ROS-, RNS-Scavenging and Anti-inflammatory Activities of Mori Fructus

서 론

(reactive oxygen species, ROS) (reactive nitrogen species, RNS)

oxidative stresss

.

,

, DNA

, , ,

, , ,

.

1-4) .

2008 2 5

2008 3 4

, 1090-1

2

(Tel:+82-54-770-1265, Fax:+82-54-770-1500, E-mail:[email protected])

桑 子

박순재, 정지천

Original Article

ROS-, RNS-Scavenging and Anti-inflammatory Activities of Mori Fructus

Soon-Jae Park, Ji-Cheon Jeong

Dept. of Internal Medicine, College of Korean Medicine, Dongguk University Objectives : Peroxynitrite (ONOO ^- ), superoxide anion radical ( O 2

- ) and nitric oxide (NO) are cytotoxic because they can oxidize several cellular components such as proteins, lipids and DNA. They have been implicated in the aging processes, and age-related diseases such as Alzheimer’s disease, rheumatoid arthritis, cancer and atherosclerosis.

The aim of this study was to investigate the ONOO ^- , NO, and O ^{2 -} scavenging and anti-inflammatory activities of Mori Fructus in ob/ob mice.

Methods : Mice were grouped and treated for 5 weeks as follows. Both the normal lean (C57/BL6J black mice) and control obese (ob/ob mice) groups received the standard chow. The experimental groups were fed a diet of chow supplemented with 7.5, 15 and 30 mg Mori Fructus per 1 kg of body weight for 14 days. For this study, the fluorescent probes, namely 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA), 4,5-diaminofluorescein (DAF-2) and dihydrorhodamine 123 (DHR 123) were used. Western blotting was performed using anti-phospho IκB-α, anti-IKK-α, anti-NF-κB (p50, p65), anti-COX-2 and anti-iNOS respectively.

Results : Mori Fructus inhibited the generation of ONOO ^- , NO and O 2

- in the lipopolysaccharide (LPS)-treated mouse kidney postmitochondria in vitro. The generation of ONOO ^- , NO and O2 ^- were inhibited in the Mori Fructus -administered ob/ob mice groups. The GSH/GSSG ratio decreased in the ob/ob mice, whereas they improved in the Mori Fructus-administered groups. Mori Fructus inhibited the expression of phospho IκB-α, IKK-α, COX-2, iNOS genes, and thereby the activation of NF-κB.

Conclusions : These results suggest that Mori Fructus is an effective ONOO ^- , O ^{2 -} and NO scavenger, and therefore it might be a potential therapeutic drug against the inflammation process and inflammation-related diseases.

Key Words : Mori Fructus , peroxynitrite, nitric oxide, reactive oxygen species, NF-κB, COX-2, iNOS

nitric oxide (NO) superoxide anion radical ( O ^{2 -} ) peroxynitrite

(ONOO ^- ) ^5,6) . ONOO ^-

,

NO

O ^{2 - 5)} .

reactive species

(RS) nuclear factor κB

(NF-κB)

7) . NF-κB family p50 p65 (heterodimer)

inhibitor κB (IκB)

8,9) . ROS, cytokines, mitogens, bacterial lipids, viral transactivators

NF-κB inducing kinase (NIK) IκB kinase

(IKK) IκB

9) , NF-κB

cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), TNF-α, vascular cell adhesion molecule-1 (VCAM-1), interleukin-6 (IL-6)

10) . NF-κB , ,

, apoptosis

9) , , , ,

, , ,

, /

10) .

(Mori Fructus) ( )

11,12)

.

13) 14)

15) .

11,16)

,

17,18)

.

in vitro ROS, NO ONOO ^- ,

(ob/ob mouse) ROS, NO ONOO ^-

, GSH(Glutathione reduced form) GSSG (Glutathione oxidized form)

.

재료 및 방법

1.

재료 1)

(Mori Fructus) ( , ,

) .

