protective eﬀects of ginseng saponins on 3-nitropropionic

ARTICLE IN PRESSDTD 5

Neuropharmacology -- (2005) ---e---

www.elsevier.com/locate/neuropharm

Protective effects of ginseng saponins on 3-nitropropionicacid-induced striatal degeneration in rats

Jong-Hoon Kima, Sunoh Kimb, In-Soo Yoona, Jong-Hwan Leea, Byung-Jun Janga,Sang Min Jeonga, Jun-Ho Leea, Byung-Hwan Leea, Jin-Soo Hanc,

Sekwan Ohd, Hyung-Chun Kime, Tae Kyu Parkf,Hyewhon Rhimb,*, Seung-Yeol Naha,)

aResearch Laboratory for the Study of Ginseng Signal Transduction and Department of Physiology and Anatomy,

College of Veterinary Medicine, Konkuk University, 143-701 Seoul, Republic of KoreabBiomedical Research Center, KIST, Seoul, Republic of Korea 136-701

cHae-Eun Biomedical Research Institute, Seoul, Republic of Korea 477-840dDepartment of Neuroscience, College of Medicine, Ewha Womans University, Seoul, Republic of Korea 158-710

eCollege of Pharmacy, Kangwon National University, Republic of Korea 200-701fDepartment of Biotechnology, College of Natural Science, Konkuk University, Seoul, Republic of Korea 380-701

Received 15 July 2004; received in revised form 14 October 2004; accepted 10 December 2004

Abstract

The precise cause of neuronal cell death in Huntington’s disease (HD) is not known. Systemic administration of 3-nitropropionicacid (3-NP), an irreversible succinate dehydrogenase inhibitor, not only induces a cellular ATP depletions but also causes a selectivestriatal degeneration similar to that seen in HD. Recent accumulating reports have shown that ginseng saponins (GTS), the major

active ingredients of Panax ginseng, have protective effects against neurotoxin insults. In the present study, we examined in vitro andin vivo effects of GTS on striatal neurotoxicity induced by repeated treatment of 3-NP in rats. Here, we report that systemicadministration of GTS produced significant protections against systemic 3-NP- and intrastriatal malonate-induced lesions in rat

striatum with dose-dependent manner. GTS also improved significantly 3-NP-caused behavioral impairment and extended survival.However, GTS itself had no effect on 3-NP-induced inhibition of succinate dehydrogenase activity. To explain the mechanismsunderlying in vivo protective effects of GTS against 3-NP-induced striatal degeneration, we examined in vitro effect of GTS against

3-NP-caused cytotoxicity using cultured rat striatal neurons. We found that GTS inhibited 3-NP-induced intracellular Ca2C

elevations. GTS restored 3-NP-caused mitochondrial transmembrane potential reduction in cultured rat striatal neurons. GTS alsoprevented 3-NP-induced striatal neuronal cell deaths with dose-dependent manner. The EC50 was 12.6G 0.7 mg/ml. These resultssuggest that in vivo protective effects of GTS against 3-NP-induced rat striatal degeneration might be achieved via in vitro inhibition

of 3-NP-induced intracellular Ca2C elevations and cytotoxicity of striatal neurons.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Ginseng saponins; 3-Nitropropionic acid; Striatum toxicity; Neuroprotection

* Corresponding authors. Research Laboratory for the Study of

Ginseng Signal Transduction and Department of Physiology, College

of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic

of Korea. Tel.: C82 2 450 4154; fax: C82 2 450 3037.

E-mail address: [email protected] (S.-Y. Nah).

0028-3908/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuropharm.2004.12.013

1. Introduction

3-Nitropropionic acid (3-NP) is a compound found incrops contaminated with fungi (Ming, 1995) and causesneurotoxicity in both animals and human (James et al.,

mailto:[email protected]

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2 J.-H. Kim et al. / Neuropharmacology -- (2005) ---e---

1980; Ludolph et al., 1991). Since treatment of 3-NPinduces a selective striatal pathology similar to that seenin Huntington’s disease (HD), it has been widely used asan agent for animal model study of HD (Beal et al.,1993; Borlongan et al., 1997; Brouillet et al., 1999). Theprimary mechanism of 3-NP-caused neurotoxicity in-volves the irreversible inhibition of mitochondrialsuccinate dehydrogenase (SDH) and leads to inhibitionof ATP synthesis (Alston et al., 1977; Coles et al., 1979).ATP exhaustion by mitochondrial dysfunction alsosubsequently couples to the slow secondary excitotox-icity by excitatory neurotransmitter (Pang and Geddes,1997). This secondary excitotoxicity in ATP deficientneurons is initiated by voltage-dependent NaC channelactivation, which is coupled to membrane depolariza-tion, Ca2C channel activation, and subsequent NMDAreceptor activation by relief of voltage-dependent Mg2C

block of the NMDA receptor (Novelli et al., 1988;Zeevalk and Nicklas, 1991). These serial cascadesinduced by 3-NP intoxication are also accompaniedwith the impaired mitochondrial Ca2C homeostasis,with intracellular Ca2C elevation via L-type and othertypes of Ca2C channel activations, and with an impairedbuffering capacity on intracellular Ca2C in astrocytesand neurons (Deshpande et al., 1997; Fukuda et al.,1998; Calabresi et al., 2001; Nasr et al., 2003).Moreover, since 3-NP-caused elevation of intracellularcalcium is known to activate calpain and caspase-9,which are involved in neuronal cell death, 3-NP-causedperturbation of calcium homeostasis in mitochondriaand the following activation of these enzymes might bethe main factors in 3-NP neurotoxicity in vivo (Fu et al.,1995; Brouillet et al., 1999; Bizat et al., 2003a, 2003b).

Ginseng saponins (GTS), which are also known asginsenosides, are active ingredients isolated from Panaxginseng C.A. Meyer, which is a well-known tonicmedicine (Nah, 1997). Recent accumulating evidenceshave shown that treatment of GTS not only attenuatesintracellular Ca2C elevation by blocking various typesof Ca2C channels like L-, N-, and P/Q-types anddepolarization-induced Ca2C influx (Nah et al., 1995;Kim et al., 1998a; Rhim et al., 2002) but also inhibitsreceptor agonist-induced intracellular Ca2C mobiliza-tion in neurons (Jeong et al., 2004). GTS also reducesglutamate/NMDA-mediated Ca2C influx in neurons(Kim et al., 1998b, 2002). Furthermore, recent reportsshowed that these GTS-mediated inhibitions on in-tracellular Ca2C elevations could be the basis of in vitroor in vivo protection against excitatory amino acids- orneurotoxins-caused neuronal cell damages. For exam-ple, GTS attenuated glutamate or kainic acid-causedcortical, hippocampal, spinal cord neuron damages inrats (Chu and Chen, 1990; Kim et al., 1998b; Lee et al.,2002a; Liao et al., 2002). GTS also attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-meth-yl-4-phenylpyridinium (MPPC)-induced dopaminergic

neuron deaths in rat or dopaminergic cell culture in mice(Kampen et al., 2003; Radad et al., 2004).

However, until now little is known about protectiveeffect of GTS against progressive striatal degenerationinduced by repeated neurotoxin insults, which could belinked to subsequent intracellular Ca2C elevations. Wetherefore examined whether systemic administrationof GTS could exert protective effects against systemic3-NP- or intrastriatal injected malonate-induced ratstriatal degeneration. We examined the ability of GTSto block 3-NP-induced intracellular Ca2C increases,3-NP-induced mitochondrial damage, and 3-NP-in-duced cytotoxicity in cultured rat striatal neurons.Herein, we present the results that in vivo protectiveeffects of GTS against 3-NP neurotoxicity are mediatedthrough in vitro inhibition of 3-NP-induced intracellularCa2C elevations and cytotoxicity of striatal neurons.

2. Materials and methods

2.1. Drugs

Fig. 1 shows the structures of the eight representativeginsenosides. GTS was kindly obtained from KoreaGinseng Corporation (Taejon, Korea). GTS containedRb1 (17.1%), Rb2 (9.07%), Rc (9.65%), Rd (8.26%), Re(9%), Rf (3%), Rg1 (6.4%), Rg2 (4.2%), Rg3 (3.8%),Ro (3.8%), Ra (2.91%) and other minor ginsenosides.GTS was diluted with bath medium or saline before use.

