Inhibition of LPS Induced iNOS, COX-2 and Cytokines Expression by $Genistein-4'-O-{\alpha}-L-Rhamnopyranosyl-(1-2)-{\beta}-D-Glucopyranoside$ through the $NF-{\kappa}B$ Inactivation in RAW 264.7 Cells

(1)

38(4) : 339 348 (2007)

339

Genistein-4 ^' -O- ^α -L-rhamnopyranosyl-(1-2)- ^β -D-glucopyranoside ^의 RAW 264.7 ^세포에서 NF- ^κ B 불활성화를 통한 LPS 에 의해 유도되는

iNOS, COX-2 ^그리고 cytokine ^{들의 발현 저해효과}

박승재¹·김지연¹·장영표¹·조영욱²·안은미³·백남인⁴·이경태¹*

1경희대학교약학대학

,

²^{경희대학교}^의과대학

,

³^{대구한의과대학교}^한방식품 ^약리학과

,

⁴^{경희대학교}^{생명과학부}

Inhibition of LPS Induced iNOS, COX-2 and Cytokines Expression by Genistein-4 ^' -O- ^α -L-Rhamnopyranosyl-(1-2)- ^β -D-Glucopyranoside through

the NF- ^κ B Inactivation in RAW 264.7 Cells

Seung Jae Park

¹

, Ji-Yeon Kim

¹

, Young Pyo Jang

¹

, Young-Wuk Cho

²

Eun-Mi Ahn

³

, Nam-In Baek

⁴

and Kyung-Tae Lee

¹^*

1

Department of pharmaceutical Biochemistry, College of Pharmacy, Kyung-Hee University, Seoul 130-701, South Korea

2

Department of Physiology, College of Medicin,e Kyung-Hee University, Seoul 130-701, South Korea

3

Department of Herbal Food Science, Daegu Haany University, Gyeongsan, 712-715, South Korea

4

Department of Life Science, Kyung-Hee University, Suwon 449-701, South Korea

Abstract −

This study were designed to evaluate the anti-inflammatory effects of genistein-4'-O-

α

-L-rhamnopyranosyl-(1-2)-

β

-D-glucopyranoside (GRG) isolated from Sophora japonica (Leguminosae) on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin (PGE

2

) production by RAW 264.7 cell line. GRG significantly inhibited the LPS-induced NO and PGE

2

production. Consistent with these observations, GRG reduced the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in a concentration-dependent manner. In addi- tion, the release and the mRNA expression levels of tumor necrosis factor-

α

(TNF-

α

) and interleukin-6 (IL-6) were also reduced by GRG. Moreover, GRG attenuated the LPS-induced activation of nuclear factor-kappa B (NF-

^κ

B), a transcription factor necessary for pro-inflammatory mediators, iNOS, COX-2, TNF-

α

and IL-6 expression. These results suggest that the down regulation of iNOS, COX-2, TNF-

^α

, and IL-6 expression by GRG are achieved by the downregulation of NF-

^κ

B activity, and that is also responsible for its anti-inflammatory effects.

Key words −

genistein-4'-O-

α

-L-rhamnopyranosyl-(1-2)-

β

-D-glucopyranoside, NF-

κ

B, LPS, Anti-inflammation, Sophora japonica

염증반응은생체나조직에물리적작용이나화학적물질

,

세균감염등의어떠한기질적변화를가져오는침습이가 해질때그손상부위를수복재생하려는기전이며

,

^일단^자

극이가해지면국소적으로

histamine, serotonine, bradykinin, prostaglandins, hydroxyeicosatetraenoic acid (HETE)

^및

leukotriene

^과^같은^혈관^활성^물질이^유리되어^혈관^투과성

이증대되면서염증을유발한다

.

^그러나^지속적인^염증반응

은오히려점막손상을촉진하고

,

^그^결과^{일부에서는}^암^발

생등의질환을유도한다

.

¹⁾

내독소로잘알려진

lipopolysaccaride (LPS)

^는^그람^음성

균의 세포외막에 존재하며

, RAW 264.7

^세포와 ^같은

macrophage

^또는

monocyte

^에서

tumor necrosis factor-alpha (TNF-

^α

), Interleukin-6 (IL-6), Interleukin-1

^β

(IL-1

^β

)

^와 ^같

은

proinflammatory cytokine

^을^{증가시키는}^것으로^알려져

있다

.

^2-6)^이러한^염증매개^물질의^형성은

NO

^생성 ^증가^및

phospholipase A

2의활성을촉진시켜

prostaglandin (PG)

^합

성을유도한다

.

^7-8)

이중

NO

^는^체내^방어기능

,

^{신호전달기능}

,

^신경독성

,

^혈

*교신저자(E-mail):[email protected] (FAX):02-966-3885

(2)

관확장등의다양한생리기능을가지고있다

.

