Inhibitory Effect of Fucoidan on TGF-β1-Induced Activation of Human Pulmonary Fibroblasts

(1)

807 Copyright © 2016 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815

서 론

특발폐섬유증

(idiopathic pulmonary fibrosis, IPF)

은아직밝 혀지지않은원인에의해폐포

,

모세혈관

,

세기관지

,

림프관등 간질조직에염증이발생하여점차조직이딱딱하게굳어지는 섬유화가특징적인통상성간질성폐렴

(usualinterstitial pneu- monia, UIP)

을보이는질환이다

(King et al., 2000).

최근에는 여러가지실험과연구를통해반복된자극으로인하여폐가조 금씩손상을받으면상처회복

(wound healing)

이지연되면서섬 유화가되어염증질환이아닌섬유성질환이유도된다는기전 이제시되었다

(Ahluwalia et al., 2014).

주로섬유모세포

(fibro- blast)

와근섬유모세포

(myofibroblast)

에의해생성되는세포외 기질

(extracellular matrix: ECM)

의과도한축적으로폐의정상

구조를파괴하여폐기능부전이유도된다

(Camelo et al., 2014;

Cottin, 2016).

특발폐섬유증의조직학적소견인통상성간질성 폐렴은초기변화로섬유모세포집단병소

(fibroblastic foci)

형 성이특징이다

(King et al., 2011; Raghu et al., 2006).

집단병소 는폐포상피세포밑의간질에위치하는섬유모세포

/

^근섬유모 세포의집합체로폐섬유화과정에서주요한위치를차지하는 것으로알려져있다

(Moore and Herzog, 2013).

따라서집단병 소가특발폐섬유증의중요한진단기준이되었고병리학적으로 도의미있는소견으로인정되고있다

.

폐에서의섬유화증은반 복적이고지속적인폐손상에대한비정상적인상처치유의결 과로서

,

정상적으로는자극에의한폐포상피세포

(alveolar epi- thelial cells)

의활성은혈관누출과혈관외응고

;

자연면역반응 활성화

;

섬유세포침윤

,

증식그리고활성화

;

세포외기질의합

후코이단에 의한 인간 폐 섬유모세포의 활성 억제 효과

임미진·이대성·최그레이스·이정민·최일환

¹

*

국립해양생물자원관 천연물연구팀, ¹인제대학교 의과대학 미생물학교실

Inhibitory Effect of Fucoidan on TGF-β1-Induced Activation of Human Pulmonary Fibroblasts

Mi-Jin Yim, Dae-Sung Lee, Grace Choi, Jeong Min Lee and Il-Whan Choi

¹

*

Natural Products Research Team, National Marine Biodiversity Institute of Korea, Seochun 33662, Korea

1

Department of Microbiology and Immunology, College of Medicine Inje University, Busan 47392, Korea

Fucoidan, one of the dominant sulfated polysaccharides extracted from brown seaweed, possesses a wide range of biological activities. Transforming growth factor-β (TGF-β) plays a pivotal role in the pathogenesis of pulmo- nary fibrosis, by stimulating the synthesis of profibrotic factors. In this study, we investigated the in vitro effects of fucoidan on collagen synthesis, α-smooth muscle actin (α-SMA) expression, and interleukin (IL)-6 production in TGF-β-stimulated human pulmonary fibroblasts. The expression of type I collagen and α-SMA was detected by Western blot, and the production of IL-6 by enzyme-linked immunosorbent assay. TGF-β1 treatment of pulmonary fibroblasts enhanced the expression of α-SMA, type I collagen, and IL-6 whereas these effects were inhibited in cells pretreated with fucoidan. The activation of Smad2/3, p38 mitogen-activated protein kinases (MAPKs), and Akt was also inhibited in fucoidan-pretreated, TGF-β1-stimulated human pulmonary fibroblasts. These data demonstrate the anti-fibrotic potential of fucoidan in TGF-β-induced human pulmonary fibroblasts, via the inhibition of Smad2/3, p38 MAPKs, and Akt phosphorylation. Our results suggest the therapeutic potential of fucoidan in the prevention or treatment of pulmonary fibrosis.