R1OR2

2012

36

OR3OH

R3R1 R2Ginsenosides

-Glc2-Glc-Glc2-Glc-Glc2-Glc-Glc2-Glc-H-H-H-H-Glc2-Glc

-H-H-H-H-O-Glc2-Rha-O-Glc2-Glc-O-Glc-O-Glc2-Rha-H

-Glc6-Glc-Glu6-Ara(pyr)-Glc6-Ara(fur)-Glc-Glc-H-Glc-H-H

Rb1Rb2RcRdReRfRg1Rg2Rg3

Fig. 1. Structures of the eight representative ginsenosides. They differ

at three side chains attached to the common steroid ring. Abbrevia-

tions for carbohydrates are as follows: Glc, glucopyranoside; Ara

(pyr), arabinopyranoside; Rha, rhamnopyranoside. Superscripts in-

dicate the carbon in the glucose ring that links the two carbohydrates.


3J.-H. Kim et al. / Neuropharmacology -- (2005) ---e---

3-NP and other chemicals were of analytical grade andpurchased from Sigma (St. Louis, MO).

2.2. Drug administrations

Male SpragueeDawley rats (300e350 g) were uti-lized. Animals were housed in groups of four per cage ina room with controlled temperature and humidity, andon a 12-h lightedark cycle with lights on at 7:00 AM.Food and water were available ad libitum throughoutthe experiments. Their care and handling were inaccordance with the highest standards of institutionalguidelines. Ninety animals were randomly divided intocontrol vehicle, 3-NP alone, and four different doses ofGTSC 3-NP group, respectively. Control vehicle groupwas administered only with saline. GTS dissolved insaline was administrated intraperitoneally (i.p.) to ratswith different doses (6.25, 12.5, 25, or 50 mg/kg, twice/day 3 h before 3-NP administration, every 12-h intervalfor 30 days, nZ 15, each group). GTS administrationbegan on day 0, followed by 3-NP (10 mg/kg, i.p., onceevery 3 days) on day 3. Fresh 3-NP powder wasdissolved in saline and the pH was adjusted to 7.4 withNaOH. On day 30, 3-NP and GTS administration werediscontinued. In malonate experiments, 40 animals wererandomly divided into saline- and GTS-treated groups.GTS (50 mg/kg, twice/day, every 12 h interval, nZ 20,each group) was preadministered for 3 days beforeintrastriatal administration of malonate. GTS wasfurther administered for a week before sacrifice.Malonate was dissolved in 0.1 M PBS (pH 7.4) at1.0 M concentration. Animals were positioned ina stereotaxic frame after anesthesia. Malonate wasinfused into the right striatum of both groups at the fol-lowing stereotaxic co-ordinates: anterior from bregma.AZ 0.8 mm; lateral to the midline, LZ�2.8 mm; andvertical below dura, VZ�6.0 mm with the nose bar set2.3 mm below the interaural line. A total of 1 ml wasinfused through a stainless-steel cannula connected viapolyethylene tubing to a 10-ml glass Hamilton syringemounted on an infusion pump at a rate of 0.25 ml/min.The cannula was left in place for a further 5 min prior towound closure. After removal of the brain, striatallesion volume was determined by TTC staining asdescribed below.

2.3. TTC staining, striatal lesion volume measurement,and cell counting

At the end of drug administration, animals weresacrificed for 2,3,5-triphenyltetrazolium chloride (TTC)staining. Brains were quickly removed and placed in ice-cold saline solution. Brains were sectioned at 2-mmintervals using rat brain matrix. Slices were thensubjected in 2% TTC for 5 min at 37 �C in the darkand removed and placed in 4% paraformaldehyde, pH

7.4 in 0.1 M phosphate buffer. For measurement oflesion volumes, serial, coronal sections (25 mm) were cutthroughout the entire striatum using a cryostat andevery fourth section was thaw-mounted on gelatin-coated slides (Maragos et al., 1999). Sections wereexposed to paraformaldehyde vapor for at least 24 h andstained for Nissle substance with cresyl violet. Usingcomputer-based image analysis (Image, NIH), lesionvolumes of control, 3-NP alone, different doses ofGTSC 3-NP, malonate alone, or GTSCmalonategroup were calculated by summing the cross-sectionalarea of the lesion in each section and multiplying thisvalue by the distance between the sections. Totalneuronal counts were performed using cresyl violet-stained sections. Cells within lesions on both sides of thebrain were measured. Six fields (400 mm2 each, two fromeach of three sections) from all six animals in controlvehicle, 3-NP alone, or GTS (100 mg/kg)C 3-NP group(nZ 6, each group) were counted. Lesions were mappedby using camera lucida drawings so that equivalentstriatal areas could be counted from control vehicleanimals. The whole of the lesion area was also used forNADPH diaphorase-positive neuron counts.

2.4. Histology

Rats were anesthetized with pentobarbital (50 ml/kg,i.p.) and perfused transcardially with 250 ml of ice-coldheparinized saline, followed by 300 ml of ice-cold 4%paraformaldehyde in 0.1 M phosphate buffer. Thebrains were dissected out of the skull and post-fixedovernight in 4% paraformaldehyde and then cryopro-tected in 30% sucrose solution for 2 days. Subsequently,the brains were frozen in 2-methylbutane (isopentane)and stored at �80 �C until they were processed forhistochemical and immunohistochemical studies. Coro-nal sections (40 mm thickness) at the level 0e1.2 mmanterior to bregma were cut with a cryostat (Leica, 1800,UK). Sections for cresyl violet staining were mounted ongelatin-coated slides and stained using a 1% solutionof cresyl violet acetate (Sigma). NADPH diaphorasestaining was performed on free-floating cryostat sectionsby incubation for 60 min in the dark at 37 �C ina reaction mixture containing 0.02 M phosphate buffer,10 mg/ml NADPH (reduced form, Sigma), and 1 mg/mlnitroblue tetrazolium (Sigma).

2.5. Immunohistochemistry

For immunohistochemistry, the coronal sections wereincubated overnight at 4 �C in PBS containing mouseglial fibrially acidic protein (GFAP) monoclonal anti-body (Sigma, 1:1000 dilution), 0.3% Triton X-100,0.5 mg/ml bovine serum albumin and 1.5% normal goatserum. Then the sections were washed 3 times, for 5 mineach in PBS solution and incubated with goat anti-mouse



IgG (Vector, 1:200) for 1 h and with avidinebiotineperoxidase complex (Vector 1:200) for 1 h at roomtemperature, respectively. The sections were reacted with0.005% 3,3#-diaminobenzidine 4HCl (DAB) and 0.01%H2O2 for 10 min. Some sections were counterstainedwith hematoxylin.

2.6. Determination of SDH activity

Measurement of SDH activity in control vehicle, GTSalone, 3-NP alone, or GTSC 3-NP group was per-formed as described by Brouillet et al. (1998). Briefly, 24rats were randomly divided into control saline, GTSalone, 3-NP alone, and GTSC 3-NP group (nZ 6, eachgroup). Control saline group was administered only withsaline. GTS dissolved in saline was administered in-traperitoneally (i.p.) to rats with dose (100 mg/kg) 3 hbefore 3-NP (25 mg/kg) administration. Animals weresacrificed after 8 h and brains were processed for SDHhistochemistry on frozen sections. The brains of eachgroup were rapidly frozen in isopentane (�25 �C) andkept at�80 �C and cut in sections of 20 mmandmountedon RNAse free poly-L-lysine coated slides and air dried.Approximately 20e30 sections in each animal wereincubated for 15 min in 0.1 M PBS at 37 �C to activateSDH. They were washed with a large volume of PBS andfollowed by incubation in 0.3 mM nitroblue tetrazolium,0.05 M sodium succinate, and 0.05 M phosphate buffer(pH 7.6) for 30 min at 37 �C. For determination ofnonspecific staining unrelated to SDH activity, adjacentsections were incubated in the same medium in whichsuccinate was omitted. Sections were rinsed in cold PBSfor 5 min, fixed in 4% paraformaldehyde, and rinsed inwater and dried at room temperature. The image of eachsection was acquired, and the quantifications wereperformed as described previously using computer-basedimage analysis (Image, NIH) (Brouillet et al., 1998).

