Anti-Inflammatory Effect of Wheat Germ Oil on Lipopolysaccharide-stimulated RAW 264.7 Cells and Mouse Ear Edema

LPS 로 유도한 RAW 264.7 세포 및 귀부종 동물 모델에 대한 밀배아유의 항염증 효과

강보경¹, 김민지¹, 정다현¹, 김꽃봉우리², 배난영¹, 박지혜¹, 박선희¹, 안동현¹*

1부경대학교식품공학과

/

2부경대학교수산과학연구소

Received: January 21, 2016 / Revised: May 20, 2016 / Accepted: May 20, 2016

서 론

염증반응은물리적

^,

^[36].

^,

histamine, prostaglandins, leukotriene, hydroxyeicosatetraenoic acid

^,

혈구가조직으로침투하게된다

^{[9, 34].}

과도하게일어나면만성염증을유도하여염증매개물질이과

도하게분비되어암

^,

전에관여하게된다

^{[4, 28].}

포중하나인대식세포는이러한염증반응에중요한역할을 하고있다

^.

lipopolysaccharide (LPS)

pro-inflammatory cytokine

tumor necrosis factor-

^(TNF-

), interleukin-1

^(IL- 1

^{), IL-6}

^{[14, 15].}

pro-inflammatory cytokine

nuclear factor-kappa B (NF-

^B)

extracellular signal-regulated kinase (ERK), p38 kinase (p38), c-Jun NH

-terminal kinase (JNK)

^mitogen- activated protein kinases (MAPKs)

^[7].

inducible nitric oxide synthase (iNOS)

cyclooxygenase-2 (COX-2)

nitric oxide (NO)

prostaglandin E

(PGE

)

Anti-Inflammatory Effect of Wheat Germ Oil on Lipopolysaccharide-stimulated RAW 264.7 Cells and Mouse Ear Edema Bo-Kyeong Kang

, Min-Ji Kim

, Da-Hyun Jeong

, Koth-Bong-Woo-Ri Kim

, Nan-Young Bae

, Ji-Hye Park

, Sun-Hee Park

, 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

This study investigated the anti-inflammatory effects of wheat germ oil (WGO) on RAW 264.7 cells. It was shown that WGO had no cytotoxicity against the treated cells or negative effect on their proliferation. WGO suppressed nitric oxide (NO) secretion considerably and had inhibitory effects on the production of LPS- induced NO and pro-inflammatory cytokines (IL-6, TNF- α, and IL-1β). In particular, the IL-6 and TNF-α inhibition activities were over 90% at 100 μg/ml concentration of the oil. WGO also inhibited the LPS- induced expression of cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor-kappa B (NF- κB), and reduced the expression of phosphorylated ERK and JNK. Moreover, the croton-oil-induced edema in mouse ears was reduced by WGO, and no mortalities occurred in mice administered 5,000 mg/kg body weight of WGO over a 2-week observation period. In conclusion, these results provide evidence for the anti- inflammatory effect of WGO that likely occurs via modulation of NF- κB and the JNK/ERK MAPK signaling pathway.

Keywords: Anti-inflammatory activity, wheat germ oil, NF- κB, 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

자가생성된다

^{. NO}

L -arginine

^nitric oxide synthase (NOS)

^,

^NO

^DNA

^[32].

^NO

iNOS, prostaglandin

^COX-2

염증성

^cytokine

자인

^MAPKs

^NF-

^B

위한핵심적인요소로인식되고있다

^[21].

과생성에관여되는효소의발현을조절할수있는물질이 염증질환예방및치료제로서주목을받고있으며

^,

^[12].

밀배아는소맥의제분과정에서얻어지는부산물로밀알의

2

3%

,

함유되어있다

^[13].

이용되고있어활용도가낮은실정이다

^.

^E

carotenoids

, tocopherol

^phenolic

^.

^[16],

^oil

것으로밝혀져있으나

^{[17, 31],}

한연구는아직보고되지않았다

^.

따라서

^,

^LPS

RAW 264.7

croton-oil

^.

