한방안이비인후피부과학회지제 31 권제 1 호 (2018 년 2 월 ) J Korean Med Ophthalmol Otolaryngol Dermatol 2018;31(1):12-21 pissn 1738-6640 eissn 2234-4020 http://www.ood.or.kr http://dx.doi.org/10.6114/jkood.2018.31.1.012 Original Article / 원저內托千金散이 RAW 264.7 대식세포주에서항염증활성에미치는영향 김태준 1 김용민 2 김희택 1* 1 세명대학교한의과대학한방안이비인후피부과학교실 2 세명대학교화장품 뷰티생명공학부 Effects of Naetakcheongeum-san on Anti-inflammatory Activities in RAW 264.7 cells Tae-Jun Kim 1 Yong-Min Kim 2 Hee-Taek Kim 1* 1 Dept. of Oriental Ophthalmology, Otolaryngology & Dermatology, College of Oriental Medicine, Semyung University 2 Dept. of Cosmetic Science & Beauty Biotechnology, Semyung University Abstract Objectives : Inflammation is one of the self-protective abilities against tissue injury, and it has clinical symptoms like redness, heat, swelling, pain, and loss of function. The purpose of this study is to examine inhibitory effects of Naetakchunkeum-san (NTCKS) on nitric oxide (NO), Prostaglandin E2 (PGE2), inducible NOS (inos), cyclooxygenase-2 (COX-2), and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), which play a major role in inflammatory response. Methods : The experiment was performed using Raw 264.7 cells pretreated with NTCKS extracts. Cell viability was determined by MTT assay. To evaluate anti-inflammatory effects of NTCKS, we examined NO and PGE 2 production in LPS-induced macrophages. We also investigated effects of NTCKS on inos, Cox-2, and ERK1/2 expression using western blot. Results : In MTT assay, no cytotoxicity of NTCKS (50, 100, 150, 200μg / ml ) on RAW 264.7 cell was found. LPS-induced NO production was decreased after treatment with NTCKS (150, 200μg / ml )(p<0.05). PGE 2 was decreased after treatment with NTCKS (150, 200μg / ml )(p<0.05). NTCKS inhibited LPS-induced expressions of inos and COX-2 in a dose-dependent manner. Increased phosphorylation of ERK1/2 by LPS was decreased by NTCKS in a dose-dependent manner. c 2018 the Society of Korean Medicine Ophthalmology & Otolaryngology & Dermatology This is an Open Access journal distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/license/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 12