2.7. Striatal neuron preparation

Striatal neurons were prepared using the methodsmodified from Keene et al. (2001) with slight modifica-tions. Briefly, the striata were isolated from 16 to 18-day-old fetal SpragueeDawley rats and incubated with0.25% trypsin in HBSS at 37 �C for 15 min. Cells werethen mechanically dissociated with fire-polished Pasteurpipettes by trituration and plated on poly-L-lysine-coated coverslips in a 35-mm culture dish or 6-wellplates. Cells were maintained in Dulbecco’s modifiedEagle’s medium (DMEM; Life Technologies, Inc.Grand Island, NY, USA) containing 10% FBS, 2% B-27 supplement, 20 mM glucose, 26.2 mM sodium bi-carbonate, 100 U/ml penicillin and 100 mg/ml strepto-mycin in a humidified atmosphere of 95% air and 5%CO2 at 37 �C. After 1 day, the medium was replacedwith a fresh defined F-12 medium supplemented with

5 mg/ml insulin, 1 mg/ml transferrin, 20 nM hydrocorti-sone, 30 nM triiodothyronine, 2 mg/ml carnitine, and15 nM selenium oxide to suppress the proliferation ofnon-neuronal cells (Lee et al., 2003). Experiments werecarried out on the cells after 7e15 days in vitro. 3-NPwas prepared as 100! stocks in saline, and wasneutralized to pH 7.4 with NaOH. The final concentra-tion of 3-NP used was 5 mM (Pang and Geddes, 1997).

2.8. Intracellular Ca2C measurement

The acetoxymethyl-ester form of fura-2 (fura-2/AM;Molecular probes, Eugene, OR) was used as thefluorescent Ca2C indicator. Striatal neurons were in-cubated for 40e60 min at room temperature with 5 mMfura-2/AM and 0.001% Pluronic F-127 in a HEPES-buffered solution composed of (in mM): 150 NaCl, 5KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, pHadjusted to 7.4 with NaOH. The cells were then washedwith HEPES-buffered solution and placed on aninverted microscope (Olympus, Japan). Cells wereilluminated using a xenon arc lamp and excitationwavelengths (340 and 380 nm) were selected by a com-puter-controlled filter wheel (Sutter Instruments, CA).Data were acquired every 2e5 s and a shutter in the lightpath between exposures was interposed to protect thecells from phototoxicity. Emitter fluorescence wasreflected through a 515-nm long-pass filter to a frametransfer cooled CCD camera, then the ratios of emittedfluorescence were calculated using a digital fluorescenceanalyzer and converted to intracellular free Ca2C

concentration ([Ca2C]i). All imaging data were collectedand analyzed using Universal Imaging software (WestChester, PA) (Kim et al., 2002).

2.9. Mitochondrial membrane potential measurement

The mitochondrial membrane potential (DJm) wasmonitored using the DJm-sensitive dye, rhodamine123(Rh123; Molecular probes, Eugene, OR) (Kannurpattiet al., 2004). In brief, striatal neurons were incubated inthe dark for 15 min with 5 mg/ml of the dye at roomtemperature. After loading, the cells were washed 3times with HEPES-buffered solution. In GTSC 3-NPgroup, GTS (100 mg/ml) were pretreated for 1 minbefore 3-NP treatment. Cells were illuminated usinga xenon arc lamp and excitation wavelengths (490 nm)were selected by a computer-controlled filter wheel(Sutter Instruments, CA). Emitter fluorescence wasreflected through a 537-nm long-pass filter to a frametransfer cooled CCD camera. Data were acquired every5 s and a shutter in the light path between exposures wasinterposed to protect the cells from phototoxicity. Allimaging data were collected and analyzed using Uni-versal Imaging software (West Chester, PA). Fluores-cent intensity at the starting point was regarded as 1.



2.10. Determination of metabolicinhibition-induced cell death

Metabolic inhibition- or mitochondrial dysfunction-induced cell death was determined by 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay (Mosmann, 1983). Cells were washed withserum-free medium, and then incubated with 3-NP(5 mM) alone or in the presence of various concen-trations of GTS for 24 h at 37 �C. Striatal neurons werepretreated with GTS for 5 min before an exposure to 3-NP. After 24 h incubation in serum-free culturemedium, the cultures were assessed for the extent ofcell viability. Cell viability was measured by thedetection of dehydrogenase activity that was retainedin living cells using a MTT assay. Aliquot (50 ml) ofMTT solution (1 mg/ml) in PBS was added directly tothe cells, and the cells were then incubated for 4 h toallow MTT to metabolize to a formazan. The superna-tant was then aspirated, and 100 ml of DMSO was addedto dissolve the formazan. The optical densities (OD)were measured with an automated spectrophotometricplate reader at a wavelength of 560 nm. Relativesurvival in comparison to untreated controls could thenbe determined.

2.11. Detection of DNA laddering and TUNEL staining

For the detection of DNA ladder, striatal neuronswere harvested in PBS (pH 7.4). The cells werecentrifuged at 12 000! g for 5 min at 4 �C. DNA wasextracted from cultured cells grown under control, 3-NPalone- or GTS and 3-NP-treated groups using a kit(GeneAll�). DNA fragmentation was detected on 1.4%agarose gel electrophoresis at 50 V for 1 h and visualizedby ethidium bromide staining. The terminal deoxynu-cleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) staining was also performed to detect the 3#OH ends resulting from DNA degradation (Garciaet al., 2002). This was conducted using the In Situ CellDeath Detection kit (Roche Diagnostics GmbH,Germany), according to the manufacturer’s instructionswith minor modifications. Briefly, the cells cultured oncover glass were divided into control vehicle, GTS(100 mg/ml) alone, 3-NP (5 mM) alone, or GTSC 3-NPgroup. In GTSC 3-NP group, GTS (100 mg/ml) waspretreated for 1 min before the 3-NP treatment. After24 h, cells were washed with PBS and fixed with 4%paraformaldehyde in PBS for 30 min and permeabilizedwith 0.25% Triton X-100 for 10 min. Again cells wererinsed with PBS and covered with a labeling reactionmixture containing terminal deoxynucleotidyl transfer-ase (TdT) for 60 min at 37 �C and the slides were rinsed3 times with PBS. And Converter-peroxidase (POD) wasadded for 30 min at 37 �C and again slides were rinsed 3times with PBS. Diaminobenzidine (DAB) was treated

for 10 min and reactions were terminated by washing thecells with PBS, and slides were mounted under glasscoverslip. A total of 600 cells in 10 different fieldsincluding TUNEL-negative and -positive neurons ineach treatment group were counted using an OlympusBX-51 light microscope with 40! magnification.

2.12. Behavioral test and survival

Forty-five animals were randomly divided intocontrol vehicle, 3-NP alone, or GTS (50 mg/kg, twice/day, i.p.)C 3-NP group (nZ 15, each group). Otherprocedures for drug administrations were same asdescribed above. Animals in each group were examinedfor sensorimotor ability 3e4 h after administration ofsaline or 3-NP at once every 3 days (Keene et al., 2001).Before dividing into each experimental group, animalswere trained on apparatus for a maximum of 180 s 3consecutive times for 3 days and animals that did notmaster this task were excluded from further experi-ments. The apparatus consisted of a bar, with a diameterof 6.0 cm, subdivided into four compartments by disks50 cm in diameter (Ugo Basile, Italy). The bar rotated atan accelerated speed from 5 to 25 revolutions/min. Foreach trial, the duration that the animal was able tospend on the apparatus prior to falling was measuredwith a trial maximum latency of 180 s. The threeseparate results were averaged and recorded. In survivalexperiments after drug administrations, 60 animals wererandomly divided into control vehicle, 3-NP alone, orGTS (50 mg/kg, twice/day, i.p.)C 3-NP group (nZ 20,each group). Other procedures were same as describedabove. Animals in each group were observed twice daily,early morning and late afternoon. Some of the animalstreated with 3-NP begin to show severe limb paralysis,dystonia, or other abnormal behaviors after 3e4 timesadministration. The criterion for euthanization was thepoint in time at which animals were unable to initiatenormal movement after being gently plodded for 2 min.

