Anti-inflammatory Effect of Sargassum coreanum Ethanolic Extract through Suppression of NF-κB Pathway in LPS Induced RAW264.7 Cells in Mice

LPS로 유도된 RAW 264.7 cell로부터 NF-κB 조절 억제와 마우스 모델을 통한 큰잎모자반 에탄올 추출물의 항염증 효과

강보경¹, 김꽃봉우리², 김민지², 박시우¹, 박원민¹, 안나경¹, 최연욱¹, 배난영¹, 박지혜¹, 안동현¹*

1부경대학교식품공학과

/

2부경대학교수산과학연구소

Received: April 29, 2015 / Revised: June 4, 2015 / Accepted: June 6, 2015

서 론

체내의염증반응은상처나세균감염등의물리적

^,

^,

다

^[10].

진시켜결과적으로통증

^,

^,

^,

^,

[11].

(machrophage)

^,

비하는사이토카인등에의해활성화되며

^,

^nitric oxide (NO)

^cytokine

을한다

^[7].

알려진

lipopolysaccharide (LPS)

tumor necrosis factor-

^(TNF-

), interleukin-1

(IL-1

⁾

^IL-6

^cytokine

^.

arachidonic acid

cyclooxygenase (COX)

leukotriene, thromboxane, prostaglandin

^nitric oxide (NO)

^,

[8, 16].

NO

NO synthase (NOS)

-arginine

^{, NOS}

constitutive NOS

inducible NOS

^.

^iNOS

pro-inflammatory cytokine

^NO

Anti-inflammatory Effect of Sargassum coreanum Ethanolic Extract through Suppression of NF-κB Pathway in LPS Induced RAW264.7 Cells in Mice

Bo-Kyeong Kang

, Koth-Bong-Woo-Ri Kim

, Min-Ji Kim

, Si-Woo Bark

, Won-Min Pak

, Na-Kyung Ahn

, Yeon-Uk Choi

, Nan-Young Bae

, Ji-Hye Park

, and Dong-Hyun Ahn

*

1

Department of Food Science & Technology/Institute of Food Science, Pukyong National University, Busan 608-737, Republic of Korea

2

Institute of Fisheries Sciences, Pukyong National University, Busan 619-911, Republic of Korea

The anti-inflammatory effect of Sargassum coreanum ethanolic extract (SCEE) was investigated using lipopolysaccharide (LPS)-induced inflammatory responses in this study. It was shown that there was no cytotoxicity in the viability of macrophages treated with SCEE when compared to the control. The production of NO was considerably suppressed by SCEE, approxi- mately up to 50% at 100 µg/ml. This significantly decreased levels of IL-6, TNF- α, and IL-1β. In addition, the expression of iNOS, COX-2, NF- κB was suppressed by SCEE treatment. In in vivo testing, the croton oil-induced mouse ear edema was attenuated by SCEE and there were no mortalities in mice administered with 5000 mg/kg body weight of SCEE over a 2 week observation period. From these results, SCEE inhibits the release of LPS-induced pro-inflammatory cytokines and mediators, suggesting that SCEE could be a potential agent for anti-inflammatory therapies.

Keywords: Sargassum coreanum, anti-inflammation, nuclear factor kappa B, ear edema

*Corresponding author

Tel: +82-51-629-5831, Fax: +82-51-629-5824 E-mail: dhahn@pknu.ac.kr

© 2015, The Korean Society for Microbiology and Biotechnology

며과잉생산된

^NO

^[12].

^COX

arachidonic acid

유리된후

prostaglandin

며

^{, COX-2}

growth factors, cytokine

lipopolysaccharide

macrophage

^monocyte

prostaglandin

^[1].

^,

^nuclear factor-kappa B (NF-

^B)

cytokine, chemokine, growth factor

transcription factor

^{[4]. NF-}

^B

^p50

^p65

iNOS, COX-2

^cytokine

^,

inhibitor NF-

^B

^(I

^B

⁾

^NF-

^B

^[17].

NF-

B

^.

최근각광받고있는천연물의하나로해조류를들수있 다

^.

