Journal ofEthnopharmacology 126 (2009) 447–454

Contents lists available at ScienceDirect

Journal ofEthnopharmacology

j ou rn a l h o m epa g e: www. el sevi er. com /l o ca te/j eth ph a rm

Chrysanthemum morifolium Ramat (CM) extract protects human neuroblastoma

SH-SY5Y cells against MPP + -induced cytotoxicity

In Su Kim

a , Sushruta Koppula a , Pyo-Jam Park a , Ee Hwa Kim b , Chan Gil Kim a , Wahn Soo Choi c ,

Kwang Ho Lee

a , Dong-Kug Choi a,∗

a Department ofBiotechnology, Konkuk University, 322 Danwol-dong, Chungju 380-701, Republic ofKorea

b Department ofAcupoint & Meridian, College ofOriental Medicine, Semyung University, Chungbuk, Republic ofKorea

c College ofMedicine, Konkuk University, Chungju, Republic ofKorea

a r t i c l e i n f o

Article history:

Received 9 April 2009

Received in revised form 18 August 2009

Accepted 14 September 2009

Available online 19 September 2009

Keywords:

Chrysanthemum morifolium (CM)

MPP +

Parkinson’s disease

ROS

Caspase

Mitochondrial dysfunction

a b s t r a c t

Ethnopharmacological relevance: Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used

in Korean and Chinese traditional herbal medicines with numerous therapeutic applications.

Aim ofthe study: To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract

against 1-methyl-4-phenylpridinium ions (MPP + ), Parkinsonian toxin through oxidative stress and

impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms.

Materials and methods: The effects of CM against MPP + -induced cytotoxicity and neuronal cell viability,

oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP)

proteolysis were evaluated by using SH-SY5Y neuroblastoma cells.

Results: CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the

elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and

PARP proteolysis.

Conclusion: These results demonstrate that CM possesses potent neuroprotective activity and therefore,

might be a potential candidate in neurodegenerative diseases such as Parkinson’s disease.

© 2009 Published by Elsevier Ireland Ltd.

1. Introduction

Parkinson’s disease (PD), the second most frequent cause of

dementia after Alzheimer’s disease, is characterized by a loss of

dopaminergic neurons in the substantia nigra. Although the eti-

ology of PD remains unclear, a number of accumulating evidence

strongly supports the involvement of oxidative stress and mito-

chondrial dysfunction (Pieczenik and Neustadt, 2007; Szeto, 2006;

Mattson, 2000). The majormitochondrial defect in PD appears to be

associated with inhibition of respiratory chain complex I. Insights

into PD pathogenesis have been experimentally achieved using

the neurotoxin 1-methyl-4-phenylpyridinium (MPP + ), which is the

active metabolite of 1-methyl-4-phenyl-2,3,6-tetrahydropyridine

(MPTP). MPP + selectively and potently inhibits complex I of the

mitochondrial electron transport chain (Singer and Ramsay, 1990)

and induces a syndrome closely resembling PD in cellular and

animal models (Eberhardt and Schulz, 2003; Przedborski and

Jackson-Lewis, 1998). Determining the cause ofthe disease, under-

standing of the molecular mechanisms in dopaminergic neuronal

∗

Corresponding author. Tel.: +82 43 840 3610; fax: +82 43 840 3872.

E-mail address: choidk@kku.ac.kr (D.-K. Choi).

cell death and developing new protective drugs have become the

primary research goals.

There is a dramatic loss of dopaminergic neurons in PD and

experimental models of PD. Activation of neuronal cell death

pathways, involving oxidative stress and mitochondrial dysfunc-

tion most likely represents the process in PD neurodegeneration

(Fiskum et al., 2003). Therefore, the suppression of dopaminergic

neuronal cell death by regulation of intracellular reactive oxygen

species (ROS) and modification ofthe apoptotic cascade may have

therapeutic benefits, which lead to alleviation of the progression

ofneurodegeneration. The herbal remedies can have recently been

demonstrated to possess neurotrophic and neuroprotective prop-

erties, which may be useful in preventing various forms ofneuronal

cell loss including the nigrostriatal degeneration seen in PD (Van

Kampen et al., 2003; Levites et al., 2001). Chrysanthemum mori-

folium Ramat (CM) has been used as a traditional herbal medicine

for thousands of years. In addition to its antioxidant activity (Kim

and Lee, 2005; Wang et al., 2001), other CM attributes include

cardiovascular protection (Jiang et al., 2004); protection against

terminal tumors (Miyazawa and Hisama, 2003); and diminished

inflammatory activity (Ukiya et al., 2001), apoptosis (Fang et al.,

2002), mutagenesis (Yen and Chen, 1996) and infection by human

immunodeficiency virus (Lee et al., 2003). However, the effect

of CM against neuronal cell injury remains uncertain. Therefore,

0378-8741/$ – see front matter © 2009 Published by Elsevier Ireland Ltd.

