2) 대한한방부인과학회지 J Korean Obstet Gynecol VOL.26 NO.2 : (2013) 금은화의 type Ⅰ interferon 억제효과및기전에관한연구 원광대학교한의과대학한방부인과학교실강용구, 유익한, 김송백, 최창민, 서윤정, 조한백

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1 2) 대한한방부인과학회지 J Korean Obstet Gynecol VOL.26 NO.2 : (2013) 원광대학교한의과대학한방부인과학교실강용구, 유익한, 김송백, 최창민, 서윤정, 조한백 ABSTRACT A Study on the Inhibitory Effect and Mechanism of Lonicera Japonica on Type Ⅰ Interferon Yong-Goo Kang, Ik-Han Ryu, Song-Baek Kim Chang-Min Choi, Yun-Jung Seo, Han-Baek Cho Dept. of Oriental Obstetrics & Gynecology, College of Oriental Medicine, Won-Kwang University Objectives: The purpose of this study was to investigate whether Lonicera japonica(lj) could inhibit LPS-induced type Ⅰ IFN production. Methods: To evaluate inhibitory effect of LJ on type Ⅰ IFN, we examined type Ⅰ IFN, IRF-1, 7 and IL-10 production on LPS-induced macrophages using real time RT-PCR. Next, we observed the interaction of type Ⅰ IFN, IRF-1, 7 and IL-10 using IL-10 neutralizing antibody. Finally we examined the activation of STAT-1, 3 using western blot. Results: LJ inhibited Type Ⅰ IFN expression of mrna and increased IL-10 expression of mrna. Also LJ inhibited the level of IRF-1, 7 mrna and the nuclear translocation of IRF-3. Further more, LJ reduced the activation of STAT-1, 3 which are involved in continuous secretion of immune cytokines. Blockade of IL-10 action caused a significant reduction of type Ⅰ IFN and IRF-1, 7 than LPS-induced LJ pretreatment. Conclusions: LJ inhibits LPS-induced production of type Ⅰ IFN by IL-10. This study may provide a clinical basis for anti-inflammatory properties of LJ. Key Words: Lonicera japonica, Type Ⅰ interferon(ifn), Interleukin-10(IL-10), Interferon regulatory factor(irf), Signal transducers and activators of transcription (STAT) " 본연구는 2011 년도원광대학교교내연구비지원으로연구되었음 " Corresponding author(han-baek Cho) : Won-Kwang Univ. Sanbon Oriental Medicine Hospital, , Sanbon-dong, Gunpo-si, Gyeonggi-do, Korea Tel : Fax : chbhan@wonkwang.ac.kr 17

