Hexane Fraction of Zingiberis Rhizoma Crudus Extract Inhibits the Production of Nitric Oxide and Pro-inflammatory Cytokines in LPS-stimulated BV2 Microglial Cells

서 론

(microglia) (central

nervous system; CNS) (immune response) (brain macrophage) .

microglia

1,2) , microglia

,

1-3) . microglia , microglia

4,5) .

6) .

腦神經小膠細胞

生成 抑制效果

정환용¹, 주예진¹, 정혜미¹, 신우진², 서운교¹

1동국대학교 한의과대학 내과학교실, ²삼세한방병원 내과

Original Article

Hexane Fraction of Zingiberis Rhizoma Crudus Extract Inhibits the Production of Nitric Oxide and Pro-inflammatory Cytokines in LPS-stimulated BV2 Microglial Cells

Hwan-Yong Jung¹, Ye-Jin Joo¹, Hye-Mi Jung¹, Woo-Jin Shin², Un-Kyo Seo¹

1Dept. of Oriental Internal Medicine, College of Oriental Medicine, Dongguk University,

2Samse Oriental Medical Hospital

Objectives: The present study is focused on the inhibitory effect of the rhizome hexane fraction extract of Zingiberis Rhizoma Crudus (ginger hexan extract; GHE) on the production of inflammatory mediators such as NO, PGE2, and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 cells, a mouse microglial cell line.

Methods: We separated the hexane fraction from Zingiberis Rhizoma Crudus's methanol extract. The inhibitory and anti-inflammatory effect of GHE was examined on microglial activation.

Results: GHE significantly inhibited the excessive production of NO, PGE2, TNF-α, and IL-1β in LPS-stimulated BV2 cells. In addition, GHE attenuated the mRNA expressions and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines.

Conclusion: The anti-inflammatory properties of GHE may make it useful as a therapeutic candidate for the treatment of human neurodegenerative diseases.

Key Words : Zingiberis Rhizoma Crudus, ginger, BV2 microglial cell, nitric oxide, pro-inflammatory cytokines

접수：2008년 8월 30일 수정：2008년 12월 26일 채택：2008년 12월 30일

교신저자：서운교(Un-Kyo Seo)

경기도 성남시 분당구 수내3동 87-2 동국대학교 분당한방병원 2내과

Tel：+82-31-710-3751, Fax：+82-31-710-3780, E-mail：[email protected]

( , Zingiberis Rhizoma Crudus) (Zingiberis officinale Rosc.) ^7,8) ,

“ , ... , ”

9) , “ ,

, , ”, “ ,

, ... , ” .

, , ,

, ,

8) .

al-Yahya ¹⁰⁾

(gastroprotective action) , Holtmann

11) , Bone ¹²⁾ , Sharma ¹³⁾ (anti-

emesis) .

6-gingerol shogaol (anti-oxidation), (anti-inflammation)

14-17)

, (anti-tumor) ¹⁸⁾ ,

(analgesic effect) ¹⁹⁾ .

,

, Grzanna ²⁰⁾

.

(n-hexan) , (microglia)

.

실 험

1. 재료 1)

(mouse microglial line) BV2

. 2)

(Zingiber Rhizoma Crudus;

Ginger)

. 3)

methanol(Merck Co, Germany), lipopolysaccharide(LPS; E. coli B0111:B4; Sigma, St. Louis, MO, USA), 3-(4,5-dimethy;lthazol-2-yl-2, 5-teterazolium bromide; MTT, Sigma), N-trityl-1,3- propanediamine acetate(Sigma), Sulfanilamide(Sigma), Acrylamide solution(BioRad Labolatories Inc, Hercules, CA, USA), anti-iNOS monoclonal antibody (SantaCruz Co, Ltd, SantaCruz, CA, USA), anti- COX-2(SantaCruz), DuoSet ELISA Development System(R&D System, Inc, Minneapolis, MN, USA)

.