⁹⁾^{포유동물에}

서분리한

nitric oxide synthase (NOS)

^는^물리^화학적^성상

에따라

Type I, II,

^및

III

^등

3

^종류의^동종^효소로^나누어

진다

. Type I (neuronal NOS, nNOS)

^과

Type II (endotheli- al NOS, eNOS)

^는^세포속에^{계속적으로}^존재하기^때문에^구

성

NOS(constitutive NOS)

^로^분류되며

,

^{상대적으로}^일부^세

포에서

LPS

^와

cytokines

^같은^특수한^{자극제들에}^노출되는

경우에만발현되는

Type II

^인^유도형

NOS (iNOS)

^로^나누

어진다

.

¹⁰⁾^이들

NOS

^중

iNOS

^에^의한

NO

^생성이^절대적으

로많으며이는병리적으로중요한작용을한다

.

^일반적인

NO

^형성은^{박테리아를}^죽이거나^종양을^제거^시키는^중요

한역할을하지만

,

^병리적인^원인에^의한^과도한

NO

^형성

은염증을유발시키게되며조직의손상

,

^유전자^변이^및^신

경손상등을유발한다

.

^11-13)

Cyclooxygenase (COX)

^는

arachidonic acid

^를

PGs

^으로^전

환시키는효소로써

COX-1

^과

COX-2

^로^분류된다

. COX-1

^은

체내에서혈소판의형성

,

^위벽보호

,

^{신장기능의}^유지등^정

상적 생체기능에 작용하지만

COX-2

^는 ^{염증매개물질인}

prostaglandins (PGs)

^를^{형성시킨다}

.

¹⁴⁾

TNF-

^α는^활성화된

macrophage, fibroblast

^및^다른^여러

세포에서생성되는데이는종양세포에영향을미치는숙주 방어인자및염증매개물질로알려져있다

. IL-6

^의^생성은

TNF-

^α나

IL-1

^β^같은^요인^외에도

LPS

^에^의해^유도된다

.

¹⁵⁾

IL-6

^는

proinflammatory cytokine

^으로써

endogenous pyrogen

^으로^작용을^하며^면역^체계와^조혈^등에^다양한^영

향을미친다

.

¹⁶⁾

Nuclear transcription factor-kappa-B (NF-

^κ

B)

^는^세포 ^분

화

,

^염증반응

,

^세포^부착^등에^관련된^여러^{유전자들의}^발

현에가장중요한역할을하는전사인자이다

.

^활성화된

NF-

κ

B

^는

iNOS, COX-2, TNF-

^α^그리고

IL-6

^등^여러^염증^매

개물질의전사를촉진한다

.

¹⁷⁾

회화나무

( Sophora japonica L.)

^는^{콩과식물에}^속하는^낙

엽교목으로그높이는

25 m

^에^달하고^가지가^퍼지며^우리

나라

,

^중국

,

^일본^등지에^날리^분포한다

.

¹⁸⁾^잎은^호생하고^화

기는

7-8

^월로^이^시기의^꽃을^{한방에서는}^괴화

(

^槐花

)

^라^하

고꽃봉오리는괴미

(

^傀米

),

^그^성숙한^열매를^괴각이라^하여

지혈

,

^토혈

,

^변혈^등에^이용되며

,

^{혈압강화효과와}^항염효과

가있는것으로알려져있다

.

^19-20)^또한^{민간에서는}^잎을^삶

은물로치질부위를수세하고생

(

^生

)

^초

(

^炒

)

^괴화를^비

(

^鼻

)

출혈

,

^세균성^치질의^치유에

,

^고혈압^환자가^상복하여^중풍

의예방에사용하여왔다

.

²¹⁾^이러한^{천연물로부터}^유효활성

성분을분리하여이들의약리작용기전을규명함으로써다 양한약품의개발가능성을부여할수있다

.

본연구진은여러천연물의추출물및분리된단일화합물 들의항염증효과를검색하고그기전을규명함으로써새로 운항염증약물의개발을시도하고있다

.

^본^{논문에서는}^이

러한 연구중 괴각으로부터 분리한

genistein-4'-O-

^α

-L- rhamnopyranosyl-(1-2)-

^β

-D-glucopyranoside (GRG)

^가

LPS

에의해활성화된

RAW 264.7

^세포에서^{항염효과를}^나타내

는기전을 연구하였으며염증에 관련된다양한단백질

mRNA

^그리고

cytokine

^의^발현을^{측정하였다}

. 재료 및 방법

재료 −시료의추출과분획에사용한유기용매는대정 화학주식회사

(Gyonggi-do, Korea)

^에서^생산한

1

^급^시약을

사용하였다

. Column chromatography

^용

silica gel

^은

Kiesel gel 60 (Merck, Germany)

^을^{사용하였다}

. TLC

^는

Kiesel gel 60 F

254와

RP-18 F

254s를사용하였고

, TLC

^상의^물질^검출

에는

UV lamp

^와

10% aq . H

2

SO

4를사용하였다

. NMR

^스

펙트럼은

Varian Inova AS 400 (Varian, USA)

^으로

, FABMS

^는

JMS-700 (JEOL, Japan)

^로 ^{측정하였다}

. Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), penicillin, streptomy- cin

^은

life Technologies Inc. (Grand Island, NY)

^에서^구입하

였다

. Genistein, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), sulfanilamide, aprotinin, leupeptin, phenylmethylsulfony- lfluride (PMSF), dithiothreitol (DTT), L-N

⁶

-(1-iminoethyl) lysine (L-NIL), NS-398, Escherichia coli lipopolysaccha- ride (LPS)

^는

Sigma Chemical Co. (CA, U.S.A.)

