Neuroprotection by chotosan, a Kampo formula, against glutamate excitotoxicity involves the inhibition of GluN2B-, but not GluN2A-containing NMDA receptor-mediated responses in primary cultured cortical neurons

Journal of Pharmacological Sciences xxx (2017) 1e4

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Neuroprotection by chotosan, a Kampo formula, against glutamate excitotoxicity involves the inhibition of GluN2B-, but not GluN2A-containing NMDA receptor-mediated responses in primary cultured cortical neurons Sachie Sasaki-Hamada a, Azusa Suzuki a, Emi Sanai a, Kinzo Matsumoto b, Jun-Ichiro Oka a, * a b

Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan Division of Medicinal Pharmacology, Institute of Natural Medicine, University of Toyama, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 June 2017 Received in revised form 11 October 2017 Accepted 19 October 2017 Available online xxx

Chotosan (CTS), a traditional herbal formula called Kampo medicine, was shown to be effective in the treatment of vascular dementia in a clinical study, and exerted protective effects against transient cerebral ischemia-induced cognitive impairment in mice. In the present study, we investigated the neuroprotective effects of CTS using primary cultured rat cortical neurons. CTS (250e1000 mg/mL) inhibited neuronal death induced by 100 mM glutamate. This glutamate-induced neuronal death was blocked by a GluN2B-, but not GluN2A-containing NMDA receptor antagonist. Therefore, the neuroprotective effects of CTS were related to an inhibition of GluN2B-containing NMDA receptor-mediated responses. © 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Keywords: Chotosan Neuroprotective effects GluN2B

Chotosan (CTS) is a Kampo formula that consists of 11 different medical herbs and gypsum fibrosum, which is generally prescribed to middle-aged and elderly patients with a weak physical constitution and symptoms related to hypertension and chronic headaches.1 CTS exhibits antioxidant and free radical scavenging activities.2 Although the effects of its major component herb, Chotoko (Uncaria geneus), or indole alkaloids contained in Chotoko on glutamate-induced neuronal death have already been examined,3e5 it currently remains unclear whether CTS exerts neuroprotective effects. Glutamate is a physiological excitatory amino acid transmitter in the central nervous system. Glutamate-mediated toxicity has been suggested to be involved in several neurological disorders including ischemia6 and neurodegenerative diseases such as Alzheimer's disease.7 Excitotoxicity is mainly associated with the excessive release of glutamate and subsequent Ca2þ influx via NMDAsubtype glutamate receptors, leading to an intracellular cascade

* Corresponding author. Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. E-mail address: [email protected] (J.-I. Oka). Peer review under responsibility of Japanese Pharmacological Society.

of cytotoxic events; i.e., the excessive activation of glutamate receptors evokes neuronal dysfunction and damage or even death.8 We herein investigated the neuroprotective effects of CTS against glutamate-induced cell death in cultured rat cortical neurons. The effects of antagonists selective for NMDA receptors containing GluN2A or GluN2B subunits on glutamate-induced cell death were also examined. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Tokyo University of Science, and conducted according to the guidelines of the National Institute of Health and the Japanese Pharmacological Society. E18 Wistar rats were purchased from Japan SLC, Inc. (Shizuoka), and efforts were made to minimize the number of animals used as well as animal pain and distress. A primary culture was performed according to our previous study9 with minor modifications. Cortical neurons were dissociated from E18 Wistar rats with 6 U/mL papain and 0.1 mg/mL DNase I at 37  C for 15 min. The brain tissue suspension was centrifuged for 5 min at 1000 rpm, and the resultant pellet was resuspended in feeding medium, which contained DMEM with 5% fetal bovine serum, 5% horse serum, and 1% penicillin-streptomycin solution. The cell suspension was seeded at 1  106 cells/mL into 24-well plates precoated with polyethyleneimine (SigmaeAldrich,

https://doi.org/10.1016/j.jphs.2017.10.009 1347-8613/© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Sasaki-Hamada S, et al., Neuroprotection by chotosan, a Kampo formula, against glutamate excitotoxicity involves the inhibition of GluN2B-, but not GluN2A-containing NMDA receptor-mediated responses in primary cultured cortical neurons, Journal of Pharmacological Sciences (2017), https://doi.org/10.1016/j.jphs.2017.10.009

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S. Sasaki-Hamada et al. / Journal of Pharmacological Sciences xxx (2017) 1e4

St. Louis, MO, USA). Cells were incubated at 37  C in a 9:1 mixture of atmospheric air and CO2, and were treated for 24 h with 10 mM cytosine arabinoside (SigmaeAldrich) 2 days after plating to remove actively dividing cells. Medium was changed on the next day, and then once a week. Cultures were used for experiments on the 10th day in vitro.