2.13. Data analysis

Data are expressed as meanG S.E.M. For statisticalcomparisons, ANOVA followed by Tukey’s test wasused for multiple comparisons and Student’s t-test forpairs of data. P! 0.05 was considered significant.

3. Results

3.1. Effect of GTS on 3-NP- or malonate-inducedrat striatal degeneration

We first examined the effects of GTS on neurotoxicityinduced by repeated treatment of 3-NP using TTCstaining method. The various dosages of GTS (6.25,



12.5, 25, or 50 mg/kg, twice/day 3 h before 3-NP, every12 h interval) were administered on day 0, followed by3-NP (10 mg/kg, i.p., once every 3 days) on day 3. Onday 30, 3-NP and GTS administration were discon-tinued. At the end of drug administration, animals weresacrificed for 2,3,5-triphenyltetrazolium chloride (TTC)staining. Fig. 2A shows representative TTC stainings ineach group. In gross TTC staining of coronal brainsections, animals treated with 3-NP alone displayedbilateral striatal lesions, whereas GTSC 3-NP grouphad a slight lesions in both sides of striatum (Fig. 2A).Thus, increasing doses of GTS from 12.5 to 100 mg/kgwith 3-NP showed dose-dependent protective effectsagainst 3-NP alone-induced rat striatal lesions. Wecould observe significant protective effects of GTSwith doses of 50 and 100 mg/kg (6.7G 2.9 and3.9G 1.9 mm3, �77.8 and �87% reduction comparedwith 3-NP alone treatment, respectively; *P! 0.001,nZ 15, each group) but there was no protective effect atdoses of 12.5 and 25 mg/kg (Fig. 2B). We examinedwhether intraperitoneal administration of GTS couldalso show protective effect against direct intrastriatalmalonate-induced lesions. As shown in Fig. 2C,intraperitoneal administration of the animals withGTS (100 mg/kg) significantly reduced the volume of

the malonate-induced striatal lesion compared with 3-NP alone-treated group (Mal:GTSCMalZ 9.5G0.4:3.2G 0.4 mm3, �66% reduction compared with 3-NP alone treatment; *P! 0.001, nZ 20, each group).The protective effects of GTS were confirmed usingcresyl violet (CV) staining, GFAP, and NADPHdiaphorase histochemistry (Fig. 3). The CV stainingshowed a marked loss of striatal cell bodies in 3-NPalone treatment group (Fig. 3B), whereas GTSC 3-NPgroup greatly attenuated 3-NP-induced cell loss. But wecould observe some degenerating cells with changes ofmorphology compared with control vehicle in GTSC 3-NP group (Fig. 3C, arrows). The protective effects ofGTS were also confirmed by normal and abnormalneuron counting (Table 1). Thus, quantification of totalneuronal loss showed that within core lesion induced by3-NP alone treatment neuronal loss was much severethan was seen in the corresponding region of lesionsfrom rats treated with GTSC 3-NP (Table 1). We couldalso observe NADPH diaphorase-containing interneur-ons with changes of morphology with decreases inlength of neuronal processes in 3-NP alone treatmentgroup but the loss of NADPH diaphorase-containinginterneurons was less pronounced or spared than otherneurons as shown in histochemical staining and neuron

Fig. 2. Effect of GTS on striatal lesions produced by systemic administration of 3-NP or intrastriatal injection of malonate. (A) Pretreatment of

ginsenosides (GTS) attenuates 3-NP alone-induced rat striatal degeneration. The representative coronal brain sections of control (Con), 3-NP alone

(3-NP), or GTS and 3-NP (GTSC 3-NP). The detailed experimental procedures for drug administration and TTC staining were described in Section

2. (B) The histograms show the percent blockade on 3-NP-induced striatal lesion by different doses of GTS (*P! 0.001, compared with 3-NP alone

treatment. nZ 15, each group). (C) The histograms show the percent blockade on intrastriatal malonate-induced lesion by GTS. (*P! 0.01,

compared with malonate alone treatment. nZ 20, each group). Data represent the meansG S.E.M.



Fig. 3. Cresyl violet (CV), GFAP, and NADPH diaphorase staining in coronal brain sections at the level of the striatum and anterior commissure

from a saline (Con), 3-NP, or GTSC 3-NP animals. These are representative photomicrographs from five rats in each group. Rats preadministered

with GTS and subsequent administration of 3-NP resulted in no demonstrable striatal lesions or neuronal loss (GTSC 3-NP) (C, F, and I). In

contrast, rats treated with 3-NP alone (3-NP) resulted in bilateral striatal lesions with marked neuronal loss (B). (E) Inset, after treatment of 3-NP

NADPH diaphorase-containing interneurons showed changes of morphology with decreases in length of neuronal processes as indicated by arrow

head but the loss of NADPH diaphorase-containing interneurons was spared than other neurons in neuron counting (Table 1). (F) Inset, co-

administration of GTS with 3-NP not only spared NADPH diaphorase-containing interneurons but also maintained the similar morphology with

that of control group (D, inset) as indicated by arrow. Scale bar, 50 mm.

counting (Fig. 3E and Table 1). This present result onsparing NADPH diaphorase-containing interneuronsafter 3-NP treatment is similar to that observed in otherprevious report (Reynolds et al., 1998). Moreover, co-administration of GTS with 3-NP not only sparedNADPH diaphorase-containing interneurons by 85% ofcontrol but also maintained the similar morphology ofNADPH diaphorase-containing interneurons with thatof control group (Fig. 3D, F and Table 1). We alsoperformed immunostainings on GFAP, a marker pro-tein of astrocytes. As shown in Fig. 3H, 3-NP treatmentalso caused a complete loss of GFAP immunoreactivityin lesion core, whereas this immunoreactivity was visible

Table 1

Neuronal counts within the striatum lesion area

Treatment Total number

of neurons

(per mm2, nZ 6)

Number of

NADPH neurons

(per mm2, nZ 6)

Control 1568.3G 45.8 27.4G 0.8

3-NP alone 108.7G 10.2* 13.8G 0.6*

GTSC 3-NP 1270.4G 80.5 23.4G 0.8

*P! 0.001 compared with GTSC 3-NP by Student’s t-test.

at the lesion border (data not shown). However,GTSC 3-NP group attenuated 3-NP-caused loss ofGFAP positive cells as much as control group (Fig. 3G,I). Thus, these results on CV staining, histochemistries onNADPH diaphorase and GFAP, and neuron countingindicate that GTS protected from 3-NP-induced striataldamages and cell deaths.

3.2. Effect of GTS on 3-NP-induced SDH inhibition

Because 3-NP is a well-known irreversible inhibitorof SDH in vivo (Brouillet et al., 1998), we tested whetherGTS treatment could interfere the 3-NP-induced in-hibition of SDH activity. As shown in Fig. 4, semi-quantitative histochemical measurements showed thattreatment of 3-NP induced a significant 29.8% in-hibition of SDH activity in the striatum compared withcontrol. However, pretreatment of GTS did notsignificantly affect 3-NP-induced inhibition of SDHactivity, suggesting that striatal SDH impairment in-duced by 3-NP was not modified by GTS treatment.GTS treatment alone also did not affect SDH activity(Fig. 4).