^에서 ^구입

하였으며

, COX-2

^와

iNOS monoclonal antibodies

^및

peroxidase conjugated secondary antibody

^는

Santa Cruz Biotechnology (CA, U.S.A.)

^에서^{구입하였다}

. iNOS, COX- 2, TNF-

^α

, IL-6

^그리고 ^β

-actin oligonucleotide primers

^는

Bioneer (Seoul, Korea)

.

^그리고

TNF-

^α

, IL- 6, prostaglandin E

2측정을 위한

kit

^는

R&D systems (MN, U.S.A.)

.

시료의 추출및분리 −괴각

1.2 kg

^에

80% MeOH

^용액

(2 L

^×

3)

^에

24

^시간^실온에서

3

^회^추출하다

.

^추출물을^여과하

고얻어진여액을모두합쳐감압농축하여

MeOH

^추출물

395 g

^을^얻었다

. MeOH

^추출물을^증류수

1

^리터에^현탁시킨

후

EtOAc

^및

n -BuOH

^로^{순차적으로}^{분획하였다}

.

^각각의^분

획물을 농축하여

EtOAc

^분획물

(9.42 g), n -BuOH

^분획물

(81.9 g)

^및^물^분획물을^얻었다

. n -BuOH

^분획물

(81 g)

^을

CHCl

3

-MeOH

^의^{혼합용매를}^용출^용매로^사용하여^기울기

용리방식으로

silica gel column chromatography (c.c)

^를^실

시하여총

11

^개의^소분획

(B1~B11)

^으로^나누었다

.

^이^중

B10

^번 ^분획을

silica gel c.c.(CHCl

3

-MeOH-H

2

O = 65:35:10)

^을 ^사용하여 ^{노란분말상의}

genistein-4'-O-

^α

-L- rhamnopyranosyl-(1-2)-

^β

-D-glucopyranoside (528 mg)

^을 ^분

리하였다

.

^이^화합물의¹

H,

¹³

C-NMR

^및

MS

^의^기존^문헌

(3)

과비교하여구조를확인동정하였다

.

²²⁾

Genistein-4'-O-

^α

-L-rhamnopyranosyl-(1-2)-

^β

-D- glucopyranoside (Fig.1)

: yellow amorphous powder, Negative FAB MS m/z : 577 ([M-H]

⁻

),

¹

H-NMR (400 MHz, pyridine- d

_5,δ_H

): 8.06 (1H, s, H-2), 7.64 (2H, d, J=8.8, H2', 6'), 7.54 (2H, d, J = 8.8 Hz, H-3', 5'), 6.74 (1H, d, J = 2.0 Hz, H-6), 6.66 (1H, d, J=2.0 Hz, H-8), 6.43 (1H, br s,rha-1), 5.58 (1H, d, J=8.0 Hz, glc-1), 1.83 (3H, d, J=6.0 Hz, rha-6),

¹³

C- NMR (100 MHz, pyridine- d

_5,δ_C

): 180.76 (C-4), 166.02 (C-7), 163.51 (C-5), 158.54 (C-9), 158.25 (C-4'), 153.59 (C-2), 131.92 (C-2', 6'), 125.23 (C-1'), 123.22 (C-3), 116.62 (C-3', 5'), 105.73 (C-10), 102.36 (rha-1'), 100.21 (C-6), 99.99 (glc-1), 94.68 (C-8), 79.40 (glc-5'), 78.76 (rha-4'), 77.75 (glc-3'), 74.18 (glc-2'), 72.76 (glc-4'), 72.53 (rha-2'), 71.35 (rha-3'), 69.94 (rha-5'), 62.16 (glc-6'), 18.98 (rha-6').

세포의 배양 −

RAW 264.7

^세포는

10% FBS

^및

penicillin (100

^µ

g/ml), streptomycin (100 U/ml)

^이 ^포함된

DMEM

^배지에서

37

^o

C, 5% CO

2

incubator

^에서^배양했다

. RAW 264.7

^세포에^{시료용액의}^여러^농도

(25, 50, 100, 200

µ

M)

^또는^양성^대조군을

1

^시간^전처리한^후

LPS (1

^µ

g/mL)

를처리하고

24

^시간^{배양하였다}

.