Key words: Fucoidan, Pulmonary fibrosis, Transforming growth factor-β, α-smooth muscle actin, Type I collagen

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://dx.doi.org/10.5657/KFAS.2016.0807 Korean J Fish Aquat Sci 49(6) 807-814, December 2016

Received 6 October 2016; Revised 3 November 2016; Accepted 7 November 2016

*Corresponding author: Tel: +82. 51. 890. 6461 Fax: +82. 51. 890. 6004

E-mail address: [email protected]

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에서지속적인섬유세포의활성화와기질생성을하게되면폐 섬유화에따른폐기능부전을유도하게된다

(Ahluwalia et al.,

2014).

이과정에서수많은사이토카인이분비되는데

,

이중에

서전환성장인자

(transforming growth factor-β, TGF-β)

가중 요한역할을하게된다

(Blobe et al., 2008). TGF-β1

^는^상처치유 와폐섬유화에중요한섬유화인자이며근섬유모세포의활성 과분화와같은섬유화기전에연관되어있다

.

폐섬유증환자의 폐조직에서

TGF-β1

의발현이증가되어있고

(Broekelmann et al., 1991; Khalil et al., 1991),

폐섬유증동물모델에서

TGF-β1

의활성을억제하면

collagen

^의^간질^축적

(interstitial accumu- lation)

을억제한다는보고가있다

(Wang et al., 1999).

Fucoidan

은미역

,

톳

,

감태

,

대황

,

다시마등의갈조류의세포 벽 구성 성분으로부터분리한 고분자황산화다당류

(sulfated polysaccharides)

로서점성이낮고용해성이우수한특징을가 지고있다

(Park et al., 2007). Fucoidan

^은^세포내^{골지체에서} 합성되어세포간조직에존재하는다당류로서

L-fucose

골격 에에스테르화황산을주성분으로하여

mannose, glucose, ga- lactose, xylose, glucuronic acid, rhamnose

등이결합되어있 다

(Park et al., 2010). Fucoidan

은항산화

,

항염증

,

항응고

,

항 알레르기

,

^{항바이러스}

,

^항암등의^다양한^생리활성^기능을^갖고 있는것으로알려져있다

(Kuznetsova et al., 2014; Cho et al., 2015).

또한림프구

,

대식세포

,

자연살해세포

,

수지상세포와같 은다양한면역세포에대해면역조절기능을가지고있는것으 로알려져있다

(Jin et al., 2014).

또한

fucoidan

은의약품

,

기능 성식품

,

^화장품의^기초^원료와^첨가제로^이용되고^있다

(Ahmed et al., 2014).

이전연구에의하면

fucoidan

이상처치유효능을가지는것 으로보고되어지지만

,

폐섬유증에관련한효능연구는보고된 바없다

(O'Leary et al., 2004).

본연구에서는

fucoidan

이특발

폐섬유증치료에효능이있는지를알아보기위해

TGF-β1

^으로

자극한인간섬유모세포

(Human pulmonary fibroblast)

에서섬 유화에밀접히관련되어있는

type I collagen

과

alpha smooth muscle actin (α-SMA)

의발현조절효능과조절기전을살펴 보고자한다

.

재료 및 방법

재료

실험에사용된인간재조합

TGF-β1

은

R&D system

사

(Min- neapolis, USA)

^의^제품을^{사용하였고}

,

^{세포독성테스트를}^위해 사용된

Cell Counting Kit-8

은

Dojindo

사

(Kumamoto, Japan)

의 제품을 사용하였다

. Fucoidan

은

Sigma (St. Louis, MO, USA)

에서구입하여사용하였다

. Western blot

실험에사용된 일차항체는

α-Smooth muscle actin (α-SMA)

와

collagen-1 (Abcam, Cambridge, UK), p-P38, p-Akt, t-Akt, p-Smad2,

USA), P38 (Santa Cruz, CA, USA), Actin (BD Biosciences, CA, USA)

이며

,

이차항체는

horseradish peroxidase

가결합된

goat-α-rabbit

과

goat-α-mouse (santacruz biotechnology Inc, Texas, USA)

를사용하였다

.

세포배양

인간섬유모세포를

Promo Cell (Germany)

에서구입하여

5%

우태아혈청이포함된섬유모세포성장배지

(fibroblast growth medium 2, Promo Cell, Germany)

에서

5% CO

₂

incubator

에 서배양하였다

.