2)

sodium dodesyl sulfate (SDS), acryla-

mide Bio-Rad (Hercules, USA), NP-40, CAPS,

protease inhibitors, DL-penicillamine, bovine serum

albumine, potassium phosphate, sodium nitroferr-

icyanide( ) dihydrate (SNP), 3-morpholinosydn-

onimine (SIN-1), 6-hydroxy-2,5,7,8-tetra methylc-

hroman-2-carboxylic acid (Trolox) Sigma-Aldrich

chemical Co. (ST. Louis, MO, USA), dihydrorh-

odamine 123 (DHR 123) 2’,7’-dichloro dihydr-

ofluorescein diacetate (DCFDA) Molecular Probes

(Eugene, OR, USA), ONOO ^- , carboxy-PTIO sodium

salt Calbiochem (EMD Biosciences, Inc., USA),

4,5-diaminofluorescein (DAF-2) Dai ichi Pure Chemical Co. (Tokyo, Japan), 1 IKK-α, p-Iκ B-α Cell Signaling Technology (Cell Signaling, USA) p50, p65, iNOS, COX-2, β-actin, histone H1 2 anti-mouse, anti-rabbit, anti-goat

Santa Cruz Biotechnology (Santa Cruz, USA) , sodium chloride, hydrogen peroxide, ethanol, methanol

. GENios (GENios- basic, TECAN, Austria) .

3)

30g , 5 ob/ob

mouse C57BL/6J mouse ( , ,

) 7

. 22 , 50%

light-dark cycle 12 ,

mouse .

2.

방법 1)

300 g 3 95%

methanol 60 24

3 .

85.20g ( 28.4%) . 2)

ether

Whatman

-70 .

4 0.1 M potassium phosphate buffer (pH 7.5)

homogenizer 4

. 600×g 10

, 8,000×g 20

mitochondria

ROS, ONOO ^- , NO GSH/GSSG

. western blot

0.1M HEPES, 1M KCl, 30mM MgCl 2 , 0.1M

PMSF buffer

ice 20 10% NP-40 12,000

rpm, 4 5 cytosol

pellet 0.1M HEPES, 1M KCl, 1M NaCl, 0.1M PMSF, 10% glycerol

buffer resuspension voltexing

ice 20 12,000 rpm,

4 10 nuclear

.

0 4 .

3) ONOO ^-

Kooy ¹⁹⁾ ONOO ^-

. 96-well microplate

, 90 mM NaCl, 5 mM KCl 100 μM diethylenetriamine penta acetic acid 10 μM DHR 123 sodium phosphate

(pH 7.4) . 10 μM ONOO ^-

,

485 nm 535 nm .

ONOO ^- ONOO ^- SIN-1

O2 ^- NO

ONOO ^- , ONOO ^-

penicillamine .

4) NO

NO indicator 4,5-diamino fluore-

scein (DAF-2) 2

NO , 490-495 nm

green triazolofluorescein

DAF-2 NO . Dimethyl sulfoxide 550

DAF-2 1 mg 50 mM phosphate

buffer (pH 7.4) 1:400 . 96-well microplate

, NO DAF-2

485 nm

535 nm 3 7

. NO SNP ²⁰⁾

carboxy PTIO .

5)

DCFDA assay ²¹⁾ .

DCFDA esterase

DCFH ,

DCFH

2’,7’-dichlorofluorescein (DCF) .

99.9% 12.5 mM DCFDA 3

600 U/ esterase -20

stock solution , 10 μM

DCFDA 600 U/ esterase (

6 U/esterase) 2’,7’-dichlorodihydrof-

luorescein (DCFH) 37 20

. 96 -well microplate

, DCFDA

485 nm

535 nm 5 7

. SIN-1

Trolox .

6)

C57 mouse , ob/ob mouse , ob/ob mouse 7.5, 15,

30 mg/kg 5

, 5 .

14 .

