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Journal of Ethnopharmacology 138 (2011) 119– 125

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

journa l h o me page: www.elsev ier .com/ locate / je thpharm

he neuroprotective effects of an extract of Gastrodia elata

hung-Fen Tsaia,1, Chuen-Lin Huangb,c,1, Yun-Lian Lind, Yi-Chao Leee, Ying-Chen Yangf,ai-Kuei Huangd,g,∗

Department of Neurology, Cardinal Tien Hospital, New Taipei City, TaiwanMedical Research Center, Cardinal Tien Hospital, Hsintien, New Taipei County, TaiwanGraduate Institute of Physiology, Department of Physiology and Biophysics, National Defense Medical Center, Taipei, TaiwanNational Research Institute of Chinese Medicine, Taipei, TaiwanDepartment of Pharmacology, College of Medicine, Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, TaiwanDepartment of Animal Science, National Ilan University, Ilan, TaiwanInstitute of Biophotonics, National Yang-Ming University, Taipei, Taiwan

r t i c l e i n f o

rticle history:eceived 22 June 2011ccepted 26 August 2011vailable online 8 September 2011

eywords:astrodia elataheochromocytoma cells2A-RAMP

a b s t r a c t

Ethnopharmacological relevance: Gastrodia elata (GE) Blume (family Orchidaceae) is a traditional Chineseherbal medicine for treating headaches, dizziness, tetanus, and epilepsy, indicating neuronal protectivefunctions.Aim of the study: To evaluate the neuroprotection of GE and its molecular mechanism in preventing serumdeprivation-induced PC12 cell apoptosis.Materials and methods: An MTT assay and Hoechst staining were used to respectively validate serumdeprivation-induced cell death and apoptosis. Cyclic (c)AMP formation and protein kinase (PK)A activitywere also measured after GE treatment. Western blotting was used to detect the phosphorylation of thecAMP response element-binding (CREB) protein. Transient transfection of a dominant negative CREB was

KAREBpoptosis

used to validate the importance of CREB.Results: GE targeted the adenosine A2A receptor (A2A-R). GE increased cAMP formation, PKA activity,and phosphorylation of the CREB protein. GE-induced CREB protein phosphorylation and protection wasblocked by a PKA inhibitor and overexpression of the dominant negative CREB, respectively.Conclusions: These results support the neuroprotective effects of GE. The protective mechanism might bemediated through an A2A-R/cAMP/PKA/CREB-dependent pathway.

. Introduction

Gastrodia elata (GE) Blume (family Orchidaceae) is officiallyisted in the Chinese Pharmacopoeia and is used to treat headaches,izziness, tetanus, epilepsy, infantile convulsions, and numbnessf the limbs, providing evidence that GE has central nervous sys-em (CNS)-protective effects. Based on these indications, a varietyf studies were carried out to purify its effective constituents andnvestigate its biological functions. Gastrodin and vanillin derivedrom GE were shown to exert sedative and anticonvulsive effects

Wu et al., 1989). Others showed that extracts of GE exertedn inhibitory effect on kainate binding to the glutamate recep-or (Andersson et al., 1995), suggesting an antiepileptic effect.

∗ Corresponding author at: National Research Institute of Chinese Medicine, 155- Li-Nung St., Sec. 2, Shipai, Peitou, Taipei 112, Taiwan. Tel.: +886 2 28201999x8061;ax: +886 2 28250743.

E-mail address: Andrew@nricm.edu.tw (N.-K. Huang).1 These authors contributed equally to this article.

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.08.064

© 2011 Elsevier Ireland Ltd. All rights reserved.

Subsequent research demonstrated that extracts of GE have anti-convulsive effects against kainate-induced seizures and epilepsyin rats (Hsieh et al., 1999) and protect against glutamate-inducedcell death in IMR-32 human neuroblastoma cells (Lee et al., 1999),supporting the CNS actions of GE.