doi:10.1016/j.jep.2009.09.017

448 I.S. Kim et al. / Journal ofEthnopharmacology 126 (2009) 447–454

the purpose of this study was to investigate the effects of CM

on MPP + -induced neurotoxicity in human dopaminergic SH-SY5Y

neuroblastoma cells, with the aim ofproviding possible therapeu-

tic application for prevention and treatment ofPD. We investigated

the neuroprotective effect of CM on neuronal cell viability, oxida-

tive damage, expression of Bcl-2 and Bax, caspase-3 activity and

poly(ADP-ribose) polymerase (PARP) proteolysis in MPP + -induced

SH-SY5Y neurotoxicity.

2. Materials and methods

2.1. Reagents

MPP + , 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 2,2-

azobis(2-amidinopropane) hydrochloride (AAPH), 1,1-diphenyl-2-

picrylhydrazyl (DPPH), (4-pyridyl-1-oxide)-N-tert-butylnitrone

(4-POBN), 3-(3,4-dimehylthiazol-2-yl)-2,5-diphenyl-tetrazolium

bromide (MTT) were obtained from Sigma–Aldrich (St. Louis,

MO, USA). Six-well and 96-well tissue culture plates and 100 mm

culture dishes were purchased from Nunc Inc. (North Aurora

Road, IL, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal

bovine serum (FBS) was from Gibco-BRL Technologies (CA, USA).

LDH assay kits were purchased from Dojindo Co. (MD, USA) and

2,7-dichlorofluorescein diacetate (DCFH-DA), propidium iodide

(PI) were supplied by BD Clontech (Terra Bella Ave, CA, USA).

The antibodies against cleaved caspase-3, PARP and ␤-actin were

obtained from Cell Signaling Co., (Boston, MA, USA). All other

chemicals used in this study were analytical grade and were

obtained, unless otherwise noted, from Sigma Chemical Co. (St.

Louis, MO, USA).

2.2. Preparation ofthe CM

Chrysanthemum morifolium Ramat (CM) was obtained from a

local market and was authenticated based on its microscopic and

macroscopic characteristics. A voucher specimen (SM-201) has

been deposited at the School of Oriental Medicine, Semyung Uni-

versity, Korea. To obtain the water extract, 90 g ofCM was added to

distilled water and extraction was performed by heating at 80 ◦ C;

it was then concentrated with a rotary evaporator and lyophilized.

The resulting powder (yield, 15.2 g) was dissolved in DMSO and

filtered through a 0.22 ␮M filter before use.

2.3. Cell culture and treatments

Human neuroblastoma SH-SY5Y cells were obtained from the

American Type Culture Collection (ATCC) and cultured in DMEM

supplemented with 10% (v/v) inactivated fetal bovine serum, and

100 U/mL penicillin/streptomycin. The cells were maintained at

37

◦ C in 5% CO 2

and 95% humidified air incubator for the indicated

time. All experiments were carried out 24–48 h after cells were

seeded. SH-SY5Y cells were pre-treated for 4 h with various con-

centrations (1, 10, 100 ␮g/mL) ofCM before incubation in medium

containing 1 mM MPP + .

2.4. Assessment ofcell viability

The cell viability was measured by quantitative

colorimetric assay using the 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) assay, showing the

mitochondrial activity of living cells as previously described

(Datki et al., 2003). MTT dissolved in phosphate-buffered saline

was added at the end of incubation to a final concentration of

0.5 mg/mL. After incubation for 4 h at 37

◦ C and 5% CO 2 , the

supernatants were removed and the formed formazan crystals

in the viable cells were measured at 550 nm using a microplate

reader (Molecular device, USA). The release of the intracellular

enzyme lactate dehydrogenase (LDH) into the medium was used

as a quantitative measurement of cell viability. The cells were

incubated in 96-well plates with the indicated concentration of

CM and 1 mM of MPP + for 48 h plus normal control group. The

cell suspension was centrifuged (4000 × g, 5 min, 4 ◦ C), and then

the supernatant was collected. LDH assay in supernatant aliquots

was performed by using the cytotoxicity assay kit according to the

manufacturer’s instructions (Takara, Japan). Absorbance was read

at 440 nm and LDH expressed cytotoxicity (%) was calculated using

the formula: (supernatant value − blank value)/[(supernatant

value − blank value) + (upper control value − blank value)] × 100%.