김태준외 2 인 : 內托千金散이 RAW 264.7 대식세포주에서항염증활성에미치는영향 Conclusions : According to above experiments, NTCKS may be applied to inflammatory diseases such as atopic dermatitis, rheumatoid arthritis, and inflammatory bowel disease. Key words : Naetakchunkeum-san; Anti-inflammation; PGE 2 ; inos; COX-2; ERK1/2 Ⅰ. 서론 염증 (Inflammation) 은상처를줄수있는자극에대한생체의방어반응으로, 임상적으로는발적, 발열, 종창, 동통, 기능장애의 5가지증상이나타난다 1). 대식세포는 nitric oxide (NO) 와 prostaglandins (PGs) 을생산하며 2), 이로인해숙주방어기전에중요한역할을하는동시에 3) 염증반응과밀접한상관관계를가진다. NO는높은반응성을가진생체생성분자로서 NO synthase (NOS) 에의해 L-arginine으로부터생성된다. 대식세포가 lipopolysaccharide (LPS) 로자극될때 inducible NOS (inos) 가발현되어많은양의 NO를생성하게된다. 이렇게생성된 NO는염증반응매개물질의역할을하게된다 1,4). PGs, 특히 prostaglandin E 2 (PGE 2 ) 는세포막에있는지방산인 arachidonic acid에 cyclooxygenase-2 (COX-2) 의자극이가해질때발생하며 4), NO와마찬가지로염증반응매개물질의역할을하게된다 5). Mitogen-activated protein kinase (MAPK) 는 extracellular signal-regulated kinase (ERK), c-jun NH 2 -terminal kinase (JNK), serine/threonine protein kinase 인 p38 MAPK 등이있으며 6,7), 이들 MAPK 는 inos 와 COX-2 의유전자발현에있어주요신호전달분자로알려져있어 8), 염증반응에있어중요한역할을하게된다. 이와같은염증반응은한의학적으로正邪抗爭의관점에서관찰할수있다. 正은正氣로邪氣에대응 Corresponding author : Hee-Taek Kim, 65, Semyung-ro, Jecheon-City, Chungbuk, Korea, (Tel: 043-649-1817, E-mail: kht8725c@naver.com) Recieved 2018/1/5 Revised 2018/2/2 Accepted 2018/2/9 하는인체의항병력및정상적인생리기능을총칭하고, 邪는邪氣로발병인자를총칭한다 9). 이러한正邪抗爭을한의학적치료강령에적용할경우扶正祛邪와같은의의를지닌다고볼수있다. 內托千金散은明代陳實功의 < 外科正宗 > 10) 에최초로기재되어있으며, 朝鮮代許浚의 < 東醫寶鑑 > 11) 에서는 治一切癰疽惡瘡能內托 이라하였다. 內托法은한의학외과치료법중內治法의한방식으로實證의치료법인透托과虛症의치료법인補托을병행하여藥物로扶正達邪 12), 즉扶正祛邪를하는것이다. 이에內托千金散은염증반응의억제에적용할수있는한의학처방으로생각하여볼수있다. 지금까지內托千金散에관한연구보고는김등 13-16) 의연구가있는데이러한연구들은주로면역조절작용 13,14), 항종양효과기전 15), 알레르기성접촉성피부염 16) 에대한실험연구였다. 이에저자는內托千金散이 RAW 264.7 대식세포주에서세포독성, NO 생성, PGE 2 생성, inos 및 COX-2 발현, ERK1/2 인산화억제에미치는영향에대하여유의한결과를얻었기에보고하는바이다. Ⅱ. 연구내용및방법 1. 재료 1) 시료의조제본실험에사용한內托千金散은東醫寶鑑에근거하여구성약물을 HMAX에서구입하여사용하였다 (Table 1). 13
한방안이비인후피부과학회지제 31 권제 1 호 (2018 년 2 월 ) Table 1. The Amount and Composition of Naetakchunkeum-san (NTCKS) 韓藥名 生藥名 用量 / 貼 (g) 金銀花 LONICERAE FLOS 4 人蔘 GINSENG RADIX 4 黃芪 ASTRAGALI RADIX 4 赤芍藥 PAEONIA RADIX RUBRA 4 當歸 ANGELICAE GIGANTIS RADIX 4 川芎 CINDII RHIZOMA 4 瓜蔞根 TRICHOSANTHIS RADIX 4 白芷 ANGELICAE DAHURICAE RADIX 4 桂皮 CINNAMOMI CORTEX 4 桔梗 PLATYCODI RADIX 4 防風 LEDEBOURIELLAE RADIX 4 甘草 GLYCYRRHIZAE RADIX 4 合計 48 조제방법은다음과같다. 內托千金散 96g을 3차증류수 2L와혼합하여 100 로 4시간동안열수추출하였으며, 여과지로여과한추출액을 rotary evaporator를이용하여 100ml까지농축하고 -80 로동결하였다. 농축한동결액을 freezing dryer system (Labconco, USA) 을이용하여 7일간동결건조하였다 (15.36g, 수율약 16%). 2. 