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Journal of Korean Medicine Rehabilitation Vol. 23 No. 3, July 2013 pissn 1229-1854 eissn 2288-4114 Original Article 김일현 최종환 이세원 송용선원광대학교한의과대학한방재활의학과교실 Anti-inflammatory Effect of Mugi-hwan Water Extract in RAW 264.7 Cells Ilhyun Kim, O.M.D., Chonghwan Choi, M.D., Sewon Lee, O.M.D., Yungsun Song, O.M.D. Department of Oriental Rehabilitation Medicine, College of Oriental Medicine, Won-Kwang University RECEIVED June 25, 2013 REVISED July 7, 2013 ACCEPTED July 11, 2013 CORRESPONDING TO Yungsun Song, Department of Oriental Rehabilitation Medicine, College of Oriental Medicine, Won-Kwang University, 142-1, Deokjin-dong 2ga, Deokjin-gu, Jeonju 561-851, Korea TEL (063) 270-1070 FAX (063) 270-1199 E-mail yssong@wku.ac.kr Objectives The aim of this study was to investigate anti-inflammatory activity of Mugi-hwan (MGH) Water Extract. Methods Cells were treated with 2 ug/ml of LPS 1 hour prior to the addition of MGH. Cell viability was measured by MTS assay. The production of NO was determined by reacting cultured medium with Griess reagent. The expression of COX-2, inos and MAPKs was investigated by Western blot, RT-PCR. The content of level of cytokines (PGE 2, IL-6, in media from LPS-stimulated Raw 264.7 cells was analyed by ELISA kit. Results MGH inhibited the production of NO, PGE 2, IL-6 as well as the expressions of inos, COX-2 in the murine macrophage, RAW 264.7 cells. MGH also had suppression effects of LPS induced MAPKs activation. Conclusions These results suggest that MGH has an anti-inflammatory therapeutic potential, which may result from inhibition of MAPK phosphorylation, thereby decreasing the expression of pro-inflammatory genes. (J Korean Med Rehab 2013;23(3):27-35) Copyright 2013 The Society of Korean Medicine Rehabilitation Key words Mugi-hwan, MAPKs, Anti-inflammatory activity 서론»»» 염증반응은상처부위에침입한미생물들에대한적대환경을만드는비특이적인방어작용이며, 인체내대부분의기관에서유발되는기본적인면역반응으로급성과만성으로나누어지는데 1), 급성염증반응은물리적자극이나외부감염등에의해수분으로부터수시간이내즉각적으로조직손상을유발하게된다. 반면만성염증반응은급성보다오래걸리며지속적이고, 단핵구나대식세포, 림프구, 혈장세포등의침윤을동반하는특징이있으며, 조직파괴와치유과정을통해혈관신생이나섬유화를유발하게된다 1,2). 대식세포는병원균과같은외부자극원의자극에의해 활성화되어 vascular amines, arachidonic acid metabolites (prostaglandins, leukotriene, lipoxin 등 ), inflammatory cytokines (TNF-α, IL-1, IL-6, IL-8, IL-12, GM-CSF), platelet activating factor, prostaglandin E 2, nitric oxide, ROS 등다양한염증매개물질들을분비함으로써면역조절을통해생체내방어기능과항상성유지기능을조절하는반면, 염증을유발하여각종폐질환이나만성기관지염, 기관지천식, 류마티스관절염, 다발성경화증, 퇴행성뇌질환, 동맥경화등의만성염증질환으로의진행에기여한다 3-6). 따라서대식세포의활성이어떻게조절되는가를이해하고, 활성화된대식세포로부터분비되는각종염증매개물질들을억제시키는것은여러원인에의해발생하는염증질환의진행을막아줄수있는유효한접근방 www.e-jkmr.org 27