In recent years, the traditional uses of many Chinese herbalmedicines were validated. Some of these medicines with reliableethnopharmacological properties were recently demonstrated topossess neurotrophic and neuroprotective abilities, such as gin-seng extract in preventing rodent models of Parkinson’s disease(PD), which might be valuable for preventing various forms ofneuronal cell loss (Van Kampen et al., 2003). Indeed, as describedabove, GE and its active principles have come to light, and exten-sive research in preventing neurotoxicity and neurodegenerationhas been initiated. For instance, GE was experimentally shown toprevent ischemia injury (Zeng et al., 2006), Alzheimer’s disease (AD;

Kim et al., 2007), and PD (An et al., 2010) and improve learning andmemory (Chen et al., 2011). These data further highlight the neu-roprotective effects of GE. However, the targets and mechanismsmostly remain elusive.

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20 C.-F. Tsai et al. / Journal of Ethn

On the other hand, adenosine, which is released frometabolically active cells by facilitated diffusion or is generated

xtracellularly by degradation of released ATP, is a potent biologicalodulator (Van Kampen et al., 2003). It is well known that adeno-

ine modulates activities of numerous cell types including variouseuronal populations, platelets, neutrophils, and smooth muscleells (Daval et al., 1991). To date, four adenosine receptors (A1-, A2A-

A2B-, and A3-R) were identified. They are involved in regulatingealth and disease (Gessi et al., 2011), including neuroprotectionnd neurodegeneration (Stone et al., 2009; Gomes et al., 2011), suchs ischemia, epilepsy, Huntington’s disease, PD, AD, and depression,nd support adenosine receptors as targets for therapeutic inter-entions in neurodegeneration (Abbracchio and Cattabeni, 1999).herefore, since our previous article showed that partially purifiedE could protect against serum deprivation-induced apoptosis ineuronal-like cells (Huang et al., 2004), in this study, we furtheremonstrated that the neuroprotection of GE targeted the A2A-R.ubsequently protective mechanisms were also examined in thistudy.

. Materials and methods

.1. Reagents and cell culture

All reagents were purchased from Sigma Chemical (St. Louis,O, USA) except where otherwise specified. CGS 21680 (CGS), ZM

41385 (ZM), SCH 58261 (SCH), dibutyl-cyclic AMP (db-cAMP), andorskolin (FK) were purchased from Tocris (Bristol, UK). H-89 and

cAMP EIA kit were purchased from Biomol (Plymouth Meeting,A, USA). An IQTM Serine/Threonine Kinase Assay kit was pur-hased from Pierce (Rockford, IL, USA). A Plasmid Midiprep Kit andipofectamineTM 2000 were purchased from Invitrogen (Carlsbad,A, USA). Plasmids were purchased from Clontech (Palo Alto, CA,SA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine

erum (FBS), and horse serum were purchased from HyCloneLogan, UT, USA). Antibodies were purchased from Cell Signal-ng Technology (Beverly, MA, USA). PC12 cells were maintained inMEM supplemented with 10% (v/v) horse serum and 5% (v/v) FBSnd were incubated in a CO2 incubator (5%) at 37 ◦C.

.2. Extraction and isolation of GE

Three-year-old GE harvested in Sichuan, China in April and Mayas purchased from a herbal medicine store and identified byr. Hsien-Chang Chang of the Bureau of Food and Drug Analysis,epartment of Health, Taipei, Taiwan. Slices (3 kg) were extractedith 4 L of 70% MeOH each time, at 60 ◦C overnight. The 70% MeOH

xtracts were combined and evaporated under reduced pressureo give 560 g of residue. The residue was chromatographed overharcoal with H2O, 25% EtOH, 50% EtOH, 75% EtOH, and 100% EtOH.ractions of the 25% and 50% EtOH eluates were concentrated andurther purified on a Diaion HP-20 column with a H2O/methanolradient to produce 50–75% MeOH eluates (9.5 g). The 75% MeOHraction was used in this study because it exhibited the highestrotection (Huang et al., 2004).

.3. MTT assay

Survival was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-iphenyl tetrazolium bromide (MTT) assay as described byosmann (1983). Cells growing on 150-mm plates were washed

times with phosphate-buffered saline (PBS) and resuspended

n DMEM. Suspended cells were plated on 96-well plates104 cells/well) and treated with the indicated reagent(s). Afterncubation for 24 h, MTT was added to the medium (0.5 mg/ml) andncubated at 37 ◦C for 2–3 h. After discarding the medium, DMSO

macology 138 (2011) 119– 125

(100 �l) was applied to the well to dissolve the formazan crys-tals derived from the mitochondrial cleavage of the tetrazoliumring. Absorbances at 570 and 630 nm in each well were mea-sured on a micro-enzyme-linked immunosorbent assay (ELISA)reader.