2.5. Isolation oftotal RNA and expression analysis

SH-SY5Y cells (1 × 10 6 cells/well) were cultured in 6-well

plates, and the total RNA was isolated by extraction with

TRIzol (Invitrogen, CA, USA). For the reverse transcription-

polymerase chain reaction (RT-PCR), 2.5 ␮g of total RNA was

reverse transcribed using a First Strand cDNA Synthesis kit

(Invitrogen, CA, USA). PCR was performed using the above-

prepared cDNA as a template. The following primers were

used for PCR: Bcl-2 sense, 5  -ACTTTGCAGAGATGTCCAGT-3  ;

Bcl-2 anti-sense, 5  -CGGTTCAGGTACTCAGTCAT-3  ; Bax sense, 5  -

CTGGACAGTAACATGGAGC-3  ; Bax anti-sense, 5  -TCTTCTTCCAG-

ATGGTGAGT-3  ; GAPDH sense, 5  -GCAGTGGCAAAGTGGAGATTG-

3  ; GAPDH anti-sense 5  -TGCAGGATGCATTGCTGACA-3  . GAPDH

was used as an internal control to evaluate the relative expressions

of Bcl-2 and Bax. RT-PCR products were electrophoresed on a 1%

(w/v) agarose gel, stained with ethidium bromide, and bands were

visualized by UV light.

2.6. Immunoblot analysis

To obtain the total cell lysate, 0.1 mL (or 0.05 mL) of RIPA

buffer [1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,

with freshly added protease inhibitor cocktail (Calbiochem, CA,

USA)] was added to the SH-SY5Y cells cultured in 6-well plates.

The cells were scraped, incubated for 10 min on ice, and cen-

trifuged at 14,000 rpm for 10 min at 4 ◦ C. The protein concentration

was determined by the DC protein assay from Bio-Rad (Her-

cules, CA, USA), and 15 ␮g of whole cell lysate was loaded for

10% SDS-PAGE. Electrophoresis was performed and the proteins

were transferred to PVDF membranes (Millipore, MA, USA) using

an electroblotting apparatus (Bio-Rad, CA, USA). The membranes

were blocked for 1 h in TBS containing 0.1% Tween-20 and 5% dry

milk, and were then incubated overnight with primary antibodies

[anti-cleaved caspase-3 1:1000 and anti-PARP 1:1000 (Cell Signal-

ing, MA, USA)] followed by incubation for 1 h with horseradish

peroxidase-conjugated secondary antibodies (1:10,000) (Santa

Cruz, CA, USA). The optical densities ofthe antibody-specific bands

were analyzed by a Luminescent Image Analyzer, LAS-3000 (Fuji,

Japan).

2.7. Flow cytometric detection ofapoptotic cells

SH-SY5Y cells (1 × 10 6 cells/well) were collected by centrifuga-

tion following MPP + exposure for 60 h and washed with ice-cold

PBS two times. The pellets were resuspended in ice-cold 70%

ethanol and fixed at 4 ◦ C for 24–48 h. Then the cells were washed

and resuspended in 1 mL of DNA staining reagent containing

50 ␮g/mL RNase, 0.1% Triton X-100, 0.1 mM EDTA (pH 7.4), and

50 ␮g/mLPI. The stainingwas stable at4 ◦ C for30 min (Telford etal.,

1991). Red fluorescence (DNA) was detected through a 563–607 nm

band pass filter by using a FACS Caliber flow cytometer (Becton

Dickinson, San Jose, CA, USA). Ten thousand cells in each sample

I.S. Kim et al. / Journal ofEthnopharmacology 126 (2009) 447–454 449

were analyzed and the percentage ofapoptotic cells accumulating

in the sub-G1 peak was calculated by Cell Quest software.

2.8. Measurement ofintracellular reactive oxygen species (ROS)

The intracellular ROS production was measured using a non-

fluorescent compound 2  ,7  -dichlorofluorescein diacetate (DCFH-

DA) as previously described (Bass et al., 1983). It measures

the formation of hydrogen peroxide generated by an oxidative

metabolic burst. Viable cells can deacetylate DCFH-DA to 2  ,7  -

dichlorofluorescin(DCFH), whichis notfluorescent. This compound

reacts quantitativelywith oxygen species within the cell to produce

a fluorescent dye 2  ,7  -dichlorofluorescein (DCF), which remains

trapped within the cell and can be measured to provide an index

of ROS level. The cells (1 × 10 6 cells/well) were collected and

loaded with 20 ␮M DCFH-DA (dissolved in DMSO) for 30 min at

37

◦ C. After washing out the excess probe, the cells were mea-

sured by FACS Caliber flow cytometer (Becton Dickinson, CA,

USA).

