Anti-Inflammatory Effect of Chondrus ocellatus Holmes Ethanol Extract on Lipopolysaccharide-induced Inflammatory Responses in RAW 264.7 Cells

Lipopolysaccharide 로 유도된 RAW 264.7 세포와 마우스모델에 대한 진두발 에탄올 추출물의 항염증 효과

배난영¹, 김민지¹, 김꽃봉우리², 박지혜¹, 박선희¹, 성낙윤³, 변의홍³, 안동현¹*

1부경대학교식품공학과

/

2부경대학교수산과학연구소

3공주대학교식품공학과

Received: March 21, 2016 / Revised: July 15, 2016 / Accepted: July 29, 2016

서 론

염증은각종부상이나상처

^,

^,

^,

cytokines, free radical, lysosomal enzyme

^[35].

^,

^,

^,

의많은만성질환에서근본적인병태생리학적기전으로알

려져있다

^[37].

면역반응이일어나지않을경우감염을유도할수있으며면 역을위한염증반응이과도하게발생하게되면기능장애와 같은조직변질순환장애와조직증식을유발한다

^[38].

^,

interleukin (IL)-6, tumor necrosis factor-

^(TNF-

^{), IL-1}

cytokine

^.

inducible nitric oxide synthase (iNOS)

nitric oxide (NO)

cyclooxygenase (COX-2)

prostaglandin E

(PGE

)

Anti-Inflammatory Effect of Chondrus ocellatus Holmes Ethanol Extract on Lipopolysaccharide-induced Inflammatory Responses in RAW 264.7 Cells

Nan-Young Bae

, Min-Ji Kim

, Koth-Bong-Woo-Ri Kim

, Ji-Hye Park

, Sun-Hee Park

, Nak-Yun Sung

, Eui-Hong Byun

, and Dong-Hyun Ahn

*

1

Department of Food Science & Technology/Institute of Food Science, Pukyong National University, Busan 48513, Republic of Korea

2

Institute of Fisheries Sciences, Pukyong National University, Busan 46041, Republic of Korea

3

Department of Food Science and Technology, Kongju University, Kongju 32588, Republic of Korea

This study aimed to investigate the anti-inflammatory effect of the ethanol extract from Chondrus ocellatus Holmes (COHEE) in RAW 264.7 cells and in a mouse ear edema model, by measuring the production of lipopolysaccharide-induced inflammatory response mediators. There were no cytotoxic effects on the proliferation of macrophages treated with COHEE compared with the control. COHEE inhibited the production of nitric oxide and pro-inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor- α, and IL-1 β]. The extract also reduced the expression of inducible nitric oxide synthase, cyclooxygenase-2, nuclear factor- κB p65, and phosphorylated mitogen-activated protein kinase in a dose-dependent manner.

In the croton-oil-induced ear edema model, COHEE decreased the formation of mouse ear edema at the highest dose compared with the control, and histological analysis revealed that the epidermal/dermal tissue thickness and mast cell numbers were reduced. Therefore, these results suggest that COHEE may be a promising topical anti-inflammatory therapeutic material through its action of modulating NF- κB and the MAPK signaling pathway.

Keywords: Chondrus ocellatus Holmes, anti-inflammatory effect, pro-inflammatory cytokines, NF-

^{B, MAPKs}

*Corresponding author

Tel: +82-51-629-5831, Fax: +82-51-629-5824 E-mail: [email protected]

© 2016, The Korean Society for Microbiology and Biotechnology

증유발인자들도생성된다

^{[26, 32].}

nuclear factor-

^{B (NF-}

^B)

^mitogen- activated protein kinase (MAPK)

^{. NF-}

^B

cytokine, chemokine, growth factor

^p50

^p65

iNOS, COX-2

^cytokine

^[10].

gram-negative bacteria

lipopolysaccharide (LPS)

^.

TLR4 (toll-like receptor 4)

^MyD88

IRAK

^TRAF6

^{, I}

^{B kinase} cascade

^cytokine

^[18].

NF-

^B

extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 kinase

mitogen-activated protein kinases (MAPKs)

NF-

B

MAPKs

[18].