3.3. Effect of GTS on 3-NP-induced sensorimotordeficits and mortality

Since striatum plays an important role in sensorimo-tor behavior, 3-NP-induced striatal lesions usuallycouple to an impairment of motor abilities (Beal et al.,1993). We investigated whether GTS-induced neuro-protection against 3-NP-induced striatal lesions alsoreduces the degree of motor impairment derived from 3-NP intoxication. For this, we performed rota-rodsensorimotor test in saline vehicle-, 3-NP alone-, orGTSC 3-NP group. In these experiments we excludedanimals showing abnormal movements by dystonia,limb paralysis, or other abnormal behaviors after 3-NPadministration in 3-NP alone and GTSC 3-NP group.3-NP alone-treated animals began to show impairedrota-rod performances after 3 times administration of 3-NP and more accumulating administration of 3-NPfurther aggravated behavioral dysfunctions (Fig. 5A).However, the animals of GTSC 3-NP group showedmuch improved rota-rod performance. Thus, co-treat-ment of GTS with 3-NP reduced sensorimotor deficitscompared with 3-NP alone-treated animals (GTSC3-NP: 3-NPZ 128G 9.8: 27G 15.8 s; **P! 0.001,nZ 15 each group, compared with 3-NP alone treat-ment after 9 times administration). The effects of GTSon 3-NP-induced mortality were also investigated. Inthis experiment, some of the animals treated with 3-NPshowed severe limb paralysis, dystonia, or otherabnormal behaviors after 3e4 times administration.Since these animals themselves lost the ability to eatfood and died if they were not paid with further cares,we considered them as dead and have euthanized them.

0.00

0.04

0.08

0.12

0.16

SDH

act

ivit

y(O

.D. u

nit/

30m

in)

Con Con+ GTS

3-NP 3-NP+ GTS

Fig. 4. Effect of GTS on 3-NP-induced SDH activity inhibition. SDH

activity in rat striatum of control vehicle, controlC ginsenosides

(100 mg/kg), 3-NP (25 mg/kg) alone, and GTSC 3-NP co-treated rats

was determined by in situ semiquantitative histochemistry as described

in Section 2. Note that treatment of ginsenosides affects neither control

SDH activity nor 3-NP-induced inhibition of SDH. Data are

meanG S.E.M. *P! 0.01 compared with control by ANOVA and

Scheffe test (nZ 6, each group).

The survival rate was significantly larger in theGTSC 3-NP group than the 3-NP alone group(GTSC 3-NP:3-NPZ 14.6:5.4, *P! 0.01, comparedwith 3-NP alone treatment after 9 times administration,nZ 20 each group,). Only 27% of animals treated with3-NP alone survived, whereas 73% of animals treatedwith GTS and 3-NP survived after 9 times administra-tion of 3-NP (Fig. 5B). Thus, treatment of GTSsignificantly extended survival compared with 3-NPalone-treated animals. On the other hand, in ginsenoside

0 6 12 18 24 30

0 6 12 18 24 30

Tim

e (s

ec)

0

50

100

150

200

250

Con

3-NP

GTS + 3-NP

Con

3-NP

GTS + 3-NP

0

20

40

60

80

100

120

Treatment (day)

Treatment (day)

B

A

Fig. 5. Effects of GTS on 3-NP-induced sensorimotor deficits and

mortality. (A) Rota-rod scores were determined 3e4 h after 3-NP

administration and quantitated as sum latencies to fall over three

trials. Control saline treated group (B) were significantly different

from GTS C 3-NP group. *P! 0.05, compared with GTS C 3-NP

animals. (nZ 15, each group). GTSC 3-NP group (,) also exhibited

significantly improved motor performance compared with 3-NP alone

group (7). (**P ! 0.001 compared with Con or GTS C 3-NP

animals, nZ 15, each group). (B) The effect of GTS on 3-NP-induced

mortality was determined. GTS C 3-NP group (,) improved survival

significantly compared with 3-NP alone treatment group (7). The

mean survival in GTSC 3-NP group was 73%, whereas in 3-NP alone

group was 27% after 9 times administration of 3-NP. *P! 0.01,

compared with 3-NP alone treatment after 9 times administration

(nZ 20, each group).



alone treatment group we could observe a slight de-crease of body weight (!5%) at dose of 100 mg/kg atthe beginning of ginsenosides administration withoutany effects on behavioral activity and mortality but thisphenomenon was disappeared soon (data not shown).

3.4. Effect of GTS on 3-NP-induced intracellular freeCa2C elevation in cultured rat striatal neurons

As a next step, we investigated the mechanism ofprotection of GTS against in vivo 3-NP-induced neuro-toxicity. We examined whether GTS could inhibit 3-NP-induced intracellular Ca2C elevation in cultured striatalneurons, since it was reported that acute treatment of3-NP induced a slow intracellular Ca2C increases incultured cortical and striatal neurons, finally resulting incell deaths (Fukuda et al., 1998). We measured in-tracellular Ca2C level using Ca2C imaging technique withfura-2. As shown in Fig. 6A, the treatment of 3-NPinduced intracellular Ca2C increases with slow timekinetics. Treatment of GTS did not prevent the initial3-NP-induced intracellular Ca2C elevation until about15 min but the presence of GTS inhibited further 3-NP-induced intracellular Ca2C elevations by inducing earlysaturation (3-NP:GTSC 3-NPZ 105.7G 5.4:78.3G3.4 nM; measured at the end of 3600 s; *P! 0.001). Asreported previously byFukuda et al. (1998), we could alsoobserve that 3-NP did not induce intracellular Ca2C

elevation in Ca2C-free medium (data not shown). Theseresults indicate that treatment of 3-NP induces extracel-lular Ca2C influx but that the presence of GTS attenuates3-NP-caused intracellular Ca2C elevation originatedfrom external Ca2C.

3.5. Effect of GTS on 3-NP-induced mitochondrialmembrane potential changes in cultured ratstriatal neurons

As a next step, we also measured the changes of3-NP-induced mitochondrial membrane potential in theabsence or presence of GTS using rhodamine (Rh123) instriatal neurons (Kannurpatti et al., 2004), since 3-NP-induced intracellular Ca2C elevation shows a possibilityof mitochondrial dysfunction. Treatment of 3-NPinduced a decrease of mitochondrial membrane poten-tial but treatment of GTS before 3-NP attenuatedsignificantly 3-NP-induced mitochondrial membranepotential reduction from ratio of 0.86G 0.01 to0.92G 0.01 (*P! 0.01, compared with 3-NP alonetreatment, measured at the end of 1200 s) (Fig. 6B).

3.6. Effect of GTS on 3-NP-induced cytotoxicity incultured rat striatal neurons

Since administration of 3-NP induces metabolicsuppression of mitochondria and cell deaths, we also

examined the preventive effect of GTS on 3-NP-inducedcytotoxicity using MTT test and DNA strand breakageassay. As shown in Fig. 7, exposure of striatal cells to 3-NP for 24 h resulted in metabolic suppression bydecreasing the mitochondrial reducing capacity onMTT and cell viability was significantly decreased by

0 1000 2000 3000 400040

60

80

100

1203-NP

GTS + 3-NP

0 200 400 600 800 1000 12000.7

0.8

0.9

1.0

1.1

GTS

GTS + 3-NP

3-NP

Time (sec)

Time (sec)

[Ca2+

] i (nM

)R

h123

Flu

ores

cenc

e

0.8

0.9

1.0

GTS GTS+ 3-NP

3-NP

Rh1

23 F

luor

esce

nce

B

A

Fig. 6. Effect of GTS on 3-NP-induced [Ca2C]i accumulation and 3-

NP-induced mitochondrial membrane potential changes in rat striatal

cells. (A) After recording basal [Ca2C]i for 2e3 min, the perfusion

solution was changed to a solution containing the 3-NP (5 mM) alone

or GTS (100 mg/ml)C 3-NP. GTS (100 mg/ml) alone was pretreated

for 1 min before GTS C 3-NP co-treatment. 3-NP induced a linear

increase in [Ca2C]i but GTS attenuated 3-NP-induced [Ca2C]iaccumulation. Inset, the representative traces after 3-NP or

GTS C 3-NP treatment. Each data point in graph represent the

meansG S.E.M., nZ 53e55, each group from 5 independent experi-

ments. (B) GTS (100 mg/ml) alone had no effect on mitochondrial

transmembrane potential, whereas 3-NP (5 mM) causes decreases in

mitochondrial transmembrane potential but the presence of GTS

attenuates 3-NP alone-induced mitochondrial transmembrane poten-

tial reduction. In cells treated with GTSC 3-NP, ginsenosides (100 mg/

ml) were pretreated for 1 min before ginsenosidesC 3-NP treatment.

