Journal of Life Science 2015 Vol. 25. No. 12. 1377~1383 ISSN (Print) 1225-9918 ISSN (Online) 2287-3406 DOI : http://dx.doi.org/10.5352/jls.2015.25.12.1377 Anti-inflammatory Effect of Scopoletin in RAW264.7 Macrophages Su-Gyeong Lee and Moon-Moo Kim* Department of Chemistry, Dong-Eui University, Busan 614-714, Korea Received July 21, 2015 /Revised October 14, 2015 /Accepted October 26, 2015 Scopoletin is a component of several plant such as Erycibe obtusifolia, Aster tataricus, Foeniculum vulgare and Brunfelsia grandiflora. It was reported to have anti-angiogenesis and anti-allergy effects. In this study, the anti-inflammatory effect of scopoletin was investigated in Raw264.7 cells, mouse macrophages. The effects of scopoletin on phagocytosis and nitric oxide (NO) production were investigated in lipopolysaccharide (LPS)-induced inflammatory responses. It was observed that scopoletin exerted inhibitory effects on both phagocytosis and NO production. In addition, scopoletin decreased the expression of inducible nitric oxide synthase (inos) and cyclooxygenase-2 (COX-2) which were related to NO and prostaglandin E2 (PGE2) production. In particular, the expression of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The expression levels of IL-1β, IL-6 were remarkably decreased by treatment with scopoletin. Furthermore, the content of TNFα produced by macrophage was decreased in the presence of scopoletin at 8 hr. These results indicate that the anti-inflammatory effect of scopoletin could exert by inhibiting the expression of pro-inflammatory cytokines in Raw264.7 cells stimulated with LPS. The above results suggest scopoletin could be a new remedial agent for anti-inflammation through inhibition of inos, COX-2, IL-1β, IL-6 and TNF-α expressions as well as supression of phagocytosis and NO production. Key words : COX-2, inos, phagocytosis, Raw264.7, scopoletin 서 최근경제가발전함에따라생활환경및식생활의변화로 인하여전세계적으로암, 당뇨병, 고혈압, 비만및혈관성질환 등의생활습관병이차지하는비율이매년증가하고있다. 이 와더불어, 현대사회의급격한산업발달로인한환경변화 그리고이에따른스트레스의증가등을포함하여다양한요 인으로인하여면역조절이상으로유발된염증이지속됨으로 써아토피, 천식등의만성염증질환이증가하고있다 [18, 37]. 염증반응은외부자극에대한생체조직의방어반응하나로 서, 물리적작용이나유해물질, 화학적자극, 세균감염등에 의한손상을수복재생하려는기전이다. 