세포독성테스트

8×10

⁴

cell/mL

로분주하고

16

시간동안 우태아혈청이없는배지로

serum starvation

시킨후

fucoidan

을농도별로처리하여

30

분동안배양후

TGF-1

을처리하여

24

시간동안배양하였다

. CCK-8

를각각

10 L

씩처리하고

37℃

에 서

1

^시간^동안^반응시킨^후

450 nm

^파장에서

SpectraMax M2 (Molecular Devices, CA, USA)

를사용하여분석하였다

. Collagen gel contraction 분석

Collagen gel contraction

은

Luo et al. (2014)

의방법에준하 여분석하였다

. 2 mg/mL Rat tail type I Collagen (BD biosci- ences, CA, USA)

^과

2×10

⁵

cells/mL

^의^{섬유모세포를}^섞은^섬 유모세포성장배지를

12 well plate

에

500 μL

씩분주하였다

. 37℃

에서

30

분동안굳힌후

fucoidan

이포함된

500 μL

의배 지를

TGF-β1

를처리하기

1

시간전에첨가하여

24

시간경과후 의

gel

의크기를

National Institutes of Health (NIH) ImageJ software

^{프로그램을}^사용하여^{측정하였다}

.

IL-6 발현 분석

8×10

⁴

cell/mL

로분주하고

16

시간동안 우태아혈청이없는배지로

serum starvation

시킨후

fucoidan

을

1

시간전처리한후

TGF-β1

를처리하여

24

시간동안배양하 였다

. BioLegend ELISA kit (San Diego, CA, USA)

^를^사용하 여

IL-6

를측정하였고

, 450 nm

파장에서

microplate reader

를 사용하여분석하였다

.

Western blot 분석

PBS

로

3

번세척후

cell lysis buffer (Mam- malian Cell-PE LB, G Biosciences, St. Louis, MO, USA)

^를 사용하여단백질을추출하였다

.

동량의단백질을

10% SDS–

polyacrylamide minigel

을사용하여분리시키고

nitrocellulose

membrane

으로옮긴다

.

이후

membrane

은적절한

primary an-

tibody

로

4℃

에서

overnight

반응시키고

secondary antibody

로 실온에서

1

^시간^반응시킨^후

ECL detection system (Thermo

Fisher Scientific Inc, MA, USA)

을사용하여확인하였다

.

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후코이단의 항섬유화 효과

809 Smad2/3 siRNA를 이용한 Western blot 분석 8×10

⁴

cell/mL

의인간섬유모세포를분주후

24

시간동안배 양한후

, 100 L

^의

transfection medium (Santa Cruz, CA, USA)

에

20 nM

과

40 nM

의

Smad2/3 siRNA (Santa Cruz, CA, USA)

와

6 L

의

transfection reagent (Santa Cruz, CA, USA)

를넣은

100 L

의

transfection medium

혼합액을세포에넣어주었다

. 6

시간경과후

2

배의우태아혈청이포함된

1 mL

의섬유모세포 성장배지를넣어주어

15

^시간^배양^하고

,

^{무혈청배지}^환경에서

1 ng/mL

의

TGF-β1

로자극후

24

시간동안배양하였다

. 통계 분석

실험결과는평균

±

표준오차로나타내었으며

,

통계는

Graph- ad Prism

의

unpaired t-test

를사용하여분석하였으며

,

대조군과

비교하여

P＜0.05

^일^때^{통계적으로}^유의^하다고^{판정하였다}

.

결과 및 고찰

특발폐섬유증은원인모르게폐조직의심한섬유화가진행 되며점차폐기능이저하되어사망하게되는질환이다

.

^진단^후 평균생존기간이

3-5

년정도되는예후가매우나쁜질병이다

(Tanaka et al., 2010).

최근연구되고있는특발폐섬유증에대한 치료에는항섬유화제제

,

항산화제

,

항응고제

,

사이토카인등을

사용하고있으나현재까지뚜렷하게효과가있는약제는보고 된바없다

(Antoniou et al., 2013).

이에본연구에서저자들은 다양한생리활성을보유한

fucoidan

을이용하여특발폐섬유증 에대한치료및억제효능이있는지확인하고자하였다

.