Arrows indicate GTS alone, 3-NP alone, or GTS C 3-NP treatment.

Each data point in graph represent the meansG S.E.M., nZ 40e50,

each group from 5 independent experiments; *P ! 0.01, compared

with 3-NP alone treatment.



51G 2% (*P! 0.001; compared with 3-NP untreatedcontrol cells). However, we could observe that pre-treatment of GTS before 3-NP significantly increasedcell viability with dose-dependent manner. Thus, cellviability was 68.7G 1.6, 77.8G 1.9, 84.4G 2.0, and92.1G 2.1% at doses of 3, 10, 30 and 100 mg/ml of GTS,respectively, but there was no significant effect at dosesof 1 mg/ml (Fig. 7). The EC50 was 12.6G 0.7 mg/ml. Inaddition, the preventive effect of GTS on 3-NP-inducedcell damage was examined using the TUNEL technique.As shown in Fig. 8A and B, in control vehicle and GTStreatment group we could observe only a slight TU-NEL-positive neurons. In contrast, 3-NP treatment for24 h induced dramatic increases in TUNEL-positiveneurons compared with control or GTS treatmentgroup, whereas the presence of GTS markedly reduced3-NP-induced TUNEL-positive neurons (*P! 0.001;compared with 3-NP treated cells). We also examinedthe effects of GTS on 3-NP-induced internucleosomalfragmentation. Treatment of 3-NP caused considerableDNA ladder formation but treatment of GTS preventedthe nuclear DNA fragmentation induced by 3-NP,demonstrating that GTS attenuates 3-NP-induced stria-tal cell death (Fig. 8C).

4. Discussion

Ginsenosides are unique saponins, which only exist inP. ginseng, with pharmacological effects in central andperipheral nervous systems (Nah, 1997). Recent studies

0

20

40

60

80

100

120

Con

3-NP

1 3 10 30 100

GTS (µg/ml)

Fig. 7. Effects of GTS on 3-NP-induced mitochondrial activity. Striatal

cells were pretreated with various indicated concentration of GTS for

5 min and then were exposed to 3-NP (5 mM) for 24 h and

mitochondrial activities were determined by MTT assay. Values are

expressed as percentage of those of untreated control. Results are

expressed as the meansG S.E.M. from 5 separate experiments of

triplicates. *P ! 0.05, **P! 0.01 compared with 3-NP treatment

alone.

showed that ginsenosides could exert in vitro and in vivoprotective actions against acute excessive stimulation ofexcitatory neurotransmitters (Chu and Chen, 1989,1990; Kim et al., 1998b; Lee et al., 2002a). The presentstudy further extended that GTS could also protectcentral nervous system from repeated neurotoxic insults.Thus, we demonstrated that intraperitoneal administra-tion of GTS exhibited protective effects against 3-NP-and malonate-induced rat striatal lesions. We could alsoobserve that GTS showed significant beneficial effects byimproving 3-NP-caused behavioral impairment and byreducing 3-NP-caused mortality (Fig. 5).

One of the main indicators of neuronal excitotoxicityor excitotoxin-induced cell death is derived from thedisturbance of intracellular Ca2C homeostasis. Recentreports showed that 3-NP-caused cellular ATP exhaus-tion is coupled to intracellular Ca2C elevation. 3-NP-induced intracellular Ca2C elevation might be mediatedvia at least two pathways; first is through voltage-dependent Ca2C channel following depolarization withNaC influx, second is through NMDA and non-NMDAreceptor activation (Deshpande et al., 1997; Fukudaet al., 1998; Lee et al., 2002b).

Herein, we present three principal findings on GTSactions through in vitro studies for explanation of theprotective effect of GTS against in vivo 3-NP neurotox-icity. Firstly, we showed that pretreatment of GTSgreatly attenuated 3-NP-induced intracellular Ca2C

elevation, although treatment of 3-NP alone induceda gradual elevation of intracellular Ca2C level afternearly 60 min as shown in present and previous reports(Deshpande et al., 1997; Fukuda et al., 1998). As thesecond piece of evidence for the neuroprotective role ofGTS, we demonstrated that pretreatment of GTSinhibited 3-NP-induced mitochondrial transmembranepotential reduction. It is known that the initialdysregulation of mitochondrial functions that precedecell shrinkage and nuclear fragmentation before apo-ptotic processes is characterized by a reduction ofmitochondrial transmembrane potential in varioussystems (Kroemer et al., 1997; Keller et al., 1998).Therefore, the reduction of mitochondrial transmem-brane potential by acute 3-NP treatment indicates thatstriatal cells might be undergoing the apoptotic pro-cesses. The presence of GTS significantly protectsmitochondrial damages of striatal neurons by inhibiting3-NP-caused mitochondrial transmembrane potentialreduction (Fig. 6B). We could also observe throughelectron microscopy that GTS protected striatal mito-chondria from in vivo 3-NP-induced mitochondrialswelling and structural alterations (data not shown).The third and last piece of evidence suggesting a pro-tective role of GTS in 3-NP-induced neurotoxicitycomes from MTT assay. Treatment of 3-NP decreasedthe reducing ability of MTT by mitochondria. Theseresults indicate that treatment of 3-NP induced a damage



M Con 3-NPGTS

+ 3-NP

B

A

0

200

400

600

C

Con GTS 3-NP GTS+ 3-NP

TU

NE

L-p

osit

ive

cells

Con GTS

3-NP GTS + 3-NP

Fig. 8. Effects of GTS on 3-NP-induced DNA strand breakage. (A) Effect of GTS on 3-NP-induced DNA strand breakage was also determined by

using TUNEL technique. Cells were divided into control vehicle (Con), GTS alone, 3-NP alone-, or GTSC 3-NP co-treated group. In GTS C 3-NP

group, GTS (100 mg/ml) was pretreated for 1 min before 3-NP treatment. Representative images of each treatment group are shown after TUNEL

staining. Arrows indicate the representative TUNEL-positive cells. The procedures for TUNEL staining were performed as described in Section 2.

(B) Represents quantification of the density of TUNEL-positive neurons. Results are expressed as the means G S.E.M. from 3 separate experiments.

*P ! 0.001 compared with 3-NP treatment alone. (C) Cellular DNA was isolated from control vehicle (Con), 3-NP (3-NP) alone-, or GTS C 3-NP

co-treated striatal cells. DNA strand breakage was determined as described in Section 2. This figure is the representatives obtained from 5

independent experiments.

on normal functions of mitochondria. However, pre-treatment of GTS protected mitochondria from 3-NPinsults with dose-dependent manner. Further, we alsoshowed that GTS attenuates both neuronal cell DNAfragmentations and TUNEL-positive neuron forma-tions induced by 3-NP.

What is the mechanism underlying the protectiveeffect of GTS against 3-NP-caused rat striatal neuro-toxicity? One possibility is that GTS-induced protectionagainst 3-NP neurotoxicity might be derived from theinhibition on 3-NP-induced Ca2C influx via L- andother types of Ca2C channel (Fig. 6A). Previous report



showed that 3-NP-induced intracellular Ca2C elevationwas mediated via a L-type and other types of Ca2C

channels (Deshpande et al., 1997). We have shown thatginsenosides inhibit L-, N-, and P/Q-types of Ca2C

channels (Nah and McCleskey, 1994; Nah et al., 1995;Kim et al., 1998a; Rhim et al., 2002). Thus, the presentstudy showed that ginsenoside-induced inhibitions onvarious types of Ca2C channels could be the basis ofattenuation of 3-NP-induced intracellular Ca2C eleva-tion. The second possibility might be derived fromginsenoside-induced attenuation of extracellular Ca2C

entry caused by NMDA receptor activation, whichmight be secondarily induced by 3-NP intoxication(Pang and Geddes, 1997; Lee et al., 2002b). In previousstudies, our group showed that ginsenosides not onlyinhibit NMDA receptor-mediated current and Ca2C

