Flower MeOH Extract of Panax Notoginseng Attenuates the Production of Nitric Oxide and Pro-inflammatory Cytokines in LPS-stimulated RA W264.7 Cells

서 론

(macrophages)

1-3) ,

nitric oxide(NO), prostaglandin E 2 (PGE 2 ), (cytokine)

2-5) , (rheumatoid arthritis) (multiple sclerosis), (asthma)

2,3,6)

.

6) . ( , Panax notoginseng (Burk.) F.

H. Chen) , ⁷⁾ “ ,

, ” , ⁸⁾ “

三七花

주예진, 정혜미, 서운교 동국대학교 한의과대학 내과학교실

Original Article

Flower MeOH Extract of Panax Notoginseng Attenuates the Production of Nitric Oxide and Pro-inflammatory Cytokines in LPS-stimulated RAW264.7 Cells

Ye-Jin Joo, Hye-Mi Jung, Un-Kyo Seo

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

Objectives: Inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokines, TNF-α and IL-1β play a critical role in inflammatory immune response. Therefore, intervention of inflammatory mediator production promises therapeutic benefit for treatment of many chronic inflammatory diseases, such as allergic asthma, rheumatoid arthritis, multiple sclerosis, septic shock and neurodegenerative diseases. In this study, the pharmacological effects of the flower MeOH extract Panax notoginseng (Notoginseng Flos; NF) on inflammation were investigated to address potential therapeutic or toxic effects.

Methods: RAW264.7 cells were treated with different concentrations of NF methanol (NF-M) extract in the presence or absence of LPS (1 / ).

Results: NF-M extract significantly inhibited LPS-induced production of NO, PGE2 and pro-inflammatory cytokines, TNF-α and IL-1β in a dose-dependent manner. In addition, NF-M extract suppressed mRNA expressions and protein levels of iNOS, COX-2 and pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells.

Conclusion: These results indicated that NF-M extract inhibits LPS-induced production of inflammatory mediators in macrophages and demonstrated that NF-M extract possesses anti-inflammatory properties in vitro.

Key Words : Notoginseng Flos, Panax notoginseng, RAW264.7, nitric oxide, pro-inflammatory cytokines

접수：2008년 8월 19일 수정：2008년 12월 29일 채택：2009년 1월 5일

교신저자：주예진(Ye-Jin Joo)

경기도 성남시 분당구 수내3동 87-2 동국대학교 분당한방병원 의국

Tel：+82-31-710-3734, Fax：+82-31-710-3780, E-mail：[email protected]

” .

,

9) , Wei ¹⁰⁾ , ¹¹⁾

, Rhule ¹²⁾

, Wang ¹³⁾ ,

Xu ¹⁴⁾ , He ¹⁵⁾ , Wun ¹⁶⁾

, . ( , Notoginseng Flos)

, ,

.

Gao ¹⁷⁾ saponin

, Wang ¹⁸⁾ ,

. ,

.

실 험

1. 재료 1)

(mouse peritoneal macrophage line) RAW264.7 American Type Culture Collection Center(ATCC) ,

. 2)

( ; Notoginseng Flos) ( )

( , )

. 3)

methanol(Merck Co, Ger-

many), lipopolysaccharide(LPS; E. coli B0111:B4;

Sigma, St. Louis, MO, USA), 3-(4,5-dimethy;lthazol -2-yl-2,5-tetrazolium 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)

(50g) grinder 80% methanol 1L

5 . Wathman No. 1 filter

paper (rotary evaporator)

-80

. (Notoginseng Flos met-

hanol extract; NF-M extract) 18.74

g(yield of 37.48%) , DMSO

.

2)

(mouse peritoneal macrophage line) RAW264.7 10% fetal bovine serum (FBS, GibcoBRL, Life Technologies Inc, Gaitherburg, MD, USA), 1% penicillin/streptomycin(GibcoBRL)

DMEM(GibcoBRL) 37 , 5%

CO .

3)

(NF-M extract) (10-100 / )

RAW264.7 24

MTT assay cell viability .

RAW264.7 (5 × 10 ⁴ cells/well) 96-well culture

plate 100 DMEM 37 ,

5% CO 2 , (1-100 /

) (NF-M extract)

24 . well 5 /

MTT 50 4

, 10 DMSO

formazan .

microplate reader 550-600nm .

(control) 100%

(cell viability; %) .