Microplate Reader(Asys, Sunnyvale, CA, USA), Deep-freezer Advantage(Queue, USA), Rotary Evaporator(Eyela, Japan), Thermal Cycler (BioRad Labolatories Inc.), Orbital Shaker (Finemould Precision IN, Co, Gyeonggi-do, korea), BioDoc It ^TM Imaging System(UVP, Cambridge, UK), UV-VIS Spectrophotometer(Shimazu, Japan) .

2. 방법 1)

(1 ) grinder

80% (methanol) 1 L 7

0 14 .

Wathman No. 1 filter paper (rotary evaporator)

. (n-hexan)

, Wathman No. 1 filter paper

. (Ginger hexan extract;

GHE) 0.85 g , 0.18% .

-80

DMSO .

2)

(mouse microglial line) BV2 10% fetal bovine serum(FBS, GibcoBRL, Life Technologies Inc, Gaitherburg, MD, USA), 1%

penicillin/streptomycin(GibcoBRL) DMEM (GibcoBRL) 37 , 5% CO 2

.

3)

BV2 (5 × 10 ⁴ cells/well) 96-well culture

plate 100 DMEM 37 ,

5% CO 2 , (0.05-1

/ ) (GHE) 24

. well 5 / MTT

50 4

, 10 DMSO

formazan .

microplate reader 550-600 nm .

(control) 100%

(cell viability; %) .

4) NO

BV2 nitric

oxide(NO) NO ^2-

Griess

. , BV2 2 × 10 ⁵ cells/ 24-well plate

30 1 /

LPS 24

(activation) . BV2

NO 100

Griess (0.1% NED & 1% sulfanilamide in 5% H 3 PO 4 ) 100 15

microplate reader 540 nm

. NO (μM) NaNO 2

.

5) PGE 2

BV2 prostaglandin

E2(PGE 2 )

(enzyme immunoassay) .

, BV2 (2 × 10 ⁵ cells/ ) 24-well plate

30 1 / LPS

16 . BV2

PGE 2 PGE 2 enzyme

immunometric assay kit(Cayman Co.)

. PGE 2 (pg/

) PGE 2 (recombinant PGE 2 protein) .

6) Enzyme-linked immunosorbent assay(ELISA) BV2

(proinflammatory cytokine) TNF-α, IL-1β DuoSet ELISA Development System(R&D Systems)

. 1× phosphate buffered saline(PBS, pH 7.2-7.4) buffer 720 / capture

monoclonal antibody(mAb) 100

96-well plate(Nunc coating plate) well . 0.05% Tween-

20 1× PBS(PBS-T) 3

1% bovine serum albumin(BSA) diluent

(blocking solution) 1

coating blocking

. Blocking solution ,

(standard

recombinant protein) 100 2

. 5 1× PBS-T

10 ⁸ / detection mAb 100 1

. plate 1×

PBS-T 3 Strepavidin-HRP mAb

30 .

1× PBS-T 3 tetramethylbensidine

(TMB) (substrate) 15 (orbital

shaker) . 1 M phosphoric acid 50 microplate reader 450 nm

.

(recombinant protein) (standard curve) .

7) Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

BV2 NO (synthease) in-

ducible nitric oxide synthase (iNOS), PGE 2

cyclooxygenase-2(COX-2) (TNF-α, IL-1β)

RT-PCR . , BV2 (2 × 10 ⁵ cells/

) 30cm culture dish

30

. LPS(1 / ) 6

. 5,000 rpm 5 TRIzol (Invitrogen ^TM )

total RNA .

mRNA oligo-(dT) primer Improm- II ^TM reverse transcriptase(Promega Co, Madison, WI,

USA) 25 10 , 42 60 ,

70 15 cDNA .

cDNA PCR BV2

mRNA cDNA 1 target-

specific primers (10 pmol) 1 10× buffer(10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 1 U Tag polymerase(Takara)

denaturation 94 30 ,

annealing 55-60 30 extension

70 60 30-35 cycles

. PCR (internal house

keeping gene) GAPDHs (Table 1).