실험방법 1) 세포배양 실험에사용된 mouse 대식세포는 RAW 264.7 cell line (ATCC, USA) 을분양받아사용하였다. RAW 264.7 cells은 37, 5% CO 2 조건에서 10% fetal bovine serum (FBS), penicillin (100U/ ml ) 및 streptomycin (100μg/ ml ) 등이포함된 DMEM 배지로배양되었다. 배양세포들은 75cm2 flask (Falcon, USA) 에서충분히증식된후배양 3일간격으로배양세포표면을 PBS 용액으로씻어준뒤 50ml flask 당 1ml의 0.25% trypsin-edta 용액을넣고실온 에서 1분간처리한다음 trypsin을버리고 37 에서 5분간보관하여세포를탈착하여계대배양하였다. 탈착된세포는 10% FBS 가첨가된 DMEM 배양액 10ml에부유시킨다음새로운배양용기 (50ml culture flask) 에옮겨 1:2 split ratio로 CO 2 배양기에서배양하였다. 2) MTT assay 세포독성유발효과를알아보기위하여 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 방법을사용하여측정하였다. 96 well plate에 1 x 10 5 cells/well 의 cell을 100μl씩넣고 37, 5% CO 2 가공급되는배양기에서 24 시간동안배양한후배지를버리고배양세포표면을 1x PBS 용액으로씻어주었다. 같은양의배지와 PBS에녹인시료를농도별로각 well에처리하고 24시간배양하였다. 배양이끝난후 PBS에녹인 1 μg / ml MTT (Sigma, USA) 를 100μl씩각 well에처리하여알루미늄호일로차광시킨뒤 2시간동안같은조건에서배양하였다. 배양액을모두제거한후 DMSO 를 100μl처리하고 37 에서 2시간방치한다음 microplate reader 를이용하여 490nm에서흡광도를측정하였다. 세포생존율은다음과같은공식으로계산되었다. Viability(%) = 100 AT/AC AT : absorbance of tested extract solution AC : absorbance of control 3) NO assay 96 well plate에 1 x 10 5 cells/well 의 cell을 100 μl씩넣고 37, 5% CO 2 가공급되는배양기에서 24시간동안배양하여세포를안정화시켰다. 안정화시킨세포에 lipopolysaccharide (LPS) 10μg / ml와內托千金散열수추출물을농도별로처리하고 24시간동안 37, 5% CO 2 가공급되는배양기에서배양한후세포배양상등액 60μl을채취하여여기에 14
김태준외 2 인 : 內托千金散이 RAW 264.7 대식세포주에서항염증활성에미치는영향 Griess 시약 100μl을혼합하여 15분동안반응시킨뒤 microplate reader 를이용하여 540nm에서흡광도를측정하였다. 4) PGE 2 생성량측정 PGE 2 의측정은 commercial competitive enzyme immunoassay kit를 R&D systems (Minneapolis, USA) 에서구입하여실험하였다. RAW 264.7 세포에內托千金散열수추출물을 1시간전처치하고 10μg / ml의 LPS를처리하여 24시간배양한후세포배양상층액을수거하여 PGE 2 측정에사용한다. 배양액을 goat anti-mouse로 coating된 96 well plate 에각각의배양액을 100μl씩 loading 한다. 여기에 primary antibody solution 50μl와 PGE 2 conjugate 50μl씩첨가하여 4 에서 overnight 시킨다. 기질용액을 200μl씩처리하여 5-20분간반응시킨후, 50μl의 stop solution 을처리하고 450nm에서흡광도를측정한다. 5) Immunoblot 측정배양한 RAW 264.7 macrophage에內托千金散열수추출물과 10μg / ml의 LPS를처리하여 18시간동안배양한후세포배양상층액을제거하고 PBS 1 ml로 3회세척하였다. Lysis buffer (25mM Tris-HCl, 1% NP 40, 150mM sodium chloride, 0.25% sodium deoxycholate, 1mM NaF, protease inhibitors) 200ml를이용하여세포를용해하였다. 용해한시료는동결, 해동과정을 3회반복하고 1800 rpm으로 20분간원심분리하여상층액만사용하였다. 단백질농도는 Bradford assay법을이용하여정량하였다. 각시료는 SDS sample buffer 를첨가하여 5분간열처리하였다. 준비된시료는 10% SDS polyacrylamide gel을이용하여전기영동한후 nitrocellulose membrane 으로이동시켰다. Nitrocellulose membrane은 5% BSA로 1시간동안 block 하고, TBST (TBS + 0.1% Tween-20) 로세척 후항체 COX-2 (Santacruz, USA), inos (Santacruz, USA), β-actin (Abcan, USA), ERK (Cell signaling, USA) 와 perk (Cell signaling, USA) 를처리하였다. 