2 Ⅰ. 서론 염증반응은활성화된면역세포에의해일어나는일련의면역반응으로 1), 면역계를동원하는생체의방어기전과상처의치유에핵심적인역할을하고있을뿐만아니라많은질병의병리발생에관련되어있는중요한과정이다 2). 대식세포는외부침입물질에대한방어기전을신속하게보내는역할을하며, 염증반응중에대식세포가활성화되면많은염증성인자들을생산하는데, 그인자들에는 TNF-α, interleukin, leukotriens, nitric oxide, interferon 등이있다 3-6). 이러한염증성인자중에 IFN은항바이러스와항박테리아반응, 면역조절의활성, 세포증식의억제, 세포사멸, 항암반응을유도하는데중추적인역할을하는 cytokine 이다 7-11). IFN-α와 IFN-β는 type Ⅰ IFN으로써바이러스나 LPS에의한 typeⅠ IFN 의유도는선천면역에서중요하며, TLR 과같은대식세포표면수용체의활성화에의해매개되어염증성 cytokine 생성에영향을미친다 12,13). IRF는바이러스에감염되면빠른시간내에활성화되는전사활성인자로, 바이러스감염시 IFN 생산을통하여면역반응을조절하게되는데 14), type I IFN의조절은 IRF-1, 7이주로관련되어있으며 15-18), 대식세포의 TLR4가 LPS와결합하여 IRF-1, 7을발현시킴으로써 IFN-α/β 을생성한다 14,19,20). 그러나 IFN-α/β 생성이지나치게되면수지상세포등항원제시세포의성숙을촉진하고세포독성 T 림프구를활성화시켜세포파괴를유 도하는한편, IFN-α/β와결합한세포에서나오는자가항원-항체반응을강화시켜염증성장염이나, 류머티즘, 피부결핵등과같은자가면역질환을야기할수있다 21-23). IL-10은면역억제와항염증작용을하는 cytokine 으로다핵호중구, 단핵구, 대식세포로부터생산되는염증성 cytokine 의생산을억제하고 IL-10의항염효과를증진시키는물질들의발현을상향조절한다 24). 따라서급성감염기간동안생긴염증반응의악화를방지하여염증반응으로생기는병변을예방할수있다 25). 금은화는忍冬科에속한多年生半常綠木質藤本인忍冬의꽃봉오리를건조한것으로性味는甘寒하며肺, 胃, 心으로歸經하고淸熱解毒, 凉散風熱효능이있 어 26) 다양한염증성질환에상용되는대 표적인항염증약물중의하나로알려져있다 27). 최근금은화에관한연구가항염증 28-31), 항균효과 32), 항암효과 33,34) 등의분야에서활발히진행되고있으나주로 PGE2, TNF-α, IL-1, IL-6, COX-2 등의 cytokine 과 NO 생성억제에관한내용들 35-38) 이보고되었고, 금은화가첨가된처방으로五味消毒飮에대한 IFN 억제효과 39) 에관하여보고된바있으나, 금은화의직접적인 IFN 억제효과에대한연구는미미한실정이다. 이에저자는금은화가 IFN-α/β의생성에어떻게관여하는지알아보고자금은화의물추출물을제조하여 LPS로유도한대식세포에서 IFN-α/β의생성에미치는기전에대해서조사하여유의성있는결과를얻었기에보고하는바이다. 18

3 J Korean Obstet Gynecol Vol.26 No.2 May 2013 Ⅱ. 실험방법 1. 재료 1) 약재실험에사용한금은화 (Lonicera japonica, LJ) 는옴니허브 ( 경북영천, 대한민국 ) 에서구입하여정선해서사용하였다. 2) 동물실험동물인 C57BL/6 mice 는 Orient Bio Co.( 경기도성남, 대한민국 ) 에서구입하였다. 3) 시약및기기세포배양에사용한 RPMI-1640, Fetal bovine serum(fbs), penicillin/streptomycin 는 Gibco BRL(Grand Island, NY, USA) 에서구입하였다. Thioglycollate(TG) 는 BD Pharmingen(San Diego, CA, USA) 에서구입하였고, Tri-zol reagent와 polymerase chain reaction(pcr) reaction kit, IL-10 중화항체는 Invitrogen cooporation(carlsbad, CA, USA) 에서구입하였으며, 실험에사용한항체인 Phospho STAT-1, 3 항체는 Cell signaling(beverly, MA, USA) 에서구입하였다. 실험에사용된 Thermo scientific 은 Tewksbury(MA, USA), Bio-rad 는 Hercules (CA, USA), Applied Biosystems 은 Invitrogen cooporation Carlsbad(CA, USA) 에서구입하였다. 2. 방법 1) 검액의제조금은화 100 g을물 1 l에약탕기 ( 대웅, 한국 ) 로 3시간가열추출한다음여과한후, 동결건조하여건조분말 g( 수득율 : 23.56%) 을얻었으며, 생리식염수에녹여 stock solution(100 mg/ml) 을제조하였다. 제조된 stock solution 은 -20 에보관하면서분석방법에적합하도록희석하여사용하였다. 2) 복강대식세포배양실험 3~4일전에실험 mouse에염증물질인 thioglycollate 2~3 ml을복강내에투여하고, RPMI % FBS medium 6~7 ml를복강내에투여한다음복강액을뽑아내원심분리한후 cell 을 counting 해서 Dish나 Plate 에깔고 5% CO 2 37 가유지되는 incubator에서 3시간배양하고, suspension 세포를버린후에부착한세포를대식세포로간주하여실험하였다. 