세포독성시험−

96 well plate

^에

1

^×

10

⁵

cells/well

^로^세포

를동일하게분주하고

24

^시간동안^배양한^후^여러^농도의

시료용액을두군으로나누어배지에희석하여첨가하였 다

. 1

^시간후^한^군에만

LPS (1

^µ

g/ml)

^를^{처리하였다}

. 24

^시

간이지난후

MTT

^시약을^넣고

4

^시간^동안^방치한^후^상등

액을제거하고형성된

formazan

^을

DMSO 100

^µ

l

^를^첨가하

여녹였다

. 30

^분^후

540 nm

^에서^흡광도를^{측정하였다}

. Nitrite

^양의^측정 ⁻

RAW 264.7

^{세포로부터}^생성된

NO

의양은

Griess

^시약을^이용하여^세포^배양액^중에^존재하

는

NO

2−의형태로서측정하였다

.

^즉^세포배양^상등액

100

µ

l

^와

Griess

^시약

[1% (w/v) sulfanilamide in 5% (v/v)

phosphoric acid

^와

0.1% (w/v) naphtylethylenediamine- HCl] 100

^µ

l

^를^혼합하여

96 well plates

^에서

10

^분^동안^반응

시킨후

540 nm

^에서^흡광도를^{측정하였다}

.

Western blot

^시험 ⁻

GRG

^를^처리한^세포^및^대조군을

PBS

^로 ^씻어낸 ^후

lysis buffer

^인

PRO-PREP (Intron Biotechnology)

^으로^단백질을^추출한^후^{원심분리하여}^상등

액을취하였다

.

^상등액을

Bradford

^시약을^사용해^단백질^농

도를정량하여

50

^µ

g

^의^단백질을^취했다

.

^추출된^단백질은

10%

^의

SDS-polyacrylamide gel

^에 ^{전기영동시킨} ^후

nitro cellulose membrane

^으로

gel

^의 ^단백질을

blot

^시켰다

. 5%

skim milk

^로^하루^밤^동안

blocking

^한^후

1:500

^의^비율로

iNOS

^와

COX-2 antibody

^를

4

^시간동안^상온에서^방치한^후

TTBS

^로

15

^분^간격으로

2

^회^{세척하였다}

. 1:1000

^의^비율로

희석한

secondary antibody

^를

1

^시간동안^상온에서^방치^시

켰다

.

^다시

TTBS

^로

15

^분^간격으로

3

^회^세척한^후

chemilu- minescence

^로^{현상하였다}

.

PGE2, TNF-

^α ^및

IL-6

^양의^측정 ⁻^{세포배양액을}^취해

각각

R&D kit

^의^지시에^따라

PGE

2

, TNF-

^α^및

IL-6

^를^정

량하였다

.

RT-PCR

^시험 ⁻

Easy Blue

^R

kits (Intron Biotechnology)

를이용하여

Kit

^의

protocol

^에^따라^전체

cellular RNA

^를^추

출하였다

.

^각각의 ^시료에서

MuLV reverse transcriptase, 1 mM dNTP

^그리고

oligo (dT

12-18

) 0.5

^µ

g/

^µ

l

^를^이용하여

1

Fig. 1.

Chemical structures of genistein and genistein-4'-O-

α

-L- rhamnopyranosyl-(1-2)-

^β

-D-glucopyranoside (GRG).

Fig. 2.

The cytotoxicity of GRG or Genistein on RAW 264.7 cells. Cells were exposed to GRG and genistein (from 6.25

µ

M to 400

µ

M) with/without LPS (1

µ

g/ml). Cytotoxicity was

assessed by 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazo-

lium bromide (MTT) assay after 24 h incubation.

(4)

µ

g

^의

RNA

^를^역전사^하여

cDNA

^를^얻었다

. cDNA

^에

Taq DNA polymerase 1 unit, 0.2 mM dNTP,

^×

10 reaction buffer

^그리고

5'

^와

3' primers 100 pmol

^을^포함한^전체^부

피

25

^µ

L

^의 ^시료를

thermal cycler (Perkin Elmer Cetus, Foster City, CA, USA)

^를 ^이용하여

PCR

^분석을 ^하였다

. PCR

^반응은

95

^o

C

^에서

2

^분간

initial denaturation

^시킨 ^후

iNOS (95

^o

C 1

^분

danaturation, 60

^o

C 1

^분

annealing

^그리고

72

^o

C 1.5

^분

extension), COX-2 (94

^o

C 1

^분

danaturaion, 60

^o

C 1

^분

annealing

^그리고

72

^o

C 1

^분

extension), TNF-

^α

(95

^o

C 1

^분

denaturation, 55

^o

C 1

^분

annealing

^그리고

72

^o

C 1

분

extension)

^그리고

IL-6 (94

^o

C 1

^분

denaturation, 56

^o

C 1

분

annealing

^그리고

72

^o

C 1

^분

extension)

^를

30

^회

amplifi-

cation

^하였다

.

