Yokukansan, a kampo medicine, protects against glutamate cytotoxicity due to oxidative stress in PC12 cells

Journal of Ethnopharmacology 134 (2011) 74–81

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Yokukansan, a kampo medicine, protects against glutamate cytotoxicity due to oxidative stress in PC12 cells Zenji Kawakami ∗ , Hitomi Kanno, Yasushi Ikarashi, Yoshio Kase Tsumura Research Laboratories, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan

a r t i c l e

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Article history: Received 14 September 2010 Received in revised form 16 November 2010 Accepted 25 November 2010 Available online 3 December 2010 Keywords: Glutamate PC12 Yokukansan Uncaria thorn Glutathione Oxidative stress

a b s t r a c t Aim of the study: Yokukansan is a traditional Japanese medicine consisted of seven medicinal herbs and has been used for treatment of neurosis, insomnia, and behavioral and psychological symptoms of dementia in Japan. The aim of the present study is to clarify the active compounds responsible for the protective effect of yokukansan against glutamate-induced cytotoxicity in PC12 cells. Materials and methods: PC12 cells which is a tool for selective evaluation of test substances against oxidative stress was used in the present study. The cell survival rates or glutathione (GSH) levels were evaluated by a MTT reduction assay or GSH assay based on the GSH reductase enzymatic recycling method, respectively. Results: Glutamate (1–17.5 mM) induced cell death of PC12 cells in a concentration- dependent manner. Yokukansan (125–500 ␮g/ml) inhibited the glutamate-induced PC12 cell death. When the effects of extracts of the seven constituent herbs in yokukansan on the cell death were examined, Uncaria thorn was found to have the highest potency in the protection. To clarify the active compounds in Uncaria thorn, the effects of seven alkaloids (rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether) on the cell death were further examined. The protective effects were found in hirsutine, hirsuteine, and geissoschizine methyl ether, which also ameliorated the glutamate-induced decrease in GSH levels. Conclusion: These results suggest that yokukansan protects against PC12 cell death induced by glutamatemediated oxidative stress, i.e., reduction of intracellular GSH level, and the effect may be mainly attributed to a synergistic effect of the hirsutine, hirsuteine, and geissoschizine methyl ether in Uncaria thorn. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Glutamate-mediated toxicity is an important mechanism of neuronal death in various pathologic conditions including ischemia (Choi and Rothman, 1990), trauma (Hayes et al., 1992), epileptic seizures (Rothman and Olney, 1987), and neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases (Monaghan et al., 1989; Coyle and Puttfarcken, 1993). To date, two mechanisms have been proposed for the glutamate neurotoxicity, i.e., glutamate receptor-mediated (Choi, 1988) and oxidative stress-mediated (Murphy et al., 1990; Froissard and Duval, 1994) neurotoxicities. The excitotoxicity of the former is mainly associated with an excessive release of glutamate and subsequent

Abbreviations: YKS, yokukansan; RT-PCR, reverse-transcription polymerase chain reaction; PC12, pheochromocytoma cell line 12; NMDA, N-methyl-d-aspartate; GSH, glutathione; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; CPG, (S)-4-carboxyphenylglycine. ∗ Corresponding author. Tel.: +81 29 889 3850; fax: +81 29 889 2158. E-mail address: kawakami [email protected] (Z. Kawakami). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.063

influx of Ca2+ via the N-methyl-d-aspartate (NMDA)-subtype glutamate receptor, leading to an intracellular cascade of cytotoxic events; i.e., the excessive activation of glutamate receptors evokes neuronal dysfunction and damage or even death (Michaels and Rothman, 1990). The oxidative glutamate toxicity of the latter type is related to a cystine/glutamate antiporter system (this system is  also called “system Xc→ ); i.e., elevated levels of extracellular glutamate inhibit cystine uptake by inhibiting the antiporter system. The inhibited uptake of cystine, which is the precursor of glutathione (GSH), leads to a marked decrease in intracellular GSH levels, resulting in the induction of oxidative stress in the cell and, ultimately cell death (Pereira and Oliveira, 2000; Penugonda et al., 2005). Yokukansan is one of the traditional Japanese medicines called “kampo” medicines in Japan. It is composed of seven kinds of dried medical herbs. This medicine has been approved by the Ministry of Health, Labor, and Welfare of Japan as a remedy for neurosis, insomnia, and irritability in children. Recently, yokukansan was reported to improve behavioral and psychological symptoms of dementia such as hallucinations, agitation, and aggressiveness in patients with Alzheimer’s disease, dementia with Lewy bodies, and other forms of senile dementia (Iwasaki et al., 2005a,b; Mizukami