The CTS extract used in this study was purchased from Tsumura Co. (Japan) in the form of a spray-dried powder extract prepared according to the standardized extraction method of medical plants registered in Japanese Pharmacopoeia XV. CTS was extracted from a mixture of 3.0 parts Uncariae Uncis cum Ramulus (the hooks and branch of Uncaria rhynchophylla MIQUEL), 3.0 parts Aurantii Nobilis

Fig. 1. Effects of CTS on glutamate- or ionomycin-induced toxicity. (A) The MTT assay for cell viability in glutamate- or ionomycin-treated primary cultured cortical neurons. Cortical neurons were pre-incubated with 100, 250, and 1000 mg/mL CTS for 48 h, followed by exposure to 100 mM glutamate (A1) or 5 mM ionomycin (A2) for 24 h. (B) Hoechst 33342 and PI double-staining in cultured cortical neurons. The glutamate treatment increased the number of PI-positive neurons that was decreased in neurons pretreated with CTS (B1). Scale bar ¼ 50 mm. Quantitative analysis of histograms expressed as a percentage of apoptotic neurons in all neurons observed under each microscopic field (B2). Results are expressed as means ± SEM. ## P < 0.01, ### P < 0.001 vs. the control group, *P < 0.05, **P < 0.01 vs. the glutamate- or ionomycin-treated group (a one-way ANOVA followed by Bonferroni's multiple comparison test). n ¼ 7e16.

Please cite this article in press as: Sasaki-Hamada S, et al., Neuroprotection by chotosan, a Kampo formula, against glutamate excitotoxicity involves the inhibition of GluN2B-, but not GluN2A-containing NMDA receptor-mediated responses in primary cultured cortical neurons, Journal of Pharmacological Sciences (2017), https://doi.org/10.1016/j.jphs.2017.10.009

S. Sasaki-Hamada et al. / Journal of Pharmacological Sciences xxx (2017) 1e4

pericarpium (the skin of Citrus unshiu MARKOVICH), 3.0 parts Pinelliae tuber (the tuber of Pinellia ternate BREITENBACH), 3.0 parts Ophiopogonis tuber (the root of Ophiopogon japonicus KERGAWLER), 3.0 parts Hoelen (the sclerotium of Poria cocos WOLF), 2.0 parts Ginseng radix (the root of Panax ginseng C.A. MEYER), 2.0 parts Saposhnikoviae radix (the root and rhizome of Saposhnikovia divaricata SCHISCHKIN), 2.0 parts Chrysanthemi flos (the flower of Chrysanthemum morifolium RAMATULLE), 1.0 part Glycyrrhizae radix (the root of Glycyrrhiza uralensis FISHER), 1.0 part Zingiberis rhizoma (the rhizome of Zingiber officinale ROSCOE), and 5.0 parts Gypsum fibrosum (CaSO4 2H2O). The yield of the CTS extract was 16.1%. Protocols for the extraction and chemical profiling of CTS were the same as those described previously10 and mass spectrometry data obtained from the extract were stored in the Wakan-Yaku Database system (WakanDB ID: LCMS:Chotosan/11000001, http://wakandb. u-toyama.ac.jp/wiki/LCMS:Chotosan/11000001), Institute of Natural Medicine, University of Toyama. CTS was dissolved in distilled water (ddw) and applied on the 7th day. [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; 0.5% final concentration; Dojindo, Kumamoto) was added to cultured cells plated on a 24-well plate at 37  C for 3 h. Formazan crystals were dissolved in DMSO. Absorbance was measured with a microplate reader (ARVO MX, PerkinElmer, Waltham, MA, USA) at a test wavelength of 570 nm and reference wavelength of 655 nm to obtain the sample signal (OD570-OD655). In experiments involving antagonists, it was added 30 min prior to the glutamate treatment. Cell death was assessed by double fluorescent staining using propidium iodide (PI) and Hoechst 33342 (SigmaeAldrich). Neurons were cultured in 24-well plates at a density of 1  106 cells/ mm2. Twenty-four hours after the glutamate or ionomycin (Merk Millipore, Darmstadt, Germany) treatment, cells were fixed in 4% paraformaldehyde for 15 min and then stained with PI (0.5 mg/mL) and Hoechst 33342 (500 mg/mL) for 60 min. Staining was imaged