2.9. Measurement offree radical scavenging activity

Free radical scavenging activity was evaluated using an electron

spin resonance (ESR) spectrometer (JEOL, Tokyo, Japan). Hydroxyl

radicals were generated by iron-catalyzed Haber–Weiss reaction

(Fenton-driven Haber–Weiss reaction), and the generated hydroxyl

radicals rapidly reacted with nitrone spin-trap DMPO (Rosen and

Rauckman, 1984). The resultant DMPO-OH adducts was detectable

with an ESR spectrometer. The reaction mixture containing 0.3 M

DMPO, 10 mM FeSO 4 , 10 mM H 2 O 2 and CM with various concentra-

tions in a PBS (pH 7.2) was incubated for2.5 min. Alkyl radicals were

generated by AAPH. The reaction mixture containing 10 mM AAPH,

10 mM 4-POBN and CM with various concentrations in PBS (pH

7.4) was incubated at 37 ◦ C in a water bath for 30 min. DPPH radical

scavenging activity was measured using the method described by

Nanjo et al. (1996). A sample solution of CM was added to 60 ␮M

DPPH in methanol solution was incubated for 2 min. Superoxide

radicals were generated by an UV-irradiated riboflavin/EDTA sys-

tem (Guo et al., 1999). The reaction mixture containing 0.8 mM

riboflavin, 1.6 mM EDTA, 0.8 M DMPO and CM with various con-

centrations was irradiated for 1 min under an UV lamp at 365 nm.

ESR spectrum was recorded using an ESR spectrometer for each

radical.

2.10. Statistical analyses

The results were expressed as mean ± SEM ofat least three inde-

pendent experiments triplicate. Statistical analysis was performed

byone-wayanalysis ofvariance (ANOVA) followed byposthocmul-

tiple comparisons using the Student–Newman–Keuls method with

the Sigma Stat 3.1 software (Systat Software Inc., San Jose, CA, USA).

P value < 0.05 was considered to be statistically significant.

3. Results

3.1. CM ameliorates MPP + -induced loss ofneuronal cell viability

The effect of CM on MPP + -induced loss of cell viability in

dopaminergic neuroblastoma SH-SY5Y cells was assessed using an

established MTTassay. The SH-SY5Ycells were pre-treated withCM

extract (1, 10, and 100 ␮g/mL) for 4 h prior to the addition of1 mM

MPP + . When the cell viability under serum free conditions was

defined as 100% survival, their viability of cells exposed to 1 mM

MPP + was decreased to 50.6 ± 2.5%, which was consistent with the

previous results (Storchetal., 2000; Chengetal., 2009). The viability

ofcell incubated with 1, 10, and 100 ␮g/mL ofCM was 56.2 ± 3.4%,

Fig. 1. Effect of CM on MPP + -induced neuronal cell death in SH-SY5Y cells. Cells

were exposed to MPP + for 48 h and cell viability was assessed by (A) MTT assay and

(B) LDH assay. Cells were treated with 1 mM MPP + in the absence or presence ofCM

(1, 10, and 100 ␮g/mL). Data are expressed as the percentage ofvalues in untreated

control cultures. Each value indicates a mean ± SEM (n = 3). # P< 0.05, compared with

control group. *P< 0.05, compared with the MPP + -treated group (one-way ANOVA

followed by Student–Newman–Keuls post hoc test).

64.8 ± 2.6% and 77.6 ± 2.7% of the control values, respectively. The

results show that the MPP + -induced loss of neuronal cell viabil-

ity can be fully attenuated by CM in a dose-dependent manner

(Fig. 1A). The level ofcell death was also assayed bya LDH assay that

detects the release ofLDH into the culture medium. The exposure of

neurons to MPP + resulted in a 1.6 ± 0.1-fold increase in the release

of LDH when compared to the control cultures. Pretreatment of

cells with 1, 10, and 100 ␮g/mL CM for 4 h significantly reduced

MPP + -induced LDH release inadose-dependentfashion(Fig. 1B). To

furtherverifythe inhibitoryeffectofCM, the cells were labeled with

propidium iodide and analyzed by flow cytometry. Fig. 2 shows

the DNA content histograms obtained after the cells were exposed

to 1 mM MPP + and with various concentrations of CM. When the

cells were incubated in medium alone, a typical single peak of

nuclei with diploid DNA content was observed (Fig. 2a) and there

was only about 2–3% cell death. In the presence of 1 mM MPP + ,

a characteristic hypodiploid DNA content peak indicative of sub-

G0–G1 apoptotic populations, was distinguishable. Treatment with

1 mM MPP + resulted in apoptotic population of34.7 ± 0.9% (Fig. 2b).