항체처리후 TBST 로 3회세척하였다. Band의시각화는 Odyssey system (LI-COR, USA) 을사용하여측정하였다. 6) 통계처리실험결과는 SPSS Window program (Ver. 12.0) 을이용하였으며, 모든측정값은 mean ± SD로나타내었고, 대조군과각실험군과의평균차이는 Student's t-test 로분석하여 p-value 가 0.05 미만일때통계학적으로유의한차이가있는것으로판정하였다. Ⅲ. 결과 1. 세포독성에미치는영향內托千金散열수추출액의농도가세포내에서독성을일으키는지확인하기위해 MTT assay 를이용하여측정하였다. 內托千金散열수추출액의대조군생존율을 100 ± 8.53% 로계산하였을때 400μg / ml을제외한모든농도에서세포독성이없음을확인하였다 (Table 2). 2. NO 생성에미치는영향內托千金散열수추출액이마우스대식세포의 NO 생성에미치는영향을관찰하였다. 內托千金散처리하지않은대조군에서는 NO 생성율이 85.3 ± 6.3μm로나타난반면에, 內托千金散 50, 100, 150 및 200 μg / ml로처리한실험군에서는 NO 생성율이 83.8 ± 3.8, 79.2 ± 4.0, 73.3 ± 6.0, 67.8 ± 5.9μm로나타났다. 內托千金散 150 및 200μg / ml처치군은 LPS 단독처리한대조군에비해서통계학적으로유의한감소가나타났다 (Table 3). 15
한방안이비인후피부과학회지제 31 권제 1 호 (2018 년 2 월 ) Table 2. The Cytotoxic Effect of Naetakchunkeumsan (NTCKS) Water-extract on RAW 264.7 Macrophage Cells by MTT Assay NTCKS Concentration ( μg / ml ) Cell Viability (% of control) Control 100 ± 8.53 50 99.83 ± 8.28 100 97.47 ± 8.79 150 95.59 ± 12.13 200 90.50 ± 12.45 400 81.48 ± 7.89* Valuses are the mean ± SD of the three independent experiments. Control : untreated with NTCKS 50, 100, 150, 200 and 400 : treated with NTCKS (50, 100, 150, 200 and 400 μg / ml ) * p < 0.05 compared to control Table 3. The Effect of NTCKS Water-extract on NO Production of RAW 264.7 Macrophage Cells Concentration ( μg / ml ) NO production ( μm ) Control 85.3 ± 6.3 LPS + 50 83.8 ± 3.8 LPS + 100 79.2 ± 4.0 LPS + 150 73.3 ± 6.0* LPS + 200 67.8 ± 5.9* 도로처리한군에서 LPS 단독처리한군에비하여통계학적으로유의한감소가나타났다 (Table 4). Table 4. The Effect of NTCKS Water-extract on Prostaglandin E2 Production of RAW 264.7 Macrophage Cells Concentration ( μg / ml ) PGE 2 production (pg/well) Control 821.24 ± 1.63 LPS + 50 827.86 ± 24.78 LPS + 100 830.27 ± 18.24 LPS + 150 767.63 ± 12.53* LPS + 200 746.35 ± 11.05* Valuses are the mean ± SD of the three independent experiments. Control : treated with LPS (10 μg / ml ) 50, 100, 150 and 200 : treated with LPS and NTCKS (50, 100, 150 and 200 μg / ml ) * p < 0.05 compared to control 4. inos 및 COX-2 발현에미치는영향 內托千金散이 inos 및 COX-2 발현에미치는영향을관찰하였다. LPS 처리에의하여증가된 inos 및 COX-2 의발현은內托千金散처리한실험군에서농도의존적으로발현이감소하였다 (Fig. 1). Valuses are the mean ± SD of the three independent experiments. Control : treated with LPS (10 μg / ml ) 50, 100, 150 and 200 : treated with LPS and NTCKS (50, 100, 150 and 200 μg / ml ) * p < 0.05 compared to control 3. PGE2 생성에미치는영향 內托千金散이마우스대식세포의 PGE 2 생성에미치는영향을관찰하였다. 