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et al., 2009). More recently, we demonstrated that yokukansan inhibited the glutamate-induced cell death of pheochromocytoma cell line (PC12) cells, a sympathetic neuron cell line derived from rat pheochromocytoma, suggesting that yokukansan possesses a neuroprotective effect against glutamate toxicity as one of its mechanisms. PC12 has been widely used to investigate NMDA receptormediated excitotoxicity and potential neuroprotective mechanisms (Mazzio et al., 2001; Crisanti et al., 2005; Lee et al., 2006; Song et al., 2006). However, although PC12 cells express NMDA receptors, the toxicity caused by glutamate is not related solely to the presence of these receptors because NMDA has no effect on PC12 cell death (Schubert et al., 1992; Froissard and Duval, 1994). Recently, Edwards et al. (2007) demonstrated that PC12 cells do not express a normal NMDA receptor profile. Pereira and Oliveira (2000), Penugonda et al. (2005), and Lo et al. (2008) also suggested that the glutamate toxicity on PC12 cells is mediated by an oxidative glutamatergic toxicity pathway that is related to the cystine/glutamate antiporter system. If so, the protective effect of yokukansan against glutamate-induced PC12 cell death reported in our previous study (Kawakami et al., 2009) might be caused by amelioration of oxidative stress due to a decrease in GSH levels via inhibition of the cystine/glutamate antiporter system. However, detailed studies regarding this hypothesis have not yet been performed. In the present study, therefore, we first confirmed that glutamate-induced PC12 cell death was due to a decrease in the GSH level resulting from inhibition of the cystine/glutamate antiporter. Subsequently, the effects of the seven constituent herbs of yokukansan on the PC12 cell death were investigated to clarify the relative importance of each constituent. Further, to clarify the essential ingredients, the effects of alkaloids isolated from the most potent constituent herb were investigated. Thus, the aim of this work is to clarify the activities of the various components of yokukansan against glutamate-induced cytotoxicity related to oxidative stress in PC12 cells.

2. Materials and methods

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0.45 ␮m pores. An aliquot (30 ␮l) of the filtrate was injected into a high-performance liquid chromatograph (Shimadzu SPD-M10AVP, Shimadzu Co., Kyoto, Japan). The chromatographic conditions were column: TSK-gel ODS-80TS (4.6˚ mm × 250 mm long, Tosoh Co., Tokyo, Japan), mobile phase: a linear gradient with 0.05 M AcONH4 , pH 3.6 (90 → 0%) and 100% CH3 CN (10 → 100%) for 60 min, column temperature: 40 ◦ C, flow rate: 1.0 ml/min, detector: diode array, and scan range: UV 200–400 nm. At least 25 compounds were identified in the three-dimensional chromatogram (Mizukami et al., 2009). 2.1.2. Seven components of Uncaria thorn Rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether isolated from Uncaria thorn were supplied by the Botanical Raw Materials Research Department of Tsumura & Co. (Ibaraki, Japan). 2.1.3. Reagents used in a real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) analysis A Qiagen RNeasy mini kit was purchased from Qiagen (Hilden, Germany). A high capacity cDNA RT Kits, TaqMan Gene Expression Master Mix and TaqMan probes for detection of NR1, NR2A, NR2B, NR2C, NR2D, NR3B, xCT, 4F2hc and GAPDH were purchased from Applied Biosystems (Foster City, CA, USA). 2.1.4. Reagents used in PC12 cell culture, MTT or GSH assay Roswell Park Memorial Institute (RPMI)-1640 medium, heatinactivated horse serum, dialyzed fetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen (Grand Island, NY, USA). Fetal bovine serum was purchased from ICN Biomedicals (Aurora, OH, USA). Dialyzed horse serum was purchased from BioWest (Nouaillé, France). Glutamate, cystine, sodium dodecyl sulfate (SDS), metaphosphoric acid (MPA), triethanolamine (TEAM), and bovine serum albumin were purchased from Sigma–Aldrich (St. Louis, MO, USA), and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Dojindo (Kumamoto, Japan). Other chemicals were purchased from commercial sources.