스테로이드계와비스테로이드계로분류할수있는데이러한 항염증제는위장장애

^,

용이한정적인문제점이있다

^[25].

부터보다안정적이고효과적인항염증제를개발할필요성 이대두되고있으며최근몇년간일상생활에서도섭취가 능한식품을항염증제로이용하기위한다양한연구가진행 되고있는실정이다

^[23].

한편

^,

^[29],

^[22],

^[11],

^[15],

^[27],

^[16]

^.

(Chondrus ocellatus Holmes)

^{[1, 2, 35]}

^.

구에서는진두발에탄올추출물

^(COHEE)

^LPS

대식세포에서

^NF-

^B

MAPKs pathway

^COHEE

^.

재료 및 방법

실험재료

부산청사포에서

²⁰¹⁴

^.

^-20

^.

진두발 에탄올 추출물 제조

분말상태의진두발에

¹⁰

95% ethanol

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

24

^.

(UNION 32R, Hanil Co., Korea)

^.

²

37

(RE200, Yamoto Co., Japan)

³⁷

^{, -20}

^.

^{, flavone}

^flavanone

^1.65

0.13%, 0.05

0.00%, 0.93

^0.00%

(data not shown).

세포배양

한국세포주은행

(KCLB 40071, Korea)

^{cell line}

murine

RAW 264.7

, Dulbecco’s Modified Eagle Medium (GIBCO, USA)

10% inactivated fetal bovine serum (FBS)

^1%

penicillin-streptomycin

³⁷

^, 5% CO

^.

세포독성 측정

Park

^[30]

^{MTT assay}

^{. 1 × 10}

^cells/ml

RAW 264.7

96-well plate

²⁰

(0.1, 1, 10, 50, 100

g/ml) COHEE

²²

. 5 mg/ml MTT (thiazolyl blue tetrazolium bromide, Sigma-Aldrich Chemical Co., USA)

²

^.

⁴

^{, 2,000} rpm

¹⁰

^dimethyl sulfoxide 100

^l

^formazan

microplate reader (Model 550, Bio-Rad, USA)

540 nm

^.

에의해계산하였다

^.

Proliferation index (%) = Sample 흡광도 / Control 흡광도 × 100

Nitric oxide

배양액내의

^nitrite

^Griess

^[24]

을이용하였다

. 2.5 × 10

cells/ml

RAW 264.7 cell

24 well plate

^{5% CO}

incubator (MCO-

15AC, Sanyo, Japan)

²⁰

^.

COHEE

0.1, 1, 10, 50, 100

^g/ml

¹

^g/ml

LPS

²⁴

^.

⁴

^{, 2,000}

rpm

10

^.

^Griess

(1% sulfanilamide + 0.1%

naphthylendiamine dihydrochloride, 1:1)

^1:1

¹⁰

microplate reader

540 nm

^.

^NO

sodium nitrite (NaNO

)

^.

Pro-inflammatory cytokines 분비량 측정

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

RAW 264.7 cell

TNF-

, IL-6

^IL-1

^cytokine

^.

RAW 264.7 cell

^{2.5 × 10}

^cells/ml

²⁴ well plate

¹⁸

^.

^{0.1, 1,} 10, 50, 100

^g/ml

^COHEE

¹

^g/ml

^LPS

¹²

^{. ELISA}

microplate

capture antibody

^anti- mouse TNF-

^{, IL-6}

^IL-1

⁴

^coating

^.

0.05% Tween 20

^PBST

^{10% FBS}

^blocking

^PBST

microplate well

²

^.

^PBST

biotinylated anti-mouse TNF-

, IL-6 detection antibody

streptavidin-horseradish peroxydase conjugate

¹

^{. IL-1}

^, biotinylated anti-mouse IL-1

detection antibody

¹

, streptavidin-horseradish peroxidase conjugate

³⁰

^.

PBST

^OPD

³⁰

^{. 2 N H}

SO

microplate reader

^{490 nm}

^.

iNOS, COX-2 및 NF-κB p65 발현량 측정

COHEE

iNOS, COX-2

^NF-

^B p65

^{RAW 264.7}

^.