^이번 ^연구에서 ^아래의^목록과 ^같은

PCR primers

^가^{사용되었으며}

Bioneer (Seoul, Korea)

^에서^구입하

였다

. : sense strand iNOS, 5'-ATT GGC AAC ATC AGG- TCG GCC ATC ACT-3', anti-sense strand iNOS,5'- GCT GTG TGT CAC AGA AGT CTC GAA- CTC-3';

sense strand COX-2, 5'-GGA GAG ACT ATC AAG ATA GT-3' anti-sense strand COX-2, 5'-ATG GTC AGT AGA CTT TTA CA-3'; sense strand TNF-

^α

, 5'-ATG AGC ACA GAA AGC ATG- ATC-3', anti-sense strand TNF-

^α

, 5'- TAC AGG CTT GTC ACT CGA ATT-3'; sense strand IL-6, 5'-GAG GAT ACC ACT CCC AAC AGA CC-3', anti-sense strand IL-6, 5'-AAG TGC ATC ATC GTT GTT

Fig. 3.

The effects of GRG on LPS-Induced NO production and iNOS protein and mRNA expressions in RAW 264.7 Cells. (a) Cells were treated with different concentrations of GRG or genistein (25

µ

M) for 1 h and then LPS (1

µ

g/ml) was added and the cells were incubated for 24 h. Control (Con) values were obtained in the absence of LPS or tested samples. L-N

⁶

-(1-iminoethyl) lysine (L-NIL) was used as an assay positive control at a concentration of 10

µ

M. (b) Lysates were prepared from control or 24 h LPS (1

µ

g/ml)-stimulated cells alone or LPS plus with different concentration (25, 50, 100, 200

µ

M) of GRG or genistein (25

µ

M).

Total cellular proteins (40

µ

g) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and detected with specific

antibodies, as described in methods. A representative immunoblot of three separate experiments is shown. Total RNA was prepared

for the RT-PCR analysis of iNOS gene expression from RAW 264.7 macrophages stimulated with LPS (1

µ

g/ml) with/without

different concentration (25, 50, 100, 200

µ

M) of GRG or genistein (25

µ

M) for 4 h. iNOS-specific sequences (807 bp) was detected

by agarose gel electrophoresis, as described in methods. PCR of

β

-actin was performed to verify that the initial cDNA contents of

the samples were similar. The experiment was repeated three times and similar results were obtained. The Western blot results have

some analogy with the RT-PCR results and these are also shown by relativc ratio graphs. The values are the mean

±

S.D. of three

independent experiments.

^#

p <0.05 vs. the control group; * p <0.05, ** p <0.01 vs. the LPS-treated group; the significances of the

difference between the treated groups was evaluated using the Student’s t -test.

(5)

CAT ACA-3';5'-GTG CTG CCT- AAT GTC CCC TTG AAT C-3'; sense strand

^β

-actin, 5'-TCA TGA AGT GTG ACG TTG ACA- TCC GT-3', anti-sense strand

^β

-actin, 5’-CCT AGA AGC ATT TGC GGT GCA CGA TG-3'.

Amplification

^후에

PCR

^반응^시킨^시료를

2% agarose gel

에서전기영동하고

ethidim bromide

^염색과

UV

^조사를^통

해확인하였다

.

NF-

^κ

B Luciferase activity

^측정 ⁻

RAW 264.7

^세포를

dish

^에 ^각각

2

^×

10

⁵

cells/dish

^농도로 ^분주한 ^후

, superfect transfection reagent (Qiagen GmbH, Germany)

^를^이용하여

NF-

^κ

B luciferase reporter plasmid DNA

^를 ^형질감염

(transfection)

^시켰다

.

^형질감염

48

^시간이^경과한^후

3~4

^×

10

⁵

cell/well

^로

12 well plate

^에^세포를^분주하고

GRG

^를

1

^시간

동안전처리한후

LPS (1

^µ

g/ml)

^를^{처리하였다}

. 24

^시간^후

세포를수집하여

luciferase assay system (Promega, U.S.A.)

와

luminometer (Perkin Elmer Cetus, U.S.A)

^를 ^이용하여

luciferase

^활성을^{측정하였다}

.

TLC

^시험 ⁻^앞^선^실험과^같은^조건으로^세포를^배양하

여

5

^×

10

⁵

cells/dish

^농도로^분주하고^시료

(50

^µ

M)

^와

LPS (1

^µ

g/ml)

^을^{처리하였다}

. LPS

^처리

24

^시간^후^배지와^세포를

분리하여모았다

. Cold methanol

^을^이용하여^단백질^제거^후

질소가스로시료를농축하고

30

^µ

l

^의

methanol

^에^녹였다

. TLC plate

^는

silica gel aluminium sheets

^를^{사용하였으며}^전

개용매는

ethylacetate-formic acid-acetic acid-water

Fig. 4.