4) NO

RAW264.7 NO

(20 / , 50 / , 100

/ ) RAW264.7 30 LPS

24 RAW264.7

NO Griess

. RAW264.7 nitric oxide(NO)

NO ^2- Griess

. , RAW264.7 2 × 10 ⁵ cells/

24-well plate

30

1 / LPS 24

(activation) . RAW

264.7 NO

100 Griess (0.1% NED

& 1% sulfanilamide in 5% H 3 PO 4 ) 100

15 microplate reader

540nm .

NO (μM) NaNO 2

. 5) PGE 2

RAW264.7 pro-

staglandin E 2 (PGE 2 )

(enzyme immunoassay)

. , RAW264.7 2 × 10 ⁵ cells/

24-well plate

30

1 / LPS 16 .

RAW264.7 PGE 2

PGE 2 enzyme immunometric assay kit(Cayman Co) protocol

. PGE 2 (pg/ ) PGE 2

(recombinant PGE 2 protein) .

6) Enzyme-linked immunosorbent assay(ELISA) RAW264.7

(proinflammatory cytokines)

(10-100 / )

LPS RAW264.7

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 coa- ting plate) well

. 0.05% Tween-20 1× PBS

(PBS-T) 3 1% bovine serum albu-

min(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)

. 1M phosphoric

acid 50 microplate reader

450nm .

(recombinant protein)

(standard curve) .

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

RAW264.7 NO

(synthease) inducible nitric oxide synthase(iNOS),

PGE 2 cyclooxygenase-2(COX-2)

(TNF-α, IL-1β)

RT-PCR . , RAW264.7

(2 × 10 ⁵ cells/ ) 30 culture dish 20 / , 50 / 100 /

30 .

LPS(1 / ) 6

.

5,000rpm 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 RAW

264.7 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).

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.

8) Western blot

RAW264.7 iNOS COX-2

Western blot . ,

RAW264.7 (2 × 10 ⁵ cells/ ) 6-well plate 20 / ,

50 / 100 / 30

. LPS(1 / ) 24

. RAW264.7

HBSS 2

50 mM HEPES, pH 7.4, 150 mM NaCl, 1% deoxycholate, 1mM 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 blue, 20% glycerol) 100 3

10-12% SDS-PAGE .

gel nitrocellulose membrane

4 , 30V 16 transfer . Me-

mbrane 10% skim milk 1

blocking , iNOS, COX-2 β-actin (monoclonal antibody) 0.05% Tween Tris-buffered saline(TBS-T) 1:500-

1000 2 . Me-

mbrane 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

RAW264.7 (cell viability; %)

100%

10 / , 20 / , 50 / , 100 / , 200

/ 500 / 100.4±1.84%(mean±

SEM), 92.41±0.51%, 92.33±0.58%, 84.56±0.21%, 61.04±1.21% 21.97±0.6% (Fig. 1),

100 / 80%

.

C ell v ia b ilit y (% )

125

100

75

50

25

0

0 10 20 50 100 200 500

NF-M extract (ug/ml)

Fig. 1.

10-100 / . p 0.05.

2. NO 생성억제효과

RAW264.7 LPS

(control) NO (4.62±0.02μM) , LPS

RAW264.7 NO

(67.9±1.41μM) , 20 /

, 50 / 100 / 52.7±0.96μM,

31.5±0.62μM 21.9±0.42μM .

100 / 6.36±0.03μM

(Fig. 2). ^* p<0.05 and ^*** p<0.001 vs. LPS-treated group, n=3.

3. iNOS 유전자 및 단백질 발현 억제효과

RAW264.7 iNOS LPS

, LPS .

50 / 100 /

(Fig. 3A).

iNOS

, LPS ,

(Fig. 3B).

4. PGE 2 생성억제효과

RAW264.7 LPS

0 20 50 100 100

- + + + -

0 + NF-M (ug/ml)

LPS (1ug/ml) iNOS GAPDH

0 20 50 100 100

- + + + -

0 + NF-M (ug/ml)

LPS (1ug/ml) iNOS Actin

AA

AA BBBB

Fig. 3.

*

***

***

70 60 50 40 30 20 10 0

N O ( uM )

LPS

0

NF-M extract (ug/ml)

20 50 100 100

- + + + -

+

Fig. 2.

(control) NO (320±7.92pg/ ) , LPS

RAW264.7 PGE 2

(2452±60.7pg/ ) , 20

/ , 50 / 100 / 1604±39.7pg/

, 1092±21.9pg/ 420±7.92pg/ ,

PGE 2 .

100 / 65.2±2.92pg/

(Fig. 4). ^** p<0.01 and ^*** p<0.001 vs. LPS-treated group, n=3.

5. COX-2 유전자 및 단백질 발현 억제효과

RAW264.7 COX-2 LPS

, LPS , 50 / 100 /

(Fig. 5A).