2.4. Hoechst staining

PC12 cells growing on 6-well plates (5 × 105 cells/well) wereserum-deprived by 2 washes of 2 ml PBS supplemented with theindicated reagent or medium. Twenty-four hours later, cells werefixed by 4% paraformaldehyde and stained with Hoechst 33258(5 �g/ml). The percentage of apoptotic cells was determined bytaking fluorescent photomicrographs and counting the numberof nuclear-condensed (apoptotic) cells versus total (1000–1500)cells in each experimental condition to determine the ratio ofapoptosis.

2.5. Western blot analysis

Cells were rinsed with ice-cold PBS and lysed in ice-cold lysisbuffer (20 mM HEPES, 1 mM DTT, 20 mM EGTA, 10% glycerol,50 mM �-glycerophosphate, 10 mM NaF, 1% Triton X-100, 1 mMPMSF, 1 mM Na3VO4, 2 �M aprotinin, 100 �M leupeptin, 2 �M pep-statin, and 0.5 �M OKA). After sonication, cell debris was removedby centrifugation at 14,000 rpm for 10 min, and the supernatantwas utilized for a Western blot analysis. Equal amounts of sam-ple were separated by 10% polyacrylamide gel electrophoresis.The resolved proteins (25 �g/lane) were then electroblotted ontoImmobilon polyvinylidene difluoride membranes (Millipore, Bed-ford, MA, USA). Membranes were blocked with 5% skim milk andthen sequentially incubated with the first and second antibodiesfor 1 h at room temperature. After washing, blots were processedfor visualization using an enhanced chemiluminescence system(Pierce). Blots were then exposed to Kodak XAR-5 film (Rochester,NY, USA) to obtain the fluorographic images.

2.6. Measurement of cAMP

The formation of intracellular cAMP was measured by followingthe instructions of the cAMP EIA kit (AK-205). In brief, after differentlengths of incubation with the indicated reagent(s), cells growingon 6-well plates at 6 × 105 cells/well were washed once with 2 mlice-cold PBS and lysed with 200 �l 0.1 N HCl. Lysates were used forthe subsequent cAMP measurement.

2.7. Measurement of PKA activity

PKA activity was measured by following the instructions ofthe IQTM Serine/Threonine Kinase Assay kit (no. 62033) contain-ing Kemptide as a PKA substrate. The fluorescent intensity wasinversely correlated with PKA activity.

2.8. Nuclear protein preparation

Cells were washed with ice-cold PBS and collected by centrifu-gation at 14,000 × g for 15 s. The pellet was lysed with 100 �l ofbuffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl,0.5 mM dithiothreitol (DTT), and 1% (v/v) protease inhibitor cocktail(containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatinA, bestatin, leupeptin, aprotinin, and trans-epoxysuccinyl-l-leucyl-

amido(4-guanidino)butane). After 10 min of incubation on ice,6 �l of 10% IGEPAL detergent was added to the cell suspension,and the tube was briefly mixed by vortexing. Following a further5-min incubation, the cell lysate was centrifuged at 14,000 × g for

C.-F. Tsai et al. / Journal of Ethnopharmacology 138 (2011) 119– 125 121

Fig. 1. Adenosine A2A receptor (A2A-R) antagonist(s) blocked Gastrodia elata (GE) in preventing serum deprivation-induced PC12 cell apoptosis. (A) After SCH or ZM treatmentfor 30 min, serum-deprived PC12 cells were treated with or without 100 �g/ml GE (or 0.1 �M CGS) for another 24 h. Cell viability was expressed as a percentage of the resultsof the MTT assay measured in the serum-containing group. (B) After treatment for 24 h, cells were fixed with paraformaldehyde and stained using Hoechst 33258. S, SF, GE,and ZM respectively indicate serum-containing, serum-free, GE-containing, and ZM-containing treatments. White arrowheads indicate apoptotic cells. Data points representthe mean ± S.E.M. of at least three independent experiments (n = 3–6). *p < 0.05, compared to the serum-deprived group. The bar represents 50 �m.