³

^PBS (phosphate buffered saline)

^{, Sheeba}

^Asha [34]

cytosol lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% deoxycholate, 5 mM phenylmethylsulfonyl fluoride, 1

g/ml aprotinin, 1% Triton X-100, 0.1% NP-40]

³⁰

⁴

lysis

, 15,520 × g

²⁰

. BCA protein assay kit (Pierce, USA)

³⁰

^l

^lysate

Laemmli [20]

10% SDS-PAGE

하였다

^.

^Towbin

^[36]

PVDF (polyvinylidene difluoride) membrane (Bio-Rad)

1

5% skim milk

TBSS (tris buffered saline, pH 7.5)

²

blocking

. iNOS, COX-2

^NF-

^B

anti-mouse iNOS, COX-2

^NF-

^B p65 (Santa Cruz Biotechnology, Inc., USA)

1:500

²

^TBSS

3

^{. 2}

HRP (horseradish peroxydase)

anti-mouse IgG

anti-rabbit IgG

^1:2,000

¹

^{, TBSS}

³

^ECL

¹

³

Gene tool (GeneGnome5, Syngene, UK)

^.

MAP kinase (JNK, ERK, p-38) 발현량 측정

MAPKs

RAW 264.7

1 × 10

cells/ml

¹⁸

^COHEE

³⁰

^MAPKs

anti-mouse c-Jun N-terminal kinase (JNK), p-JNK, extracellular signal-regulated kinase (ERK), p-ERK, p38 protein kinase (p38)

p-p38 (Cell Signaling Technology Inc., USA)

^1:500

^.

iNOS, COX-2

^.

귀부종 측정 및 조직 관찰

COHEE

in vivo

^Kim

^[14]

^Saraiva

^[33]

^.

⁸

^{, ICR}

COHEE

^{10, 50}

250 mg/kg· body weight

²⁰⁰

^l

^,

2.5% croton oil

²⁰

^l/ear

^.

⁵

croton oil

^.

^ICR

오른쪽 귀에 진두발 에탄올 추출물을

100 mg/ml

20

^l

¹⁵

, 5% croton oil

²⁰

^l

^{. 6}

, diethylether

^,

10% formaldehyde

⁷²

^.

hematoxylin-eosin

toluidine-blue

^.

^.

Edema formation (%)

= Sample

^/Control

^{× 100}

통계 처리

모든실험결과에대한유의차검정은

SAS software (ver.

9.3, SAS Institute, Inc., USA)

^, Duncan's multiple range test

p < 0.05

^.

결과 및 고찰

RAW 264.7 cell

COHEE

(0.1, 1, 10, 50, 100

^{g/ml) RAW} 264.7 cell

^MTT assay

^.

^,

^PBS

^(Fig.

1).

^COHEE

^0.1

¹⁰⁰

^g/ml

^.

^,

^[1]

성을나타내지않은결과와유사하다

^[20].

Nitric Oxide 생성 억제 효과

NO

-arginine

^NOS

-citrulline

^.

^,

^,

neurotransmitter,

apoptosis

^.

LPS, interferon-

^(IFN-

γ

^{), IL-1}

^TNF-

^NO

성되면

^shock

^,

직손상

, mutagenesis,

^,

^,

^{[19, 27].}

^NO

^LPS

RAW 264.7 cell

NO

^Griess

^{. LPS}

RAW 264.7 cell

^NO

(Fig. 2), LPS

^NO

27.42

^0.36

^M

^,

^PBS

3.04

^0.07

^M

⁹

^.

(0.1, 1, 10, 50, 100

^g/ml)

서

^COHEE

^{, LPS}

농도의존적으로

^NO

^.

^,

⁵⁰

^g/ml

^35%

^.

NO

^{[1, 19]}

^.