^[18],

^[21],

^[9]

^,

fucoidan, phycocolloids, phlorotannins

^,

^,

^[5].

(Sargassum coreanum)

^.

재료 및 방법

실험 재료

본실험에사용한큰잎모자반

(Sargassum coreanum)

^-20

^C

^.

에탄올 추출방법

큰잎모자반건조분말에

¹⁰

^95%

^,

(H-0820, Dongwon Science Co., Busan, Korea)

²⁴

^.

(UNION 32R, Hanil Co., Incheon, Korea)

1,977

^g

¹⁰

^,

²

^.

³⁷

^C

(RE200, Yamoto Co.,

Tokyo, Japan)

^,

-20

C

^.

실험 동물

ICR

⁽

⁸

^,

⁾

^(Orient Co., Seongnam, Korea)

^,

^Balb/c

⁽

¹⁰

⁾

^.

20

2

C,

50

10%, 12

¹

^.

대식세포인

RAW 264.7

^(KCLB 40071)

^{, DMEM}

^{100 mg/ml} inactivated fetal bovine serum

10 mg/ml penicillin- streptomycin

³⁷

^{C, 5% CO}

^.

⁸⁰

^90%

, 20 passages

^.

세포 독성 측정

시료의 세포독성을 평가하기 위해

tetrazolium-based colorimetric assay (MTT assay)

. RAW 264.7 cell 1

¹⁰

^cells/ml

well plate

²⁰

^{, 1}

^g/ml

^LPS

(0.1, 1, 10, 50, 100

^g/ml)

³⁷

^{C, 5% CO}

incubator(MCO- 15AC, Sanyo, Osaka, Japan)

²⁴

^.

^{, 5 mg/ml}

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT)

²

⁴

^{C, 1,318}

^g

¹⁰

(UNION 32R, Hanil Co., Incheon, Korea)

^.

^,

^well

^DMSO

microplate reader (Model 550, Bio-rad, Richmond, USA)

^{540 nm}

(absorbance at 540 nm (Abs540))

^.

^.

Proliferation Index(%) =

(sample Abs540 / control Abs540) × 100

Nitric Oxide 생성량 측정

NO

^nitrite

^griess

[10]. Raw 264.7 cell

^DMEM

^2.5

¹⁰

^cells/ml

24 well plate

^{5% CO}

incubator (MCO-15AC, Sanyo, Osaka, Japan)

²⁰

^.

¹

^g/ml

^LPS

0.1, 1, 10,

50, 100

^g/ml

²⁴

^.

^,

^griess

^{(10 mg/ml} sulfanilamide + 1 mg/ml naphthylendiamine dihydrochloride, 1:1)

¹⁰

, microplate reader (Model 550, Bio-rad, Richmond, USA)

540 nm

^.

^NO

sodium nitrite (NaNO

)

^.

염증 관련 cytokines 분비량 측정

세포배양액내의

^TNF-

^{, IL-6}

^IL-1

^cytokine

ELISA kit (Mouse ELISA set, BD Bioscience, San Diego, USA)

^.

^ELISA microplate

capture antibody

anti-mouse TNF-

^{, IL-6}

^IL-1

⁴

^C

^coating

^.

0.5 mg/ml Tween 20

^PBST

100 mg/ml FBS

^blocking

^{. PBST}

^,

microplate

^NO

²

^.

^PBST

biotinylated anti-mouse TNF-

^{, IL-6} detection antibody

streptavidin-horseradish peroxidase conjugate

¹

^{. IL-1}

, biotinylated anti-mouse IL-1

detection antibody

¹

, streptavidin-horseradish peroxidase conjugate

³⁰

^.

^,

PBST

, o-phenylenediamine-dihydrochloride (OPD)

³⁰

^{. 2 M} H

SO

, microplate reader (Model 550, Bio-rad, Richmond, USA)

^{490 nm}

^.

iNOS, COX-2 및 NF-κB 발현량 측정

배양이끝난세포를수집하여

³

PBS (phosphate buffered saline)

, lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10 mg/ml deoxycholate, 5 mM phenylmethylsulfonyl fluoride (PMSF), 1

g/ml aprotinin, 10 mg/ml Triton X-100, and 1 mg/ml NP-40)

30

⁴

^C

^lysis

^{, 7,908}

^g

²⁰

^.