2.1. Drugs and reagents

2.2. Preparation of PC12 cells and primary cultured neurons

2.1.1. Yokukansan and seven constituent herbs The dry powdered extracts of yokukansan and its seven constituent medicinal herbs used in the present study were supplied by Tsumura & Co. (Tokyo, Japan). Yokukansan is composed of seven dried medicinal herbs: 4.0 parts Atractylodis Lanceae rhizome (rhizome of Atractylodes lancea De Candolle, Compositae), 4.0 parts Poria sclerotium (sclerotium of Poria cocos Wolf, Polyporaceae), 3.0 parts Cnidium rhizome (rhizome of Cnidium officinale Makino, Umbelliferae), 3.0 parts Japanese Angelica root (root of Angelica acutiloba Kitagawa, Umbelliferae), 2.0 parts Bupleum root (root of Bupleurum falcatum Linné, Umbelliferae), 1.5 parts Glycyrrhiza root (root and stolon of Glycyrrhiza uralensis Fisher, Leguminosae), and 3.0 parts Uncaria thorn (thorn of Uncaria rhynchophilla Miquel, Rubiaceae). The seven medical herbs were extracted with purified water at 95 ◦ C for 1 h, and the extraction solution was separated from the insoluble waste and concentrated by removing water under reduced pressure. Spray-drying was used to produce a dried extract powder. The yield of the extract was about 15.9%. We have already reported a three-dimensional highperformance liquid chromatographic analysis of the ingredients of a yokukansan extract (Mizukami et al., 2009). In brief, the dried extract (1.0 g) of yokukansan was dissolved in 20 ml methanol under ultrasonication for 30 min and then centrifuged at 3000 rpm for 5 min. The supernatant was filtered through a membrane with

2.2.1. PC12 cells PC12 cells obtained from Dainippon Sumitomo Pharma (Osaka, Japan) were maintained at 37 ◦ C in 95% air and 5% CO2 with 95% relative humidity in RPMI-1640 medium supplemented with 5% fetal bovine serum, 10% heat-inactivated horse serum, 50 U/ml penicillin, and 50 ␮g/ml streptomycin until used in experiments. 2.2.2. Primary culture neurons Primary cortical neuronal culture was performed according to the method of Arimatsu and Hatanaka (1986), and Perry et al. (2004) with minor modification. In brief, neopallia in 18-dayold Sprague Dawley rat embryos (Charles River Laboratories, Yokohama, Japan) were dissociated with 9 unit/mL papaine (Worthington, Lakewood, NJ, USA) and 0.01% DNase I (Sigma–Aldrich) (37 ◦ C, 15 min). The dissociation was terminated by addition of horse serum and the tissue fragments were centrifuged at 1000 rpm for 5 min. The pellet was resuspended in DMEM/Ca- and Mg-free phosphate-buffered saline [PBS(−)] (1:1) and then mechanically disrupted by pipetting. The cell suspension was passed through lens paper to remove possible cell clumps. The cells were resuspended in Neurobasal media with B27 supplement (Invitrogen) and seeded onto poly-d-lysine-coated 100 mm culture dishes (BD Falcon, Franklin Lakes, NJ, USA) at a density of 105 cells/cm2 for

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Fig. 1. Expression profiles of NMDA receptor and System Xc− in PC12 cells and primary cultured neurons. Each subunit mRNA expression level of NMDA receptor (NR1, NR2A, NR2B, NR2C, NR2D, NR3B) or System Xc− (xCT, 4F2hc) was analysed by a real-time PT-PCR analysis. GAPDH was used as endogenous control gene. Data are presented as the mean value of duplicate determinations. ND: not detected.

Fig. 3. Relationship between concentration of glutamate or cystine in the medium and cellular glutathione levels. PC12 cells were incubated for 24 h in media containing various concentrations of glutamate (A: 10 and 17.5 mM) or cystine (B: 10 and 0 ␮M). The control media contained 208 ␮M cystine and no glutamate. Each value, calculated as a percentage of the cellular glutathione (GSH) level in control PC12 cells, represents the mean ± S.E.M. (n = 6). ** P < 0.01 and *** P < 0.001 vs. corresponding control: one-way ANOVA + Dunnett’s test.