염증반응은급성염 증과만성염증으로나누어지는데특히만성염증이라고불리 는지속적인염증반응은오히려점막손상을촉진한다. 또한 만성염증반응은급성보다오래걸리고지속적이며단핵구나 대식세포, 림프구, 혈장세포등의침윤을동반하는특징이있 으며, 조직을파괴하고 [7, 15, 40], 동맥경화, 당뇨병, 관절염 및암과같은각종만성염증성질환을유발한다 [22]. 염증은 *Corresponding author *Tel : +82-51-890-1511, Fax : +82-51-890-2620 *E-mail : mmkim@deu.ac.kr This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (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. 론 염증세포인대식세포가외부의자극으로인해활성화되어염증매개물질을분비하면서시작되는데, 염증세포는동물체내모든조직에분포하며인체내에서선천적면역반응을담당하고인체면역체계에서중요한역할을하는백혈구인대식세포 (macrophage) 가주로관여한다. 이렇게자극된대식세포는천식, 기관지염, 관절염, 다발성경화증, 동맥경화증, 뇌졸중, 알츠하이머병이나파킨슨병과같은퇴행성뇌질환및바이러스감염으로인한염증질환등을유발하고, 질환을악화시키게된다 [1]. 대식세포를그람음성균주의세포벽조각인지질다당체인 lipopolysaccharide (LPS) 로자극하게되면 tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) 와같은 pro-inflammatory cytokine을증가시키며, nitric oxide (NO), prostaglandin E2 (PGE2) 등의염증매개물질을분비한다. 또한염증상태에서는 cyclooxygenase-2 (COX-2) 와 NO synthase (NOS) 가유도되어과량의 PGE2, NO 등이생성되며이것이질병악화의원인이된다 [21]. NO는염증의대표적인지표물질로, 일반적인 NO는박테리아를죽이거나종양을제거하는중요한역할을하지만염증에서의 NO생성은염증을유발시켜조직의손상, 유전자변이및신경손상을일으킨다. NO는 L-arginine 에서 NO synthase (NOS) 에의해합성된다. NOS에는 endothelial NOS (enos), neuronal NOS (nnos), inducible NOS (inos) 의세가지형태가있으며, 이들중 inos에의한 NO 생성이병을악화시키는주요원인이된다 [29, 36]. 염증을매개하는염증성 cytokine은면역세포에서생성된단백질중재자로외부항원에대한여러면역세포
1378 생명과학회지 2015, Vol. 25. No. 12 간의협력을중재한다. 그래서이들의생성과분비는면역반응조절에있어서매우중요하다. 현재 12가지이상의 cytokine 들이규명되었고그중에서 IL-1, IL-6 및 TNF-α 등을중심으로그기능들이알려져있다 [26]. TNF-α는주로급성염증반응에관여하고, 면역세포를조절하며, 인슐린저항성을유도하여당대사를저해시킨다 [11]. 또한 IL-6와같은다른염증성 cytokine을증가시킨다 [12]. 여기서 IL-6는대식세포에서합성되며 T 세포와 B 세포의분화에관여하며 IL-1과협동적으로작용한다 [27]. 또한 JAK-STAT (Janus kinase-signal transducer and activator of transcription) 신호전달과정을활성화시켜백혈구와후천성면역 (acquired immunity) 과관련된림프구인 T 및 B 세포를자극하여급성염증을만성염증으로전환시킨다 [14, 39]. 이러한매개인자의과잉된생산에의한지속적인염증반응은점막손상을촉진하고, 일부는동맥경화, 당뇨병, 관절염및암과같은각종만성염증성질환을유도한다 [22]. 이와같이 NO 및염증성 cytokine은염증반응과밀접하게관련되어있어이들의생성을조절할수있는물질이염증질환의예방및치료제로주목받고있다. 특히현재사용되고있는항염증제가위염, 신장염및심장질환같은문제를일으킨다고보고되어, 그사용이일부제한되고있다 [12, 23]. 보다안정적인항염증제가필요한실정이므로본연구에서는노화를억제한다고알려진 Brunfelsia grandiflora 유래의 scopoletin을이용하여 LPS로유도한 RAW 264.7 세포의염증반응을조사하고자한다. Scopoletin (7- Hydroxy- 6- methoxycoumarin) 은 coumarin유도체로 Erycibe obtusifolia, Aster tataricus 그리고 Foeniculum vulgare 같은많은약제식물에함유되어있다 [31]. 