따라 서인간섬유모세포를이용하여

collagen

^젤의^수축성^분석

(Rat tail type I Collagen gel contraction), IL-6

발현분석

, α-SMA

발현분석

, collagen-1

발현분석및억제기전을분석함으로써 Fig. 1. The effect of fucoidan on Human pulmonary fibroblasts

viability. The cells were treated with various fucoidan concentrations (1-20 μg/mL) for 24 h. Cell viability was assessed using the CCK-8 assay, and the results are expressed as the percentage of surviving cells relative to the fucoidan untreated cells. Each value indicates the mean±S.D. and is representative of results obtained from three independent experiments.

0 20 40 60 80 100 120

C ell v iab ilit y (%)

Fucoidan (mg/mL)

0 1 5 10 20

No cell NT TGF-β-

TGF-β/F1 TGF-β/F5 TGF-β/F10

0 20 40 60 80 100 120

G el a rea (%)

* *

TGF-β (1 ng/mL) Fucoidan (mg/mL)

-

1 5 10

0 + 0

+ +

+ A)

B)

Fig. 2. Effect of fucoidan on TGF-β1-induced fibroblasts contrac- tile activity in rat tail type-1 collagen gel. (A) Human pulmonary fibroblasts (2×10⁵ cells/mL) were cultured in rat tail type I collagen gels (2 mg/mL) and pre-treated with fucoidan for 24 h. Cells were exposed to TGF-β1 (1 ng/mL). TGF-β1 treatment decreased the size of the gel while fucoidan pre-treatment blocked the reduction in gel size. (B) A summation of the percentage of gel surface area in each well of Fig. 2A to the control gel surface area. All values are mean±S.D. for three separate experiments, each performed in triplicate. *P<0.05; compared with the values of TGF-β1 treated group.

0 20 40 60 80

C ell v iab ilit y (%)

Fucoidan (mg/mL)

0 1 5 10 20

No cell NT TGF-β-

TGF-β/F1 TGF-β/F5 TGF-β/F10

0 20 40 60 80 100 120

G el a rea (%)

* *

-

1 5 10

0 + 0

+ +

+ A)

B)

(4)

fucoidan

의섬유화억제효능을평가하였다

.

먼저본연구에들어가기앞서

,

인간섬유모세포에서의

fucoi- dan

의세포독성을측정하였다

. Fig. 1

에서와같이

fucoidan

은

20 g/mL

^{농도에서도}^독성이^없어서^이후^실험은

20 g/mL

^이하 의농도를실험에사용하였다

.

다음으로

, fucoidan

에의한수축 능억제효능을분석을하였다

(Fig. 2).

근섬유모세포의과도한 수축능은심장

,

폐

,

간

,

콩팥

,

피부등의여러장기에서섬유화를 유발한다

(Hinz, 2007).

근섬유모세포의과도한수축능은응축 섬유

(stress fibers)

^의^특징인^분화된^{근섬유모세포의}

α-SMA

^에 의해유도된다

(Tomasek et al., 2002).

현재

,

근섬유모세포의과 도한수축능은

collagen gel

수축분석법을이용한다

. TGF-β1

를처리한

gel

에서는처리하지않은대조군보다수축이많이되 는것을확인할수있었다

.

이러한

gel

의수축정도는

fucoidan

의처리에의해억제되었고이억제수준은

fucoidan

^의^농도에 비례하여증가하였다

.

이러한결과는

fucoidan

이폐의섬유화 억제후보물질로서의가능성을가지는것을의미한다

.

TGF-β

는

TGF-β1, -β2, -β3

의아형이있으며이중

TGF-β1

이섬유화기전에가장연구가많이되어있다

. TGF-β

는다기능

적사이토카인으로세포증식및분화

,

세포자살에작용하고

,

세 포외기질단백질분해를억제하는

PAI-1 (plasminogen acti- vator inhibitor-1)

활성과

Type І collagen

과같은세포외기질 단백질축적을증가시켜섬유화유도에핵심인자로작용한다

(Guo et al., 2005; Leask and Abraham, 2004). TGF-β1

은정 상폐조직의기관지상피세포와섬유모세포에서생산되는주 된사이토카인이다

(Coker et al., 1996). TGF-β1

는섬유모세포 를근섬유모세포로의 분화를촉진하고

,

분화된근섬유모세포 의지표인

α-SMA

^을^발현을^증가^시킨다

(Hu et al., 2003).