24 h, then the media was changed to Neurobasal media with B27 supplement minus AO (Invitrogen) for 3 weeks. 2.3. Real-time RT-PCR analysis to examine the expression profile of NMDA receptor subunits and System Xc− subunits in PC12 cells and primary cultured neurons Total RNA was isolated from the PC12 cells or primary cultured neurons using the Qiagen RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. In brief, the cultured cells were lysed in RLT buffer containing guanidine-thiocyanate. The lysate was mixed with same volume of 70% ethanol. The mixture was infused into RNeasy Mini spin column for adsorption of total RNA. The total RNA adsorbed in the column was finally eluted with 50 ␮L of RNase-free water. The RNA concentration was determined spectrophotometrically at 260 nm.

Fig. 2. Effects of glutamate, MK801, NMDA, cystine, and (S)-4-carboxyphenylglycine on PC12 cell death. PC12 cells were incubated for 24 h in media containing various concentrations of glutamate (1–17.5 mM), glutamate (17.5 mM) + MK801 (10 and 100 ␮M), NMDA (62.5 and 125 ␮M), cystine (0–100 ␮M), or (S)-4carboxyphenylglycine (CPG: 0–1000 ␮M). The control media contained 208 ␮M cystine and no glutamate. Each value, calculated as percentage of the MTT activity in control (Cont) PC12 cells, represents the mean ± S.E.M. (n = 6). ** P < 0.01 and *** P < 0.001 vs. control, and NS (no significance) vs. 17.5 mM glutamate: one-way ANOVA + Dunnett’s test.

RT was carried out using High Capacity cDNA RT Kits (Applied Biosystems) according to the manufacturer’s protocol with total RNA samples and run on TAK-TP400 Thermal Cycler (TaKaRa, Shiga, Japan). In brief, 2500 ng of total RNA was added to the final reaction mixture (50 ␮L) containing 1x TaqMan RT buffer, 8 mmol/L dNTP Mix, 2.5 ␮mol/L Ramdom Hexamers, 0.4 U/␮L RNase Inhibitor, and Multiscribe Reverse Transcriptase. RT was carried out at 25 ◦ C for 10 min, 37 ◦ C for 120 min, and 85 ◦ C for 5 min. After RT, real-time PCR was performed with the TaqMan Gene Expression Master Mix (Applied Biosystems) and run on ABI PRISM 7900HT sequence detection system (Applied Biosystems). After an initial denaturation at 50 ◦ C for 2 min and at 95 ◦ C for 10 min, 40 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C were performed. Each subunit mRNA expression level of NMDA receptor (NR1, NR2A, NR2B, NR2C, NR2D, NR3B) or cystine antiporter (xCT, 4F2hc) was analysed by Ct method using GAPDH as endogenous control gene.

Fig. 4. Effects of l-buthionine sulfoximine and ethacrynic acid on PC12 cells. PC12 cells were incubated for 24 h in media containing various concentrations of l-buthionine sulfoximine (30–300 ␮M) and ethacrynic acid (100–200 ␮M). Each value, calculated as percentage of the MTT activity in control PC12 cells, represents the mean ± S.E.M. (n = 6). *** P < 0.001 vs. corresponding control: one-way ANOVA + Dunnett’s test.

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Fig. 5. Effects of yokukansan on PC12 cell death induced by glutamate, cystine deficiency, and (S)-4-carboxyphenylglycine. (A) PC12 cells were incubated for 24 h in media containing 17.5 mM glutamate (G) + various concentrations of yokukansan (YKS: 125–500 ␮g/ml). The control media (C) did not contain glutamate. (B) PC12 cells were incubated for 24 h in media containing various concentrations of YKS (125–500 ␮g/ml) in a cystine-deficient condition (CD). Control medium (C) contained 208 ␮M cystine. (C) PC12 cells were incubated for 24 h in media containing 1000 ␮M (S)-4-carboxyphenylglycine (CPG) + various concentrations of YKS (125–500 ␮g/ml). The control media (C) did not contain CPG. Each value, calculated as percentage of the MTT activity in control PC12 cells, represents the mean ± S.E.M. (n = 6). A significant decrease in the survival rate (††† P < 0.001) was found in G, CD, or CPG group compared to the corresponding control (C). The G-, CD-, or CPG-induced decrease in the survival rate was inhibited by YKS. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. G, CD, or CPG: one-way ANOVA + Dunnett’s test.

