Protective Effects of Celastrol, the Triterpenoid Component of Celastrus Orbiculatus, on Dopaminergic Neuronal Cells in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned Parkinson's Disease Rats

서 론

(neurodegenerative disorders)

이갑득

¹

, 김광진

²

, 박용기

^2,3

1

동국대학교 과학기술대학 나노소재화학과,

²

동국대학교 한의학대학 한의학연구소,

³

동국대학교 한방신약개발센터

Original Article

Protective Effects of Celastrol, the Triterpenoid Component of Celastrus Orbiculatus, on Dopaminergic Neuronal Cells in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned

Parkinson's Disease Rats

Kap-Duk Lee

¹

, Kwang-Jin Kim

²

, Yong-Ki Park

²

1Department of Department of Nanomaterial Chemistry, College of Science & Technology Dongguk University, Gyeongju, 780-714 Korea.

2Oriental Medicine Research Institute and ³Oriental Medicine Drug R&D Center, College of Oriental Medicine, Dongguk University, Gyeongju, 780-714 Korea.

Objectives: The aim of this study was to determine whether celastrol, the triterpenoid component of Celastrus orbiculatus, offers neuroprotection against Parkinson's disease (PD) in mice administered 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine(MPTP).

Methods: We examined how celastrol affected MPTP-induced neuronal loss of tyrosine hydroxylase (TH)-positive dopaminergic neurons in substantia nigra pars compacta (SNpc) in the midbrain of mice. C57BL/6J mice were divided into four groups: (1) saline-saline, (2) saline-celastrol, (3) MPTP-saline, and (4) MPTP-celastrol. The mice were injected intraperitoneally (i.p.) with four administrations of MPTP (18 / ) at 2 h intervals and then i.p. administered celastrol (3 / ) two times at 12 h after last celastrol administration. Expression of TH on the SNpc of brain tissues were analyzed at 7 days after the treatments by immunohistochemistry and Western blot.

Results: Immunohistochemical analysis using TH antibody showed that celastrol provided significantly protective effects against MPTP-induced loss of TH-positive dopaminergic neurons in the SNpc region of the midbrain of mice.

Our Western blot study also showed that celastrol significantly inhibits the MPTP-induced neuronal damage via the up-regulation of TH protein levels in MPTP mice.

Conclusions: The present results suggest that it may be possible to use celastrol for the prevention of nigral degenerative disorders including PD, caused by exposure to toxic substances.

Key Words : Celastrus orbiculatus, celastrol, dopaminergic neuron, neuroprotection, Parkinson's disease.

접수：2008년 6월 30일 수정：2008년 9월 4일 채택：2008년 9월 8일

교신저자 : 박용기(Yong-Ki Park)

경주시 석장동 707번지 동국대학교 한의과대학 본초학교실, 한의학연구소 한방신약개발센터 Tel : +82-54-770-2661, Fax : +82-54-770-2281, E-mail : [email protected] 이 논문은 동국대학교 교내연구비 지원 및 BT 특성화사업단 연구기금에 의한 것으로 이에 감사드립니다.

1) .

,

(Alzheimer’s disease), (Parkinson’s disease),

(Amyotrophic lateral sclerosis;ALS), (Huntington’s disease) ,

(oxidative stress) (free radical species)

(antioxidant defence mechanism) (imbalance) (cell apoptosis)

2-4) .

(substantia nigran pars compactra; SNpc)

(striatum) (tremor),

(bradykinesia), (rigidity), (postural instability), (akinesia)

5,6) .

(oxidative stress), β-amyloid (aggregation)

6) .

L-dopa(levodopa)

,

L-dopa ,

.

,

7-9) .

10-14)

.

100 , ,

15) .

(Celastrus orbiculatus) (Ce- lastraceae)

C. flagellaris, C. stephanotifolius

, ,

, (Celastri caulis)

16) .

, ,

, , celastrol,

kaempferol-7-α-L-rhamnoside, kaempferol-3-β-D-rh- amnoside kaempferol-3,7-α-L-dirhamnoside, quercetin -3,7-α-L-dirhamnoside, kaemp ferol-3-P-coumaroyl

glucoside , 42-

58% ^16,17) . ,

, ,

, , , ,

. , (Tripterygium regelii SPRAGUE et TAKEDA, T. wilfordii var regelii (SPRAGUE et TAKEDA) MAKINO)

, , , ,

16,17)

. Celastrol

, , , .

celastrol diterpenoid triptolide tripdiolide , celacinine, celab- enzine, celafurine, wilfordine, wilforine, wilforgine, wilfortrine, wilforzine alkaloid

16) . , ,

, ,

. C.

paniculatus

( )

17) , Mal-

19) .