^BCA protein assay kit (Pierce, IL, USA)

^{, 30}

^l

^lysate

10% SDS-PAGE

^.

polyvinylidene difluoride (PVDF) membrane (Bio- rad, CA, USA)

^{70 mA}

¹

³⁰

^, 50 mg/ml skim milk

TBST (tris buffered saline;

pH7.5)

²

^blocking

^.

iNOS, COX-2

^NF-

^B

anti-mouse iNOS, COX-2

^NF-

^B

^1:500

²

^TBST

³

^{. 2}

HRP (horse radish peroxidase)

anti-mouse IgG

anti-rabbit IgG

^1:2000

¹

^{, TBST}

³

enhanced chemiluminescence (ECL)

^1-3

Gene tool (Syngene software)

^.

귀 부종 측정 및 조직 관찰

ICR

^{10, 50}

250 mg/kg·body weight

²⁰⁰

^l

^.

^,

25 mg/ml croton oil

²⁰

^l/ear

^.

croton oil

⁵

^croton oil

^.

조직관찰은

^ICR

^{100 mg/}

ml

²⁰

^l

¹⁵

, 50 mg/ml croton oil

²⁰

^l

^{. 6}

, diethylether

^,

100 mg/ml formaldehyde

⁷²

^.

hematoxylin-eosin

toluidine-blue

^.

단기 독성 평가

Balb/c

⁴

⁶

300, 2000

5000 mg/kg·body weight

^{. 6}

²

^.

통계처리

모든실험결과에대한유의차검정은

SAS software (SAS Institute, Inc., Cary, NC, USA)

, Duncan's multiple range test

p < 0.05

^.

결과 및 고찰

세포 독성 측정

대식세포로부터염증매개물질의억제효과는세포독성으

로인해

^RAW264.7

수있으므로이러한가능성을배제하고자

RAW 264.7 cell

^MTT

assay

^.

0.1, 1, 10, 50

¹⁰⁰

^g/ml

^,

농도에서유의적인차이를보이지않음을관찰하였다

^{(Fig. 1).}

Nitric oxide 생성 억제 효과

병리적인조건하에서

^NO

^[19].

^NO

RAW 264.7

^LPS

^,

을각농도별로

(0.1, 1, 10, 50

¹⁰⁰

^g/ml)

^NO

^griess

^.

^(Fig.

2A),

^{, NO}

^LPS

^.

¹⁰

^g/

ml

25%

^.

염증 관련 cytokines 생성 억제 효과

인체에서염증반응이진행되기위해서는

NO

PGE

cytokine

^,

^cytokine

^IL-6, TNF-

^IL-1

^{[20]. IL-6}

류의세포에서분비되며다양한기능을가지는대표적인

^pro-

inflammatory cytokine

요한역할을한다

^.

^TNF-

식세포나림프구등백혈구에의해생성되는

^cytokine

정상상태에서는만들어지지않으며

^LPS

^{[14]. IL-1}

IL-6, TNF-

^cytokine

^NO

^,

^T

^, B

^{NK cell}

^cytokine

^[15].

따라서추출물이염증성

^cytokine

Fig. 1. Effect of SCEE on the proliferation of RAW 264.7 cells.

Proliferation index = (sample Abs540/control Abs540) × 100. ND means no significant difference.

Fig. 2. Inhibitory effect of SCEE on the production of nitric oxide (A), IL-6 (B), TNF- α (C), and IL-1β (D) in RAW 264.7 cells. RAW

264.7 cells were treated with the indicated concentrations of SCEE (0.1, 1, 10, 50, and 100 μg/ml) in the presence or absence of LPS

(1 μg/ml) for 24 h. Culture supernatants were then isolated and analyzed using the Griess reagent for nitric oxide and ELISA kit for cyto-

kines. a-g indicates significant differences (p < 0.05).

알아보기위하여

RAW 264.7

^LPS

^,

농도별로처리하여

^ELISA

^.

(Fig. 2B,C,D),

cytokine (IL-6, TNF-

^IL-1

⁾

^.