2.4. Examination of effects of test substances on glutamate-induced PC12 cell death The maintained PC12 cells were seeded into 96-well microplates (10,000 cells/well in 100 ␮l medium) in RPMI-1640 supplemented with 5% dialyzed fetal bovine serum, 10% dialyzed horse serum, 50 U/ml penicillin, and 50 ␮g/ml streptomycin without phenol

red. The cultures were incubated at 37 ◦ C in 95% relative humidity with a mixture of 5% CO2 and 95% air. In order to examine the effects on NMDA receptors, the medium was renewed 48 h after seeding with fresh culture media including various concentrations of NMDA or glutamate, and then the cells were incubated for 24 h. In addition, in order to examine the effect on the cystine/glutamate antiporter, the medium was renewed with a custom-designed RPMI-1640 medium lacking in cystine (CSTI, Sendai, Japan) supplemented with various concentrations of cystine. The effects of test substances on glutamate- or antiporterinduced cell death were evaluated by renewing media including a fixed concentration of glutamate or the cystine-deficient media plus various concentrations of test substances. After incubation, the cell survival rates or glutathione levels were evaluated by a MTT reduction assay or GSH assay, respectively.

2.5. MTT reduction assay for evaluation of cell survival

Fig. 6. Effects of seven constituent herbs of yokukansan on glutamate-induced PC12 cell death. PC12 cells were incubated for 24 h in the media containing 17.5 mM glutamate (G) + each constituent herb (200 ␮g/ml): Atractylodis Lanceae rhizome (ALR), Poria sclerotium (PS), Cnidium rhizome (CR), Uncaria thorn (UT), Japanese Angelica root (JAR), Bupleurum root (BR), and Glycyrrhiza root (GR). The control media (C) did not contain glutamate. Each value, calculated as percentage of the MTT activity in control PC12 cells, represents the mean ± S.E.M. (n = 6). ††† P < 0.001 vs. control (C), and * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. glutamate (G): one-way ANOVA + Dunnett’s test.

The MTT reduction assay (Sakai et al., 1994) was performed as follows: 20 ␮l of 5 mg/ml MTT dissolved in phosphate-buffered saline (PBS) was added to each well and incubated at 37 ◦ C for 5 h. The reaction was stopped by the addition of 100 ␮l of a solubilization solution (10% SDS in 0.01 N HCl), and the blue formazan formed from MTT by the reaction was dissolved by additional incubation for 18 h at 37 ◦ C. Absorbance of the formazan solution was measured using a microplate reader at a test wavelength of 540 nm and a reference wavelength of 690 nm. The specific MTT absorbance of test substance-treated sample (ODtest ) or control sample (ODcontrol ) was defined by subtracting the corresponding blank absorbance (ODBL ) which test substance or control solution had. Each ODBL was determined in the experimental condition without cultured cells. Cell survival (%) of test substance against control was calculated by the following formula: Cell survival (%) = [(ODtest − ODtest BL )/(ODcontrol − ODcontrol BL )] × 100.

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Fig. 7. Effects of seven constituents of Uncaria thorn on glutamate-induced PC12 cell death. PC12 cells were incubated for 24 h in the media containing 17.5 mM glutamate (G) + various concentrations of each constituent (3–100 ␮M): rhynchophylline (RP), isorhynchophylline (IRP), corynoxeine (CX), isocorynoxeine (ICX), geissoschizine methyl ether (GME), hirsuteine (HTE), and hirsutine (HIR). The control media (C) did not contain glutamate. Each value, calculated as percentage of the MTT activity in control PC12 cells, represents the mean ± S.E.M. (n = 6). ††† P < 0.001 vs. corresponding control (C), and ** P < 0.01 and *** P < 0.001 vs. glutamate (G): one-way ANOVA + Dunnett’s test.