또한최근에노화진행의속도를늦추는데탁월한효능이보고된식물중남미기원인가지과의꽃관목에속하는 Brunfelsia grandiflora에도 scopoletin이함유되어있다고밝혀졌다 [13, 33]. Scopoletin은항염증, 항알레르기 [5] 그리고신혈관생성억제등다양한생물학적활성을가지고있다 [30]. 또한 scopoletin은복강내상처에서특히과발현된염증성 cytokine을하향조절하는효과를가지는것으로보고되어졌다 [32]. 하지만, 이와같은효과의정확한기전은아직밝혀진바가없어 Raw264.7 세포주에서 scopoletin이가지는항염증효과에대하여연구하고자하였다. 재료및방법세포배양및재료세포배양을위한 Dulbecco s Modified Eagle s Medium (DMEM), Trypsin-EDTA, penicillin / streptomycin / amphotericin ( 각각 10,000 U/ml, 10,000 μg/ml 및 2,500 μg/ml), fetal bovine serum (FBS) 시약은 Gibco BRL, Paisley, Scotland 로부터구입하였다. Raw264.7 세포는 American Type of Culture Collection, Manassas, USA으로부터제공받았다. Raw264.7 세포는 5% CO 2 및 37 에서 95% 이상의습도를유지한배양기에서 10% fetal bovine serum, 2 mm glutamine and 100 μg/ml penicillin -streptomycin을포함하는 DMEM 배지에서배양하였다. MTT assay Hansen [17] 의방법에따라 Raw264.7세포에대한 scopoleitn의세포독성을 MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) 를이용하여측정하였다. Nitric oxide assay Raw264.7 세포로부터생성된 NO의양은 Griess 시약을이용하여세포배양액중에존재하는 NO - 2 의형태로서측정하였다. Raw264.7 세포를 DMEM배지를이용하여 1 10 5 cell/well 로조절한후 24 well plate에접종하고 37, 5% CO 2 의습윤배양기에서배양하였다. 세포에 scopoletin을 1,2,4,8 and 16 μm 농도를전처리하고 1 hr 뒤 LPS를 1 μg/ml 처리한뒤 24 hr 배양하였다. 세포배양상등액과동량의 Griess 시약을혼합하여 96 well plate 에서 10분동안반응시킨후 540 nm에서흡광도를측정하고그비를 % 값으로환산하였다. Phagocytosis assay Raw264.7 세포를분리하여 DMEM배지를이용하여 24well plate에 24 hr 배양하였다. 세포에 scopoleitn을 1, 2, 4, 8 그리고 16 μm 농도를전처리하고 1 hr 후배양액을제거하였다. PBS 1 g/100 ml에녹인뒤 30분간가열과정을거쳐활성화시킨 zymosan A 10 μl와 NBT (nitroblue tetrazolium) 20 μl 그리고 PBS 470 μl를처리하였다. 충분히섞어준뒤 620 nm에서흡광도를측정하고그비를 % 값으로환산하였다. Western blot analysis Raw264.7 세포에용출완충용액 (50 mm Tris HCl, ph 7.5, 0.4% Nonidet P-40, 120 mm NaCl, 1.5 mm MgCl 2, 2 mm phenylmethylsulfonyl fluoride, 80 μg/ml leupeptin, 3 mm NaF and 1 mm DTT) 을첨가하여 4 에서 30 min 동안처리하였다. 10 μg의세포용출액을 10% Tris HCl gel에서전기영동후단백질을전기적으로 nitrocellulose membrane으로전이시켰다. 그다음 10% skim milk를 nitrocellulose membrane에전처리하고목적단백질에대한 1차항체 (anti-inos, anti- COX-2, anti-il-1β, anti-tnfα 그리고 anti β-actin) 를처리한다음 2차항체를처리후, chemiluminescent ECL kit (Amersham Pharmacia Biotech Piscataway, USA) 를사용하여목적단백질을검출하였다. Western blot의 band는 LAS3000 image analyzer (Fujifilm Life Science, Tokyo, Japan) 를이용하여관찰하였다.
