Effects of Sotosaja-hwan on the Generation of ROS, RNS, and on the Expression of NF-${\kappa}B$-dependent Proteins in ob/ob Mouse

서 론

reactive oxygen

species (ROS) reactive nitrogen species (RNS) cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), XOD, NADPH oxidase

小 絲子丸 κ

방용석, 정지천 동국대학교 한의과대학 내과학교실

Original Article

Effects of Sotosaja-hwan on the Generation of ROS, RNS, and on the Expression of NF-κ κ κ κB-dependent Proteins in ob/ob Mouse

Yong-Suk Bang, Ji-Cheon Jeong

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

Objectives: Peroxynitrite (ONOO ), superoxide anion radical ( O2 ) 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, diabetes, obesity and atherosclerosis. The aim of this study was to investigate the ONOO , NO, O2 scavenging and NF-κB related anti-inflammatory activities of Sotosaja-hwan 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 have received standard chow. The experimental groups were fed with a diet of chow supplemented with 30 and 90 mg Sotosaja-hwan 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, anti-iNOS, anti-VCAM-1 and anti-MMP-9 antibodies, respectively.

Results: Sotosaja-hwan inhibited the generation of ONOO , NO and O2 in the lipopolysaccharide (LPS)-treated mouse kidney postmitochondrial fraction in vitro. The generation of ONOO , NO, O2 and PGE2 were inhibited in the Sotosaja-hwan-administered ob/ob mice groups. The GSH/GSSG ratio was decreased in the ob/ob mice, whereas the ratio was improved in the Sotosaja-hwan-administered groups. Sotosaja-hwan inhibited the protein expression levels of phospho-IκB-α, IKK-α, NF-κB (p50, p65), COX-2, iNOS, VCAM-1 and MMP-9 genes.

Conclusions: These results suggest that Sotosaja-hwan is an effective ONOO , O2 and NO scavenger and has NF-κ B related anti-inflammatory activity in ob/ob mice. Therefore, Sotosaja-hwan might be a potential therapeutic drug against the inflammation process and inflammation-related diseases.

Key Words : Sotosaja-hwan, peroxynitrite, nitric oxide, reactive oxygen species, IKK-α, IκB-α, NF-κB, COX-2, iNOS

접수：2008년 7월 29일 수정：2008년 12월 30일 채택：2009년 1월 9일

교신저자：정지천(Ji-Cheon Jeong)

경북 경주시 석장동 1090-1 동국대학교 경주한방병원 2내과

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

. (molecular inflammation)

, ROS iNOS nitric oxide (NO) peroxynitrite (ONOO )

1,2) . NO superoxide anion

radical ( O 2 ) ONOO

,

NO O 2

3) .

(vas- cular endothelial cell) (smooth muscle cell)

4) . cytokine

.

, nuclear factor κB (NF-κB)

5) .

NF-κB , , , apoptosis

6) , , , ,

, ,

, , /

7) . NF-κB family p50 p65

(heterodimer) , inhi-

bitor κB (IκB)

6,8) .

ROS, cytokines, mitogens, bacterial lipids, viral transactivators

NF-κB inducing kinase (NIK) IκB kinase (IKK) IκB

6) , NF-κB

COX-2, iNOS, tumor necrosis factor-α (TNF-α), vas- cular cell adhesion molecule-1 (VCAM-1), interle- ukin-6 (IL-6)

7) . COX-2 prostaglandin E 2 (PGE 2 ) ,

9,10)

. ROS, NO PGE 2

iNOS COX NF-κB

.

< > ¹¹⁾

, < > ¹²⁾

. , , ,

,

13) .

, .

in vitro ROS, NO ONOO ,

(ob/ob mouse) ROS, NO, ONOO PGE 2

NF-κB

.

재료 및 방법

1. 재료 1)

( ,

, ) , 1

.

2)

sodium dodecyl sulfate (SDS), acrylamide Bio-Rad (Hercules, USA), NP-40, CAPS, protease inhibitors, DL-penicillamine, bovine serum albumine, potassium phosphate, sodium nitroferricyanide( ) dihydrate (SNP), 3-morpholinosydnonimine (SIN-1), 6-hydroxy-2,5,7,8-tetra methylchroman-2-carboxylic acid (Trolox) Sigma-Aldrich chemical Co. (ST.

Louis, MO, USA), dihydrorhodamine 123 (DHR 123) 2’,7’-dichloro dihydrofluorescein 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, IκB-α, p-IκB-α Cell Signaling Technology (Cell Signaling, USA) p50, p65, iNOS, COX-2, VCAM-1, β-actin, histone H1 2 anti-mouse, anti-rabbit, anti-goat IgG 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

.

25.05 g

( 8.35%)

. 2)

C57BL/6J mouse , ob/ob

mouse , ob/ob mouse

4 ,

5 .

30, 90 mg/kg 14 .

3)

ether

Whatman

-70 .