Pro-inflammatory cytokines 생성 억제 효과

COHEE

pro-inflammatory cytokines

RAW 264.7

IL-6, TNF-

^IL-1

cytokine

^ELISA

^{. Pro-} inflammatory cytokine

Fig. 1. Effect of Chondrus ocellatus Holmes ethanol extract on

proliferation in RAW 264.7 cells. Proliferation index (%) = (sample absorbance / control absorbance) × 100. ND: not significantly dif- ferent.

Fig. 2. Effect of Chondrus ocellatus Holmes ethanol extract on

nitric oxide production in RAW 264.7 cells. Cells were incubated

in the presence of LPS (1 μg/ml) alone or in combination with

COHEE (0.1, 1, 10, 50, and 100 μg/ml) for 24 h. The culture media

of the treated cells were used to measure NO levels.

Means

with different superscripts are significantly different (p < 0.05).

는 매개물로 정상조직에서도 발현되지만 과도한

^pro- inflammatory cytokine

^PGE

^NO

^,

^[9].

^IL-6

T cell, monocyte, macrophage, synovial fibroblast

pro-inflammatory cytokine

^B

^T

^[4].

^TNF-

^LPS

^T

프구의활성과성장을조절하여염증부위로의

^cytokine

내피세포부착분자의발현을증가시키며염증부위에서생 성이증가된다

^.

eicosanoid,

^,

^[3].

^IL-1

^{T cell}

B cell

^[21].

pro-inflammatory cytokine

^.

^LPS

RAW 264.7 cell

^COHEE

(Fig. 3), IL-6, TNF-

IL-1

^LPS

^2081.30

^20.32

^{M, 2334.41}

19.81

^{M, 84.28}

^11.74

^M

^PBS

^122, 233

⁸⁴

^{, COHEE}

^{. IL-6}

^TNF-

^{50, 100}

^g/ml

^LPS

처리구에비해약

^50%

^(Fig.

3A, 3B). IL-1

^LPS

량을

^COHEE

결과를확인하였다

(Fig. 3C).

¹⁰⁰

^g/ml

^LPS

^93%

¹⁰⁰

^g/ml

^94%

^[12]

^.

iNOS, COX-2 및 NF-κB 발현 억제 효과

LPS

^cytokine

^NF-

^B

성화시켜

^iNOS

의

^NO

^,

^,

^,

^.

^COX-2

arachidonic acid

^PGE

, thromboxane A2, prostacyclin

Fig. 3. Effect of Chondrus ocellatus Holmes ethanol extract on

the production of IL-6 (A), TNF- α (B), and IL-1β (C) in RAW 264.7 cells. Cells were incubated in the presence of LPS (1 μg/

ml) alone or with various concentrations of COHEE (0.1, 1, 10, 50,

and 100 μg/ml). The levels of pro-inflammatory cytokine in the

cell culture media were measured by ELISA.

Means with differ-

ent superscripts are significantly different (p < 0.05).

관생성을유도하는데관여한다

^{[7]. NF-}

^B

^promoter

iNOS, COX-2

에관여하는것으로알려져있다

^{[5]. COHEE}

매개인자를생합성하는효소인

^iNOS

COX-2, NF-

^B

western blot

사한결과

^{, COHEE}

효과적으로억제하는것을확인하였다

^{(Fig. 4).}

^COHEE

^NF-

^B

^iNOS

^COX-2

^NO

pro-inflammatory cytokine

^.

MAP kinase (JNK, ERK, p-38)

MAPKs

하나인데그경로들중

ERK, JNK/stress-activated protein kinase (SAPK), p38 MAPK

^.

외부자극에반응해서상위

MAPK kinases

가일어남으로써활성화된다

^{. MAPKs}

^LPS

^TNF-

^kinase,

^,

^,

cytokine

^[31].

MAPKs

, RAW 264. 7 cell

^LPS

p-p38, p-ERK, p-JNK

^{(Fig. 5).}

^COHEE

^NF-

^B

^MAPKs

^.

귀부종 억제 효과 및 조직 관찰

진두발에탄올추출물이염증반응의하나인부종의완화 효과및조직내의

mast cell

^{10, 50}

250 mg/kg

croton oil

^.