3) MTT 분석대식세포의생존율은밀집세포의미토콘드리아탈수소효소에의해자줏빛 formazan 생성물로변하는 MTT 환원을바탕으로 MTT 분석법으로측정했다. 세포들을 RPMI 배지에서 cells/well 의밀도로현탁하여 100, 250, 500 μg/ml 의농도로금은화물추출물을처리하였다. 24시간배양후, 5 mg/ml의농도로 MTT 용액을첨가하고다시 30분동안배양하였다. MTT-formazan 생성물은 DMSO 200 μl를첨가함으로써용해했다. formazan 의양은解液을 96-well plate에 loading 하여, spectrophotometer(md, USA) 를이용하여 540 nm에흡수되는양을측정함으로써결정했다. 세포의생존율은어떠한처치도가하지않은 control cells 과의비율로나타내었다. [ 즉, viability(%) =100 (absorbance of treated sample) /(absorbance of control)] 4) RNA 추출 Total RNA는 Tri-zol 시약을이용하여추출하였다. 먼저배양한세포에금 19

4 은화물추출물을전처리하고 LPS로자극한후 1시간배양한세포를 PBS로 2회씻은다음 PBS를 1 ml 씩가해세포를포집한후, 원심분리하여위에 PBS는버리고바닥에남은세포를 Tri-zol reagent (Invitrogen, USA) 용액을 1 ml 넣어서세포를용해시키고 100 μl의 chloroform 용액을가하여두세번잘섞어준뒤 12,000RPM 에서 15분간원심분리하여맨위의상층액을취하였다. 그후 2-propanol 과 1:1로섞고 12,000RPM에서 10분간원심분리하여위에상층액은버리고남은침전물을 80% ethanol로 2회씻고침전물을건조시킨뒤침전물에 DEPC 처리한증류수를 15 μl씩넣어 RNA를용해시키고정량하였다. 추출한 RNA는 reverse transcriptase PCR의 protocol을사용하여 cdna로합성하였다. 간단히기술하자면역전사반응을위하여 1 mg의 total RNA에 0.5 mg의 oligo-(dt) 을넣고 70 에서 10분간변성시켰다. 1X single strand buffer, 0.5 mm DTT, 500 mm dntps, 200 Unit reverse transcriptase를첨가하고 42 에서 1시간동안반응시켰다. 그후에 PCR은각각의 tube에 1 ml cdna, 1X PCR buffer, 1 mm MgCl2, 200 mm dntps, 0.2 mm 의 primer 를넣고 PCR 조건인 92 에서 30초, 58 에서 45초, 72 에서 30초를 30cycle을반복하였다. 5) Primers and probes Forward(f) 와 reverse(r) primer는 Invitrogen(Carlsbad, CA, USA) 에서합성하였고 TaqMan probe는 MWG-Biotech AG(Ebersberg, Germany) 에서합성하였으며 TaqMan minor groove binder(mgb) probe(applied Biosystems, USA) 는 Primer Express 1.5 software(applied Biosystems, USA) 를사용하여디자인하였다. 6) 면역염색법마우스복강내에서추출한대식세포에금은화물추출물을 3시간동안전처리한군과처치하지않은대조군에 500 ng/ml LPS로 37 에서 1시간동안자극하고세포를고정시키기위해 PBS로 3회세척한후 4% paraformaldehyde용액에 30분간배양하였다. 그뒤 0.1% TritonX-100 용액으로상온에서 15분간배양하였다. 세척을하고 blocking serum으로 1시간 blocking 한뒤 1:100으로희석한 1차항체 IRF-3 antibody(zymed, , Invitrogen, Carlsbad, CA92008, USA) 를넣어하룻밤동안배양하고다음날 1:50으로희석한 Alexafluor 568 형광이붙어있는 2차항체 goat anti-rabbit IgG(Invitrogen, USA) 로 4시간동안암실에서반응시키고핵염색을위하여 1:1000으로희석시킨 DAPI 로 30분간염색시켜형광현미경으로관찰하였다. 7) Western blot analysis 복강에서추출한마우스대식세포를 60 mm culture dish에 cells/ml로세포를배양하고 serum free media(rpmi1640) 로 12시간 starvation 시키고금은화물추출물을 1시간전처리한뒤에 500 ng/ml 의 LPS로자극하여 cold PBS로 3회세척하였다. 4시간후, cell 을수확하여얻은뒤 5000RPM 으로 5분동안원심분리하여그상층액을버리고 cell pellet 을수거한후 lysis buffer(lysis buffer 1 ml + phosphotase inhibitor 10 μl + protase inhibitor 10 μl) 를넣어단백질을 lysis 시켜서 15000RPM으로 20분동안원심분리하여찌꺼기를가라앉히고단백질을정량 20