4 0.1 M

potassium phosphate buffer (pH 7.5)

homogenizer 4 .

600 × g 10 ,

8,000 × g 20

mitochondria

韓藥名 생약명 중량

蓮子肉 Nelumbinis Semen 60 g

絲子 Cuscutae Semen 150 g

白茯 Poria 30 g

山 藥 Dioscoreae Rhizoma 60 g

Total Amount 300 g

Table 1.

ROS, ONOO , NO GSH/GSSG . western blot 0.1 M HEPES, 1 M KCl, 30 mM MgCl2, 0.1 M PMSF buffer

ice 20 10%

NP-40 12,000 rpm, 4 5

cytosol pellet 0.1

M HEPES, 1 M KCl, 1 M NaCl, 0.1 M PMSF,

10% glycerol buffer resuspe-

nsion vortexing ice 20

12,000 rpm, 4 10

nuclear .

0 4 .

4) ONOO

Kooy ¹⁴⁾ ONOO

. 96 well microplate

(2, 10, 50 / ) , 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

penicillamine .

5) NO

NO indicator 4,5-diamino fluorescein

(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

(2, 10, 50 / )

, NO DAF-2

485 nm 535

nm 3 7 ¹⁵⁾ . NO

carboxy PTIO .

6)

DCFDA assay ¹⁶⁾ .

DCFDA esterase

DCFH , DCFH

2’,7’-dichlorofluorescein (DCF) . 96 well

microplate (2, 10, 50

/ ) , 0.125 mM

DCFDA

485 nm 535 nm 5

7 .

Trolox .

7) GSH/GSSG

GSH/GSSG Gaitonoide ¹⁷⁾

. 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) Prostaglandin E 2

Prostaglandin E 2

(PGE 2 ) serum PGE 2 immunoassay

kit (R&D SYSTEMS) .

9) 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 .

10)

Lowry ¹⁸⁾ Bradford

protein assay kit .

11)

t-test .

성 적

1. 마우스 신장 균질액에서 ONOO ^－ , NO 및

ROS 생성에 미치는 영향

LPS

ONOO , NO ROS . ONOO

10, 37, 76% ONOO penicillamine

(Fig. 1A).

NO 7,

29, 75% NO carboxy-PTIO

In hib itio n ( % )

100

0

2 10 50

90 80 70 60 50 40

10 20 30

Concentration (ug/ml)

Penicillamine Sotosajahwan

In hib itio n ( % )

100

0

2 10 50

90 80 70 60 50 40

10 20 30

Concentration (ug/ml)

Penicillamine Sotosajahwan

AA

AA BBBB

In hib itio n ( % )

120

0 2 10 50

20

Concentration (ug/ml)

Penicillamine Sotosajahwan 100

40 60 80

C C C C Fig. 1.

NO (Fig. 1B).

ROS

9, 25, 72%

ROS trolox

O 2 (Fig. 1C).

2. ONOO ^－ , NO 및 ROS 생성에 미치는 영향

ONOO 13.28±0.77%

31.98±2.75% .

30, 90 /

30.44±2.02, 26.98±1.71% , 90 / (Fig. 2A).

NO 12.11±0.81%

22.87±1.92% .

22.09±1.81, 19.14±1.29% , 90 / (Fig. 2B).

ROS 27.34±1.65%

65.87±4.01% .

62.34±3.77, 56.01±3.07% , 90

/ (Fig. 2C).

3. GSH/GSSG 비율에 미치는 영향

GSH GSSG 0.18±0.01%

0.1±0.01%

. 0.24±0.02, 0.32±0.02%

, 30, 90 / (Fig. 3).

O N O O

g en er at io n ( % )

0 ob/ob

90 35

30 25 20 15

5 10

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

40

a)**

b)**

O N g en er at io n ( % )

0 ob/ob

90 25

20 15 10 5

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

30

a)**

b)**

A AA

A BBBB

R O S g en er at io n ( % )

0 ob/ob

90 70

60 50 40 30 20

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

80 a)**

b)**

10

C C C C Fig.

Fig.

Fig.

Fig. 2.2.2.2.

4. PGE 2 생성에 미치는 영향

PGE 2

283.24±19.81 pg/

369.54±25.79 pg/ .

353.19±25.47, 316.88±23.53 pg/

, 90 / (Fig. 4).

5. IKK- α및 phospho I κ B- α에 미치는 영향

NF-κB IKK-α

(Fig. 5A).

Phospho IκB-α

(Fig. 5B).

6. NF- κ B 단백질 양에 미치는 영향 NF-κB

MAPK .

MAPK phospho-ERK phospho-p38

(Fig. 6).

P G E

(p g /m l)

0 ob/ob

90 500

350 300 250 200 150

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

450 a)**

b)*

50 100

Fig.

Fig.

Fig.

Fig. 4.4.4.4.