1 opharmacology 138 (2011) 119– 125

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Fig. 2. Gastrodia elata (GE) activated the cAMP/PKA-dependent pathway. (A) Cellswith or without ZM pretreatment for 30 min following 100 �g/ml GE or 10 �MFK (30 min) treatment for the indicated intervals were lysed for analysis of cAMP.*p < 0.05, compared to the serum-containing group. (B) After H-89 treatment for30 min, serum-deprived PC12 cells were treated with or without 100 �g/ml GE,10 �M FK, or 100 �M db-cAMP for another 24 h. Cell viability was expressed as apercentage of the results of the MTT assay measured in the serum-containing group.*p < 0.05, compared to the respective treatment and serum-deprived group. (C) Cellswith or without H-89 pretreatment for 30 min following 100 �g/ml GE or 10 �M FKtreatment for the indicated intervals were lysed for analysis of PKA activity. *p < 0.05,compared to the serum-containing group. Data points represent the mean ± S.E.M.of at least three independent experiments (n = 3–6).

22 C.-F. Tsai et al. / Journal of Ethn

5 s. The pellet (containing nuclei) was re-suspended in buffer con-aining 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM

gCl2, 0.5 mM DTT, 0.2 mM EDTA, and 1% (v/v) protease inhibitorocktail. The suspension was incubated on ice for 30 min, vortexedeveral times during this period, and then centrifuged for 5 mint 15,000 × g. The supernatant containing the nuclear protein wassed for the Western blot analysis.

.9. Transient transfection

The control (pEGFP), dominant negative CREB (pCMV-KCREB),nd dominant positive CREB (pCMV-CREB) vectors were preparedsing a PureLinkTM Hipure Plasmid Midiprep kit. The pEGFP vectoras used as a control which did not harbor the cytomegalovirus

CMV) promoter region. The dominant positive pCMV-CREB over-xpressed a normal CREB protein. However, the pCMV-KCREBector expressed a mutant variant of the human CREB proteinhat contains mutations in its DNA-binding domain. KCREB actss a dominant repressor by forming an inactive dimer with CREB,locking its ability to bind the cAMP-regulated enhancer ele-ent (CRE). Each vector was transfected into PC12 cells using

ipofectamine 2000 following the manufacturer’s protocol. Beforeransfection, cells were incubated with Hank’s balanced salt solu-ion for 10 min. The transfection efficiency was typically >50%.ransfected cells were harvested for the MTT assay at 24 host-transfection.

.10. Statistical analysis

Results were analyzed by one- or two-way analysis of varianceANOVA) according to which was suitable. Differences between

eans were assessed by the Student–Newman–Keuls method andere considered significant at p < 0.05.

. Results

.1. A2A-R antagonists blocked the protection of GE against serumeprivation-induced apoptosis in PC12 cells

The partially purified GE extract was shown to prevent serumeprivation-induced cell death in PC12 cells (Fig. 1A). This protec-ion was blocked by two A2A-R antagonists, ZM and SCH (Fig. 1A).he protection afforded by CGS, an A2A-R agonist, was used as aositive control. ZM blocked the protection of GE against serumeprivation-induced apoptosis (Fig. 1B).

.2. GE increased cAMP formation and PKA activity

GE significantly increased cAMP formation (Fig. 2A) which waslocked by ZM, an A2A-R antagonist (Fig. 2A). FK, which directlyctivates adenylyl cyclase and increases cAMP formation, was useds a positive control. The protection afforded by GE and two PKActivators, db-cAMP and FK, was blocked by H-89, a PKA inhibitorFig. 2B). GE significantly increased PKA activity which was blockedy H-89 (Fig. 2C). FK resulted in PKA activation and was used as aositive control.

.3. GE increased CREB phosphorylation

GE increased the phosphorylation of CREB (p-CREB) duringerum deprivation (Fig. 3A). GE-induced p-CREB was blocked by-89 (Fig. 3B). H-89-blocked CGS-increased CREB phosphorylationas used as a positive control (Fig. 3C).