7) GSH/GSSG

GSH/GSSG Gaitonoide ²²⁾

. GSH 0.1 M

phosphate buffer OPT 30

485 nm

535 nm , GSSG

NEM 30

0.5 N NaOH OPT 25

485 nm 535 nm

8) Western blot analysis

cytosol, nuclear

. sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE)

, nitrocellulose membrane . membrane 5% skim milk

antibody 1 antibody 2 . membrane 0.1% Tween 20

TBST 60 2 antibody 2

, TBST 60 membrane ECL solution

X-ray film .

9)

Lowry ²³⁾ Bradford

protein assay kit .

10)

t -test .

성 적

1.

마우스 신장 균질액에서

및

^- 생성에 미치는 영향

LPS

ONOO ^- , NO O2 ^- . O2 ^-

2, 10, 50 /

3, 18, 63% .

O2 ^- trolox

O2 ^- (Fig. 1A). NO

7, 21, 61%

NO carboxy-PTIO

NO (Fig. 1B). ONOO ^-

7, 21, 68% ONOO ^- penicillamine

(Fig. 1C).

2. ROS, NO

및

^- 생성에 미치는 영향

ROS 28.77±1.73%

58.10±3.88% .

2

Concentration (㎍/ml) 90

80 70 60 50 40 30 20 10 0

In h ib itio n (% )

10 50

Trolox

Mori Fructus Carboxy PTIO

Mori Fructus

Penicill amine Mori Fructus

2

Concentration (㎍/ml) 90

80 70 60 50 40 30 20 10 0

In h ib itio n (% )

10 50

120 100 80 60

20

Concentration (㎍/ml)

2 10 50

40

0

In h ib itio n (% )

Fig. 1.

70 60 50 40 30 20 10 0 O

g e n e ra tio n (% )

Mori Fructus (mg/kg) C57

0 ob/ob

0 ob/ob

7.5 ob/ob

15 ob/ob

30

b)*

b)**

35 30 25 20 15 10 5 0

N o g e n e ra tio n (% )

Mori Fructus (mg/kg) C57

0 ob/ob

0 ob/ob

7.5 ob/ob

15 ob/ob

30

b)**

Mori Fructus (mg/kg) C57

0 ob/ob

0 ob/ob

7.5 ob/ob

15 ob/ob

30

b)**

O N O O g e n e ra tio n (% )

45 40 35 30 25 20 15 10 5 0

Fig. 2.

7.5, 15, 30 /

55.64±3.21, 49.72±2.96, 42.15±2.83%

, 15, 30 /

(Fig. 2A). NO 15.68±

0.97% 31.09±2.09%

. 30.48±1.92, 27.84±1.67,

22.79±1.54% , 30 /

(Fig. 2B). ONOO ^- 12.38±0.68%

36.79±2.32% . 35.42±

2.29, 33.28±2.03, 27.46±1.72% ,

30 / (Fig. 2C).

3. GSH/GSSG

비율에 미치는 영향

GSH GSSG 0.19±0.01%

0.07±0.01%

. 0.22±0.02, 0.25±0.02,

0.32±0.02% , 15, 30 /

(Fig. 3).

4. IKK-

α에 미치는 영향

NF-κB IKK-

α

Mori Fructus (mg/kg) C57

0 ob/ob

0

ob/ob 7.5

ob/ob 15

ob/ob 30 0.4

G SH / G SS G ra tio

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

b)**

b)**

b)*

a)**

Fig. 3.

C57 0

ob/ob 0

ob/ob 7.5

ob/ob 15

ob/ob 30 Mori Fructus (mg/kg) p-IκB-α

β-actin IKK-α

β-actin

A rb itr a ry d e n sit y o f IK K - a le ve l(% ) A rb itr a ry d e n sit y o f p - l

B - a e ve l(% )

200 180 160 140 120 100 80 60 40 20

0 C57 0

ob/ob 0

ob/ob 7.5

ob/ob 15

ob/ob 30 Mori Fructus (mg/kg)

250 200 150 100 50 0

b)*

a)**

b)** b)**

Fig. 4. α κ α

α κ α

α κ α

(Fig. 4A). phospho IκB-α

(Fig. 4B).