Following treatment with CM (1, 10, and 100 ␮g/mL), the pro-

portion of apoptotic cells was reduced to 31.6 ± 1.4%, 23.6 ± 3.3%,

and 14.6 ± 2.7%, respectively, in a concentration-dependent man-

ner (Fig. 2d–f).

450 I.S. Kim et al. / Journal ofEthnopharmacology 126 (2009) 447–454

Fig. 2. Effect of CM against MPP + -induced neurotoxicity in cultured SH-SY5Y cells by flow cytometric DNA analysis. (a) Control cells and (b) cells exposed to 1 mM MPP +

alone; (c) cells exposed to 100 ␮g/mL CM alone; (d–f) cells pre-treated with 1, 10, and 100 ␮g/mL of CM, respectively, in the presence of 1 mM MPP + . Bar ( ) represents a

sub-G0/G1 or hypodiploid DNA fraction. The results are representative ofone ofsix independent experiments.

3.2. CM suppresses MPP + -induced oxidative stress

Accumulating evidence suggests that oxidative damage occurs

in the Parkinsonian brain, as well as in vitro and in vivo exper-

imental models of PD (Zhou et al., 2008; Choi and Suk, 2007).

Overproduction of ROS can cause severe impairment of cellular

functions, and may contribute to the apoptotic process found in

PD (Kehrer and Smith, 1994; Zhou et al., 2008). There is substantial

evidence that the production of ROS is involved in MPP + -induced

apoptotic mechanisms (Cassarino et al., 1999; Domingues et al.,

2008). Therefore, we measured ROS generation in SH-SY5Y cells

exposed to 1 mM MPP + by fluorometric analysis using DCFH-DA.

Cells exposed to MPP + displayed an obvious increase in DCF signal

at 24 h when compared to the control cultures, and the ROS pro-

duction in cells incubated with 1, 10, and 100 ␮g/mL of CM were

suppressed to 94.1 ± 2.5%, 81.6 ± 2.8%, and 66.6 ± 4.5% as compared

to that in the MPP + -treated group, respectively (Fig. 3A). Histogram

plots also showed that CM significantlysuppressed ROS generation,

as evident by a shiftto the left ofthe fluorescence intensity (Fig. 3B).

3.3. Free radical scavenging activities ofCM by ESR

Free radical scavenging activities of CM were examined using

ESR spectroscopy. Hydroxyl radicals generated in the Fe 2+ /H 2 O 2

system were trapped by DMPO, forming spin adducts that were

detected by ESR, and the typical 1:2:2:1 ESR signal of the DMPO-

OH adducts was observed. These results may have been due to

the paramagnetic impurities contained in unpurified commercial

DMPO. The height of the third peak of the spectrum represents

the relative amount ofDMPO-OH adducts. As shown in Fig. 4A, CM

scavenged hydroxyl radicals, with the scavenging activity increas-

ing in a dose-dependent manner. The alkyl radical spin adduct of

4-POBN/free radicals was generated from AAPH at 37

◦ C for 30 min,

and the decrease ofESR signals was observed with the dose incre-

ment of CM (Fig. 4B). These results were firm evidence of CM

alkyl radical scavenging activity. DPPH is a stable free radical, and

accepts an electron or hydrogen radical to become a stable dia-

magnetic molecule, which has been used to evaluate free radical

scavenging activity of natural antioxidants. The capacity of CM to

scavenge DPPH was measured by ESRspectrometry, and the results

are shown in Fig. 4C. CM exhibited DPPH scavenging activity in a

dose-dependent manner, and completely eliminated generation of

DPPH at 0.5 mg/mL. The ESR signals of the superoxide radical are

shown in Fig. 4D. CM scavenged superoxide radical up to 32% at

1 mg/mL.

3.4. CM affects the expression ofBcl-2 and Bax in MPP + -treated

cells

The Bcl-2 family consists of both apoptotic and anti-apoptotic

members, and the balance between these proteins plays a pivotal

role in the cellular apoptotic machinery (Cory and Adams, 2002).