C.-F. Tsai et al. / Journal of Ethnopharmacology 138 (2011) 119– 125 123

Fig. 3. Gastrodia elata (GE) increased phosphorylation of CREB. (A) Cells with orwithout 100 �g/ml GE treatment for the indicated times during serum deprivationwere harvested and analyzed by Western blotting. (B) Cells pretreated with 10 �MH-89 for 30 min followed by 100 �g/ml GE treatment for the indicated times dur-ing serum deprivation were harvested and analyzed by Western blotting. (C) Cellspib

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Fig. 4. Gastrodia elata (GE) prevented serum deprivation-induced apoptosis throughan adenosine A2A receptor (A2A-R)/cAMP/PKA/CREB-dependent pathway. (A) Cellspretreated with 10 �M H-89 for 30 min followed by 100 �g/ml GE or 10 �M FKtreatment for the indicated times during serum deprivation were harvested, andthe nuclear fractions were analyzed by Western blotting. (B) Cells were tran-siently transfected with a control vector or with a vector overexpressing CREB orKCREB. One day post-transfection, cells were subjected to an MTT assay. Cell via-bility was expressed as a percentage of the results of the MTT assay measuredin the serum-containing control group. Data points represent the mean ± S.E.M.of at least three independent experiments (n = 3–6). *p < 0.05 compared to thecontrol vector-transfected and serum-deprived group. (C) GE antagonized serumdeprivation-induced apoptosis through acting on the A2A-R and modulation via acAMP/PKA/CREB-dependent pathway. CGS was used as a positive control to activatethe A2A-R which was blocked by ZM. FK was used as a positive control to induce PKA

retreated with 10 �M H-89 for 30 min followed by 0.1 �M CGS treatment for thendicated times during serum deprivation were harvested and analyzed by Westernlotting.

.4. GE-mediated CREB activation played an important role inegulating apoptosis

H-89 blocked GE-increased nuclear CREB phosphorylationFig. 4A). FK was also used as a positive control. Overexpressionf the dominant negative mutant of CREB (KCREB) counteractedhe protection by GE and CGS (Fig. 4B). GE prevented serumeprivation-induced apoptosis by acting on the A2A-R which

nvolved increased cAMP formation, PKA activity, and CREB phos-horylation (Fig. 4C).

. Discussion

Apoptosis plays an important role during neuronal develop-ent, toxicity, and stress, and may underlie various neurodegener-

tive disorders. We utilized serum deprivation-induced apoptosisn neuronal-like PC12 cells as a model to identify the neuroprotec-ive effects from traditionally used herbal extracts. We previouslyound that the methanol extract of GE exhibited significant pro-ection against serum-deprived apoptosis (Huang et al., 2004). Inhis study, we further demonstrated the target and neuroprotective

echanism of GE.The PC12 cell line is a commonly used model for studying neu-

onal differentiation and cell death. Apoptosis may occur whenriggered by deprivation of either serum (Rukenstein et al., 1991)r trophic factor/nerve growth factor (NGF) (Batistatou and Greene,991; Lindenboim et al., 1995). Protection by GE was primarily

dentified using an MTT assay (Fig. 1A). Two A2A-R antagonists,M and SCH, were found to block the protection by GE (Fig. 1A),emonstrating the target site of GE. The protection mediated by2A-R activation in this system was confirmed by CGS that waslso blocked by A2A-R antagonists (Fig. 1A) (Huang et al., 2001). Inddition, GE suppressed serum deprivation-induced apoptosis thatas blocked by ZM as also monitored by Hoechst staining (Fig. 1B).

hese data indicated that GE acting on the A2A-R is required for itsrotection.