G S H / G S S G ra tio

0 ob/ob

90 0.35

0.3 0.25 0.2 0.15 0.1

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

0.4

a)**

b)**

0.05

b)**

Fig.

Fig.

Fig.

Fig. 3.3.3.3.

7. 염증 촉진 인자의 발현에 미치는 영향 COX-2, iNOS

, (Fig. 7).

VCAM-1 MMP-9

A rb itr ar y d en sit y o f IK K - a le ve l (% )

0 ob/ob

90 180

160 140 120 100 80

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

200 a)**

b)**

20 40

b)*

60

β-actin

A rb itr ar y d en sit y o f p - Ik B - a le ve l (% )

0 ob/ob

90 300

250 200

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

350

a)**

b)**

50

b)**

150 100

lκB-α

AAA

A BBBB

Fig.Fig.

Fig.Fig. 5.5.5.5. α κ α

α κ α

α κ α

A rb itr ar y d en sit y o f p 50 le ve l (% )

0 ob/ob

90 450

350 300

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

500

a)**

b)**

50

b)**

200 150 400

250

100

Histone H1

A rb itr ar y d en sit y o f p 65 le ve l (% )

0 ob/ob

90 250

200

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

350

a)**

b)**

50

b)**

150 100 300

Histone H1

A AA

A BBBB

Fig.

Fig.

Fig.

Fig. 6.6.6.6. κ

κ

,

(Fig. 8).

0 ob/ob

90 300

250

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

350 a)**

b)**

50

b)**

150 100

A rb itr ar y d en sit y o f iN O S le ve l (% )

200

β-actin

0 ob/ob

90 400

350

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

450 a)**

b)**

50

b)**

250

100

A rb itr ar y d en sit y o f C O X - 2 le ve l (% )

300

150 200

β-actin

AAA

A BBBB

Fig.Fig.

Fig.Fig. 7.7.7.7.

0 ob/ob

90 160

140

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

180 a)**

b)**

20

b)**

100

40

A rb itr ar y d en sit y o f V C A M - 1 le ve l (% )

120

60 80

β-actin

0 ob/ob

90 400

350

C57 0

ob/ob 30 ob/ob Sotosaja-hwan (mg/kg) 0

450

a)**

b)**

50

b)**

250

100

A rb itr ar y d en sit y o f M M P - 9 le ve l (% )

300

150 200

β-actin

A AA

A BBBB

Fig.

Fig.

Fig.

Fig. 8.8.8.8.

고 찰

, , ,

, ,

.

, , ,

11-13)

.

, .

,

. Cytokine

.

, NF-κB

.

NF-κB IL-1β, IL-6 TNF-α

COX-2 iNOS

5) . NF-κB

.

, NF-κB

19,20)

. ROS

ob/ob ONOO , NO ROS

,

. ob/ob 6

ob homo (ob/ob)

. 3

80

g (30-40 g) 3

.

2

21,22)

. ROS

, ROS

23,24)

. LPS

ONOO

. NO ROS

.

ob/ob mouse ROS NO

. ONOO

ob/ob mouse

. GSH/GSSG

ob/ob mouse

.

ob/ob mouse GSH GSSG

GSH

GSSG . ROS

glutathione (GSH) glutathione peroxidase H 2 O 2

glutathione (GSSG) , GSSG glutathione reductase NADPH

GSH ²⁵⁾ .

GSSG GSH ROS

. PGE 2

, c-AMP

.

NOS, COX signaling

proteins NO PG

, , ,

,

9,10)

. ob/ob mouse

PGE 2

. IKK-α

ob/ob mouse

. NF-κB

IκB-α ob/ob mouse

.

NF-κB ob/ob

mouse p50, p65

. NF-κB

. COX-2

26) .

mitogen PG

,

, , Crohn's disease, helicobacter

pylori ,

27,28)

.

iNOS L-arginine NO

, iNOS

NO ,

, .

iNOS NO

. shock cytokine

. iNOS

, , , ,

29) . VCAM-1 IL-1, IL-6, TNF-α cytokine

.

30-32)

. Matrix metalloproteinases (MMPs) (extracellular matrix, ECM)

(basement membrane)

family . MMP

, cytokines free radicals , ,

, MMP-9 , ,

,

33-35)

.

ob/ob mouse COX-2, iNOS

.

VCAM-1 MMP-9

.

ROS RNS

, PGE 2

.

,

ROS RNS IKK pathway

IκB NF-κB

, NF-κB COX-2, iNOS VCAM-1

MMP-9

.

결 론

ROS, NO, ONOO

PGE 2 NF-κB

. LPS

ROS, NO ONOO

. ob/ob mouse ROS, NO, ONOO PGE 2

. ob/ob mouse GSH/GSSG

. ob/ob

mouse IKK-α, phospho IκB-α NF-κB

(p50, p65) . ob/ob mouse

COX-2, iNOS, VCAM-1 MMP-9

.

ROS, NO, ONOO

PGE 2 NF-κB

.

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