COX-2

, LPS ,

(Fig. 5B).

6. 전염증성 사이토카인 생성억제효과

RAW264.7 TNF-α

29.25±1.74pg/

, LPS 1029±21.13pg/

. LPS TNF-α

0 20 50 100 100

- + + + -

0 + NF-M (ug/ml)

LPS (1ug/ml) COX-2 GAPDH

0 20 50 100 100

- + + + -

0 + NF-M (ug/ml)

LPS (1ug/ml) COX-2 Actin

AA

AA BBBB

Fig. 5.

***

***

3000

0 P G E

(p g /m l)

LPS

NF-M extract (ug/ml)

20 50 100 100

- + + + -

+ 2000 **

1000

Fig. 4.

20 / , 50 / 100

/ , 723.2±8.69pg/ , 323.2±8.69

pg/ 105.6±9.34pg/ .

100 /

35.57±3.91pg/ (Fig. 6A).

IL-1β

29.25±1.74pg/ ,

LPS 229.3±21.13pg/ .

LPS IL-1β

20 / , 50 / 100 / ,

216.5±6.85pg/ , 84.52±5.51pg/ 67.67±2.01pg/

. 100

/ 32.63±5.12pg/

(Fig. 6B). ^* p<0.05, ^** p<0.01 and ^*** p<0.001 vs. LPS- treated group, n=3.

7. 전염증성 사이토카인의 유전자 발현 억제효과

RAW264.7 TNF-α IL-1β

LPS

, LPS , 50 / 100

/ (Fig. 7).

0 20 50 100 100

- + + + -

0 + NF-M (ug/ml)

LPS (1ug/ml) TNF-α

IL-1β

GAPDH

Fig. 7.

α β

***

***

1250

0

TN F - a (p g /m l)

LPS

0

NF-M extract (ug/ml)

20 50 100 100

- + + + -

+ 750 **

500 1000

250

*

**

250

0

IL - b ( p g /m l)

LPS

0

NF-M extract (ug/ml)

20 50 100 100

- + + + -

+ 150

100 200

50

AA

AA BBBB

Fig. 6.

α β

고 찰

,

TNF-α, IL-1, IL-6 eicosanoids, reactive oxygen species(ROS), NO, superoxide(O ^2- )

.

, , ,

,

, ,

19,20)

.

,

.

( ; Panax notoginseng) (

: Araliaceae) ( ,

Panax notoginseng (Burk.) F. H. Chen) ,

, , ,

, .

7) “ . ,

, , , ,

.”, “ , , ,

, ” ,

,

8) “ , ,

, . ,

” ,

.

21) “ ,

, , .”

.

, , , , , , ,

, , ,

. ( ; Notoginseng Flos)

, , , ,

22) .

23-29)

, ,

ginsenoside Rc, Rb2, Rb3 protopanax-

adial saponin ¹⁴⁾ ,

(colon cancer cell)

18) .

.

NO L-argi-

nine NO (NO synthase pathway;

NOS) iNOS

(free radical) ,

,

, LPS IFN-γ, β-amy- loid

1-3) .

NO

11,30,31)

. NO

, NO

shock

.

NO iNOS

, iNOS

iNOS

NO .

. iNOS

, NO

iNOS promoter activity iNOS

19) .

, PGE 2 prostaglandin endoperoxide synthase

COX-2 NO

4,5) , COX-2

.

PGE 2 COX-2

, COX-2

PGE 2

.

. Bacterial endotoxin LPS

inflammagen , (tumor necrosis factor; TNF)

TNF-α, IL-1β

32) . TNF-α IL-1β LPS

. LPS

,

20) . LPS

NO, TNF-α, monocyte chemoattractant protein-1(MCP-1), macrophage inflammatory peptide -1α(MIP-1α), IL-1β, IL-6, IL-8, IL-18, macrophage colony-stimulating factor(M-CSF)

32,33)

.

ROS, NO

30,31,34,35)

. TNF-α IL-1β , TNF-α

TNF-α (lung)

TNF-α mRNA

TNF-α

36,37)

.

RAW264.7

TNF-α IL-1β ,

.

,

.

NO, PGE 2

(TNF-α, IL-1β)

.

.

결 론

(RAW264.7 cells)

.

1. RAW264.7

100 / .

2. LPS RAW264.7

NO , NO

iNOS

.

3. LPS RAW264.7

PGE 2 , PGE 2

COX-2

.

4. LPS RAW264.7

TNF-α IL-1β

, .

RAW264.7

LPS NO

TNF-α, IL-1β

.

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