內托千金散을처치하지않은대조군의 PGE 2 생성은 821.24 ± 1.63pg/well 로나타난반면內托千金散을 50, 100, 150 및 200μg / ml로처리한실험군에서는각각 827.86 ± 24.78, 830.27 ± 18.24, 767.63 ± 12.53, 746.35 ± 11.05 pg/well 로나타났다. 內托千金散 150 및 200μg / ml농 Fig. 1. The Effect of NTCKS Water-extract on inos and COX-2 Expression NC : untreated with LPS and NTCKS LPS : treated with LPS (10 μg / ml ) 50 μg, 100 μg, 150 μg, 200 μg : treated with LPS and NTCKS (50, 100, 150, 200 μg / ml ) 16
김태준외 2 인 : 內托千金散이 RAW 264.7 대식세포주에서항염증활성에미치는영향 5. ERK1/2 인산화억제에미치는영향 內托千金散이 ERK1/2 인산화억제에미치는영향을관찰하였다. LPS 처리에의하여증가된 ERK1/2 의발현은內托千金散처리한실험군에서농도의존적으로인산화가억제되었다 (Fig. 2). Fig. 2. The Effect of NTCKS Water-extract on ERK1/2 Expression NC : untreated with LPS and NTCKS LPS : treated with LPS (10 μg / ml ) 50 μg, 100 μg, 150 μg, 200 μg : treated with LPS and NTCKS (50, 100, 150, 200 μg / ml ) Ⅳ. 고찰 염증은조직손상에대한자가방어능력중의하나이며, 이는임상적으로통증, 열감, 발적, 기능저하의증상을보이는것이다 17). 염증은양날의칼과같아서적절한염증반응은내재면역반응을바탕으로적응면역반응을매개하여외인적 / 내인적인자로부터생체를보호하는필수불가결한반응이나, 과도하고부적절한염증반응은세포및조직의괴사및패혈증에의한사망과더불어각종만성질환의원인이된다 18). 이와같은염증반응은한의학에서는 < 黃帝內徑素問刺法論 > 19) 에 正氣存內邪不可干 이라하여정상적인장부기능을바탕으로하는正氣와각종발병인자를지칭하는邪氣와의싸움인正邪相爭, 즉正邪抗爭으로보아正氣가약할때는補益을위주로하는扶正法을사용하고, 邪氣가강할때는淸熱, 解毒등의祛邪法을사용하여왔다 20). 한의학피부외과치료법중에는가볍고얕은부위에발생한적은瘡에사용하는外治法과국부와전체가 같이重한瘡에주로사용하는內治法이있다. 이內治法의한방식으로實證에사용하는透托法과正氣가虛할때사용하는補托을병행하여扶正達邪 12), 즉扶正祛邪를하는것이內托法이다. 이와같은內托法은正邪抗爭상황인염증반응에서扶正祛邪의治法으로서응용하여볼수있다. 內托千金散은 < 東醫寶鑑 > 11) 에서 治一切癰疽惡瘡能內托 이라하여內托法을따르는처방으로염증반응억제에유의미성이있을것으로생각되나, 지금까지는주로면역조절작용 13,14), 항종양효과기전 15), 알레르기성접촉성피부염 16) 등에대한연구가진행되었다. 이에본연구에서는內托千金散의염증반응에있어주요한역할을하는 NO, PGE 2, inos, COX-2, ERK1/2 인산화에대한억제효과를살펴보았다. MTT assay는세포연구에서광범위하게사용되며, 이는생체외세포증식과생존능력분석에유용성이있다 4). 內托千金散열수추출액의농도가세포내에서독성을일으키는지확인하기위해 MTT assay 를이용하여측정하였다. 內托千金散열수추출액은 400μg / ml을제외한모든농도에서 90% 이상의세포생존율을확인할수있었다 (Table 2). NO는 NOS에의해 L-arginine으로부터생성되는자유라디칼이다. 체내에서소량생성되는 NO는혈관확장, 신경전달, 병원체에대한세포파괴등과같은정상적인생리기능을담당하지만, 대식세포에서 inos 에의해과다생성된 NO는염증반응과관련되어있으며 1), 과다생성된 NO의파생물인 peroxynitrite 와 nitrogen dioxide 또한염증반응에주요한역할을한다 21). 內托千金散열수추출액이마우스대식세포의 NO 생성에미치는영향을관찰하기위하여 LPS만처리한대조군과內托千金散을농도별로처리한실험군을비교한결과, 內托千金散 150 및 200μg / ml농도로처리한실험군에서대조군에비해 NO 생성이통계적으로유의하게감소하였다 (Table 3). Prostaglandin 은탄소수 20개의불포화지방산조직인 eicosanoid 의한종류로탄소수 20개의불포화 17