Journal of Life Science 2015, Vol. 25. No. 12 1379 통계처리각실험은 3회이상반복실험을통하여그결과를얻어각각의시료농도에대해평균 ± 표준편차로나타내었다. 각시료농도군에대한유의차검정은대조군과비교하여 Student s test 한후 p<0.05 값을통계적으로유의성있는결과로간주하였다. 결 과 Scopoletin 의세포독성 scopoletin의대식세포에독성을미치지않는농도를결정하기위하여 MTT assay를수행하였다. Fig. 1에서보는바와같이 scopoletin은농도가증가하더라도세포독성이나타나지않았다. 따라서 scopoletin은 16 μm 농도이하의범위에서는세포독성이없는것으로관찰되었다. Scopoletin 의 NO 생성억제효과 LPS에의해 Raw264.7 세포로부터생성되는 NO에대한 scopoletin의억제효과를세포배양액으로부터 Griess assay 방법에의해측정하였다. Fig. 2에서보는바와같이 scopoletin의 1, 2, 4, 8 그리고 16 μm 처리농도에서 NO의생성억제효과를측정한결과 8 μm 이상의농도로처리한세포군에서 NO의생성함량은대조군와비교했을때약 20% 정도 NO의생성이억제되는것으로나타났다. Fig. 2. Effect of of scopoletin on nitric oxide in Raw 264.7 cells. The cells cultured in phenol red and serum-free media were pretreated with different concentrations of of scopoletin for 1 hr and stimulated with a 1 μg/ ml of LPS for 48 hr. Conditioned medium was mixed with an equal amount of the griess reagent, and the absorbance was measured at 550 nm. The values obtained in this study were compared with value of LPS group and were calculated as percentage. Data are given as means of values ± SD from three independent experiments. Level of significance was identified statistically (**, p<0.01; ***, p<0.001) using Student s t test. Scopoletin 의대식세포의식균작용억제효과 Scopoletin의 zymosan A에의해활성화된 Raw264.7 세포의식균작용억제능을조사하였다. Fig. 3에서보는바와같이 NBT 발색법을이용하여식균작용억제능을조사한결과 zymosan A를처리하에서 scopoletin은 1, 2 and 4 μm 농도에서 Fig. 1. Effect of scopoletin on viability of Raw 264.7 cells. Raw 264.7 cells were treated with scopoletin at 1, 2, 4, 8 and 16 μm and cell viability was determined using MTT assay after 48 hr. Data are given as means of values ± S.D. from three independent experiments. Level of significance was identified statistically using Student s t test. Fig. 3. Inhibitory effect of scopoletin on phagocytosis induced by zymosan A. Phagocytosis of RAW264.7 cells was evaluated by NBT. Cells cultured were pretreated with different concentrations of scopoletin for 1 hr and stimulated with zymosan A for 6 hr. The values obtained in this study were compared with value of zymosan group as control and were calculated as percentage. Data are given as means of values ± SD from three independent experiments. Level of significance was identified statistically (**, p<0.01) using Student s t test. 는식균작용을억제시키지못하였으나, 8 μm 이상의농도에서약 10% 정도의식균작용억제능을나타내었다.