^따 라서

,

폐섬유모세포에서의

α-SMA

와

collagen-1

의발현표현 조절기전을이해하면폐섬유화의발생과진행을억제할수있 는치료법을제시할수있을것이다

. TGF-β1

에의한폐섬유모 세포의섬유화유도유무와

fucoidan

에의한섬유화억제효과 를확인하고자

α-SMA

^와

collagen-1

^의^단백질^발현^정도를^확 인하였다

.

인간섬유모세포에

fucoidan

을

30

분동안전처리하 고

TGF-β1

의자극후

24

시간동안배양한세포로부터

α-SMA

와

collagen-1

항체를이용하여

Western blot

방법으로발현억 제효과를확인하고분석하였다

. Fig. 3A

에서와같이

TGF-β1

IL-6 (pg/ml)

0 200 400 600 800 1000 1200 1400 1600

**

** **

-

1 5 10

0 + 0

+ + + α-SMA

Collagen-1

Actin

Fucoidan (mg/mL) 0 0 1 5 10

0 0.5 1 1.5

0 0.4 0.8 1.2

a-SMACollagen-1

- 1 5 10

0 +0

+ + +

**

Relative intensity (%) control

-

1 5 10

0 + 0

+ +

+

t-Smad2 p-Smad2 p-Smad3

t-Smad3

A)

-

1 5 10

0 + 0

+ + + 0

0.4 0.8 1.2

*

** **

p-Smad3

0 0.4 0.8 1.2

** *

p-Smad2

Relative intensity (%) control

TGF-β (1 ng/mL) Smad 2/3 siRNA (nM)

-

20 40

0 + 0

+ +

Collagen-1 α-SMA

Actin

B)

*

**

0 0.5 1

Collagen-1α-SMA

-

20 40

0 + 0

+ +

*

Relative intensity (%) control

A)

p-AKT

t-AKT p-P38

t-P38 TGF-β (1 ng/mL) Fucoidan (mg/mL)

-

1 5 10

0 + 0

+ + +

**

0 0.4 0.8 1.2

-

1 5 10

0 + 0

+ + +

p-AKTp-P38

Relative intensity (%) control

Collagen-1

Actin α-SMA- TGF-β (1 ng/mL) SB203538 (mM)

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

0 +

10

B)

*

**

*

**

0 0.4 0.8 1.2

TGF-β (1 ng/mL) SB203538 (mM)

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

0 + 10 α-SMACollagen-1

** *

** **

Relative intensity (%) control

0 100 200 300 400 500 600

IL-6 (pg/mL)

#

**

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

0 +

10

C)

Fig. 3. The effect of fucoidan on α-SMA, collagen-1, and IL-6 production by TGF-β1-stimulated human pulmonary fibroblasts.

The Cells were seeded at 8×10⁴ cells/mL and incubated with various fucoidan concentrations (1, 5, and 10 µg/mL) for 1 h prior to TGF-β1 stimulation (1 ng/mL) (A) After treating with TGF-β1 for 24 h, the cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed using antibodies against α-SMA and collagen-1. Actin was used as an internal control for the western blot analyses. (B) After incubating for 24 h, the culture supernatant was collected, and the quantity of IL-6 was measured using an ELISA. Each value indicates the mean ± S.D. and is representative of results obtained from three independent experiments. (^#P<0.05 vs. control group; **P<0.01 vs. TGF-β1 group). Control, untreated cells.

(5)

후코이단의 항섬유화 효과

811

에의해

α-SMA

와

collagen-1

의단백질의발현이증가되었고

, fucoidan

에의해농도의존적으로그발현이감소되었다

.

인터루킨

-6 (interleukin-6, IL-6)

는면역및염증반응의조절 을포함한여러기능을갖는다면발현성사이토카인이다

. IL-6

는혈관내피세포

,

단핵구

,

대식세포및섬유모세포등에서생 성되어급성염증반응을촉진하거나억제시킨다

(Fries et al., 1994).