5 J Korean Obstet Gynecol Vol.26 No.2 May 2013 하였다. 동일한양의단백질에 sampling buffer(4x) 를같이넣어섞은다음그샘플을 10% SDS-PAGE에전기영동한후 membrane 에옮기고나서 5% skin milk 로 2시간동안 blocking 하였다. STAT-1, 3의 phosphorylation과 STAT-1, 3를 ECL detection 용액 (Amersham, Carlsbad, CA, USA) 으로확인하였다. 2. IFN-α/β의 mrna 생성에미치는영향대식세포에금은화물추출물을 3시간전처리한다음, LPS로 1시간자극하고 real time RT-PCR 의방법을이용하여확인한결과, 금은화물추출물은 IFN-α/β 의 mrna 발현을농도의존적으로억제하였다 (Fig. 2). 3. 통계처리실험결과에대한통계처리는 SPSS version 10.0을이용하여 Student's t-test 에준하였고 p-value 가 0.05 미만일경우유의한것으로판정하였다. Ⅲ. 결과 1. 세포독성대식세포에금은화물추출물을처리하여 24시간후에세포의생존율을측정한결과금은화물추출물은대식세포에독성을나타내지않았다 (Fig. 1). Fig. 1. Cytotoxic Effect of LJ on Murine Peritoneal Macrophages. Macrophages were incubated with LJ as indicated dose for 24 h. The cell viability was measured by MTT assay as described in materials and methods. Data are mean of three independent experiments. The similar results were obtained from three additional experiments. Fig. 2. Effect of LJ on LPS-induced IFN-α/β Expression. The cells were pre-treated with LJ water extract at indicated concentration for 3 h, and then incubated with LPS for 1 h. IFN-α/β mrna expression were examined by real time RT-PCR. * P<0.05 : significant as compared to saline. P<0.05 : significant as compared to LPS. Data represent the mean±s.e. of three independent experiments. 21

6 3. IRF-1, 7의 mrna 생성에미치는영향금은화의면역조절기전을조사하기위하여 IFN-α/β의생성에중요한역할을한다고알려진 IRF-1, 7의 mrna 생성을측정하였다. real time RT-PCR을이용하여 IRF-1, 7에금은화물추출물이미치는영향을조사한결과금은화물추출물은 IRF-1, 7의 mrna 생성을유의성있게억제하였다 (Fig. 3). 4. IRF-3의핵이동억제에미치는영향금은화의면역조절기전을알아보기위하여 IFN-α/β의생성에중요한역할을한다고알려진 IRF-3가핵으로이동하는지를관찰하였다. 면역염색법을이용하여금은화물추출물이 IRF-3에미치는영향을조사한결과금은화물추출물은 IRF-3가핵으로이동하여 IFN 을생성하는과정을억제하였다 (Fig. 4). Fig. 4. The Effect of LJ on LPS-induced IRF-3 Translocation. The cells were pre-treated with LJ water extract at indicated concentration for 3 h, and then incubated with LPS for 1 h. Nuclear translocation of IRF-3 was examined using immunostaining. Data represent the mean±s.e. of three independent experiments. Fig. 3. The Effect of LJ on LPS-induced