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and analyzed microscopically using BZ-9000 and software BZ-HIC (Keyence, Osaka). In order to assess apoptotic nuclei and dead/ dying neurons, three visual fields were randomly selected from each well. The significance of differences was evaluated using a one-way ANOVA followed by the Bonferroni-adjusted post hoc test. In all cases, significance was set at P < 0.05. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). We evaluated the protective effects of CTS on glutamateinduced neuronal death using the MTT assay, and calculated cell viability. Exposure to glutamate (100 mM) reduced cell viability to approximately 66%. CTS at a concentration of 250e1000 mg/mL significantly inhibited glutamate-induced cell death (Fig. 1A1). CTS (250e1000 mg/mL) also significantly inhibited neuronal death induced by ionomycin (5 mM) (Fig. 1A2) Hoechst 33342 and PI double-staining was performed to further evaluate the neuroprotective effects of CTS on glutamate-induced neuronal death in cultured cortical neurons. Hoechst 33342 and PI are frequently used fluorescent nucleic acid dyes. Hoechst 33342 and PI emit blue and red fluorescence, respectively, when bound to double-stranded DNA. Since Hoechst 33342 penetrates the cell membrane of necrotic and living cells, whereas PI cannot enter living cells, PI is typically used to detect cell apoptosis and necrosis. The glutamatetreated group underwent cell death or apoptosis (Fig. 1B1), and the treatment with CTS significantly decreased the number of apoptotic and necrotic cells (Fig. 1B2). Glutamate-induced neuronal death was prevented by AP5 (100 mM) (a specific NMDA receptor antagonist), but not CNQX (20 mM) (a specific non-NMDA receptor antagonist) (Fig. 2). The specific GluN2A- and GluN2B-containing NMDA receptor antagonists NVP-AAM077 (Novartis Pharma, Basal, Switzerland) and ifenprodil tartrate (Wako Pure Chemical Industries, Osaka),

Fig. 2. Effects of the pretreatment with antagonists of non-NMDA or NMDA receptors on glutamate-induced toxicity in the MTT assay. Effects of the pretreatment with CNQX (20 mM) (A) or AP5 (100 mM) (B) on glutamate-induced toxicity in primary cultured cortical neurons. Results are expressed as means ± SEM. ### P < 0.001 vs. the control group, *P < 0.05 vs. the glutamate-treated group, ns: non-significant (a one-way ANOVA followed by Bonferroni's multiple comparison test). n ¼ 9e16.

Please cite this article in press as: Sasaki-Hamada S, et al., Neuroprotection by chotosan, a Kampo formula, against glutamate excitotoxicity involves the inhibition of GluN2B-, but not GluN2A-containing NMDA receptor-mediated responses in primary cultured cortical neurons, Journal of Pharmacological Sciences (2017), https://doi.org/10.1016/j.jphs.2017.10.009

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S. Sasaki-Hamada et al. / Journal of Pharmacological Sciences xxx (2017) 1e4

Fig. 3. Effects of the pretreatment with antagonists of GluN2A- or GluN2B-containing NMDA receptors on glutamate-induced toxicity in the MTT assay. (A, B) Effects of the pretreatment with NVP-AAM077 (50 nM) (A) or ifenprodil (10 mM) (B) on glutamate-induced toxicity in primary cultured cortical neurons. (C) Effects of the pretreatment with a low concentration of ifenprodil (1 mM) on the neuroprotective effects of CTS (100 mg/mL). Results are expressed as means ± SEM. ### P < 0.001 vs. the control group, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the glutamate-treated group, ns: non-significant (a one-way ANOVA followed by Bonferroni's multiple comparison test). n ¼ 12e19.

respectively, were used to identify the roles of NMDA receptor subunits in excitotoxicity. The IC50 values of NVP-AAM077 are 14 nM and 1.8 mM for GluN2A and GluN2B, respectively,11 while those of ifenprodil is 0.34 and 146 mM for GluN2B and GluN2A, respectively.12 Ifenprodil (10 mM), but not NVP-AAM077 (50 nM) abolished excitotoxicity (Fig. 3A and B). Moreover, a low concentration of ifenprodil (1 mM) and CTS (100 mM) synergistically exhibited the neuroprotective effects in the MTT assay (Fig. 3C). We previously demonstrated that CTS improves diabetesinduced cognitive deficits by modulating GluN2B-containing NMDA receptor expression.13 Rhynchophylline, a constituent of Uncaira species, was reported to reverse GluN2B mRNA and protein levels.14 Further studies are required to clarify detailed mechanisms. Since CTS inhibited ionomycin-induced neuronal death, and because glutamate neurotoxicity is mediated by the Ca2þ influx induced by stimulation of NMDA receptors, the neuroprotective effects of CTS may result from the inhibition of the neurotoxic process subsequent to Ca2þ influx through GluN2B-containing NMDA receptors. In conclusion, CTS exerted neuroprotective effects against glutamate-induced cell death in cultured rat cortical neurons. These results provide further evidence for the beneficial effects of CTS on neurodegenerative disease.

Conflict of interest There is no conflict of interest to disclose.

Acknowledgements This work was in part supported by Grants-in-Aid for the 2012 and 2013 Cooperative Research Project I from Institute of Natural Medicine, University of Toyama (to J.-I.O. and K. M.).

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Please cite this article in press as: Sasaki-Hamada S, et al., Neuroprotection by chotosan, a Kampo formula, against glutamate excitotoxicity involves the inhibition of GluN2B-, but not GluN2A-containing NMDA receptor-mediated responses in primary cultured cortical neurons, Journal of Pharmacological Sciences (2017), https://doi.org/10.1016/j.jphs.2017.10.009