1380 생명과학회지 2015, Vol. 25. No. 12 Scopoletin 의염증관련 cytokine 의단백질발현조절효과 scopoletin의염증관련 cytokine의단백질수준에서의발현억제효과를조사하였다. scopoletin로전처리된 Raw264.7 세포에 LPS로자극하였고, β-actin으로단백질의정량화를나타내었다. NO를생성하는 inos의발현을조사한결과 4 μm 이상의농도에서 inos의단백질발현수준을공시험군과비슷한수준까지억제하였다. COX-2의단백질수준에서의발현억제효과를조사한결과 scopoletin은 COX-2의발현을 LPS 군보다억제하였지만많은효과를나타내지못하였다. 또한 scopoletin은급성염증과만성염증을유발한다고알려진 IL-6 와이와협동적으로작용하는 IL-1β의단백질발현조절효과를조사한결과, scopoletin 처리군에서 IL-6과 IL-1β 둘다 4와 8μM 농도에서단백질발현수준이감소하였다. Scopoletin 의시간에따른 TNFα 단백질발현조절효과 TNFα는활성화된 macrophage에서분비되어염증반응을일으키는 cytokine으로 sample 처리후 8 hr 및 24 hr을방치하여단백질발현조절효과를조사하였다. Fig. 5에서보는바와 Fig. 5. Effect of scopoletin on protein expressions of TNFα and COX-2 in Raw264.7 cells. The cells were treated with scopoletin at 1, 2, 4, 8 and 16 μm prior to stimulation of cells with LPS at 1 μg/ml for 24 hr. In case of TNFα, the cells were treated for 8 hr and 24 hr with scopoletin at 1, 2, 4, 8 and 16 μm prior to stimulation of cells with LPS at 1 μg/ ml, respectively. Aspirin was used as a positive control. Western blot analysis of cell lysates was performed using antibodies as indicated. The expression level of β-actin was used as a control for normalization of target proteins. The values of scopoletin treatment groups obtained in this study were compared with value of LPS group. Data are given as means of values ± SD from three independent experiments. Level of significance was identified statistically (**, p<0.01; ***, p<0.001) using Student s t test. 같이 scopoletin 은 8 μm 농도에서 LPS 단독처리군에비하여 TNFα 의발현을억제하는것으로나타났다. 고 찰 Fig. 4. Effect of scopoletin on protein expressions of inos, IL-6 and IL-1β in Raw264.7 cells. The cells were treated with scopoletin at 1, 2, 4, 8 and 16 μm prior to stimulation of cells with LPS at 1 μg/ml for 24 hr. Dexamethason was used as a positive control. Western blot analysis of cell lysates was performed using antibodies as indicated. The expression level of β-actin was used as a control for normalization of target proteins. The values of scopoletin treatment groups obtained in this study were compared with value of LPS group. Data are given as means of values ± SD from three independent experiments. Level of significance was identified statistically (**, p<0.01; ***, p<0.001) using Student s t test. 염증은생체나조직에물리적작용이나화학적물질, 세균감염등의어떠한기질적변화를가져오는침습이가해질때그손상부위를재생하려는기전으로 [3] 염증반응이지속적으로또는과도하게일어나면여러염증성질환이나다양한질환의원인이된다 [6]. 식균작용 (Phagocytosis) 은선천성, 적응성면역반응으로병원균감염에대한첫번째방어라인으로작용한다 [37]. 식균작용이일어날때 macrophage의막이항원을완전히둘러싸서 phagosome이되면 macrophage 안으로들어가서 lysosome과융합되어 phagolysosome을형성하여안에있는항원을분해한다 [2]. 또한식균작용은염증매개체의분비와항원의침입으로인해자극된다 [16]. 따라서염증
Journal of Life Science 2015, Vol. 25. No. 12 1381 매개체의분비가증가할수록식균작용도증가하므로만성적으로지속되는염증을억제하기위해서는식균작용을억제시켜야한다. 본연구에서 scopoeltin은 LPS로자극된 Raw264.7 세포에서농도가증가함에따라 macrophage의식균작용을감소시키는것으로나타났다. 식균작용이계속지속된다면염증이만성적으로일어나고있는것인데, scopoletin은이러한식균작용을감소시켜만성적으로일어나는염증을억제시킬것으로사료된다. 이전연구에서대식세포는선천면역뿐만아니라획득면역등다양한숙주반응에관여하여항상성유지에관여하는것으로알려져있으며, 염증반응시에는 NO와 cytokine을생산하여감염초기에생체방어에중요한역할을한다 [9, 19]. 