2.6. Determination of GSH levels

3. Results

PC12 cells (1.84 × 106 cells/16 ml medium) were grown on 100 mm culture dishes and treated with glutamate and components of Uncaria thorn for 24 h. The cells were scraped off the dishes using a rubber policeman and collected by centrifugation. The cells were suspended in 125 ␮l of PBS, and an equal volume of the MPA reagent was added. The mixture was stood at room temperature for 5 min and centrifuged for 10 min at 8000 × g. The supernatant was mixed with TEAM reagent, and the GSH level in the supernatant was measured using a GSH assay kit based on the glutathione reductase enzymatic recycling method (Anderson, 1985; Eyer and ´ 1986) according to the manufacturer’s instructions Podhradsky, (Cayman Chemical, Ann Arbor, MI, USA). The protein concentration was measured by the method of Lowry et al. (1951) with bovine serum albumin as the standard. GSH levels were normalized to cellular protein, and then the levels were expressed as a percentage of the control.

3.1. Characteristics of PC12 cell death

2.7. Statistical analysis The real-time RT-PCR analysis data are presented as the mean value of duplicate determinations. Other data are presented as the mean ± S.E.M. The statistical significance of differences between groups was assessed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The significance level in each statistical analysis was accepted at P < 0.05.

To characterize the expression profile of NMDA receptor subunits and System Xc− subunits in PC12 cells, the subunit mRNA expression was compared between PC12 cells and the primary cultured neurons. A real-time RT-PCR analysis of NMDA receptor subunits showed the absence of NR2A and NR2B in PC12 cells compared to those in the neurons (Fig. 1A). On the other hand, the expression of System Xc- subunit (xCT and 4F2hc) was detected obviously in both PC12 cells and neurons though the amount of each expression was different between both cells (Fig. 1B). The effects of glutamate, MK801, and NMDA on the death of PC12 cells are shown in Fig. 2. Addition of glutamate (1–17.5 mM) to the medium decreased the survival rate (i.e., induced cell death) in a concentration-dependent manner. However, the 17.5 mM glutamate-induced cell death was not inhibited by the NMDA receptor antagonist MK801 (10 and 100 ␮M), and NMDA (62.5 and 125 ␮M) did not induce cell death in PC12 cells. On the other hand, the elimination of cystine from the culture medium, or addition of (S)-4-carboxyphenylglycine (CPG), a cystine/glutamate antiporter inhibitor, to the culture medium induced the cell death. Fig. 3 shows the effects of glutamate and cystine deficiency on the intracellular GSH level of PC12 cells. The addition of glutamate (10 and 17.5 mM) to the medium (A) or the removal of cystine from

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4. Discussion

Fig. 8. Effects of constituents of Uncaria thorn on glutamate-induced decrease in glutathione level. PC12 cells were incubated for 24 h in media containing 17.5 mM glutamate (G) + 30 ␮M or 100 ␮M of geissoschizine methyl ether (GME), hirsuteine (HTE), or hirsutine (HIR). The control media (C) did not contain glutamate. Each value, calculated as percentage of the cellular glutathione (GSH) level in control PC12 cells, represents the mean ± S.E.M. (n = 6). Glutamate (G) significantly decreased the GSH level (††† P < 0.001) compared to control (C). The decrease in the GSH level was inhibited by GME, HTE, or HIR. ** P < 0.01 and *** P < 0.001 vs. glutamate (G): one-way ANOVA + Dunnett’s test.

the medium (B) decreased in intracellular GSH level of PC12 cells in a concentration-dependent manner. Fig. 4 shows the effects of l-buthionine sulfoximine, a GSH synthesis inhibitor, and ethacrynic acid, a GSH depletor, on PC12 cells. Both substances induced the cell death in a concentrationdependent manner. 3.2. Effects of yokukansan and constituents on glutamate-induced PC12 cell death The effects of yokukansan (125–500 ␮g/ml) on the glutamate-, cystine deficiency- and CPG-induced PC12 cell death are shown in Fig. 5A–C, respectively. Glutamate, cystine-deficiency or CPG significantly decreased the survival rate of PC12 cells compared with the corresponding control. Yokukansan inhibited their effects in a concentration-dependent manner. When the effects of the seven constituents (200 ␮g/ml) of yokukansan on the cell death were examined (Fig. 6), the highest potency of protective effect (i.e., inhibition of glutamate-induced cell death) was found in Uncaria thorn, and followed by Glycyrrhiza root. No protective effects were observed for Cnidium rhizome, Japanese Angelica root, Bupleurum root, Atractylodis Lanceae rhizome and Poria sclerotium. 3.3. Effects of seven alkaloids in Uncaria thorn on glutamate-induced cell death and decrease in GSH level The effects of seven alkaloids (rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine and geissoschizine methyl ether) contained in Uncaria thorn on glutamate-induced cell death of PC12 cells are shown in Fig. 7. The protective effects were found in geissoschizine methyl ether, hirsuteine, and hirsutine, which also ameliorated the glutamateinduced decrease in GSH levels (Fig. 8).