IRF-1, 7 Expression. The cells were pre-treated with LJ water extract at indicated concentration for 3 h, and then incubated with LPS for 1 h. IRF-1, 7 mrna expression were measured using real-time RT-PCR. * P<0.05 : significant as compared to saline. P<0.05 : significant as compared to LPS. Data represent the mean±s.e. of three independent experiments. 5. IL-10을통한면역조절에미치는영향금은화의 IFN-α/β의생성억제기전을알아보기위하여, IL-10의 mrna 발현을조사한결과 IL-10은금은화물추출물을전처리하였을때생성이증가하였다 (Fig. 5A). 또한 mrna 수준에서금 22

7 J Korean Obstet Gynecol Vol.26 No.2 May 2013 은화물추출물의처리시간에따른 IL-10 의생성 (Fig. 5A) 과 IFN-α/β, IRF-1의 억제효과가일치하였다 (Fig. 5B, 5C). A) B) C) Fig. 5. IL-10 Has Association in LPS- induced IFN-α/β Inhibition of LJ. (A) The cells were pre-treated with LJ water extract (500 μg/ml) for indicated times, then IL-10 mrna expression was measured by real time RT-PCR. (B and C) The cells were pre-treated with LJ water extract (500 μg/ml) for indicated times, and then incubated with LPS for 1 h. IFN-α/β and IRF-1, 7 mrna expression were examined by real time RT-PCR. * P<0.05 : significant as compared to saline. P<0.05 : significant as compared to LPS. Data represent the mean±s.e. of three independent experiments. 6. 중화항체처리후 IL-10을통한면역조절에미치는영향금은화물추출물이 IL-10을통한면역조절기전에미치는영향을더욱정확하게검증하기위해 IL-10 중화항체인 IL-10 neutralizing antibody를이용하여 실험한결과, 금은화물추출물만을처리했을때 IFN-α/β, IRF-1, 7의생성이억제되는것에반해 (Fig. 6A, 6B), IL-10 중화항체를처리했을때는 IFN-α/β, IRF-1, 7의억제효과가나타나지않았다 (Fig. 6A, 6B). 23

8 A) B) Fig. 6. IL-10 Has Association in LPS-induced IFN-α/β Inhibition of LJ. The cells were pre-treated with neutralizing antibody (10 μg/ml) for 1 h, then treated with LJ water extract for 3 h, and then incubated with LPS for 1 h. IFN-α/β (A) and IRF-1, 7 (B) mrna expression were examined by real time RT-PCR. nil-10 : IL-10 neutralizing antibody. * P<0.05 : significant as compared to saline. P<0.05 : significant as compared to LPS. Data represent the mean±s.e. of three independent experiments. 7. STAT에미치는영향 LPS로자극한대식세포에서는 2~3시간부터인산화가진행되어신호전달이시작되는것에비해금은화물추출물을전처리한곳에서는 STAT-1, 3 모두인산화되지않았다 (Fig. 7). Phosphorylation. The cell were pre-treated with LJ water extract at indicated concentration for 3 h, and then incubated with LPS for 4 h. Western blot analysis was performed to detect the phosphorylation of STAT-1, 3 (pstat-1, 3). STAT-1, 3 were loading control. Similar results were obtained in three independent experiments. Ⅳ. 고찰 Fig. 7. Effect of LJ on STAT-1, 3 금은화 (Lonicera japonica) 는忍冬科 (Caprifoliaceae) 에속한忍冬 (Lonicera japonica Thunb) 의花雷를건조한것으로여름철꽃이피기전에채취한것을약재로사용하며, 性은寒하고, 味는甘 24