5. NF-

κ

단백질 양에 미치는 영향 NF-κB

NF-κB subunit p50 p65

(Fig. 5).

6.

염증 촉진 인자의 발현에 미치는 영향

COX-2 iNOS

, (Fig. 6).

C57 0

ob/ob 0

ob/ob 7.5

ob/ob 15

ob/ob 30 Mori Fructus (mg/kg) 0 C57

0 ob/ob

0 ob/ob

7.5 ob/ob

15 ob/ob

30 Mori Fructus (mg/kg)

0 p50

Histone H1

p65 Histone H1

A rb itr a ry d e n sit y o f p 5 0 le ve l ( % )

600 500 400 300 200 100

A rb itr a ry d e n sit y o f p 6 5 le ve l ( % )

700 600 500 400 300 200 100

b)**

b)**

a)**

b)**

b)**

b)*

Fig. 5. κ

κ

C57 0

ob/ob 0

ob/ob 7.5

ob/ob 15

ob/ob 30 Mori Fructus (mg/kg) 0 C57

0 ob/ob

0 ob/ob

7.5 ob/ob

15 ob/ob

30 Mori Fructus (mg/kg)

0 iNOS

β-acti n

COX-2 β-acti

iN O S le ve l ( % ) A rb itr a ry d e n sit y o f C O X - 2 le ve ll (% ) A rb itr a ry d e n sit y o f n

700 500

600 500 400 300 200 100

450 400 350 300 250 200 150 100 50

Fig. 6.

고 찰

11,12)

.

.

11,16)

,

, ²⁴⁾

25) .

.

carotene, vitamin (B1, B2, C), nicotinic acid, ,

11,12)

, ¹³⁾

14) monoamine

oxidase (MAO)

15) .

(vascular endothelial cell) (smooth muscle

cell) ,

,

26) .

.

, NF-κ

B

.

NF-κB IL-1β, IL-6 TNF-α

COX-2 iNOS

27) . ROS

ob/ob ROS, NO ONOO ^-

,

. ob/ob Jackson Lab.

6

ob homo (ob/ob)

. 3

80 g

(30-40 g) 3 .

2

28,29)

. ROS

, ROS

30,31)

. LPS

ROS

NO .

ONOO ^-

. ob/ob mouse ROS NO

. ONOO ^-

ob/ob mouse

. GSH/GSSG

ob/ob mouse

. ob/ob mouse GSH GSSG

GSH

GSSG .

oxygen free radical

glutathione (GSH) glutathione peroxidase

H 2 O 2 glutathione

(GSSG) , GSSG glutathione redu-

ctase NADPH GSH

32) . GSSG

GSH oxygen free radical

. IKK- α

ob/ob mouse

. NF-κ

B IκB-α ob/ob mouse

. NF-κB

ob/ob mouse p50, p65

. NF-κB

. COX-2

33) . mitogen

PGs ,

, , Crohn’s

disease, helicobacter pylori ,

34,35)

.

iNOS L-arginine NO

, iNOS

NO ,

,

. iNOS

NO

. shock cytokine

. iNOS

, ,

, ,

36) .

ob/ob mouse COX-2, iNOS

.

ROS RNS

.

,

ROS RNS IKK pathway

IκB

NF-κB COX

-2, iNOS NF-κB binding site

.

결 론

ROS, NO ONOO ^-

.

LPS ROS, NO ONOO ^-

. ob/ob mouse ROS, NO ONOO ^-

. ob/ob

mouse GSH/GSSG

.

ob/ob mouse IKK-α,

phospho IκB-α NF-κB(p50, p65)

. ob/ob mouse

COX-2 iNOS

.

ROS, NO ONOO ^-

.

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