The effects of GRG on LPS-Induced PGE

2

and COX-2 protein and mRNA expressions in RAW 264.7 Cells. (a) Effect of the GRG and genistein on PGE

2

production by LPS-induced RAW 264.7 macrophage for 24 h. 10

µ

M of NS-398 was as a positive control in the assay. (b) Lysates were prepared from control or 24 h LPS (1

^µ

g/ml)-stimulated cells alone or LPS plus with different concentration (25, 50, 100, 200

^µ

M) of GRG or genistein (25

^µ

M). Total cellular proteins (40

^µ

g) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and detected with specific antibodies, as described in methods. A representative immunoblot of three separate experiments is shown. Total RNA was prepared for the RT-PCR analysis of COX-2 gene expression from RAW 264.7 macrophages stimulated with LPS (1

µ

g/ml) with/without different concentration (25, 50, 100, 200

µ

M) of GRG or genistein (25

µ

M) for 4 h. COX-2-specific sequences (721 bp) was detected by agarose gel electrophoresis, as described in methods. PCR of

β

-actin was performed to verify that the initial cDNA contents of the samples were similar. The experiment was repeated three times and similar results were obtained. The Western blot results have some analogy with the RT-PCR results and these are also shown by relativc ratio graphs. The values are the mean

^±

S.D. of three independent experiments.

^#

p <0.05 vs. the control group;

* p <0.05, ** p <0.01 vs. the LPS-treated group; the significances of the difference between the treated groups was evaluated using

the Student’s t -test.

(6)

(30:1.2:1.2:1.5)

^로^전개한^다음^말리고

AlCl

3를분무한후

UV (254 nm)

^로^{확인하였다}

.

통계학적분석 −실험치의값은

mean

^±

S.D.

^로^나타냈으

며분석은

Student’s t -test

^로^그^유의성을^{나타내었다}

.

결 과

GRG

^의^{세포독성에}^대한^효과 ⁻

GRG

^와

GRG

^의

agly- cone

^인

genistein

^의

RAW 264.7

^세포에^대한^{세포독성을}^측

정하기위해

MTT assay

^를^{수행하였다}

. GRG

^와

genistein

^모

두농도의존적으로

RAW 264.7

^세포의^생존^능력을^감소시

켰으며

(Fig. 2a) LPS (1

^µ

g/ml)

^는^세포의

viability

^에^영향이

없음을확인하였다

. GRG

^의

IC

50는

284.25

^µ

M

^로^확인되었

으며

genistein (IC

50

:79.69

^µ

M)

^에^비해

RAW 264.7

^세포에

서세포독성이낮음을확인하였다

(Fig. 2b).

GRG

^의

Nitrite

^형성^및

iNOS

^단백질과

mRNA

^발현저

해 −

LPS

^에^의해^활성화된

RAW 264.7

^세포의^배양액^중

에생성된

nitrite

^의^양을

Griess

^시약을^사용하여

GRG

^의

NO

^{생성저해효과를}^{조사하였다}

. GRG

^는^농도^{의존적으로}

NO

^생성을^{저해하였으며}

(Fig.3a) 100

^µ

M

^에서

NO

^생성을

46.9%

^{저해하였다}

. genistein

^은

25

^µ

M

^에서

NO

^생성을

39.4%

^{저해하였으며}^양성^{대조군으로는}

L-arginene

^과의^기

질경쟁에의하여

iNOS

^저해제로^알려진

L-NIL (10

^µ

M)

^을

사용하였다

. GRG

^에^의한^염증인자

(NO)

^의 ^생성^억제와

iNOS

^발현의^상관성을^알아^보기 ^위하여

Western blot

^과

RT-PCR

^로

iNOS

^단백질과

mRNA

^발현을^{조사하였다}

. LPS

에의해

iNOS

^단백질이^뚜렷하게^{증가하였으며}

, GRG

^에^의

해

iNOS

^단백질의^발현이^농도^{의존적으로}^{저해되었다}

.

^β

- actin

^의

band density

^비율에^따라

iNOS

^단백질의^발현 ^정

도를보정하였을때

GRG

^의

aglycone

^인

genistein

^은

25

^µ

M

에서

iNOS

^단백질^발현을

68.7%

^{저해하였으며}

GRG

^는

50

^µ

M

^농도에서

iNOS

^단백질^발현을

68.9%

^저해함을^확

인하였다

. GRG

^에^의한

iNOS mRNA

^{발현저해는}^농도^의

존적이며단백질발현저해와상관성있게나타났다

(Fig.3b).

GRG

^의

PGE2

^형성^및

COX-2

^단백질과

mRNA

^발현

저해효과 −

GRG

^가

RAW 264.7

^세포에서

LPS

^처리에^의

한

PGE

2의생성을농도의존적으로유의성있게감소시키는 것을 확인할수있었으며

(Fig. 4a) IC

50는

105.97

^µ

M

^로^확

인되었다

.

^양성^{대조군으로}^사용한

NS398 (5

^µ

M)

^과

GRG

의

aglycone

^인

genistein (25

^µ

M)

^에서

PGE

2의생성을뚜렷

Fig. 5.