9 J Korean Obstet Gynecol Vol.26 No.2 May 2013 하며, 淸熱解毒, 凉血止血, 散風熱의효능이있어 26) 癰腫疔瘡, 腫瘍, 喉痺, 丹毒, 疥癬, 楊梅惡瘡, 五種尸疰, 瘰癧, 痔漏, 熱毒血痢, 風熱感冒, 溫病發熱등을치료 하는 40) 대표적인항염증약물이다 41). 알 콜또는물을용매로추출하여사용되며 41) 주성분으로는 luteolin, inositol, saponin, tannin 과 ginnol, sterols, glycoside, chlorogenic acid, flavonoid 화합물및 isochlorogenic acid 등이있다 42-44). 한의학적으로금은화가배합된방제를보면 東醫寶鑑 에는金銀花散, 內托千金散, 仙傳化毒湯, 連翹敗毒散, 天花散, 淸熱消毒飮, 托裏消毒飮등 45) 이수록되어瘰癧, 癰疽, 疔疽, 天疱瘡, 便癰, 乳癰등넓은의미의化膿性炎症병변치료에응용되었다 46). 염증반응이일어나면대식세포는외부침입물에대한방어기전으로많은염증성인자들을생산하게되는데 3,4) 염증성매개물질이과량생산되면, 과도한면역반응을야기하게되어인체의각종질환을악화시키는요인이된다. 따라서염증반응을억제하는것은염증성질환을치료하는데중요하다 47,48). LPS(Lipopolysaccharide) 는그람음성세균의균체내독소로외부세포막을구성하는성분으로이전부터대식세포의활성화인자이며내독소성쇼크의원인물질로알려져있다 49). LPS 자극에의해서대식세포가활성화되면 TNF-α, IL-1, IL-6, IL-10, NO, IFN 5,6,49) 같은물질들을생산한다. 이연구는금은화가염증반응에서발생되는 type Ⅰ IFN 생성의억제에미치는효과를확인하기위하여, LPS로활성화된대식세포에서생성되는 IFN-α/β 와, IFN의전사활성인자인 IRF, 항염증 cytokine 으로알려진 IL-10, IFN에의해활성화되는 STAT 기전에미치는영향을관찰하였다. 금은화물추출물이세포생존률에미치는영향을관찰하기위하여대식세포에금은화물추출물을 100, 250, 500 μg/ml 농도로처리한결과모든농도에서세포의생존율에영향을주지않았다 (Fig. 1). 바이러스에감염되면 IFN에의해면역반응을유도하게된다. IFN은크게 type Ⅰ, Ⅱ 로구별되며 IFN-α와 IFN-β 를포함하는 type Ⅰ IFN은선천성면역반응을구성하는요소로써, 바이러스감염에대한첫번째방어기작으로알려져있다 50). 그러나 IFN-α/β의과다생성은면역계의말초내성 (peripheral tolerance) 을깨뜨릴수있어자가면역질환을유발할수있다. 실제로암이나감염환자들에게치료목적으로쓰인 IFN-α/β로인해 systemic lupus erythematosus(sle), 인슐린의존성당뇨병, 쇼그렌증후군등자가면역질환이발생했다는보고가있어 23) IFN-α/β와같은염증매개물질의과량생산을억제하는것은각종면역질환및염증성질환의치료에중요하다. 이연구에서금은화물추출물이 IFN-α/β의 mrna 수준의발현을농도의존적으로억제함을알수있었다 (Fig. 2). 이는금은화물추출물이 IFN-α/β 을억제함으로써염증반응을감소시킬수있다는것을보여주고있다. Interferon regulatory factor(irf) 는지금까지 IRF-1부터 IRF-9까지총 9개의유전자가 IRF계로밝혀져있으며 14,51) 바이러스감염시 IFN 생산으로감염된세포에서면역반응을조절한다 14). IRF-1 25