한방안이비인후피부과학회지제 31 권제 1 호 (2018 년 2 월 ) 지방산인 arachidonic acid로부터생합성된다 22). 이는그구조상의불포화성또는측쇄에따라 prostaglandin E, F, A, B 등의여러유도체들이알려져있다 23). 이중 COX-2 에의해생성되는 PGE 2 는 NO와마찬가지로손상된부위나조직에서통증과발열의전달에주로관여하는중요한염증매개물질이다 5). 內托千金散열수추출액이마우스대식세포의 PGE 2 생성에미치는영향을관찰하기위하여 LPS만처리한대조군과內托千金散을농도별로처리한실험군을비교한결과, 內托千金散 150 및 200μg / ml농도로처리한실험군에서대조군에비해 PGE 2 생성이통계적으로유의하게감소하였다 (Table 4). NO synthase (NOS) 는 endothelial NOS (enos), neuronal NOS (nnos), inducible NOS (inos) 의세가지주요한 isoform이있다 24). 이중 inos 는 murine macrophage, 내피세포, 평활근세포및심근세포등많은세포에서 8) interferon-γ (IFN-γ), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), LPS 로자극될때발현되며 25), Ca 2+ 비의존성경로를통해서장시간에걸쳐증가하여다량의 NO를형성한다 8). Arachidonic acid를 prostaglandin 으로변화시키는효소에는세포구성요소로서의 COX-1 과유도성인 COX-2 가있다 23). COX-1 은대부분의조직에있는관리단백질로일반적인생리기능을가진 PGs들을생산하며, 이에비해 COX-2 는염증매개체인 PGE 2 를생산하며염증반응을매개한다 26). 內托千金散열수추출액이 inos 및 COX-2 발현에미치는영향을관찰하기위하여 LPS 처리에의하여증가된 inos 및 COX-2 의발현이內托千金散처리한실험군에서농도별로어떠한양상인지관찰한결과 inos 및 COX-2 의발현은內托千金散농도의존적으로감소하였다 (Fig. 1). ERK는 JNK, serine/threonine protein kinase 인 p38 MAPK 와함께 MAPK superfamily 를형성한다 6). MAPK 는인산화를통하여 nuclear factor-κ B(NF-κB), activator protein-1(ap-1), activating transcription factor-2, camp-responsive element binding protein 을포함한다양한전사인자들을활성화시키는핵심신호전달분자로보고되고있다 27,28). 또한대식세포를매개한염증반응에있어 NO 생성효소인 inos, prostaglandin 생합성의속도조절단계효소인 COX-2 의유전자의발현에있어서도 MAPK 는주요신호전달분자의역할을하기에 7) 염증반응에서중요한의미를지닌다. 內托千金散열수추출액이 ERK1/2 인산화억제에미치는영향을관찰하기위하여 LPS 처리에의하여증가된 ERK1/2 의발현이內托千金散처리한실험군에서농도별로어떠한양상인지관찰한결과 ERK1/2 의발현은內托千金散농도의존적으로인산화가억제되었다 (Fig. 2). 이상으로본연구에서는內托千金散이 RAW 264.7 대식세포주에서 LPS 처리에서생성된 NO, PGE 2, inos 및 COX-2 에대한억제효과와 ERK1/2 의인산화억제효과가있다는결과를얻을수있었다. 염증은그발생과정에있어일반적인매개체들을이용한다. 이매개체들은서로발생의선후가있기는하지만, 그매개체들을각각논의하기는어렵다 29). 이런측면에서염증반응의주요한매개체들에대해억제효과를보인內托千金散은향후아토피피부염, 류마티스관절염, 염증성장질환 30) 등의각종염증성질환에있어효과적인치료처방으로활용될수있을것으로사료된다. Ⅴ. 결론內托千金散이 RAW 264.7 대식세포주에서세포독성, NO 생성, PGE 2 생성, inos, COX-2 발현, 및 ERK1/2 인산화억제에미치는영향에대하여다음과같은결과를얻었다. 1. 內托千金散추출물은 400μg / ml을제외한모든농도에서세포독성이없었다. 18
김태준외 2 인 : 內托千金散이 RAW 264.7 대식세포주에서항염증활성에미치는영향 2. 內托千金散추출물은 150 및 200μg / ml농도에서 NO 생성을통계적으로유의하게감소시켰다. 3. 內托千金散추출물은 150 및 200μg / ml농도에서 PGE 2 생성을통계적으로유의하게감소시켰다. 4. 內托千金散추출물은 LPS에의하여증가된 inos 및 COX-2 의발현을농도의존적으로감소시켰다. 5. 內托千金散추출물은 LPS에의하여증가된 ERK1/2 의발현을농도의존적으로인산화를억제시켰다. 