Fig. 4. Effect of Chondrus ocellatus Holmes ethanol extract 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 COHEE (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. Means with different superscripts (a-e) are sig-

nificantly different (p < 0.05).

의해손상된부위를복구시키기위해염증반응을일으켜혈 관확장

^,

^protease

histamine

^{[8, 33].}

Croton oil

, phorbal esters, 12- O-tetradecanoylphorbol-13-acetate (TPA)

^.

^PGE

leukotriene B

(LTB

)

^,

^{mast cell}

^[6].

특히혈관이확장되고조직내로면역세포들이침윤되는현 상은

protein kinase C (PKC)

^,

phospholipase A2 (PLA2)

arachidonic acid

prostaglandin

leukotriene

^.

^pro- inflammatory cytokine

^chemokine

^[32].

croton oil

(Fig. 6), croton oil

여부종이발생한대조군과비교시

^COHEE

든농도에서유의적으로귀두께가감소하였으며

^,

^,

Fig. 5. Effect of Chondrus ocellatus Holmes ethanol extract on MAPKs expression in RAW 246.7 cells. The levels of p-p38, p-ERK, and p-JNK in the cytosolic protein were determined by western blot analysis. RAW 264.7 cells were treated with the indicated concen- trations of COHEE (0.1, 1, 10, 50, and 100 μg/ml) and LPS (1 μg/ml) for 30 min, and the proteins were detected using specific antibodies.

Means with different superscripts (a-e) are significantly different (p < 0.05).

Fig. 6. Inhibition of Chondrus ocellatus Holmes ethanol extract

against croton oil-induced mouse ear edema.

Means with

different superscript are significantly different (p < 0.05).

재사용되고있는합성스테로이드제인

prednisolone

⁵⁰ mg/kg

250 mg/kg body weight

^30%

^.

(Fig. 7), croton oil

^COHEE

prednisolone

^.

COHEE

toluidine-blue

^{mast cell}

^.

[12],

^[13]

250 mg/kg body weight

prednisolone 50 mg/kg body weight

mast cell

^/

^.

250 mg/kg body weight

^46%

^[17].

요 약

본연구는

COHEE

으로써

pro-inflammatory cytokine

^iNOS, COX-2, NF-

^B

^MAPKs

^.

MTT assay

^COHEE

하였다

^{. COHEE}

^NO

소하였으며

IL-6, TNF-

^IL-1

^.

¹⁰⁰

^g/ml

^LPS

구인대조구에비하여

IL-6, TNF-

^IL-1

^{66%, 43%}

^93%

^.

^NF-

^B

^MAPKs

지확인하기위하여발현량을관찰한결과

^{, COHEE}

^LPS

처리에의해현저히증가한단백질의발현을농도의존적으 로유의성있게억제하였다

^.

^COHEE

croton oil

250 mg/kg

prednisolone 10 mg/kg

^{mast cell}

^.

^COHEE

^NF-

^B

^iNOS

^COX-2

NO, IL-6, TNF-

IL-1

하였으며

^,

^{, COHEE}

^.

Acknowledgments

This research was a part of the project titled Yeongnam Seagrant funded by Ministry of Oceans and Fisheries, Korea.

References

1. Bae, NY, Kim MJ, Kim KBWR, Ahn NK, Choi YU, Park JH, et al. 2015.

Anti-inflammatory effect of ethanol extract from Grateloupia

elliptica Holmes on lipopolysaccharide-induced inflammatory

responses in RAW 264.7 cells and mice ears. Korean Soc. Biotech-

Fig. 7. Photomicrograph of transverse sections of mice ears sensitized with topical application of 5% croton oil (v/v) in acetone

(a-c) or vehicle acetone (d, non-inflamed), stained with hematoxylin-eosin (A) and toluidine-blue (B) examined under light

microscopy (magnification: 200×). Treatments: vehicle 2% Tween 80 (a), prednisolone 0.08 mg/ear (b) and Chondrus ocellatus Holmes

ethanol extract 20 μl/ear (c). The numbers 1 and 2 indicate dermis and epidermis, respectively and the arrows indicate mast cell infil-

tration.