influx but also attenuate kainate-induced hippocampalneuron deaths (Lee et al., 2002a; Kim et al., 2002). Thus,these GTS-induced limiting actions on extracellular 3-NP-induced Ca2C influx via Ca2C channels and sub-sequent Ca2C influx via secondarily NMDA receptoractivation might help not to aggravate 3-NP-inducedintracellular Ca2C unbalance. Moreover, these contri-butions of GTS might help to diminish ATP consump-tion needed for maintaining intracellular ionic balancesin striatal cells under 3-NP or malonate insults andfinally ameliorate 3-NP- or malonate-induced neurotox-icity. Similarly, MK801, a NMDA receptor antagonist,or riluzole, which inhibits neuronal voltage-dependentCa2C and NaC channel activity, not only attenuated 3-NP-induced Ca2C elevation but also exhibited neuro-protective effects against 3-NP-induced neurotoxicity(Guyot et al., 1997; Lee et al., 2002b). The lastpossibility is that GTS-induced neuroprotection against3-NP neurotoxicity might be derived from the attenu-ation of oxidative stress caused by glutamate in striatalcells under 3-NP insults, since ginsenosides inhibitglutamate mediated-overproduction of NO and malo-nyldiadehyde and prevented a decrease of superoxidedismutase activity in glutamate-treated cortical neurons(Chu and Chen, 1989, 1990; Kim et al., 1998b).

However, it is unlikely that the protective effects ofGTS are due to direct interaction with 3-NP, activationof SDH, or blocking action of SDH inhibitor, since GTSitself had no effect on SDH activity and GTSadministered 3 h before 3-NP treatment also did notaffect 3-NP-induced inhibition of SDH activity (Fig. 4).GTS could not reverse malonate-induced inhibition ofSDH activity (Joo and Han, 1976). It is also possible tosay that GTS treatment may accelerate the eliminationof 3-NP, rendering the neurotoxin less active. Here, thispossibility can be clearly ruled out, since GTS is active invitro where 3-NP concentrations remain stable, GTS isalso active against malonate which is injected directlyinto the striatum, and finally 3-NP-induced inhibition ofSDH activity was not affected by GTS treatment

(Figs. 2, 4, and 7). Taken together, the main contrib-uting factors on GTS-induced protection against 3-NPand malonate neurotoxicity could be due to theinhibitory effects on intracellular Ca2C elevations thatmight be due to Ca2C channel and NMDA receptoractivations.

In summary, using a rodent HD model system thatshows a selective striatal lesion by 3-NP or malonatetreatment, we obtained results suggesting that GTS hasneuroprotective effects against 3-NP- or malonate-induced striatal neurotoxicity. As a mitochondrial toxin,3-NP produces selective basal ganglia lesions anddelayed dystonia when human ingested it by accident(James et al., 1980; Ludolph et al., 1991). Moreover,since the pathological symptoms in striatum induced bylong-term treatment of 3-NP share many commoncharacteristic features observed in human HD patientsand transgenic mouse HD models (i.e. disturbances inmitochondrial Ca2C homeostasis and changes in mito-chondrial membrane potentials) (Sawa et al., 1999;Panov et al., 2002), the present findings further suggestthat treatment of GTS might be a novel preventivestrategy against neurodenegeration in HD.

Acknowledgements

This work was supported by the Ministry of Scienceand Technology through the Bio-Food and Drug Re-search Center at Konkuk University, Chungju, Korea.

References

Alston, T.A., Mela, L., Bright, H.F., 1977. 3-Nitropropionate, the

toxic substance of Indigofera, is a suicide inactivator of succinate

dehydrogenase. Proceedings of the National Academic of Sciences,

USA 74, 3767e3771.

Beal, M.F., Brouillet, E., Jenkins, B.G., Ferrante, R.J., Kowall, N.W.,

Miller, J.M., Storey, E., Srivastava, R., Rosen, B.R., Hyman, B.T.,

1993. Neurochemical and histological characterization of striatal

excitotoxic lesions produced by the mitochondrial toxin 3-nitro-

propionic acid. Journal of Neuroscience 13, 4181e4192.

Bizat, N., Hermel, J.M., Humbert, S., Jacquard, C., Creminon, C.,

Escartin, C., Saudou, F., Krajewski, S., Hantraye, P., Brouillet, E.,

2003a. In vivo calpain/caspase cross-talk during 3-nitropropionic

acid-induced striatal degeneration: implication of a calpain-medi-

ated cleavage of active caspase-3. Journal of Biological Chemistry

278, 43245e43253.Bizat, N., Hermel, J.M., Boyer, F., Jacquard, C., Creminon, C.,

Ouary, S., Escartin, C., Hantraye, P., Kajewski, S., Brouillet, E.,

2003b. Calpain is a major cell death effector in selective striatal

degeneration induced in vivo by 3-nitropropionate: implications for

Huntington’s disease. Journal of Neuroscience 23, 5020e5030.

Borlongan, C.V., Koutouzis, T.K., Sandberg, P.R., 1997. 3-Nitro-

propionic acid animal model and Huntington’s disease. Neurosci-

ence and Biobehavioral Reviews 21, 289e293.

Brouillet, E., Guyot, M.C., Mittoux, V., Altairac, S., Conde, F.,

Palfi, S., Hantraye, P., 1998. Partial inhibition of brain succinate

dehydrogenase by 3-nitropropionic acid is sufficient to initiate

striatal degeneration in rat. Journal ofNeurochemistry 70, 794e805.



Brouillet, E., Conde, F., Beal, M.F., Hantraye, P., 1999. Replicating

Huntington’s disease phenotype in experimental animals. Progress

in Neurobiology 59, 427e468.

Calabresi, P., Gubellini, P., Picconi, B., Centonze, D., Pisani, A.,

Bonsi, P., Greengard, P., Hipskind, R.A., Borrelli, E.,

Bernardi, G., 2001. Inhibition of mitochondrial complex II induces

a long-term potentiation of NMDA-mediated synaptic excitation

in the striatum requiring endogenous dopamine. Journal of

Neuroscience 21, 5110e5120.

Chu, G.X., Chen, X., 1989. Protective effect of ginsenosides on acute

cerebral ischemia-reperfusion injury of rats. Chinese Journal of

Pharmacology and Toxicology 3, 18e23.

Chu, G.X., Chen, X., 1990. Anti-lipid peroxidation and protection of

ginsenosides against cerebral ischemia-reperfusion injury of rats.

Acta Pharmacologica Sinica 11, 119e123.

Coles, C.J., Edmondson, D.E., Singer, T.P., 1979. Inactivation

of succinate dehydrogenase by 3-nitropropionate. Journal of

Biological Chemistry 254, 5161e5167.

Deshpande, S.B., Fukuda, A., Nishino, H., 1997. 3-Nitropropionic

acid increases the intracellular Ca2C in cultured astrocytes by

reverse operation of the NaCeCa2C exchanger. Experimental

Neurology 145, 38e45.

Fu, Y., He, F., Zhang, S., Huang, J., Zhang, J., Jiao, X., 1995. 3-

Nitropropionic acid produces indirect excitotoxic damage to rat

striatum. Neurotoxicology and Teratology 17, 333e339.

Fukuda, A., Deshpande, S.B., Shimano, Y., Nishino, H., 1998.

Astrocytes are more vulnerable than neurons to cellular Ca2C

overload induced by a mitochondrial toxin, 3-nitropropionic acid.

Neuroscience 87, 497e507.

Garcia, M., Vanhoutte, P., Pages, C., Besson, M.J., Brouillet, E.,

Caboche, J., 2002. The mitochondrial toxin 3-nitropropionic acid

induces striatal neurodegeneration via a c-Jun N-terminal kinase/

c-Jun module. Journal of Neuroscience 22, 2174e2184.

Guyot, M.C., Palfi, S., Stuzmann, J.M., Maziere, M., Hantraye, P.,

Brouillet, E., 1997. Riluzole protects from motor deficits and

striatal degeneration produced by systemic 3-nitropropionic acid

intoxication in rats. Neuroscience 81, 141e149.

James, L.F., Hartley, W.J., Williams, M.C., Van Kampen, K.R., 1980.