또한

,

폐섬유모세포의증식을유도하고

,

폐섬유증에서 분비가증가된다고알려져있다

(Pedroza et al., 2011). TGF-β1

로자극한인간섬유모세포에서

fucoidan

^의

IL-6

^분비^억제^효 능을알아보기위하여

, 1

시간전처리한후

, 1 ng/mL

의

TGF-β1

를

24

시간동안처리하였다

. TGF-β1

자극에의해분비가증

가된

IL-6

는

fucoidan

의 처리농도상승에의존적으로 그분 비량이유의하게감소하는것을확인하였다

(Fig. 3B).

따라서

,

TGF-β

수용체이후의신호전달체계를조절할수있으면폐섬

유화의발생과진행을차단할수있는가능성이있다

. TGF-β

의수용체를매개로하는생물학적활성은

Smad

의존 적인활성과

Smad

신호기전에비의존적인

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

와 같은

mitogen activated protein (MAP) kinases

와

phosphoinositide 3-kinase/Akt (PI3K/Akt)

^세포내^신호^전달 경로를통해조절된다

(Pan et al., 2016).

저자들은

fucoidan

이 위와같은세포내신호전달경로를차단하는효능이있는지

IL-6 (pg/ml)

0 200 400 600 800 1000

1200 **

** **

-

1 5 10

0 + 0

+ + + α-SMA

Collagen-1

Actin

0 0.5 1 1.5

0 0.4 0.8 1.2

a-SMACollagen-1

- 1 5 10

0 +0

+ + +

**

Relative intensity (%) control

-

1 5 10

0 + 0

+ +

+

t-Smad3

A)

-

1 5 10

0 + 0

+ + + 0

0.4 0.8 1.2

*

** **

p-Smad3

0 0.4 0.8 1.2

** *

p-Smad2

Relative intensity (%) control

-

20 40

0 + 0

+ +

Collagen-1 α-SMA

Actin

B)

*

**

0 0.5 1

Collagen-1α-SMA

-

20 40

0 + 0

+ +

*

Relative intensity (%) control

A)

p-AKT

t-AKT p-P38

-

1 5 10

0 + 0

+ + +

**

0 0.4 0.8 1.2

-

1 5 10

0 + 0

+ + +

p-AKTp-P38

Relative intensity (%) control

Collagen-1

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

0 +

10

B)

*

**

*

**

0 0.4 0.8 1.2

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

** *

** **

Relative intensity (%) control

0 100 200 300 400 500 600

IL-6 (pg/mL)

#

**

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

0 +

10

C)

Fig. 4. The effect of fucoidan on Smad2/3 signaling pathway. The Cells were seeded at 8×10⁴ cells/mL and incubated with various fucoidan concentrations (1, 5, and 10 µg/mL) for 1 h prior to TGF-β1 stimulation (1 ng/mL). (A) After treating with LPS for 30 min, the cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed using antibodies against Smad2 and Smad3. (B) The production of α-SMA and collagen-1 were blunted by siRNA-Smad2/3 transfection in TGF-β1-induced human pulmonary fibroblasts. siRNA-Smad2/3 transfected and non-transfected human pulmonary fibroblasts were stimulated with TGF-β1 (1 ng/mL) for 24 h. Each value indicates the mean±S.D. and is representative of results obtained from three independent experiments. (*P<0.05 and **P<0.01 vs. TGF-β1 group).

(6)

임미진

ㆍ

이대성

ㆍ

최그레이스

ㆍ

이정민

ㆍ

최일환

812 IL-6 (pg/ml)

0 200 400 600 800 1000

** **

-

1 5 10

0 + 0

+ + + Collagen-1

Actin

0 0.5 1 1.5

0 0.4 0.8 1.2

a-SMACollagen-1

- 1 5 10

0 +0

+ + +

**

Relative intensity (%) control

-

1 5 10

0 + 0

+ +

+

t-Smad3

A)

-

1 5 10

0 + 0

+ + + 0

0.4 0.8 1.2

*

** **

p-Smad3

0 0.4 0.8 1.2

** *

p-Smad2

Relative intensity (%) control

-

20 40

0 + 0

+ +

Collagen-1 α-SMA

Actin

B)