^,

50

^g/ml

^,

^{39, 45}

^31%

^{, 100}

^g/ml

LPS

^,

^{49, 51}

^48%

^.

RAW 264.7

^cytokine

^.

iNOS, COX-2 및 NF-κB 발현 억제 효과

NO

^NOS

-arginine

-citrullin

^NO

^,

^NOS

^iNOS

^,

^[13].

일어나면관련세포에서

^iNOS

의

^NO

^,

^NO

^,

^,

^,

켜부종등의염증반응을촉진시킨다

^[23].

^{, COX-2}

^PGE

^{[3], NF-}

^B

^promoter

^cytokine

lipopolysaccharide (LPS)

^COX-2

^iNOS

^[2].

따라서염증반응에서

iNOS, COX-2

^NF-

^B

. RAW 264.7

^0.1

¹⁰⁰

^g/ml

^iNOS

(Fig. 3), LPS

^,

^.

⁵⁰

¹⁰⁰

^g/ml

^PBS

^.

^,

^NF-

^B

^COX-2

^iNOS

PGE

NO

다고사료된다

^.

귀 부종 억제 효과 및 조직 관찰

피부는외부환경에의해손상된부위를복구시키려는

Fig. 3. Effect of SCEE on LPS-induced iNOS, COX-2, and NF- κB p65 expression in RAW 246.7 cells. The levels of iNOS, COX-2

in the cytosolic protein and the p65 subunit of NF- κB in nuclear protein were determined by a western blot analysis. RAW 264.7 cells

were treated with the indicated concentrations of SCEE (0.1, 1, 10, 50, and 100 μg/ml) and LPS (1 μg/ml) for 18 h or 30 min and the

proteins were detected using specific antibodies. One of the similar results from three separate experiments is represented.

일련의생체과정으로서혈관확장

^,

[6].

prednisolone 10

^{50 mg/kg}

^{10, 50}

250 mg/kg

200

^l

, croton oil

^.

(Fig. 4), control

^.

^,

250 mg/kg

positive control

prednisolone

, prednisolone 10 mg/kg

^.

, croton oil

마우스귀조직에서

croton oil

출물을

100 mg/ml

prednisolone

였다

(Fig. 4B).

, toluidine-blue

mast cell

(Fig. 4C),

mast cell

^.

기능성식품소재로써이용가능성을알아보기위해

^World

Health Organization (WHO) [22]

^300, 2000

5000 mg/kg

²⁰⁰

^l

²

(Table 1).

4

^{, 30}

¹

^{, 2}

^0%

^.

^.

Fig. 4. SCEE-mediated inhibition of croton oil-induced mouse ear edema (A). Photomicrographs of transverse sections of mice ears

sensitized with topical application of 50 mg/ml croton oil in acetone (a-c) or acetone alone (d, non-inflamed), stained with hematoxylin-

eosin (B) or toluidine-blue (C). Photomicrographs recorded under light microscopy (magnification: 200 ×). Treatments: vehicle 20 mg/ml

Tween 80 (a), prednisolone 0.08 mg/ear (b), SCEE 20 μl/ear (c), and acetone (d). The numbers 1 and 2 indicate dermis and epidermis,

respectively. a-c indicates significantly different results (p < 0.05).

요 약

본연구에서는큰잎모자반의항염증효과를알아보기위 해

^LPS

RAW 264.7

^.

cytokine (IL-6, TNF-

^IL-1

⁾

^,

^,

^, 100

^g/ml

^LPS

48%

^.

^iNOS, COX-2

^NF-kB

^, LPS

^,

^.

⁵⁰

¹⁰⁰

^g/

ml

^PBS

염증효과를가짐을확인하였다

^.

^,

250 mg/kg

prednisolone 10 mg/kg

^.

, croton oil

로부종을유발한마우스귀조직에서

croton oil

한경우에비해 추출물을

100 mg/ml

prednisolone

얇아진것을확인하였으며

^,

mast cell

^.

^,

^.

Acknowledgments

This research was supported by Basic Science Research Pro- gram through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2012R1A6A1028677).

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