Field and experimental studies in cattle and sheep poisoned by

nitro-bearing Astragalus or their toxins. American Journal of

Veterinary Research 41, 377e382.

Jeong, S.M., Lee, J.H., Kim, S., Rhim, H., Lee, B.H., Kim, J.H.,

Oh, J.H., Lee, S.M., Nah, S.Y., 2004. Ginseng saponins induce

store-operated calcium entry in Xenopus oocytes. British Journal of

Pharmacology 142, 585e593.Joo, C.N., Han, J.H., 1976. The effect of ginseng saponins on chicken’s

hepatic mitochondrial succinate dehydrogenase, malate dehydro-

genase and a-ketoglutarate dehydrogenase. Korean Biochemical

Journal 9, 43e51.

Kampen, J.V., Robertson, H., Hagg, T., Drobitch, R., 2003. Neuro-

protective actions of the ginseng extract G115 in two rodent models

of Parkinson’s disease. Experimental Neurology 184, 521e529.

Kannurpatti, S.S., Sanganahalli, B.G., Mishra, S., Joshi, P.G.,

Joshi, N.B., 2004. Glutamate-induced differential mitochondrial

response in young and adult rats. Neurochemistry International 44,

361e369.Keene, C.D., Rodrigues, C.M., Eich, T., Chhabra, M.S., Linehan-

Steer, C., Abt, A., Krens, B.T., Steer, C.J., Low, W.C., 2001. A bile

acid protects against motor and cognitive deficits and reduces

striatal degeneration in the 3-nitropropionic acid model of

Huntington’s disease. Experimental Neurology 171, 351e360.

Keller, J.N., Guo, Q., Holtsberg, F.W., Bruce-Keller, A.J.,

Mattson, M.P., 1998. Increased sensitivity to mitochondrial

toxin-induced apoptosis in neural cells expressing mutant preseni-

lin-1 linked to perturbed calcium homeostasis and enhanced

oxyradical production. Journal of Neuroscience 18, 4439e4450.

Kim, H.S., Lee, J.H., Koo, Y.S., Nah, S.Y., 1998a. Effects of

ginsenosides on Ca2C channels and membrane capacitance in rat

adrenal chromaffin cells. Brain Research Bulletin 46, 245e251.

Kim, Y.C., Kim, S.R., Markelonis, G.J., Oh, T.H., 1998b. Ginseno-

sides Rb1 and Rg3 protect cultured rat cortical cells from

glutamate-induced neurodegeneration. Journal of Neuroscience

Research 53, 426e432.

Kim, S., Ahn, K., Oh, T.H., Nah, S.Y., Rhim, H., 2002. Inhibitory

effect of ginsenosides on NMDA receptor-mediated signals in rat

hippocampal neurons. Biochemical and Biophysical Research

Communication 296, 247e254.

Kroemer, G., Zamzami, N., Susin, S.A., 1997. Mitochondrial control

of apoptosis. Immunology Today 18, 44e51.

Lee, J.H., Kim, S.H., Kim, D., Hong, H.N., Nah, S.Y., 2002a.

Protective effect of ginsenosides, active ingredients of Panax

ginseng, on kainate-induced neurotoxicity in rat hippocampus.

Neuroscience Letters 325, 129e133.

Lee, W.T., Yin, H.S., Shen, Y.Z., 2002b. The mechanism of neuronal

death produced by mitochondrial toxin 3-nitropropionic acid: the

role of N-methyl-D-aspartate glutamate receptors and mitochon-

drial calcium overload. Neuroscience 112, 707e716.

Lee, J.H., Choi, S., Kim, J.H., Kim, J.K., Kim, J.I., Nah, S.Y., 2003.

Effects of ginsenosides on carbachol-stimulated formation of

inositol phosphates in rat cortical cell cultures. Neurochemical

Research 28, 1307e1313.

Liao, B., Newmark, H., Zhou, R., 2002. Neuroprotective effects of

ginseng total saponin and ginsenosides Rb1 and Rg1 on spinal cord

neurons in vitro. Experimental Neurology 173, 224e234.

Ludolph, A.C., He, F., Spencer, P.S., Hammerstad, J., Sabri, M.,

1991. 3-Nitropropionic acid B exogenous animal neurotoxin and

possible human striatal toxin. Canadian Journal of Neurological

Sciences 18, 492e498.

Maragos, W.F., Tillman, P.A., Chesnut, M.D., Jakel, R.J., 1999.

Clorgyline and deprenyl attenuate striatal malonate and 3-nitro-

propionic acid lesions. Brain Research 834, 168e172.

Ming, L., 1995. Moldy sugarcane poisoning e a case report with

a brief review. Clinical Toxicology 33, 363e367.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and

survival: application to proliferation and cytotoxicity assays.

Journal of Immunological Methods 16, 55e63.

Nah, S.Y., 1997. Ginseng; recent advances and trends. Journal of

Ginseng Research 21, 1e12.Nah, S.Y., McCleskey, E.W., 1994. Ginseng root extract inhibits

calcium channels in rat sensory neurons through a similar path, but

different receptor, as m-type opioids. Journal of Ethnopharmacology

42, 45e51.

Nah, S.Y., Park, H.J., McCleskey, E.W., 1995. A trace component of

ginseng that inhibits Ca2C channels through a pertussis toxin-

sensitive G protein. Proceedings of the National Academy of

Sciences, USA 92, 8739e8743.

Nasr, P., Gursahani, H.I., Pang, Z., Bondada, V., Lee, J.,

Hadley, R.W., Geddes, J.W., 2003. Influence of cytosolic and

mitochondrial Ca2C, ATP, mitochondrial membrane potential,

and calpain activity on the mechanism of neuron death induced by

3-nitropropionic acid. Neurochemistry International 43, 89e99.

Novelli, A., Reilly, J.A., Lysko, P.G., Hennebery, R.C., 1988.

Glutamate becomes neurotoxic via the N-methyl-D-aspartate

receptor when intracellular energy levels are reduced. Brain

Research 451, 205e212.

Pang, Z., Geddes, J.W., 1997. Mechanisms of cell death induced by the

mitochondrial toxin 3-nitropropionic acid: acute excitotoxic necro-

sis and delayed apoptosis. Journal of Neuroscience 17, 3064e3073.

Panov, A.V., Gutekunst, C.A., Leavitt, B.R., Hayden, M.R.,

Burke, J.R., Strittmatter, W.J., Greenamyre, J.T., 2002. Early

mitochondrial calcium defects in Huntington’s disease are a direct

effect of polyglutamines. Nature Neuroscience 5, 731e736.



Radad, K., Gille, G., Moldzio, R., Saito, H., Ishge, K., Rausch, W.D.,

2004. Ginsenoside Rb1 and Rg1 effects on survival and neurite

growth of MPPC-affected mesencephalic dopaminergic cells.

Journal of Neural Transmission 111, 37e45.Reynolds, D.S., Carter, R.J., Morton, A.J., 1998. Dopamine

modulates the susceptibility of striatal neurons to 3-nitropropionic

acid in the rat model of Huntington’s disease. Journal of

Neuroscience 18, 10116e10127.Rhim, H., Kim, H., Lee, D.Y., Oh, T.H., Nah, S.Y., 2002. Ginseng

and ginsenoside Rg3, a newly identified active ingredient of

ginseng, modulate Ca2C channel currents in rat sensory neurons.

European Journal of Pharmacology 436, 151e158.

Sawa, A., Wiegand, G.W., Cooper, J., Margolis, R.L., Sharp, A.H.,

Lawler Jr., J.F., Greenamyre, J.T., Snyder, S.H., Ross, C.A., 1999.

Increased apoptosis of Huntington disease lymphoblasts associated

with repeat length-dependent mitochondrial depolarization.

Nature Medicine 5, 1194e1998.

Zeevalk, G.D., Nicklas, W.J., 1991. Mechanisms underlying initiation

of excitotoxicity associated with metabolic inhibition. Journal of

Pharmacological Experimental Therapeutics 257, 870e878.

protective eﬀects of ginseng saponins on 3-nitropropionic

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