The effects of GRG and genistein on LPS-induced TNF-

^α

release and mRNA expression in RAW 264.7 cells. Cells were

treated with different concentrations of GRG or genistein (25

µ

M) for 1 h and then LPS (1

µ

g/ml) was added and the cells were

incubated for 24 h. Control (Con) values were obtained in the absence of LPS or tested samples. Total RNA was prepared for the

RT-PCR analysis of TNF-

α

gene expression from RAW 264.7 macrophages stimulated with LPS (1

µ

g/ml) with/without different

concentration (25, 50, 100, 200

^µ

M) of GRG or genistein (25

^µ

M) for 4 h. TNF-

^α

-specific sequences (351 bp) was detected by

agarose gel electrophoresis, as described in methods. PCR of

β

-actin was performed to verify that the initial cDNA contents of the

samples were similar. TNF-

α

release results have some analogy with the RT-PCR results and these are also shown by relativc ratio

graphs. The values are the mean

±

S.D. of three independent experiments.

^#

p <0.05 vs. the control group; * p <0.05, ** p <0.01 vs. the

LPS-treated group; the significances of the difference between the treated groups was evaluated using the Student’s t -test.

(7)

하게저해하는것을확인하였다

. GRG

^에^의한

PGE

2생성 저해와

COX-2

^발현의^상관성을^알아보기^위하여

Western blot

^과

RT-PCR

^로

COX-2

^단백질과

mRNA

^발현을^조사하

였다

. GRG

^는

COX-2

^단백질과

mRNA

^의^발현을^농도^의존

적으로저해하였으며

GRG

^의

aglycone

^인

genistein (25

^µ

M)

에서도

COX-2

^단백질과

mRNA

^의^발현이^뚜렷하게^저해되

는것을확인하였다

(Fig. 4b).

GRG

^의

TNF-

^α와

IL-6

^의^형성^및

mRNA

^발현저해^효

과 −

GRG

^가

RAW 264.7

^세포에서

LPS

^에 ^의한

pro- inflammatory cytokine

^의^형성을^{억제하는지}^알아보기^위해

ELISA

^와

RT-PCR

^을^이용하여

TNF-

^α와

IL-6

^의^생성 ^및

mRNA

^발현을^{측정하였다}

. LPS

^처리에^의한

TNF-

^α와

IL- 6

^의^생성이

25, 50, 100

^그리고

200

^µ

M GRG

^에서^모두^유

의성감소되는것을확인하였다

. GRG

^는

25

^µ

M

^에서

TNF-

α의생성을

42.3%

^저해하여^같은^농도의

genistein (41.2%

저해

)

^과^비슷한^효과를^{나타내었다}

(Fig. 5a). LPS

^에^의한

IL-6

^생성은

GRG

^의

100

^µ

M

^농도에서

56.7%

^저해^되었으며

GRG

^의

aglycone

^인

genistein (25

^µ

M)

^에서는

66.2%

^가^저해

됨을확인하였다

(Fig. 6a).

^또한

GRG

^는

LPS

^에^의한

TNF-

α와

IL-6 mRNA

^의^발현을^유의성^있게^저해하며

TNF-

^α

와

IL-6

^생성^{저해효과와}^상관성이^있음을^{확인하였다}

(Figs.

5b and 6b).

GRG

^의

NF-

^κ

B

^활성^저해^효과⁻

LPS

^에^의해^유도되는

iNOS, COX-2, TNF-

^α

,

^그리고

IL-6

^의발현에

NF-

^κ

B

^의^활

성이 중요한역할을한다고보고되어있으므로²³⁾

GRG

^가

LPS

^에^의한

NF-

^κ

B

^의^활성화를^{억제하는지}^알아보기^위해

luciferase assay

^를^{수행하였다}

. RAW 264.7

^세포에^일시적

으로

pNF-

^κ

B-luc plasmid

^를

transfection

^시키고

GRG

^또는

genistein

^을^처리한^군과^처리하지^않은^대조군에

LPS (1

µ

g/ml)

^로^자극을^가한다

. GRG

^가

LPS

^에^의해^유도된

NF-

κ

B

^의존적인

luciferase

^효소의^발현을^농도^{의존적으로}^유

의성있게감소시키는것을확인하였다

(Fig. 7).

TLC

^에^의한^세포와^배지^내^시료의^측정 ⁻

^{In vitro}

^상에

서

GRG

^의^항염증^효과가^당이^떨어진^형태인

genistein

^에

의한것인지확인하기위해세포와배양액중의

GRG

^와^그

aglycone

^인

genistein

^을^각각^{확인하였다}

. genistein

^을^처리한

경우에는세포와배양액에서모두

genistein

^{표준물질과}^같

은위치에서

spot

^이^확인^되었다

.

^그러나

GRG

^를^처리한^세

포와배양액에서는

genistein

^이^존재하지^않음을^{확인하였으}

며배지에서는

GRG

^표준^물질과^같은^위치에서

spot

^이^확

Fig. 6.