재료 및 방법

실험재료

본실험에사용한밀배아유

(Gooworl, Korea)

⁴

^.

palmitic acid (C16) 9.47%, stearic acid (C18) 3.79%, oleic acid (C18:1) 27.16%, linoleic acid (C18:2) 52.3%, linolenic acid (C18:3) 3.6%

^.

실험동물

생후

⁸

^{, ICR}

^(Orient

Co., Korea)

^,

생후

¹⁰

^{, Balb/c}

에사용하였다

^.

²⁰

²

^,

⁵⁰

^{10%, 12}

¹

^.

⁽

2014-01).

세포배양

마우스의대식세포주인

RAW 264.7

(KCLB40071)

, DMEM (GIBCO, USA)

10% inactivated fetal bovine serum (FBS)

^1%

penicillin-streptomycin

³⁷

^, 5% CO

incubator (MCO-15AC, Sanyo, Japan)

^.

⁸⁰

^90%

, 20 passages

^.

세포독성측정

시료의세포독성을평가하기위해

^{MTT assay}

. RAW 264.7

^{1 × 10}

^cells/ml

^96-well plate

²⁰

^,

(0.1, 1, 10, 50, 100

^g/ml)

³⁷

^{, 5% CO}

incubator

22

^.

^{, 5 mg/ml}

^MTT

²

⁴

, 2,000 rpm

¹⁰

(UNION 32R, Hanil Co., Korea)

^.

^,

^well

^DMSO

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

540 nm

^.

해계산하였다

^.

Proliferation index (%) = sample 흡광도 /control 흡광도 × 100

Nitric oxide

NO

^nitrite

^griess

. RAW 264.7

^DMEM

^{2.5 × 10}

^cells/ml

24-well plate

³⁷

^{, 5% CO}

incubator

¹⁸

^.

, RAW 264.7

¹

^{g/ml LPS}

(0.1, 1, 10, 50, 100

^g/ml)

²⁰

^.

^,

^griess

^(1%

sulfanilamide + 0.1% naphthylendiamine dihydrochloride,

1

¹⁾

¹⁰

, microplate

reader

^{540 nm}

^.

^NO

sodium nitrite (NaNO

)

.

염증관련 cytokine 분비량 측정

세포배양액내의

^TNF-

, IL-6, IL-1

^cytokine

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

^.

, ELISA microplate

^anti- mouse TNF-

^{, IL-6}

^IL-1

^mAb

coating

^.

0.05% Tween 20

^PBST

^{, 10% FBS}

^blocking

^{. PBST}

^,

microplate

²

^.

^PBST

biotinylated anti-mouse TNF-

^{, IL-6 mAb}

streptavidin-horseradish peroxidase conjugate

¹

^{. IL-1}

, biotinylated anti-mouse IL-1

^detection antibody

¹

, streptavidin- horseradish peroxidase conjugate

³⁰

^.

^,

^PBST

^{, OPD}

microplate reader

490 nm

^.

iNOS, COX-2 및 NF-κB p65 발현량 측정

배양이 끝난 세포를 수집하여

³

PBS (phosphate

buffered saline)

^{, iNOS}

^COX-2

cytosolic 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]

^{, NF-}

^{B p65}

nucleus lysis buffer (10 mM HEPES, 100 mM NaCl, 1.5 mM MgCl

, 0.1 mM EDTA, 0.1 mM dithiothreitol)

³⁰

4°C

^lysis

, 12,000 rpm

²⁰

^.

^BCA protein assay kit (Pierce, USA)

^, 30

^l

^lysate

10% SDS-PAGE

^.

PVDF (polyvinylidene difluoride) membrane (Bio- Rad)

^{200 mA}

¹

^{, 5% skim} milk

TBSS (tris buffered saline; pH 7.5)

²

^blocking

. iNOS, COX-2

NF-

^{B p65}

^anti- mouse iNOS, COX-2

^NF-

^{B p65}

^1:500

²

^TBSS

³

^{. 2}

horseradish peroxidase

anti-mouse IgG

anti-rabbit IgG

^1:2,000

¹

^TBSS

³

ECL

¹

³

^Gene tool (GeneGnome5, Syngene, UK)

^.