10 은 IFN을유도하고 52,53) IRF-3, 7은바이러스감염에반응하여 type Ⅰ IFN 발현을조절한다. IRF-3, 7은세포질에단량체 (monomer) 로존재하고있다가바이러스감염시인산화되어이량체 (homodimer) 및이종체 (heterodimer) 를형성하고핵으로이동하여, type Ⅰ IFN 유전자프로모터의 interferon stimulated response element(isle) 에결합하여 IFN의전사를활성화시킨다 52,54-56). 금은화물추출물이 IFN-α/β 생성을억제하는기전을알아보기위하여금은화물추출물이 IRF-1, 7의 mrna 발현에미치는영향을조사한결과, 금은화물추출물은 IRF-1, 7의 mrna 발현을유의성있게억제하였다 (Fig. 3). 또한 IRF-3가핵으로이동하는것을막는다는것을보여주었다 (Fig. 4). 이는금은화가 IFN-α/β의생성을억제하는기전이 IRF-1, 3, 7 과관련되어있다는것을나타낸다. IL-10 은염증세포의침윤혹은그활성화를감소시키는중요한항염증성 cytokine 이다 57). 생체내에서 IL-10은면역억제역할과부분적으로림프구의성장촉진역할을동시에수행하고있다. 또한단핵세포 MHC class Ⅱ 발현을감소시키며 58), 면역억제기능으로는 Th1 type 세포와단핵구에의해생산되는 pro-inflammatory cytokine(il-1, TNF-α, IL-6, IL-18) 의생산을억제함 59-61) 으로써염증세포의화학적주성 (chemotaxis) 을방해하여염증을억제하는기능을한다 62). 정등 63) 은금은화의성분중하나인 luteolin이 LPS로유도한 IFN-α/β를억제하는기전과 IFN 전사활성인자인 IRF 와의연관성을증명하였으나 IL-10과의 연관성은밝혀내지못하였다. 이에금은화가항염증 cytokine 인 IL-10의생성에영향을미치는영향을알아보기위하여 IL-10의 mrna 생산량을관찰하였다. 금은화물추출물을처리한지 2시간이후부터 IL-10의 mrna 생산량이유의성있게증가하는것을관찰할수있고, 3시간경과하였을때가장많은증가량을보였다 (Fig. 5A). 또한 IL-10의생산량이증가하는 2~3시간사이에 IFN-α/β 와 IRF-1, 7의생산량이유의성있게억제되는것을볼수있다 (Fig. 5B, 5C). 이는금은화가 IFN-α/β 생산을억제하는기전에 IL-10이관련될수있는가능성을보여준다. 이에금은화가 IFN-α/β 생산을억제하는기전이금은화에의해생산유도된 IL-10과관련이있을것이라는가설을세우고이를검증하기위하여, IL-10 중화항체를사용하여그기능을억제시킨후 IFN-α/β와 IRF-1, 7의 mrna 생산에미치는영향을조사하였다. 실험결과 IL-10의기능을억제하지않은상태에서는 IFN-α/β와 IRF-1, 7의생성이억제되는것에비해 (Fig. 6A, 6B), IL-10 중화항체를사용하여 IL-10의기능을억제시켰을때에는 IFN-α/β 와 IRF-1, 7의생성이억제되지않는것을관찰하였다 (Fig. 6A, 6B). 이는금은화가 IFN-α/β 와그전사활성인자인 IRF-1, 7 생성을억제하는기전에 IL-10이관련이있다는것을보여준다. 이결과는기존에 luteolin을통한연구에서 luteolin 전처리는 IL-10의생성을증가시키지만, IL-10의기능이억제된상태하에서는 IFN-α/β의생성에미치는영향이없었다는결과 63) 와는상이한것으로, 금은화의다른성분혹은아 26

11 J Korean Obstet Gynecol Vol.26 No.2 May 2013 직밝혀지지않은성분이 IL-10을통한 typeⅠ IFN의억제효과에기여할것으로판단되며향후이에대한추가연구가필요하다고생각된다. Cytokine 이목적세포에서생물학적반응을유발하려면세포표면에위치한 cytokine 수용체에결합한후세포내신호전달을통해핵내에위치한특정유전인자를활성화시켜야한다 64). STAT는세포질내에서는비활성으로있다가여러가지 cytokine이나호르몬의자극으로인산화를통해활성화되고, 이합체 (dimer) 를형성한후핵내로이동하여유전자의전사를조절하는단백질군이다 65). 일반적인 cytokine 과 peptide 호르몬들의세포내신호전달은다양한단백키나제연쇄반응을통해다단계로작용하는여러분자들을경유하여이루어지는것에반하여, JAK-STAT 경로는신호전달과정에서다른분자들의참여를제외시킴으로써매우신속하고정확하게특정 ligand 에대한반응을유발시킬수있는독특한신호전달경로로써주목받고있다 66-68). 특정 cytokine 이그에대항하는수용체와결합하게되면, JAK 이수용체에있는 tyrosin 의인산화를유도하게되고, 이렇게인산화된 tyrosin 은 STAT 단백이수용체에결합하도록도와준다. 이후 STAT 단백은스스로인산화되어 p-stat(phosphorylated STAT) 의형태로수용기에서떨어져나와이량체를형성하여핵내로이동하여표적유전자발현에관여하게된다 69). 대식세포에서 IFN-α/β가생성되어분비되면 STAT 경로가활성화되어, 염증반응중에 cytokine 의지속적인분비에관여한다. 이에금은화물추출물이 IFN-α/β 에의해서활성화되는 STAT 경로의활 성여부를알아보기위해실험한결과, LPS 로자극한대식세포에서금은화물추출물을전처리한군에서는 STAT-1, 3 모두인산화하지않았다 (Fig. 7). 이결과는금은화물추출물이 type Ⅰ IFN 의생성억제에관여하고, 그이후의기전인 STAT signaling의활성화또한감소시킨다는것을보여준다. 