fig. 6C shows the effects of microinjection of ketanserin, a 5-HT2A receptor antagonist, into the medial prefrontal cortex on freezing behavior induced by contextual fear conditioning in mice. A single administration of ketanserin at a dose of 5 nmol 40 min prior to the test session significantly suppressed freezing behavior. 93
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Fig. 6. (A) Experimental schedule. (B) Schematic diagram of microinjection sites within the prefrontal cortex of mice used in the present study. The injection sites are indicated by asterisks. (C) Effects of microinjection of ketanserin into the medial prefrontal cortex on freezing behavior induced by contextual fear conditioning in mice. V: vehicle, KTS: ketanserin. Values are the mean ± SEM of 7 mice. *p < .05 (Student's t-test).
the synergistic effect of fluvoxamine and yokukansan, the present study set treatment period under which yokukansan alone will not develop a significant anxiolytic-like effect (Fig. 3). A growing body of evidence has shown that yokukansan improves behavioral and psychological symptoms, such as excitement, aggression, hallucinations, insomnia, anxiety, wandering and depression, in patients with Alzheimer's disease and other forms of dementia, without severe adverse effects (Matsuda et al., 2013). Recently, it has also been reported that yokukansan alleviates preoperative anxiety without undesirable sedation, compared to diazepam (Arai et al., 2014; Wada et al., 2017). These clinical effects are supported by the finding in preclinical studies that yokukansan reduces anxiety in animal models (Mizoguchi et al., 2010; Yamaguchi et al., 2012; Shoji and Mizoguchi, 2013). In addition to these previous reports, the present findings provide the new insight that yokukansan may be effective for enhancing the effect of fluvoxamine in the treatment of anxiety disorders. There are now believed to be seven kinds of 5-HT receptor families, 5-HT1–7, that are comprised of 14 structurally and pharmacologically distinct 5-HT receptor subtypes (Hoyer et al., 2002). Among them, the 5-HT1 and 5-HT2 receptor subtypes have received particular attention due to their implication in emotional modulation and in several psychiatric disorders. Indeed, 5-HT1A receptor agonist as well as 5-HT2 receptor antagonist are known to be effective in the treatment of anxiety and depression (Jones and Blackburn, 2002). Therefore, the
present study tried to clarify the changes in the expression levels of 5HT1A and 5-HT2 receptors in brain regions of mice that had been subjected to the fear conditioning paradigm. The results showed that the expression level of 5-HT2A receptor is specifically decreased in the prefrontal cortex of mice that had been administered yokukansan and fluvoxamine (Fig. 4). This finding is in good agreement with a previous report (Egashira et al., 2008). Another report indicated that isolation stress significantly increased 5-HT2A receptor density in the prefrontal cortex, and repeated treatment with yokukansan significantly downregulated this increase (Ueki et al., 2015b). Thus, yokukansan may attenuate the function of cortical 5-HT2A receptor. These findings may explain the mechanism of the effectiveness of yokukansan for psychiatric disorders, since it has been reported that cortical 5-HT2A receptor may play a critical role in the expression of anxiety (Aznar and Klein, 2013). For example, cortical 5-HT2A receptors have been shown to play a role in modulating the anxiety response in a transgenic mouse model, where selective restoration of 5-HT2A receptor signaling in the cortex normalized conflict anxiety-like behavior in 5-HT2A receptor knockout (htr2a-/-) mice (Weisstaub et al., 2006). In contrast, a recent behavioral pharmacological study demonstrated that microinjection of ketanserin, a selective 5-HT2A receptor antagonist, into the infralimbic and prelimbic cortices had anxiolytic-like effects in the elevated plusmaze test and a contextual fear conditioning paradigm (León et al., 2017). The present study confirmed this report in that the freezing 94
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Author contributions
behavior of mice induced by contextual fear conditioning was suppressed by the microinjection of ketanserin into the medial prefrontal cortex before the test session (Fig. 6). The blockade of 5-HT2A receptor may make it difficult for 5-HT to bind to the 5-HT2A receptor, and may also create an environment amenable to other receptor subtypes that are important for the expression of an anxiolytic effect such as 5-HT1A receptor. Taken together, the present findings, along with previous reports, suggest that reduction of cortical 5-HT2A receptor-mediated signaling may be involved, at least in part, in the enhancement of the anxiolytic-like effect of fluvoxamine by repeated pretreatment with yokukansan. Additionally, it has been reported that the downregulation of 5-HT2A receptors is suggested to be induced by a synergistic effect of Bupleuri Radix, Uncariae Uncis cum Ramulus, Angelicae Radix and Glycyrrhizae Radix among the seven constituent components of yokukansan (Ueki et al., 2015b). Thus, these multiple herbal medicine containing a large number of ingredients may be responsible for the synergistic effects of yokukansan and fluvoxamine on anxiety. In contrast to 5-HT2A receptor, no significant changes in the expression of cortical 5-HT1A receptor were observed in the present study in mice that had been repeatedly treated with yokukansan (Fig. 5). However, it has been reported that repeated treatment of yokukansan did not change protein or mRNA levels of 5-HT1A receptor in the prefrontal cortex of mice, but significantly increased the density of receptor as detected by a receptor binding assay (Ueki et al., 2015a). Furthermore, enhancement of the 8-OH-DPAT-induced decrease in rearing behavior of mice, a behavior that is considered to reflect the function of postsynaptic 5-HT1A receptor in the cerebral cortex (Bert et al., 2006), was also observed under repeated treatment with yokukansan, suggesting enhancement of the function of cortical 5-HT1A receptor (Ueki et al., 2015a). Otherwise, an in vitro binding assay revealed that yokukansan had a partial agonistic effect on 5-HT1A receptor (Terawaki et al., 2010), and geissoschizine methyl ether, an indole alkaloid derived from Uncariae Uncis cum Ramulus, has been identified as the active ingredient responsible for the partial agonism of 5-HT1A receptors by yokukansan (Nishi et al., 2012). Additionally, there is a report suggesting that feurlic acid, an active ingredient contained in Cnidii Rhizoma, also possesses agonist activity to 5-HT1A receptors (Zeni et al., 2012). It is well known that activation of 5-HT1A receptor consistently reduces freezing behavior of mice subjected to a fear conditioning paradigm (Inoue et al., 2011). Therefore, it is also possible that yokukansan may positively affect cortical 5-HT1A receptor-mediated signaling and enhance the anxiolytic-like effect of fluvoxamine. Further detailed studies will be needed to evaluate this hypothesis.
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5. Conclusion The present study found that repeated treatment with yokukansan synergistically enhances the anxiolytic-like effect of fluvoxamine in the contextual fear conditioning paradigm in mice in conjunction with a decrease in 5-HT2A receptor-mediated signaling in the prefrontal cortex. Therefore, the combination of yokukansan and fluvoxamine may prove useful as a complementary and integrative medicine for augmentation therapy in treatment-resistant anxiety disorders.
Acknowledgements We would like to thank Tsumura & Co. (Tokyo, Japan) for kindly supply the extract of yokukansan and its component analysis data.
Conflict of interest The authors declare that they have no conflicts of interest to disclose. 95
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