감사의글이논문은 2016학년도세명대학교교내학술연구비지원에의해수행된연구임 Ⅵ. References 1. Jeong MY, Park HJ, Jeong JH, Kim JY, Kang JM, Lee NK, et al. Inhibitory effect of Angelica gigas Nakai extract on nitric oxide production in RAW 264.7 cells. J Korean Oriental Med. 2007;28(2):155-65. 2. Shin JS, Noh YS, Kim DH, Cho YW, Lee KT. Mangiferin isolated from the rhizome of Anemarrhena asphodeloides inhibits the LPS-induced nitric oxide and prostagladin E 2 via the NF-κB inactivation in inflammatory macrophages. Natural Product Sciences. 2008;14(3):206-13. 3. Lee KH, Jung JH, Kim EH, Lee JH. Inhibitory effect of Smilacis Glabrae Rhizoma on nitric oxide production in the macrophage cell line RAW 264.7. Journal of Meridian&Acupoint. 2009;26(3):69-76. 4. Im NK, Jung YS, Choi JH, Yu MH, Jeong GS. Inhibitory effect of the leaves of Rumex crispus L. on LPS-induced nitric oxide production and the expression of inos and COX-2 in macrophages. Natural Product Sciences. 2014;20(1):51-7. 5. Choi YJ, Roh JD. Effects of Angelica Gigantis Radix pharmacopuncture on nitric oxide and prostaglandin E 2 production in macrophage. Journal of Pharmacopuncture. 2011;14(3): 81-90. 6.Kim YS, Lee SG, Lee KS. Effect of Cirsii Japonici Herba on LPS-induced inflammation in mouse BV2 microglial cells. Korean J. Orient. Int. Med. 2008;29(4):1048-60. 7. Park SM, Byun SH, Kim YW, Cho IJ, Kim SC. Inhibitory effect of Mori Folium ethanol extract on pro-inflammatory mediator in lipopolysaccharide-activated RAW 264.7 cells. Kor J Herbology. 2012;27(3):31-8. 8. 8. Kim TY. Effect of Gagam-danguieumja through regulation of MAPK on LPS-induced inflammation in RAW 264.7 cells. Korean J Orient Int Med. 2013;34(4):339-48. 9. Faculties of Korean Medical pathology in the Korean Medical Colleges. Korean Medical Pathology. Seoul:Iljungsa. 2004:109-12. 10. Chen SG. Waikezhengzong. Beijing:People s Medical Publishing House. 1983:259. 11. Heo J. Donguibogam. Hadong:Donguibogam Publisher. 2006:1543-45. 12. Ko WS, Kim KJ, Kim NK, Kim YB, Kim JH, et al. Text of Traditional Korean Dermatology & Surgery. Busan:Seonu. 2007: 111-21. 13. Kim HT, Roh SS. Effect of Naetakchungumsankamibang on skin tumor induced by 19
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김태준외 2 인 : 內托千金散이 RAW 264.7 대식세포주에서항염증활성에미치는영향 30. Lee SE, Lee JH, Kim JK, Kim GS, Kim YO, Soe JS, et al. Anti-inflammatory activity of medicinal plant extracts. Korean J. Medicinal Crop Sci. 2011;19(4):217-26. 21