The effects of GRG and genistein on LPS-induced IL-6 release and their mRNA expression in RAW 264.7 cells. Cells were

treated with different concentrations of GRG or genistein (25

µ

M) for 1 h and then LPS (1

µ

g/ml) was added and the cells were

incubated for 24 h. Control (Con) values were obtained in the absence of LPS or tested samples. Total RNA was prepared for the

RT-PCR analysis of IL-6 gene expression from RAW 264.7 macrophages stimulated with LPS (1

µ

g/ml) with/without different

concentration (25, 50, 100, 200

µ

M) of GRG or genistein (25

µ

M) for 4 h. IL-6-specific sequences (142 bp) was detected by agarose

gel electrophoresis, as described in methods. PCR of

β

-actin was performed to verify that the initial cDNA contents of the samples

were similar. IL-6 release results have some analogy with the RT-PCR results and these are also shown by relativc ratio graphs. The

values are the mean

^±

S.D. of three independent experiments.

^#

Inhibition of LPS Induced iNOS, COX-2 and Cytokines Expression by $Genistein-4'-O-{\alpha}-L-Rhamnopyranosyl-(1-2)-{\beta}-D-Glucopyranoside$ through the $NF-{\kappa}B$ Inactivation in RAW 264.7 Cells

38(4) : 339 348 (2007)

339

Genistein-4 ' -O- α -L-rhamnopyranosyl-(1-2)- β -D-glucopyranoside 의 RAW 264.7 세포에서 NF- κ B 불활성화를 통한 LPS 에 의해 유도되는

iNOS, COX-2 그리고 cytokine 들의 발현 저해효과

,

,

,

Inhibition of LPS Induced iNOS, COX-2 and Cytokines Expression by Genistein-4 ' -O- α -L-Rhamnopyranosyl-(1-2)- β -D-Glucopyranoside through

the NF- κ B Inactivation in RAW 264.7 Cells

Seung Jae Park

, Ji-Yeon Kim

, Young Pyo Jang

, Young-Wuk Cho

Eun-Mi Ahn

, Nam-In Baek

and Kyung-Tae Lee

Department of pharmaceutical Biochemistry, College of Pharmacy, Kyung-Hee University, Seoul 130-701, South Korea

Department of Physiology, College of Medicin,e Kyung-Hee University, Seoul 130-701, South Korea

Department of Herbal Food Science, Daegu Haany University, Gyeongsan, 712-715, South Korea

Department of Life Science, Kyung-Hee University, Suwon 449-701, South Korea

This study were designed to evaluate the anti-inflammatory effects of genistein-4'-O-

-L-rhamnopyranosyl-(1-2)-

-D-glucopyranoside (GRG) isolated from Sophora japonica (Leguminosae) on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin (PGE

) production by RAW 264.7 cell line. GRG significantly inhibited the LPS-induced NO and PGE

(TNF-

) and interleukin-6 (IL-6) were also reduced by GRG. Moreover, GRG attenuated the LPS-induced activation of nuclear factor-kappa B (NF-

B), a transcription factor necessary for pro-inflammatory mediators, iNOS, COX-2, TNF-

and IL-6 expression. These results suggest that the down regulation of iNOS, COX-2, TNF-

, and IL-6 expression by GRG are achieved by the downregulation of NF-

B activity, and that is also responsible for its anti-inflammatory effects.

genistein-4'-O-

-L-rhamnopyranosyl-(1-2)-

-D-glucopyranoside, NF-

B, LPS, Anti-inflammation, Sophora japonica

,

,

histamine, serotonine, bradykinin, prostaglandins, hydroxyeicosatetraenoic acid (HETE)

leukotriene

.

,

.

lipopolysaccaride (LPS)

, RAW 264.7

macrophage

monocyte

tumor necrosis factor-alpha (TNF-

), Interleukin-6 (IL-6), Interleukin-1

(IL-1

)

proinflammatory cytokine

.

NO

phospholipase A

prostaglandin (PG)

.

NO

,

,

,

.

nitric oxide synthase (NOS)

Type I, II,

III

3

. Type I (neuronal NOS, nNOS)

Type II (endotheli- al NOS, eNOS)

NOS(constitutive NOS)

,

LPS

cytokines

Type II

NOS (iNOS)

.

NOS

iNOS

NO

.

NO

,

Genistein-4 ^' -O- ^α -L-rhamnopyranosyl-(1-2)- ^β -D-glucopyranoside ^의 RAW 264.7 ^세포에서 NF- ^κ B 불활성화를 통한 LPS 에 의해 유도되는

iNOS, COX-2 ^그리고 cytokine ^{들의 발현 저해효과}

Inhibition of LPS Induced iNOS, COX-2 and Cytokines Expression by Genistein-4 ^' -O- ^α -L-Rhamnopyranosyl-(1-2)- ^β -D-Glucopyranoside through

the NF- ^κ B Inactivation in RAW 264.7 Cells