뿐만아니라대식세포는 cytokines을생산하여감염초기에생체방어에중요한역할을한다고보고되었다 [28, 29]. 일반적으로 NO의형성은박테리아를죽이거나종양을제거시키는중요한역할을하지만, 염증상태에서 inos에의해과잉생산된 NO는혈관투과성, 부종등의염증반응을촉진시킬뿐만아니라염증매개체의생합성을촉진하여염증을심화시키는것으로알려져있다 [35, 38]. PGE2는통증, 발열에관여하는염증매개체로염증반응과면역반응에관여하고신생혈관 (angiogenesis) 을촉진시켜암발생에깊이관여하고있는것으로알려져있다 [24]. Scopoletin의 NO와 PGE2 생성억제에대한효과는쥐의귀염증모델에서의연구에서보고된바가있고 [10]. PGE2는본연구와같은 cell인 Raw264.7에서도억제효능을나타냈다 [20]. 본연구에서 LPS로자극된 Raw264.7 세포에서 scopoletin은 NO의생성을억제하는것으로나타났다. 또한 NO와 PGE2는각각 NOS와 COX-2에의해서생산된다 [34]. COX는 arachidonic acid를 prostaglandins로전환하는효소로 COX-1과 COX-2로나뉘어지는데, 이는다양한세포들에서각각다른발현경향을나타낸다. COX-1은정상적인생체기능에작용하는반면, COX-2는염증부위에서발현이된다. COX-2에의해생성되는 PGE2는통증, 발열에관여하는염증매개체로서염증반응, 면역반응등에반응한다 [24]. 또한 NO 의합성은 3가지 NOS에진행된다. NOS의종류에는 neuronal NOS(nNOS/NOS I), endothelial NOS(eNOS/NOS III) 그리고 inducible NOS(iNOS/NOSII) 의크게세가지가있다. inos 는 L-arginine을세포내산화반응에의해 N-hydroxy- L-arginine을중간체로하여 NO를생성하는반응을촉매하게된다 [28]. 본연구에서 scopoeltin은 LPS로자극된 Raw264.7 세포에서농도가증가함에따라 inos와 COX-2의발현을감소시키는것으로나타났다. 이는 scopoletin이 NO와 PGE를감소시키는것이단백질발현감소에서기인한것으로사료된다. 염증의초기단계에서항원을침입자로인식하여인체의면역세포에신호전달을하여면역기능이활성화되도록하거나외부침입자를직접제거하게된다. 면역세포들은 interleukin 과같은염증성 cytokine을분비하여면역, 염증반응을일으키 게된다 [11]. 염증성 cytokine은면역세포에서생성되는단백질중재자로외부항원에대한여러면역세포간의협력을중재하므로이들의생성과분비는면역반응조절에있어서매우중요하다. 현재 12가지이상의 cytokine들이규명되었으나대부분최근에밝혀진것으로기능이많이알려져있지않으나그중에서 IL-1, IL-2, IL-6 및 TNF-α 등을중심으로그기능들이알려져있다 [26]. TNF-α는주로급성염증반응에관여하고, 면역세포를조절하며, 인슐린저항성을유도하여당대사를저해시킨다 [4]. 또한 TNF-α는 interleukins 발현을증가시켜염증반응진행에서중요한역할을할뿐아니라대사증후군, 비만, 인슐린저항성의발생에도관여한다 [41]. IL-1β는다양한염증성자극요인에의하여활성화된단핵구, 대식세포, B 세포, 수지상세포등에서분비되는것으로, T 세포를활성화시키고 B 세포의성숙을돕는역할을하여염증을심화시키며, 염증반응시시상하부에작용하여발열증상을유도하는 cytokine이다 [8, 25]. 본연구에서 scopoeltin은 LPS로자극된 Raw264.7 세포에서농도가증가함에따라 IL-1 β, IL-6을억제하는것으로나타났다. 또한 TNF-α의발현을 8 hr 및 24 hr에서조사한결과 8h에서발현이감소하는것으로나타났다. 이는 scopoletin이염증성 cytokine인 IL-1β, IL-6, 그리고 8 hr에발현이일어나는 TNF-α의발현을감소시켜염증을억제시키는것으로사료된다. 따라서 scopoletin이과도하게일어나는식균작용과 NO와 PGE2의생성을 inos 와 COX- 2 단백질수준에서억제하여염증의진행을막고, 염증반응을활성화시키는 IL-1β, TNF-α, IL-6 같은염증성매개인자의발현을억제하여만성염증을억제하는것으로사료된다. 이러한연구결과를바탕으로 scopoletin이만성염증을억제시키는새로운생의학소재로이용될수있으리라기대된다. 감사의글 이논문은 2015년도정부 ( 미래창조과학부 ) 의재원으로한국연구재단의기초연구사업지원을받아수행된것임 (No. 2013R1A1A1A05005160). 동시에이연구는 2015년한국여성과학기술인지원센터의지원을받아연구되었습니다. References 1. Albina, J. and Reichner, J. 1995. Nitric oxide in inflammation and immunity. New Horiz 3, 46-64. 2. Alvarez, M. and Casadevall, A. 2006. Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages. Curr. Biol. 16, 2161-2165. 3. Barrett, J. T. 1983. Textbook of immunology: an introduction to immunochemistry and immunobiology: Mosby. 4. Bradley, J. 2008. TNF mediated inflammatory disease. J. Pathol. 214, 149-160. 5. Cheng, A. S., Cheng, Y. H. and Chang, T. L. 2012. Scopoletin
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