MAPKs 측정

MAPKs

기 위해

RAW 264.7

^DMEM

1 × 10

cells/ml

¹⁸

^LPS

(0.1, 1, 10, 50, 100

^g/ml)

³⁰

^,

iNOS, COX-2

^NF-

^{B p65}

. p-JNK, p-ERK

p-p38

anti-mouse p- JNK, p-ERK

^p-p38

^1:500

^.

귀 부종 측정 및 조직관찰

밀배아유의

in vivo

^, croton oil

^ICR

^.

^{10, 50}

250 mg/kg body weight

²⁰⁰

^l

^ICR

^.

2.5% croton oil

20

^l/ear

^.

croton oil

⁵

, croton oil

^.

^{100 mg/}

ml

²⁰

^l

15

5% croton oil

²⁰

^l

^{. 6}

diethyl ether

^,

^10%

formaldehyde

⁷²

^.

hematoxylin-eosin

toluidine-

blue

^.

같은식에의해계산하였다

^.

Edema formation (%) = Sample 귀 두께 /Control 귀 두께 × 100

단기독성평가

단기독성평가는식품의약품안전처의제

^2014-136

^“

^”

OECD test guide line [29]

5,000 mg/kg body weight

^Balb/c

300, 2,000

5,000 mg/kg body weight

^,

⁴

⁶

^.

⁶

^{, 2}

^.

통계처리

모든실험에대한통계처리는

SAS program (Statistical

analytical system V8.2, SAS Institute Inc., USA)

one way ANOVA

^,

^Duncan

p < 0.05

^.

결과 및 고찰

세포독성 측정

밀배아유의항염증효능을탐색하기앞서밀배아유가

^RAW 264.7

^{, 100}

^g/

ml

^MTT

^{. MTT}

^0.1

¹⁰⁰

^g/ml

^PBS

^0.1

¹⁰⁰

^g/ml

(Fig. 1).

^,

^.

Nitric oxide

LPS

RAW 264.7

^NO

^,

^nitrite

^{. NO}

^NOS

L -

arginine

^,

성신경전달계및혈관이완등의여러생물학적과정에관 여하며

^[19]

^iNOS

^NO

[24, 25]. LPS

RAW 264.7

^NO

^55.8

^M

^,

¹⁰

¹⁰⁰

^g/ml

^NO

^.

⁵⁰

¹⁰⁰

^g/ml

^NO

36.6

^34.7

^M

^36%

^40%

^{(Fig. 2).}

sandalwood essential oil

²⁵⁰

^g/ml

^30%

^NO

^[31], Camellia japonica oil

¹⁰⁰

^g/ml

^LPS

^NO

^[20],

배아유에의한

^NO

^.

oleic acid

linoleic acid

^,

^NO

의분비량감소에효과가있는것으로보고되고있다

^{[5, 30].}

따라서

^,

^NO

^.

인체에서염증반응이진행되기위해서는면역반응에서필 연적으로염증성사이토카인이동반되는데

^,

^cytokine

^TNF-

, IL-6, IL-1

^{[23]. LPS}

^TNF-

^TNF-

^IL-1

^IL-6

^{[2]. TNF-}

(intracellular adhesion molecule-1)

(vascular cell adhesion molecule-1)

^,

^{[1]. IL-6}

pro-inflammatory/anti- inflammatory

^cytokine

^,

관절염과열과같은병적증상을나타나게된다

^{[33]. IL-1}

는미생물감염에대해염증반응의초기와발달에중요한 요소로서작용한다

^[18].

^,

^pro- inflammatory cytokine

^.

RAW 264.7 cell

^LPS

pro-inflammatory cytokine

^TNF-

, IL-6, IL-1

^(Fig.

3).