8) Western blot

BV2 iNOS COX-2

Western blot . , BV2 (2 ×

10 ⁵ cells/ ) 6-well plate

30

. LPS(1 / ) 24

. BV2

HBSS 2

50 mM HEPES, pH 7.4, 150 mM NaCl, 1%

deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 / aprotinin lysis buffer 0.1 lysis

30 . 20

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

Primers Sequences

iNOS Sense 5'-GAC CAG ATA AGG CAA GCA C-3' Antisense 5'-CTT GTC TTT GAC CCA GTA GC-3' COX-2 Sense 5'-ATG CTC CTG CTT GAG TAT GT-3' Antisense 5'-CAC TAC ATC CTG ACC CAC TT-3'

TNF-α Sense 5'-TTC TGT CTA CTG AAC TTC GGG GTG AT C GGT CC-3' Antisense 5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3' IL-1β Sense 5'-ATG GCA ACT GTT CCT GAA CTC AAC T-3'

Antisense 5'-CAG GAC AGG TAT AGA TTC TTT CCT TT-3' GAPDH Sense 5'-CTC GTG GAG TCT ACT GGT GT-3'

Antisense 5'-GTC ATC ATA CTT GGC AGG TT-3' Table 1.

blue, 20% glycerol) 100 3

10-12% SDS-PAGE .

gel nitrocellulose membrane

4 , 30 V 16 transfer .

Membrane 10% skim milk 1

blocking , iNOS, COX-2 β-actin (monoclonal antibody) 0.05%

Tween Tris-buffered saline(TBS-T)

1:500 1000 2

. Membrane TBS-T 3

anti-rabbit IgG conjugated HRP (1:1,000)

1 enhanced chemilumine-

scence(ECL) kit(Amersham) X-ray film .

9)

3

± (standard error of mean; SEM)

SPSS p

0.05 .

결 과

1. 세포독성검정

BV2

(cell viability; %)

100% 2 / ,

5 / , 10 / , 20 / , 50 / 100 / 98.1±1.17%(mean±SEM), 98.61±0.26

%, 99.6±0.08%, 66.5±0.59%, 42.76±2.58% 25.96

±0.58% (Fig. 1).

10 / 90%

, 1-10 / .

2. NO 생성억제효과 BV2

(control) NO

(6.51±0.02 μM) , LPS BV2

NO (53.22±1.12

μM) (Fig. 2). LPS

BV2 NO

1 / 10 /

44.3±3.04 μM 16.7±0.18 μM

( ^* p<0.05 and ^*** p<0.001 vs. LPS-treated group,

n=3.) NO (Fig.

2). 10 /

7.16±0.23 μM LPS

BV2 NO

.

100

50

0

0 2 5 10 20 50 100

GHE (ug/ml)

C ell v ia b ilit y (% )

Fig. 1.

BV2 NO

.

3. iNOS 유전자 및 단백질 발현 억제효과

, BV2 iNOS

, LPS (Fig. 3A). LPS iNOS

10 / .

, iNOS

, LPS ,

10 / (Fig.

3B). BV2

iNOS

NO .

4. PGE 2 생성억제효과 BV2

(control) NO

GHE (ug/ml) GHE (ug/ml)

LPS (1ug/ml) LPS (1ug/ml)

iNOS GAPDH

iNOS β-actin

10

10 1

+

- +

- +

10

10 1

+

- +

- +

AA

AA BBBB Fig. 3.

***

* 60

50 40 30 20 10 0

0 0 10 1 10

- + - + +

LPS (1 ug/ml)

GHE (ug/ml)

N O ( uM )

Fig. 2.

(116.2±14.22 pg/ ) , LPS

BV2 PGE 2

(1407±66.3 pg/ ) (Fig. 4).