The ratio between the pro- and anti-apoptotic members ofthe Bcl-

2 family could be instrumental in the cellular decision between

survival and cell death (Nicotra and Parvez, 2002). Bcl-2 family

members are involved in cell death processes caused by MPP + ,

with Bcl-2 being an anti-apoptotic protein while Bax exhibits pro-

apoptotic activity (Cory and Adams, 2002; O’Malley et al., 2003).

In this study, we investigated whether CM had any effect on the

expression ofBcl-2 and Bax in MPP + -treated cells using expression

analysis. As shown in Fig. 5, Bax expression was increased signif-

icantly in MPP + -treated cells compared with that of the control

cells, a finding which is consistent with previous reports (Cheng

et al., 2009; Gao et al., 2008). However, CM treatment suppressed

Bax mRNAexpression in a dose-dependent manner. In contrast, the

level ofBcl-2 in the MPP + -treated group was significantlydecreased

compared with that of the control cells, while expression of Bcl-

2 was recovered following CM treatment. The Bax/Bcl-2 ratio in

cells exposed to 1 mM MPP + was 3.6-fold higher than the control

group, while in cells pre-treated with 1, 10, and 100 ␮g/mL CM,

the ratio decreased in a dose-dependent fashion, suggesting that

CM treatment shifted the balance between pro- and anti-apoptotic

I.S. Kim et al. / Journal ofEthnopharmacology 126 (2009) 447–454 451

Fig. 3. Effects ofCM on ROS generation in SH-SY5Ycells exposed to MPP + . Cells were exposed to 1 mM MPP + with or without different concentration ofCM. (A) ROS generation

was detected by fluorometric analysis using DCFH-DA. Fluorescence intensity of DCF was measured after SH-SY5Y cells were exposed to 1 mM MPP + in the absence of CM

or in the presence of1, 10, and 100 ␮g/mL CM. (a) Control cells; (b) cells exposed to 1 mM MPP + only; (c) cells exposed to 100 ␮g/mL CM only; (d–f) cells pre-treated with 1,

10, 100 ␮g/mL CM, respectively, in the presence of1 mM MPP + . (B) Histograms depict one representative set of results from three different experiments. Cells were treated

with 1 mM MPP + in the absence or presence ofCM for 48 h at 37 ◦ C.

members towards cell survival (Fig. 5B). CM treatment alone did

not significantly alter the Bax/Bcl-2 ratio.

3.5. CM suppresses MPP + -induced caspase-3 expression and

PARP proteolysis

Caspases are the molecular machinery that drives apoptosis

(Grütter, 2000). As caspase-3 is crucial biomarker of the neuronal

apoptosis and acts as an apoptotic executor(Hartmann et al., 2000),

its expression was investigated. SH-SY5Y cells exposed to 1 mM

MPP + displayed an increase in caspase-3 expression, in agree-

ment with previous studies (Kitamura et al., 1998; Cheng et al.,

2009). However, treatment with 1, 10, and 100 ␮g/mL CM effec-

tively attenuated MPP + -induced cleaved caspase-3 expression in

a dose-dependent manner (Fig. 6). Caspase-3 is the major effec-

tor of PARP cleavage during apoptosis (Lazebnik et al., 1994; Le

et al., 2002). Therefore, we further examined PARP cleavage. An

earlier report indicated that MPP + induces an obvious increase

in PARP proteolysis at 48 h when compared to the control cul-

tures (Kitamura et al., 1998). Cleavage of PARP to yield an 85-kDa

452 I.S. Kim et al. / Journal ofEthnopharmacology 126 (2009) 447–454

Fig. 4. Effect ofCM on the free radical scavenging activity. (A) Left: relationship between the signal intensity ofDMPO-OH and the various concentrations ofCM. Right: ESR

spectra ofDMPO-trapped hydroxyl radicals recorded. (B) Left: relationship between the signal intensity ofthe POBN-alkyl radicals and the various concentrations ofCM. Right:

ESR spectra of 2-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN)-trapped alkyl radicals recorded. (C) Left: relationship between the signal intensity of DPPH radical and the

various concentrations ofCM. Right: ESR spectra ofDPPH radicals recorded. (D) Left: relationship between the signal intensity ofDMPO-OOH and the various concentrations

ofCM. Right: ESR spectra of5,5-dimethyl-pyrrokine N-oxide (DMPO)-trapped superoxide radicals recorded.