*

**

0 0.5 1

Collagen-1α-SMA

-

20 40

0 + 0

+ +

*

Relative intensity (%) control

A)

p-AKT

t-AKT p-P38

-

1 5 10

0 + 0

+ + +

**

0 0.4 0.8 1.2

-

1 5 10

0 + 0

+ + +

p-AKTp-P38

Relative intensity (%) control

Collagen-1

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

0 +

10

B)

*

**

*

**

0 0.4 0.8 1.2

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

** *

** **

Relative intensity (%) control

0 100 200 300 400 500 600

IL-6 (pg/mL)

#

**

-

1 10 0

0 + 0

+ + +

0 0 1

0 0

LY294002 (mM)

0 +

10

C)

Fig. 5. The effect of fucoidan on P38 MAPK and Akt pathway activation induced by TGF-β1. (A) Human pulmonary fibroblasts were pretreated with fucoidan and then cultured in 1 ng/mL TGF-β1 for 1 h. The levels of p-P38 and p-Akt were determined by Western blot analysis. Cells were pretreated with SB203580 (1 or 10 µM) and LY294002 (1 or 10 µM) 30 min prior to TGF-β1 stimulation. (B) After incubating for 24 h, the production of α-SMA and collagen-1 were determined by western blot analysis. (C) After incubating for 24 h, the culture supernatant was collected, and the IL-6 quantity was measured using an ELISA. (^#P<0.05 vs. control group; *P<0.05 and **P<0.01 vs. TGF-β1 group). Control, untreated cells.

를조사하였다

.

^{인간섬유모세포에}

fucoidan

^을

1

^시간^동안^전 처리하고

TGF-β1

의자극을준후

30

분이경과한세포로부터

Smad2

와

Smad3

의인산화신호기전을

Western blot

방법을이

용하여확인하였다

(Fig. 4A). TGF-β1

^의^자극에^의해

Smad2

와

Smad3

의인산화가촉진되었고

,

증가된인산화는

fucoidan

에의해감소되었다

. Fucoidan

의이러한세포내신호전달경

(7)

로차단효능을직접적으로검증하기위하여

small interfering RNA (siRNA)

를 사용해

Smad

유전자를

knock-down

시킨 후

α-SMA

와

collagen-1

의발현변화를관찰하였다

(Fig. 4B).

siRNA

도입후

α-SMA

와

collagen-1

단백질발현이감소한것 을확인하였다

.

또한

, Smad

신호기전에비의존적인세포내신 호전달경로인

MAPK

^와

Akt

^를^{확인하였다}

. Fig. 5A

^에서와^같 이

,

인간섬유모세포에

TGF-β1

의처리로

P38

과

Akt

의인산화 가유도되었다

.

증가된

P38

과

Akt

의인산화는

fucoidan

의처리 에의해감소되는것을확인할수있었다

.

또한

, P38

과

Akt

특 이억제제는

α-SMA

와

collagen-1

단백질의발현과

(Fig. 5B)

세포밖으로분비된

IL-6

^단백질의^발현양을^{감소시키는}^것을

확인할수있었다

(Fig. 5C).

따라서

,

본연구에서

fucoidan

의생 리활성효능은

Smad

의존적인활성과

Smad

신호기전에비의 존적인

p38 MAPK

과

Akt

신호전달경로를통해조절됨을확 인하였다

.

이와같이

, TGF-β1

의세포내신호전달경로에대한

fucoidan

^의^차단^효능은^폐^섬유화증^억제에^유용한^치료법이

될수있음을제시한다

.

이상의결과에서

fucoidan

이

TGF-β1

으로자극한인간폐섬 유모세포에서폐섬유증표지단백질

(α-SMA, collagen-1, IL-

6)

발현과세포내신호전달경로를차단하는효능을보유하

고있음을확인하였다

.

^따라서

,

^본^실험을^통하여

fucoidan

^은 폐섬유화증을차단할수있는항

-

섬유화물질로서의가능성 을제시하였다

.

추가적으로폐섬유화증실험동물을이용하여

fucoidan

의효능을검증하여야할것이다

.

사 사

본논문은

2011

년도인제대학교학술연구조성비보조에의

한것임

.

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