한의학에서사용되는대표적인항염증약물인금은화물추출물로대식세포의 type I IFN 억제와그기전에미치는영향을검증한결과금은화물추출물은 IRF-1, 3, 7의억제를통해 type Ⅰ IFN 생산을감소시키는효과를보여주었으며, 이러한억제기전에 IL-10이관여하고있음을알수있었다. 이는이전정등 63) 의연구에서 luteolin의 IFN-α/β 억제효과가 IL-10 경로를통한것이아니라는결과와는상이하였다. 또한금은화물추출물은 STAT-1, 3의인산화를억제하여염증반응중에 cytokine 의지속적인분비에도관여하는것을알수있었다. 따라서금은화는 type I IFN 생성억제기전을이용한항염증약물로써의개발가능성이있다고판단되며, 향후금은화의 type I IFN 생성억제기전에미치는영향에대한세밀하고다양한방면으로의추가연구가필요하다고사료된다. Ⅴ. 결론 금은화가 IFN-α/β의생성에미치는영향을알아보고자금은화물추출물을제조하여 LPS로유도한대식세포에서 27

12 IFN-α/β의생산에미치는기전에대해서조사하여다음과같은결론을얻었다. 1. 금은화물추출물은 IFN-α/β 의 mrna 의발현을농도의존적으로억제하였다. 2. 금은화물추출물은 IRF-1, 7의 mrna 의발현을농도의존적으로억제하고, IRF-3가핵으로이동하여 IFN-α/β를생성하는과정또한억제하였다. 3. 금은화물추출물은 IL-10의분비를증가시키며, 금은화물추출물처리시간에따른 IL-10의생성효과와 IRF-1, 7, IFN-α/β 생성억제효과가일치하였다. 4. IL-10의중화항체처리를했을때 IRF-1, 7, IFN-α/β 생성억제효과가유의성있게감소됨으로써 IRF-1, 7, IFN-α/β 생성억제에 IL-10이관여함을확인할수있었다. 5. IFN-α/β가관여하는 STAT-1, 3의인산화를조사하여, 금은화물추출물이 LPS로유도된 IFN-α/β의생성억제에관여하고그이후의기전인 STAT-1, 3의활성화를감소시키는것을확인하였다. 투고일 : 2013년 4월 24일 심사일 : 2013년 5월 2일 게재확정일 : 2013년 5월 9일 참고문헌 1. Abul K, Abbas AK. Cellular and Molecular Immunology. 6th edition. philadelphia:saunders Elsevier : 대한병리학회. 병리학. 서울 : 고문사 : Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cellular Signal. 2001;13: Kubes P, Mccafferty DM. Nitric oxide and intestinal inflammation. American J Medicine. 2000;109: Rietschel ET, et al. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994; 8(2): Galdiero F, et al. Release of cytokines induced by Salmonella typhimurium porins. Infect Immun. 1993;61(1): Platanias LC, Fish EN. Signaling pathways activated by interferons. Exp Hematol. 1999;27: Sen GC. Viruses and interferons. Annu Rev Microbiol. 2001;55: Biron CA. Interferons alpha and beta as immune regulators - a new look. Immunity. 2001;14(6): Chawla-Sarkar M, et al. Apoptosis and interferons: role of interferonstimulated genes as mediators of apoptosis. Apoptosis. 2003;8: Pestka S, et al. Interferons, interferon -like cytokines, and their receptors. Immunol Rev. 2004;202: Honda K, et al. Type I Interferon Gene Induction by the Interferon Regulatory Factor Family of Transcription Factors. Immunity. 2006;25: Bogdan C. The function of type I Interferons in antimicrobial immunity. 28

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