fragment was detected using polyclonal antibody against full

length PARP (116 kDa), as well as cleaved PARP fragments. PARP

cleavage was attenuated in a dose-dependent manner by treat-

ment with CM (Fig. 6). Western blot analysis ofthe protein levels of

cleaved caspase-3 and PARP was performed to provide an estimate

Fig. 5. Effects of CM on the expression of Bcl-2 and Bax in SH-SY5Y. Cells were

treated with 1 mM MPP + in the absence or presence of CM, and total RNA was

collected for semi-quantitative RT-PCR. The levels of Bax and Bcl-2 were quan-

titated by densitometric analysis (A) and the Bax/Bcl-2 ratio was determined

(B). Each value indicates a mean ± SEM (n = 3).

# P< 0.05, compared with control

group. *P< 0.05, compared with MPP + treated group (one-way ANOVA followed by

Student–Newman–Keuls post hoc test).

Fig. 6. CM inhibits MPP + -induced cleavage of caspase-3 and PARP. Cells were

exposed to 1 mM MPP + with or without various concentration of CM for 48 h, and

the cleavage ofcaspase-3 and PARP were detected in the cell lysate by Western blot

analysis.

ofthe relative level ofexpressionofthese proteins. The proteinlevel

ofcleaved caspase-3 and PARP in the control cells was set at 100%.

The level of cleaved caspase-3 protein following treatment with

1 mM MPP + markedly increased to 219.4 ± 41.6%, but decreased to

191.9 ± 36.6%, 159.7 ± 28.6%, and 145.2 ± 27.5% in cells treated with

1, 10, and 100 ␮g/mL CM, respectively. The level of cleaved PARP

protein following treatment with 1 mM MPP + markedly increased

to 243.4 ± 43.9%, but decreased to 219.5 ± 39.8%, 166.5 ± 22.4%, and

148.4 ± 26.9% in cells treated with 1, 10, and 100 ␮g/mLCM, respec-

tively.

4. Discussion

PD is a chronic, progressive, neurodegenerative disease with

no effective treatment. Although several approved drugs such as

levodopa and other dopaminergic medications do alleviate PD

symptoms, use of these drugs for only a few years is associ-

ated with debilitating side effects (Rascol et al., 2003; Kostic et

al., 1991), and none seems to definitively stop the progression

of the disease. Thus, the development of effective neuroprotec-

tive drugs is urgently needed. A variety of medicinal plants have

long been used in traditional Oriental medicine as crude extracts

and mixtures, in order to prevent or alleviate neurological symp-

toms (Packer et al., 2004; Houghton and Howes, 2005). These

extracts are shown to relieve neurological symptoms inexperimen-

tal animal models, as well as reducing in vitro activity. Increasing

I.S. Kim et al. / Journal ofEthnopharmacology 126 (2009) 447–454 453

evidence supports the beneficialeffects ofmedicinalplants onsome

neurodegenerative diseases such as PD and Alzheimer’s disease

(Packer et al., 2004; Houghton and Howes, 2005). Chrysanthemum

morifolium Ramat (CM) has long been used as traditional herbal

medicine for treatment ofdisease for thousands ofyears. However,

to date, the neuroprotective effects ofCM and the possible molec-

ular mechanisms that underlie its action remain to be investigated.

In this study, we demonstrate that CM protects human dopamin-

ergic SH-SY5Y cells against MPP + -induced cytotoxicity in several

aspects.

Oxidative damage occurs in the Parkinsonian brain (Alam et al.,

1997; Nagatsu and Sawada, 2006), and overproduction ofROS can

cause severe impairment ofcellular functions, are also involved in

apoptotic mechanisms and may contribute to the apoptotic process

found in PD (Kehrerand Smith, 1994). Ourpresentresults also show

that SH-SY5Y cells exposed to 1 mM MPP + significantly increase

their production of ROS, and that CM treatments suppress the

MPP + -induced accumulation ofROS dose dependently and attenu-

ate MPP + -induced SH-SY5Y death. It is interesting to compare our

results with those of Fang et al. (2002), who analyzed the effect

and the mechanism of CM on apoptosis of bovine smooth mus-

cle cells. In the previous study, the number of apoptotic cells was

reduced in the CM treatment group in a concentration-dependent

manner through inhibition of antioxidant enzymes. Furthermore,

it was recently reported that CM improves the antioxidant defense

system and protects brain and liver against lead-induced oxida-

tive damage in mice (Xia et al., 2008). Both the previous and

present observations support the suggestion that CM has cytopro-

tective effects byregulation ofoxidative stress. ESRhas been widely

used as a powerful determinative method for a variety of radicals

because of its accuracy and short time consumption (Sachindra

et al., 2007). Presently, the antioxidant activity of CM was deter-

mined by scavenging free radicals including DPPH, superoxide,

hydroxyl and alkyl radicals by ESR spectrometry. The data suggest

that CM is a powerful antioxidant with radical scavenging activity

for DPPH, superoxide, hydroxyl and alkyl radicals, supporting the

earlier reports (Kim and Lee, 2005; Wang et al., 2001).