Since signaling of the A2A-R sequentially involves couplingith GS�, activation of adenylyl cyclase, formation of cAMP, and

activation which was reversed by H-89. Solid and dashed lines respectively indicatestimulatory and inhibitory effects.

stimulation of PKA (Fredholm et al., 2005), we next measured andfound that GE did increase cAMP formation (Fig. 2A). Pretreatmentwith ZM blocked this phenomenon, indicating an A2A-R-mediatedevent. The importance of cAMP formation after GE treatment wasconfirmed by directly treating db-cAMP (a cAMP analogue) andFK (Fig. 2A and B). Furthermore, this protection (including GE)was blocked by H-89 (Fig. 2A), a PKA inhibitor, confirming the

importance of GE-mediated cAMP formation and drawing atten-tion to PKA. We further identified and found that GE activated PKA(Fig. 3C). H-89 pretreatment blocked this phenomenon (Fig. 3C),

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24 C.-F. Tsai et al. / Journal of Ethn

upporting the importance of this signal transduction pathway.herefore, a cAMP/PKA-dependent pathway is required for protec-ion by GE. Currently, PKA-mediated transcription factors includeREB, activating transcription factor-1, AP-2, nuclear factor (NF)-B, etc. (Daniel et al., 1998). Among them, the signaling pathway ofKA/CREB is well known for its antiapoptotic effect (Shaywitz andreenberg, 1999); we thus characterized whether GE could activateREB.

The transcriptional activation of CREB is crucially dependent onhe phosphorylation of Ser133 by PKA (Gonzalez and Montminy,989) or by other kinases, such as Ca2+-activated calmodulininases, ribosomal S6 kinase 2, or mitogen-activated proteininase-activated protein kinase 2 (Beitner-Johnson et al., 1998).n its active form, CREB was shown to regulate many aspects ofeuronal functioning, including neuronal excitation (Moore et al.,996), development (Imaki et al., 1994) and long-term synapticlasticity (Silva et al., 1998). Recent evidence suggests that CREBight also be involved in an active process of neuroprotection

Walton and Dragunow, 2000) or that its disruption in the brainight lead to neurodegeneration (Mantamadiotis et al., 2002), sug-

esting a pivotal role of CREB in preventing cell death (Finkbeinert al., 1997). Since the protection by GE was mediated through

PKA-dependent pathway, the downstream target was subse-uently assayed and revealed that CREB phosphorylation increasedfter GE treatment (Fig. 3A). Pretreatment with H-89 blocked GE-nduced CREB activation (Fig. 3B), indicating that GE may have theotential to prevent serum-deprived PC12 cell death through theKA/CREB signaling pathway. CGS-induced CREB activation, whichas also blocked by H-89 (Fig. 3C), was monitored to re-confirm

he A2A-R-mediated event. In addition, since CREB is a nuclear tran-cription factor, we thus extracted the nuclear fraction and furtheronfirmed GE-mediated CREB activation (Fig. 4A). Thus, in order toonfirm the role of CREB as a protective mechanism of GE, dominantositive and negative vectors of CREB were applied. As expected,he protection by GE (including CGS) was attenuated by KCREBverexpression (Fig. 4B), demonstrating the pivotal roles of the2A-R and CREB in mediating protection by GE.

. Conclusions

Validation of traditionally used herbal medicines through cur-ent pharmacological investigations is important. However, owingo the existence of multiple components or constituents, the target-ng and mechanisms of these medicines can be diverse and difficulto identify. Taking GE as an example, the anti-convulsion, anti-sychosis, and anti-depression effects were respectively attributedo neuromodulation of GABAergic (Ha et al., 2000; Shin et al.,011a), serotonergic (Shin et al., 2011b), and monoaminergic (Chent al., 2009) systems. In this study, we demonstrated that the neuro-rotective effect of GE may act on the adenosinergic system (A2A-R).ince the adenosinergic system is also involved in modulating thebove described convulsions, psychosis, depression, etc. (Fredholmt al., 2005; Gessi et al., 2011; Gomes et al., 2011; Stone et al.,009), understanding the targeting of the neuroprotective effectsf GE requires further investigations. Taken together, we demon-trated that the neuroprotective effect of GE acts on the A2A-R and isodulated through a cAMP/PKA/CREB-dependent pathway. These

ndings support the neuroprotective effects of GE and also extendhe therapeutic potential of GE.

cknowledgements

This work was supported by Cardinal Tien Hospital (CTH-8-1-2A23), the National Research Institute of Chinese MedicineNRICM98-DBCM-08), and the National Science Council, Taiwan

macology 138 (2011) 119– 125

(NSC98-2323-B-077-001). We thank Mr. D.P. Chamberlin for criti-cally editing the manuscript.

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