LPS BV2

PGE 2 1 / 10

/ 1149±29.01 pg/ 375.2

±6.55 pg/ , ( ^* p<0.05 and

** p<0.01 vs. LPS-treated group, n=3.) PGE 2

(Fig. 4).

10 / 129.42±0.72

pg/ LPS BV2

PGE 2

.

BV2 PGE 2

.

5. COX-2 유전자 및 단백질 발현 억제효과

, BV2 COX-2

, LPS (Fig. 5A). LPS COX-2

10 / .

GHE (ug/ml) GHE (ug/ml)

LPS (1ug/ml) LPS (1ug/ml)

COX-2 GAPDH

COX-2 GAPDH

10

10 1

+

- +

- +

10

10 1

+

- +

- +

AA

AA BBBB Fig. 5.

**

* 1500

0

0 0 10 1 10

- + - + +

LPS (1 ug/ml)

GHE (ug/ml) P G E

(p g /m l)

1000

500

Fig. 4.

, COX-2

, LPS ,

(Fig. 5B).

BV2 COX-2

PGE 2

.

6. 전염증성 사이토카인 생성억제효과

, BV2 TNF-α

24.56±0.99 pg/

, LPS 867.9±3.23 pg/

. LPS TNF-α

1 / 10 /

518.9±6.62 pg/ 139.3±1.43 pg/

( ^*** p<0.001 vs. LPS-treated group, n=3.) TNF-α

(Fig. 6A).

10 / 57.44±4.51 pg/

LPS BV2

TNF-α

. IL-1β

8.48±0.99 pg/

, LPS 160.1±12.98 pg/

. LPS IL-1β

1 / 10 / ,

GHE (ug/ml)

LPS (1ug/ml) TNF-α IL-1β

10

10 1

+

- +

- +

GAPDH

Fig. 7.

α β

***

***

900

0

0 0 10 1 10

- + - + +

GHE (ug/ml) LPS (1 ug/ml)

TN F - a (p g /m l)

800 700 600 500 400 300 200 100

***

***

900

0

0 0 10 1 10

- + - + +

GHE (ug/ml) LPS (1 ug/ml)

IL - 1b ( p g /m l)

100

A A A

A BBBB

Fig. 6.

α β

135.7±0.13 pg/ 31.6±0.05 pg/ , ( ^*** p<0.001 vs. LPS-treated group, n=3.) IL-1β

(Fig. 6B).

33.58±2.95 pg/

LPS BV2

IL-1β .

BV2 TNF-α IL-1β

.

7. 전염증성 사이토카인의 유전자 발현 억제효과

, BV2 TNF-α IL-1β

, LPS

. LPS

TNF-α IL-1β 10 /

.

BV2 TNF-α IL-1β

.

고 찰

(homeostasis) NO, PGE 2 TNF-α, IL-1β, IL-6

, IL-4, IL-10, IL-13, tumor growth factor-β(TGF-β)

(anti-inflammatory cytokine)

21,22)

. (brain macrophage)

(microglia) (resid-

ent immune cell)

TNF-α,

IL-1, IL-6 (proinflammatory

cytokine) eicosanoids, PGE 2 , reactive oxygen species (ROS), NO, superoxide(O ^2- )

,

(Alzheimer's disease)

(Parkinson's disease) (neur-

odegenerative disease), (stroke)

21,24,25)

.

,

(non-steroidal anti-inflammatory drugs; NSAIDS)

21,23)

. ,

(bioactive components)

21,22)

. ( , Zingiberis Rhizoma Crudus) (Zingiberaceae)

(Zingiberis officinale Rosc.) ⁸⁾ .

9) “ , , . ,

. , , ,

, , .”

, , ,

.

25) “ . .

. . . .

... . .

. .” ,

, , . ,

, , ,

, ,

. , ,

, , , ⁸⁾ .

hydrocarbon , ketone ,

alcohol zingiberene, α-cadinene ,

6-gingerol

7,8,26)

. 6-gi-

ngerol shogaol , ,

,

14,15,27,28)

.