The Bax/Bcl-2 ratio may better predict the cell decision between

survival and death, and any shift in the balance of pro- and anti-

apoptotic members may affect cell death (Cory and Adams, 2002).

Our findings show that MPP + has a profound effect on the expres-

sion of Bcl-2 family members in SH-SY5Ycells. MPP + upregulates

Bax expression, while affects the suppression of Bcl-2 expression.

Consequently, the ratio of the pro-apoptotic Bax to the anti-

apoptotic Bcl-2 increases significantly upon treatment with MPP + ,

which correlates well with a previous study (Blum et al., 2001).

Our results show that treatment with CM reduces the expression

ofpro-apoptotic Baxand increased the expression ofanti-apoptotic

Bcl-2 significantlyin a dose-dependent manner, therebyameliorat-

ing the MPP + -induced Bax/Bcl-2 ratio elevation in SH-SY5Y cells.

In general, apoptosis is driven from activation of a family of cys-

tein proteases called caspases, which cleave a critical set ofcellular

proteins to initiate apoptotic cell death, and cell death in PD most

likely occurs through caspase-dependent pathways. It has been

reported that caspases-3 can act directly on its substrate, PARP,

causing hydrolysis (Nicholson et al., 1995). PARP, which is a down-

stream target of caspase-3 is an abundant nuclear enzyme and

normally functions in DNA repair, but extensive PARP activation

can promote cell death (Fernandes-Alnemri et al., 1994; Oliver

et al., 1998). It was previously reported that proteolytic cleav-

age of PARP occurs during 2,2  ,5,5  -tetrachlorobiphenyl-induced

apoptosis in human neuronal SK-N-MC cells (Hwang et al., 2001),

staurosporine-induced apoptosis in dopaminergic neurons (Kim et

al., 1999), catechol-thioether-induced apoptosis inhumanSH-SY5Y

neuroblastoma cells (Mosca et al., 2008) and MPP + -induced apop-

tosis in cerebellar granule neurons (Pu et al., 2003). Presently, CM

treatments effectively attenuated MPP + -induced capase-3 expres-

sion and PARP cleavage in a dose-dependent manner, indicating

that protective effect of CM associated with the inhibition of the

downstream apoptotic signaling pathways, which prevented the

activation of PARP proteolysis. Based on these observations, CM

maymodulate the expressionofBcl-2 familyproteins inresponse to

MPP + treatment, regulatingasuccessionofmitochondria-mediated

downstream molecular events including the activation of PARP.

The effects of CM might be caused by compounds included in CM

extracts suchas ruteolin, apigenin, dicaffeoylquinicacids and triter-

pene, which displayvarious biological activities such as antioxidant

and anti-inflammation activity (Wruck et al., 2007; Elsisi et al.,

2005; Kim and Lee, 2005; Yasukawa et al., 1996). These findings,

taken together, support the contention that CM-mediated neu-

roprotection is due, in part, to inhibition of the mitochondrial

apoptotic pathway. The anti-oxidative and anti-apoptotic proper-

ties of CM might play a major role in rendering such protective

action against MPP + -induced cytotoxicity.

In summary, this study reports for the first time to our

knowledge that CM protects against MPP + -induced cytotoxicity

in SH-SY5Y human neuroblastoma cells and exerts neuroprotec-

tive activity. However, further studies on mature primary neurons

and animal models of PD and comparison with known anti-

parkinsonian agents should be considered. Based on the protective

effects of CM on MPP + -induced cell injury and its long history of

safe use as a traditional herbal medicine, CM might be a potential

therapeutic candidate in treating neurodegenerative disease such

as PD.

Acknowledgements

This research was financially supported by the Ministry of

Education, Science Technology (MEST) and Korea Institute for

Advancement of Technology (KIAT) through the Human Resource

Training Project for Regional Innovation, and also supported by the

grant ofthe Korean Ministry ofEducation, Science and Technology

(The Regional Core Research Program/Chungbuk BIT Research-

Oriented University Consortium).

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