, esquiterpene

esters, celastrol, celaphanol A

20) benzoylated flavan-3-ol glycoside

21) , celastrol

23) 22-24)

.

celastrol

(microglia)

22) . MPTP celastrol

, .

실 험

1. 동물 및 재료

1)

MPTP

25) 15-17

20-30g C57BL/6 .

12

. , NIH

(2000 ) .

2)

celastrol(Microsource, Gaylordsville, CT, USA), MPTP-HCl (Fluka, Buchs, Switzerland), lipopolysaccharide(LPS; E. coli B0111:

B4; Sigma, St. Louis, MO, USA), 3-4,5-dimethy;lthazol -2-yl-2,5-teterazolium bromide(MTT, Sigma), Sulfani- lamide(Sigma), N-trityl-1,3-propanediamine acetate(NED;

Sigma), Acrylamide solution (BioRad Labolatories

Inc., Hercules, CA, USA), anti-TH monoclonal antibody (Zymed Lab. Inc. CA, USA), FITC-labeled anti-TH antibody(Zymed Lab. Inc.), anti-actin monoclonal antibody (Sigma), anti-rabbit IgG conjugated HRP antibody(SantaCruz Biotech., Santa Cruz, CA, USA), OCT compound(Tissue-Tek, Sakura Finetek, Torrance, CA, USA), Blotting NC membrane (Hybond C pure, Amersham Life Science, Buckinghamshire, UK), ECL Western Blotting Detection Reagents (Amersham Life Science), avidine-biotin-horseradish peroxidase complex(Vectastain Elite, Vector Laboratories, Burl- ingame, CA, USA), 3,3‘-diaminobenzidine tetrahydro- chloride (DAB; Sigma), Bradford solution(BioRad Laboratories, Hercules, CA, USA) .

2. 방법 1)

(24 ) 4 ( ;

salinesaline, celastrol ; saline-celastrol, MPTP

; MPTP-saline, MPTP celastrol

; MPTP-celastrol) ,

(Intraperitoneal injection) . MPTP MPTP(calculated as free base)

0.9% (saline) 18 /

0.5

, 2 4 .

MPTP 12 24 3

/ celastrol 2 .

0.5 0.9% MPTP

. 2) Tyrosine hydrolase

MPTP saline 7

(SNpc)

. 4% paraformald-

ehyde 2-3 30% sucrose

4 .

OCT compound

-70 cryostat cryocut microtome (LEICA CM. 3050S, Germany)

SNpc striatum (rostrocaudal

extent) 30 coronal sections

.

(immuno-histoche-

mistry) slide PBS 3

5% normal goat serum 0.3% Triton X-100

PBS 1 blocking .

2% normal goat serum 0.15% Triton X-100

PBS 1,000 anti-TH antibody

4 overnight PBS 3

. biotinylated secondary antibody(goat

anti-rat, 1:1000) 1 PBS

3 avidine-biotin-horseradish peroxidase complex (Vectastain Elite) 1

. slide 0.1M Tris

buffer(TB, pH 7.4) 3 , 0.004%

H2O2 0.05% DAB(Sigma) TB

2-5 , 100% alchol xylene

DAKO ^® fluorescent mounting medium

mounting .

,

. , slide cresylviolet

striatum .

slide PBS 3 5% normal goat serum 0.3% Triton

X-100 PBS 1 blocking

. slide 2% normal goat serum 0.15 % Triton X-100 PBS 2,000

FITC-labeled anti-TH antibody 4

2 PBS 3 .

slide 100% alchole xylene mounting medium

. SNpc

TH (TH-positive cells)

.

3)

SNpc TH celastrol

Western blot . , basal ganglia, diencephalon, midbrain

150mM KCl homogenizer

800xg 10

.

Bio-Rad Bradford

.

30 2× sample buffer(100mM Tris- HCl, pH 6.8, 200mM dithithreitol, 4% SDS, 0.2%

bromophenol blue, 20% glycerol) 100 3 10% SDS-polyacrylamide gel

. gel nitro-

cellulose membrane 4 , 30V 16 transfer . Membrane 10% skim milk

1 blocking , TH actin

(monoclonal antibody) 0.05% Tween Tris-buffered saline(TBS-T) 1:1000

4 overnight . Membrane

TBS-T 3 anti-rabbit

IgG conjugated HRP (1:1,000) 1

ECL x-ray film

.