^0.1

¹⁰⁰

^g/ml

0.1

¹

^g/ml

^LPS

^{, 10}

¹⁰⁰

^g/ml

1916.5 pg/ml, 108.9 pg/ml

79.9 pg/ml

^LPS

TNF-

(2400.7 pg/ml)

25%, 95%, 97%

Fig. 1. Effect of wheat germ oil on proliferation of RAW 264.7

cells. Proliferation index (%) = sample O.D./control O.D. × 100.

ND : not significantly different.

Fig. 2. Inhibitory effect of wheat germ oil on the production

of nitric oxide in RAW 264.7 cells.

Means with different

superscripts are significantly different (p < 0.05).

억제되었다

^{. IL-6}

^{, LPS}

235.9 pg/ml

^{, 0.1}

^g/ml

^{135.8 pg/}

ml

^42%

^{, 50}

¹⁰⁰

^g/ml

40.3 pg/ml

13.5 pg/ml

^82.9%

^94.3%

^{. IL-1}

^LPS

22.5 pg/ml

^,

^{, 50}

^g/ml

13.2 pg/ml

^41%

^{, 100}

^g/ml

10.3 pg/ml

54%

^.

^LPS

로인해증가된전염증성

^cytokine

50

¹⁰⁰

^g/ml

^.

^NF-

^{B p65}

MAPKs (p-ERK, p-JNK)

^western blot

(Fig. 4, 5)

⁵⁰

¹⁰⁰

^g/ml

서

^LPS

나

^{, NF-}

^B

^MAPKs

. Sandalwood essential oil

Camellia japonica oil

²⁵⁰

^g/ml

cytokine

[20, 31],

LPS

^cytokine

^.

^,

pro-inflammatory

cytokine

절할수있을것으로사료된다

^.

iNOS, COX-2 및 NF-κB p65 발현 억제 효과

LPS

RAW 264.7

^iNOS

^COX-2

^NF-

^{B p65}

Western blot

^.

^NOS

^,

³

neuronal NOS (nNOS), endothelial NOS (eNOS),

^iNOS

^[27].

^nNOS

^eNOS

^iNOS

^LPS,

cytokine

현되며염증반응에관여하게된다

^{[35]. COX}

arachidonic acid

prostaglandin

^,

발현하는

^COX-1

^,

시세포분열인자나

^cytokine

^COX-2

^{[8]. NF-}

^B

p65

^p50

heterodimer

^cytosol

^{, I}

^B

^.

^LPS

^cytokine

^I

^B

^NF-

^B

^iNOS, COX-2

^cytokine

^{[11, 26].}

RAW 264.7

^0.1

¹⁰⁰

^g/ml

iNOS, COX-2, NF-

^{B p65}

^{. iNOS}

^{, LPS}

Fig. 3. Inhibitory effect of wheat germ oil on production of

TNF- α, IL-6, and IL-1β in RAW 246.7 cells.

Means with differ-

ent superscripts are significantly different (p < 0.05).

가하였으며

^{, 0.1}

⁵⁰

^g/ml

^LPS

^{, 100}

^g/ml

^iNOS

^.

^{, Fig. 2}

에서의

^NO

^iNOS

을확인하였다

(Fig. 4). COX-2

^LPS

^{, 0.1}

¹⁰

^g/ml

^{, 50}

^g/ml

¹⁰⁰

^g/ml

^{. NF-}

^{B p65}

¹⁰

50

^g/ml

^{, 100}

^g/ml

^.

^NF-

^B signaling

iNOS, COX-2

^cytokine

^.

^oleic acid

^NF-

^B

^iNOS

^COX-2

^[30],

^NF-

B signaling pathway

^.

MAPKs 발현 억제 효과

MAPKs

serine/threonine kinase

p38, ERK, JNK

분류된다

^{. MAPKs}

LPS

전염증성

^cytokine

^iNOS

^[22].

MAPKs pathway

^LPS

^NF-

^B

중요한역할을하는것으로알려져있다

^[3].

^,

유가

^MAPKs

^{, LPS}

에의해증가하는

^MAPKs

Western blot

^.