Sarcoma-180

29) .

oleoresin, gingerol, shogaol (natural killer cell)

15) .

,

. NO

L-arginine NO (NO synthase

pathway; NOS) iNOS (free radical)

, ,

, LPS interferon- gamma(IFN-γ), β-amyloid

1-3) . , NO (pro- inflammation) (anti-inflammation)

, NO

shock

. NO

11,14,30)

.

NO iNOS

, iNOS

(transcription) iNOS

NO .

. iNOS

, NO iNOS promoter

activity iNOS (transc-

ription) ²⁰⁾ .

, PGE 2 COX-2 NO

4,31)

. COX-2

(inhibitor)

, COX-2

, PGE 2

. COX-2

(transcription)

PGE 2 .

.

Bacterial endotoxin LPS (tumor

necrosis factor; TNF)

inflammagen TNF-α, IL-1β

32) . TNF-α IL-1

β LPS microglia

. LPS

,

21) . , TNF-α microglia

,

TNF-α

mRNA TNF-α

21,23)

.

BV2

TNF-α IL-1β ,

.

,

.

NO, PGE 2

.

.

결 론

,

(BV2 microglial cells)

. 1. BV2

10 / .

2. BV2

NO .

3. NO iNOS

.

4. BV2

PGE 2

.

5. PGE 2 COX-2

.

6. BV2

TNF-α IL-1β . 7. TNF-α IL-1β

. LPS (BV2 microglial cell)

NO, PGE 2 , TNF-α, IL-1β ,

.

참고문헌

1. Orr CF, Rowe DB, Haliday GM. An inflam- matory review of Parkinson's disease. Progress in neurobiology. 2002;68:325-40.

2. Norton WT, Aquino DA, Hozymi I, Chiu FC, Brosnan CF. Quantative aspects of reactive gliosis: a review. Neurochemical research. 1992;

17:877-85.

3. Vila M, Jackson-Lewis V, Guegan C, Wu DC, Teismann P, Choi DK, Tieu K, Prezeborski S.

The role of glial cells in Parkinson's disease.

Current opinion in neurology. 2001;14:483-9.

4. Gonzalez-Scarano F, Baultuch G. Microglia as mediators of inflammatory and degenerative disease. Annual review of neuroscience 1999;34:

307-21.

5. Cross AK, Woodroofe MN. Immunoregulation of microglial functional properties. Microscopy research and technique. 2001;54:10-7.

6. Seo WG, Pae HO, Oh GS, Chai KY, Kwon TO,

Yun YG, Kim NY, Chung HT. Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW264.7 cells.

Journal of ethnopharmacology. 2001;76:59-64.

7. , ,

11 . :( ) . 1985:1207.

8. . . :

. 2007:375-7.

9. . . : . 1982:

1620-5.

10. Al-Yahya MA, Rafatullah S, Mossa JS, Ageel AM, Parmar NS, Tariq M. Gastroprotective activity of ginger zingiber officinale rosc, in albino rats.

The American journal of Chinese medicine.

1989;17(1-2):51-6.

11. Holtmann S, Clarke AH, Scherer H, H hn H. The antimotion sickness mechanizm of ginger. Acta oto-laryngologica. 1989;108:168-14.

12. Bone ME, Wilkinson DJ, Young JR, McNeil J, Charlton S. Ginger root-a new antiemetic, The effect of ginger root on postoperative nausea and vomiting after major gynaccological surgery.

Anaesthesia. 1990;45:669-71.

13. Sharma SS, Kochupillai V, Gupta SK, Seth SD, Gupta YK. Antiemetic efficacy of ginger(Zingiber officinale) against cisplatin-induced emesis in dogs. Journal of Ethnopharmacology. 1997;57:

93-6.

14. Chung YK. Components of essential oil and antimicrobial of ginger. Journal of Korean Society Applied Biological Chemistry. 1997;40(6):514-8.