4)

3 ±

(standard deviation; SD)

ABI prism ANOVA Student t-test

p 0.05

.

실험성적

1. 줄무늬체의 신경세포 보호효과

Celstrol

cresyl violet

(Fig. 1), ,

striatum

(Fig. 1. A), MPTP(18 / )

(Fig. 1.

B), MPTP celastrol ,

(Fig. 1.

C). celastrol MPTP

.

2. 흑색 치밀부의 도파민 신경세포 보호효과

Celstrol(3 / ) (dopaminergic neuron)

TH (Fig 2),

(SNpc) TH

(TH-positive dopaminergic cells) (Fig 2A,B), MPTP(18 / ) TH

(Fig 2. C,D). , celastrol (Fig 2. E,F), TH

.

, SNpc TH

(Fig 3), MPTP(MPTP-

AAA

A BBBB

C CC

C DDDD

EEE

E

F F F F

Fig. 2.

A A A A B B B B C C C C

Fig. 1.

N um b er o f TH - p o sit iv e ce lls

10000 8000 6000 4000 2000 0

p < 0.001

MPTP Celastrol

- -

- +

+ -

+ +

Fig. 3.

saline group)

celastrol (MPTP-celastrol group) . , TH

(saline-saline group) celstrol(saline-

celastrol group) SNpc

TH

(Fig 4. A), MPTP(18 / ) (MPTP -saline group)

(Fig. 4. C), celastrol (MPTP-celastrol group)

(Fig. 4. D).

celastrol MPTP

TH .

3. TH 단백질 발현에 대한 celstrol 의 효과

Celstrol SNpc TH

TH

Western blot . ,

Fig. 5 (Saline-Saline

group) celastrol(Saline-Celastrol group)

TH

, MPTP

(MPTP-Ssaline group) TH

. MPTP

TH celastrol

(MPTP-Celatrol group) .

celastrol MPTP

TH

.

고 찰

.

.

, , , ,

, (SNpc)

(striatum) ,

(Lewy body) A A

A A B B B B

C C C

C D D D D

Fig. 4.

TH

Actin

S-S S-C M-S M-C

Fig. 5.

4,26,27)

.

glutathione ,

,

, .

, 10

.

.

, (ginseng)

(MPTP-induced PD mice/rats)

28) , (ginkgo biloba)

(OHDA-induced PD rats) levodopa

29) , (Thunder God Vine; Tripterygium wilfordii

Hook F) trichloride MPP+

30) ,

(polyphenolic compounds) (anti- oxidant activities)

31) . Celastrol

, , ,

, celastrol

(neuroinflammation) (microglia)

22) . celastrol microglia

microglia

.

, ,

, ,

. celastrol

diterpenoid triptolide tripdiolide , alkaloid celacinine, celabenzine, celafurine, wilfordine, wilforine, wilforgine, wilfortrine,

wilforzine ^6,7) .

,

, ,

, ,

.

16) ,

nuclear

factor-kappaB (NF-kB) quinone

methide triterpenoid celastrol

32) ,

celastrol (fatty acid ester)

24,32)

. MPTP

celastrol TH

. MPTP

(nigrostriatal dop- aminergic pathway)

26) . , MPTP

monoamine oxidase 1-

methyl-4-phenylpyridinium(MPP+) , MPP+

I(complex I) ATP

,

34) . MPTP

(blood-brain barrier, BBB) mon-

oamine oxidase(MAO)-B MPP+

25) . MPTP MPP+

,

, superoxide dismutase(SOD)

glutathione peroxidase . ,

cathecholamine

cathecholamine rate-

limiting tyrosine hydroxylase (TH; tyrosine

3-monoxygenase) tyrosine

, L-dopa(L-3,4-dihydroxyphenyl alanine) aromatic amino acid decarboxylase

(AADC) norepinephrine

epinephrine

26) . TH N-terminal C-terminal

60kDa

serine/ threonine kinase

tyrosine, L-dopa, catecholamine,

25) . TH

. MPTP

TH ,

celastrol

(Fig. 1), MPTP TH

(Fig. 2). , TH

MPTP TH

celastrol (Fig. 3).

TH celastrol Western

blot MPTP

TH celastrol

(Fig. 5).

, celastrol

TH

.

, ,

E

,

4-5) .

L-dopa

. ,

28-34)

.

quinone methide triterpenoid

celastrol TH

.

결 론

MPTP (C57BL/6)

(Celastrus orbiculatus) quinone methide triterpenoid

celastrol TH

.

1. Celastrol MPTP

.

2. Celastrol MPTP

TH TH

.

, celastrol

, celastrol

.

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