(Fig. 5), LPS

^p-p38, p-ERK

^p-JNK

^,

의한발현량변화를보면

^{, p-p38}

제효과가거의없었으나

^{, 10}

¹⁰⁰

^g/ml

^{. p-ERK}

^0.1

^g/ml

^,

^{. p-JNK}

^{, 10}

^g/ml

^{, 100}

^g/ml

Fig. 4. Inhibitory effects of WGO on the protein expression of iNOS, COX-2 and NK- κB p65 in RAW 264.7 cells. The levels of iNOS

and COX-2 in the cytosolic protein, and the NF- κB p65 in nuclear protein were determined by western blot analysis. RAW 264.7 cells

were treated with the indicated concentrations of WGO (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. For quantification, the expression data were normalized to the β-actin signal. Data

are obtained from three independent experiments and expressed as means ± SD. Means with different letters (a-e) above the bars are

significantly different (p < 0.05).

발현억제가가장컸다

^{. Kim}

^[20]

^LPS

ERK, p38, JNK

Camellia japonica oil

²⁵⁰

^g/ml

^,

^JNK

^ERK

^.

^,

^p-ERK

p-JNK MAPKs

^.

귀 부종 억제 효과 및 조직 관찰

피부염증에서일어나는대표적반응으로염증반응시혈 관확장

^,

^,

정물질의항염증효능을증명할수있다

^[10].

아유를

^{10, 50}

250 mg/kg

²⁰⁰

^l

, croton oil

^,

Fig. 6

^{. Control}

^,

Fig. 5. Inhibitory effects of wheat germ oil on the protein expression of p-p38, p-ERK, and p-JNK in RAW 264.7 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 concentrations of WGO (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. For quantification, the expression data were normalized to to the β-actin signal. Data are obtained from three inde- pendent experiments and expressed as means ± SD. Means with different letters (a−g) above the bars are significantly different (p < 0.05).

Fig. 6. Inhibition of wheat germ oil (WGO) against croton oil-

induced mouse ear edema.

Means with different superscript

are significantly different (p < 0.05).

의적으로귀두께가감소한것을확인하였다

^.

^,

^{250 mg/kg}

^{, positive} control

prednisolone 10 mg/kg

^.

, croton oil

^,

배아유를

100 mg/ml

가얇아진것을확인하였다

(Fig. 7A).

^mast cell

toluidine-blue

(Fig. 7B), prednisolone

mast cell

^.

linoleic acid

oleic acid

pumpkin seed oil

^test

positive control

^,

^,

^cell

^,

^[6].

^,

^.

단기독성평가

본연구에서밀배아유의항염증효과를확인하였으며

^,

야하기때문에

^,

^.

300, 2,000

^5,000 mg/kg

²⁰⁰

^l

²

(Table 1),

5,000 mg/kg

^{, 2}

^0%

^.

^.

요 약

밀배아유가

^LPS

자들에 미치는 영향을 살펴보기 위해

^{, NO}

cytokine

^iNOS

^COX-2

^{, NF-}

^{B p65}

^MAPKs

^.

^ICR

croton oil

^.

^,

^LPS

^NO

^cytokine

^,

^{, IL-6}

^TNF-

¹⁰⁰

^g/ml

^90%

^{. LPS}

^iNOS

^COX- 2

¹⁰⁰

^g/ml

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), stained with (A) hematoxylin-eosin and (B) toluidine-blue examined under light microscopy (magnification: 200×). Treatments: vehicle 2% Tween 80 (a), prednisolone 0.08 mg/ear (b) and wheat germ oil 20 μl/ear (c). The num- bers 1 and 2 indicate dermis and epidermis, respectively and the arrow in (B) means infiltration of mast cells.

Table 1. Mortality of mice treated orally with wheat germ oil.

Days after treatment

0 2 4 6 8 10 12 14

Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

300 mg/kg·body weight 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

2,000 mg/kg·body weight 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

5,000 mg/kg·body weight 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5