We previously demonstrated that yokukansan inhibited glutamate-induced PC12 cell death, suggesting that yokukansan has a neuroprotective effect against glutamate neurotoxicity as one of its mechanisms. However, two mechanisms have been proposed for the glutamate toxicity, i.e., glutamate receptor-mediated (Choi, 1988) and oxidative stress-mediated neurotoxicities (Murphy et al., 1990; Froissard and Duval, 1994). Recently, many scientists suggest that PC12 cells do not express a normal profile of NMDA receptors, and that the cytotoxicity is mediated by the oxidative glutamatergic toxicity pathway, which is related to the System Xc− (Schubert et al., 1992; Froissard and Duval, 1994; Pereira and Oliveira, 2000; Penugonda et al., 2005; Edwards et al., 2007; Lo et al., 2008). The competition by glutamate for the System Xc− induces an imbalance in the homeostasis of cysteine, which is the precursor of GSH; i.e., the inhibition of cystine uptake by exposure to high glutamate concentrations is supposed to give rise to an inability to maintain intracellular GSH levels to protect against oxidative injury of the cells (Pereira and Oliveira, 2000). Therefore, we first examined the expression of profile of NMDA receptor- and System Xc− -subunit mRNAs in PC12 cells, and then the results were compared with those in the primary cultured neurons. Our realtime RT-PCR analysis confirmed the absence of NR2A and NR2B subunits in PC12cells. This expression profile is consistent with previous report (Vazhappilly et al., 2010). On the other hand, the expression of System Xc− subunit such as xCT and 4F2hc (Bassi et al., 2001; Iemata et al., 2007) was confirmed obviously in both PC12 cells and neurons though the amount of each expression was different between both cells. We next investigated the characteristics of the PC12 cells using NMDA receptor- and System Xc− -related substances. It has been reported that 100 ␮M NMDA is a sufficient to induce cell death in neuronal cells (Zhu et al., 2003), but cell death in PC12 cells was not observed at 125 ␮M, and even at 1000 ␮M (data not shown). In addition, though 100 ␮M MK-801 was reported to effectively block glutamate-induced neuronal cell death (Lysko et al., 1989), the death of PC12 cells was not blocked by that concentration of MK-801 in the present study. These results suggest that the glutamate-induced cell death of the PC12 cells used in the present study was not due to the cell death caused by stimulation of NMDA receptors, and also support previous reports that PC12 cells do not express functional NMDA receptors (Schubert et al., 1992; Froissard and Duval, 1994; Pereira and Oliveira, 2000; Penugonda et al., 2005; Edwards et al., 2007; Lo et al., 2008). On the other hand, the elimination of cystine from the culture medium, and addition of CPG, the System Xc− inhibitor, to the culture medium induced the cell death. Both glutamate and cystine deficiency decreased GSH level in PC12 cells. Induction of the cell death by GSH reduction was also confirmed by induction of cell death treated with l-buthionine sulfoximine, a GSH synthesis inhibitor, and ethacrynic acid, a GSH depletor. These lines of results suggest that the System Xc− and GSH are closely related to the glutamate-induced PC12 cell death, and also suggested that PC12 cells is a valuable tool for selective evaluation of test substances against oxidative stress through the System Xc− . Yokukansan inhibited the glutamate-induced PC12 cell death in a concentration-dependent manner. Because similar protective effect was observed on cystine deficiency- and CPG-induced cell death, it is suggested that the protection by yokukansan is regulated by the increase in intracellular GSH level than the effect to the antiporter. Among the seven constituents, the highest potency against the protective effect was found in Uncaria thorn. Uncaria thorn has been reported to protect against oxidative damage of NG108-15 cells by H2 O2 (Mahakunakorn et al., 2004). In the present study, the effects of seven alkaloids in Uncaria thorn (rhynchophylline,