15. F Zakaria-Rungkat, Nurahman, E Prangdimurt, Tejasari. Antioxidant and Immunoenhancement Activities of Ginger (Zingiber officinale Roscoe) Extracts and Compounds in In Vitro and In Vivo Mouse and Human System. Nutraceuticals &

Food 2003;8(1):96-104.

16. Lee BS, Ko MS, Kim HJ, Kim DH. Seperation of 6-gingerol from ginger and antioxidative

activity. Korean Journal of Biotechnology and bioengineering. 2006;21:484-8.

17. Park KK, KS Chun, JM Lee, SS Lee, YJ Surh.

Inhibitory effects of [6]-gingerol, a major pungent principle of ginger, on phorbol ester-induced inflammation, epidermal ornithine decarboxylase activity and skin tumor promotion in ICR mice.

Cancer letters. 1988;129:139-44.

18. Eguchi A, Murakami A, Ohigashi H. Novel bioassay system for evaluating anti-oxidative activities of food items: use of basolateral media from differentiated Caco-2 cells. Free Radical Research 2005;39:1367-75.

19. Lantz RC, Chen GJ, Sarihan M, Sólyom AM, Jolad SD, Timmermann BN. The effect of extracts from ginger rhizome on inflammatory mediator production. Phytomedicine. 2007;14: 123-8.

20. Grzanna R, Phan P, Polotsky A, Lindmark L, Frondoza CG. Ginger extract inhibits beta-amyloid peptide-induced cytokine and chemokine expression in cultured THP-1 monocytes. Journal of alternative and complementary medicine. 2004;10(6):1009 -13.

21. McGeer PL, Yasojima K, McGeer EG. Inflammation in Parkinson's disease. Advances in neurology.

2001;86:83-93.

22. Hunot S, Dugas B, Faucheux B, Hartmann A, Tardieu M, Debre P, Agid Y, Dugas B, Hirasch EC. FceRII/CD23 is expressed in Parkinson's disease and induces, in vitro, production of nitric oxide and tumor necrosis factor-a in glial cells.

The Journal of neuroscience. 1999;19:3440-7.

23. Gonzalez-Scarano F, Baultuch G. Microglia as mediators of inflammatory and degenerative disease.

Annual review of neuroscience. 1999;34:307-21.

24. Hanada T, Yoshimura A. Regulation of cytokine signaling and inflammation. Cytokine & growth factor reviews. 2002;3(4-5):413-21.

25. . . : . 1981:

75-6.

26. Qureshi S, Shah AH, Tariq M, Ageel AM. Studies on herbal aphrodisiacs used in Arab system of medicine. The American journal of Chinese medicine. 1989;17(1-2):57-63.

27. Suekawa M, Ishige A, Yuasa K, Sudo M, Aburada M, Hosoya E. Pharmacological Studies on ginger I, Pharmacological actions of pungent constituents, (6)-gingerol and (6)-shogaol. Journal of pharmacobio-dynamics. 1984;7:836-48.

29. Nakamura H, Yamamoto T. The active part of the [6]-gingerol molecule in mutagenesis. Mutation research. 1983;122(2):87-94.

29. Park KY, Lee SJ, Lee KI, Rhee SH. The antitumor effect in sarcoma-180 tumor cell of mice admin-

istered with Japannes apricot, garlic or ginger Doenjang. Korean Journal of Food and Cookery Science. 2006;5(21):599-606.

30. Shoji N, Iwasa A, Takemoto T, Ishida Y, Ohizumi Y. Cardiotonic principles of ginger(Zingiber officinale Rosc.). Journal of pharmaceutical sciences.

1982;71:1174.

31. Lee YL, Shih K, Bao P, Ghirnikar RS, Eng LF.

Cytokine chemokine expression in contused rat spinal cord. Neurochemistry international. 2000;

36(4-5):417-25.

32. Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cellular Signaling.

2001;13:85-94.