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isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether) on glutamate-induced cell death were examined, and the results showed that hirsutine, hirsuteine, and geissoschizine methyl ether possess a neuroprotective effect. These three components also ameliorated the decrease in GSH levels induced by glutamate. These results suggest that yokukansan protects PC12 cell death induced by glutamatemediated oxidative stress, i.e., reduction of intracellular GSH level, and the effect may be mainly attributed to a synergistic effect of the hirsutine, hirsuteine, and geissoschizine methyl ether in Uncaria thorn. In the previous in vivo study, oral administration for 28–37 days of yokukansan (1.0 g/kg) significantly ameliorated cerebral neuronal death observed in thiamine-deficient rats (Ikarashi et al., 2009; Iizuka et al., 2010). The present in vitro study showed that yokukansan (125–500 ␮g/mL concentration) and three compounds (hirsutine, hirsuteine, and geissoschizine methyl ether: 3–100 ␮M) inhibited glutamate-induced PC12 cell death in a concentrationdependent manner. We have confirmed that hirsutine, hirsuteine, and geissoschizine methyl ether are contained 0.013, 0.015 and 0.014% in yokukansan, i.e., 1.0 g of yokukansan contains 130 ␮g, 150 ␮g and 140 ␮g of hirsutine, hirsuteine, and geissoschizine methyl ether, respectively. However, it is difficult to compare effective dose or concentration directly, because the experimental conditions are different between in vivo and in vitro studies. Therefore, it is very important to verify whether in vitro results are reflected in vivo. We are thinking that the detailed in vivo experiments on blood levels, brain levels and blood-brain barrier permeability and synergy or interactive effect of these compounds after administration of yokukansan to animals will be necessary in the future. To date, it has been shown that Uncaria thorn blocks NMDAinduced currents in cortical neurons and reduces NMDA-induced neuronal death (Sun et al., 2003; Lee et al., 2003). Oxyindole alkaloids such as isorhynchophylline, isocorynoxeine, and rhynchophylline and indole alkaloids such as hirsuteine and hirsutine contained in Uncaria thorn are known for their protective effects against glutamate-induced neuronal death in cultured cerebellar granule cells (Shimada et al., 1999). Rhynchophylline and isorhynchophylline have also been reported to be antagonists of the NMDA receptor, which reduces the maximal current responses evoked by NMDA and glycine, but shows no interaction with the polyaminebinding site, Zn2+ site, proton site, or redox modulatory site on the NMDA receptor (Kang et al., 2002). Recently, we demonstrated that yokukansan bound potently to NMDA receptors, in particular to its glutamate and glycine recognition sites (Kawakami et al., 2009). These findings include the possibility that yokukansan may contribute to the neuroprotective effect as an NMDA receptor antagonist. Our present study using PC12 cells shows that yokukansan protects against oxidative stress-mediated cell death in addition to the action on the NMDA receptors. Recently, we have reported that yokukansan ameliorated thiamine deficiency-induced decreases in the glutamate uptake, and the protein and mRNA levels of glutamate transporter (Kawakami et al., 2009). Because the ameliorative effect of yokukansan was completely abolished by the glutamate transporter inhibitor DL-threo-␤-hydroxy-aspartic acid (TBHA), and thiamine deficiency-induced down-regulation of glutamate transport is ameliorated by yokukansan, the mechanism is thought to be closely related to glutamate transporter activity (Kawakami et al., 2009) i.e., it is suggested that yokukansan also possesses a neuroprotective effect against glutamate-induced excitotoxicity by ameliorating the dysfunction of glutamate uptake in astrocytes. In conclusion, the traditional Japanese medicine yokukansan consists of a combination of seven medicinal herbs. Although additional studies are required to clarify the detailed underly-

ing mechanisms, the present study newly suggests, at least, that yokukansan may possess a neuroprotective effect against the glutamate-induced oxidative cytotoxicity. The protective effects may be attributed to alkaloids such as the geissoschizine methyl ether, hirsuteine and hirsutine in Uncaria thorn, a constitutent of yokukansan.

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