김일현 최종환 이세원 송용선 법이될수있다 6,7). 戊己丸 (Mugi-hwan, MGH) 은宋代 太平惠民和劑局方 8) 에처음기록된처방으로黃連, 吳茱萸, 白芍藥으로구성되어있으며 東醫寶鑑 9) 에濕痢를치료하는데, 濕痢란腹脹身重하고黑豆汁이나赤黑渾濁한설사를하는것이다. 戊己丸에대한실험적연구는거의없는실정이나, 조성약재인黃連, 吳茱萸, 白芍藥에대한연구는비교적많이이루어졌다. 黃連추출물은 Helicobacter pylori의성장을억제하고 10), LPS에의해유도된 nitric oxide의생성을억제하며 11) 黃連에함유된 berberine 은항염증효과를가지는것으로알려져있다 12). 吳茱萸의주요성분으로알려진 evodiamine 은염증에대한효과가보고되어있으며 13), 白芍藥추출물은항혈전작용 14), 항고지혈작용 15), 항산화작용 16), 혈관확장작용 17) 등이보고되어있다. 이에저자는戊己丸의항염증효과를조사하기위하여 LPS로자극된설치류의대식세포주인 RAW 264.7 세포에서세포가방출하는 NO와 PGE 2 생성량과 inos, COX-2 의발현및 IL-6에미치는영향을알아보고그기전을밝히고자실험하여유의한결과를얻었기에보고하는바이다. 재료및방법»»» 1. 재료 1) 시약 LPS (E. coli lipopolysaccaride), RPMI-1640 배지는 Sigma Chemical Co. (St. Luis, MO, USA) 로부터구입하 Table I. Primer Sequences for RT-PCR cdna Primer sequence inos forward reverse 5'-CATGTTCTCTGGGAAATCGTGG-3' 5'-AACGCACTAGGTTTGCCGAGTA-3' COX-2 forward reverse 5'-CACTCAGTTTGTTGAGTCATTC-3' 5'-GATTAGTACTGTAGGGTTAATG-3' β-actin forward reverse 5-ATGAAGATCCTGACCGAGCGT-3' 5'-AACGCAGCTCAGTAACAGTCCG-3' 였고, 우태아혈청 (Fetal bovine serum, FBS) 및항생제는 Gibco BRL Co. (Grand Island, NY, USA) 로부터구입하였다. 조직배양 plate와직경 100 mm petri-dish 는 Nunc Inc. (Rochester, NY, USA) 로부터구입하여사용하였다. PGE2, IL-6은효소결합면역측정 (ELISA) 키트를 R&D Systems Inc. (Minneapolis, MN, USA) 로부터구입하였으며, COX-2, inos, MAPKs (ERK, JNK, p38) 는 Santa Cruz Biotechnology Inc. (Santacruz, CA, USA) 에서구입하였다. 2) 약재 戊己丸의구성약재인黃連, 吳茱萸, 白芍藥은대학한약국 ( 익산, 한국 ) 에서구입하여사용하였다. 3) Primer 준비 RT-PCR 을위해여러개의 primer 를사용하였다. Primer 의구성은다음과같다 (Table I). 2. 방법 1) 戊己丸물추출 戊己丸물추출물은 3차증류수 (100 g/l) 로 2시간환류추출하여준비하였다. 그추출물은 0.45μm 필터로걸렀고, 감압하에동결건조 (lyophilized) 하였으며, 4 o C에서보관하였다. 동결건조된추출물은실험하기위하여인산완충식염수 (phosphate-buffered saline. PBS) 에용해하였다. 본실험에사용한戊己丸은 東醫寶鑑 9) 처방을기준으로하였으며, 黃蓮, 吳茱萸, 白芍藥각 1:1:1의비율로구성되어있으며 (Table II) 총 45 g에서 D.W 500 ml씩 2회 4.94 g을추출하여수득률 10.97% 이다. 2) 세포배양 설치류대식세포주 (murine macrophage cell line, RAW 264.7) 는한국세포주은행에서구입하여사용하였 Table II. The Composition of Mugi-hwan Herbal name Pharmacognostic name Weight (g) 黃蓮 Rhizoma Coptidis 15.0 吳茱萸 Fructus Evodiae 15.0 白芍藥 Radix Paeoniae Alba 15.0 28 J Korean Med Rehab 2013;23(3):27-35

다. 대식세포주는항생제및항균제 (penicillin G 100 U/ml, streptomycin 100 U/ml) 를첨가하고 10% 열처리우태아혈청 (heat inactivated FBS) 을첨가한완전한 RPMI 1640 배지에서 5% CO 2 의습한대기, 37 o C의온도조건에서배양하였다. 3) NO 생성량측정 DMSO 로녹인 PA를여러농도 (1~100μM) 로처리하고여기에 LPS (200 ng/ml) 를각각주입한다음상기대식세포주 RAW 264.7을 24시간배양하였다. 세포배양액의최종 DMSO 농도는 0.1% 이하였다. 배양한대식세포주의상층액을수집하여 Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylene diamine dihydrochloride in 2.5% phosphoric acid solution) 와동량으로주입한후 10분간실온에서방치하였다. 아질산의농도는 ELISA 리더기를이용하여 570 nm에서흡광도를측정하여결정하였다. 세포가없는배양액은아질산이 0~30μM 로나타나, 이값을표준으로하여여러실험군의아질산값의흡광도를측정하였다. 4) MTS 분석 RAW 264.7 세포 (5 10 4 cells/well) 를 96-well culture plate에 100μl의 RPMI 1640 배지와함께하룻밤배양한다음, MGH 추출물 (50, 100, 250, 500μg/ml) 을각각처리하여 24시간배양하였다. 각 well에 5 mg/ml 농도의 MTS 용액을 50μl 씩넣은후 2시간동안배양하면서환원반응을유도하여발색정도를 microplate reader를이용하여 550 nm에서흡광도를측정하였으며, 세포독성 (cell toxicity) 은세포만배양한비처리군의생존도 100% 를기준으로약물처리군의상대적인세포생존도를계산하였다. 5) ELISA RAW 264.7 세포를 RPMI 1640 배지를이용하여 1.5 10 5 cells/ml 로조절한후 24 well plate에접종하고, 5% CO 2 항온기에서 18시간전배양하였다. 이후배지를제거하고 MGH를각각 100, 250μg/ml 농도로 1시간동안처리한후, 그람-음성박테리아내독소인 LPS (200 ng/ml) 로 RAW 264.7 대식세포주를자극한후세포부유액을원심분리하여세포들을침전시켜상층액을수집하고, 상층액 내 IL-6 생성량을 ELISA kit (R&D Systems Inc., Minneapolis, MN, USA) 를이용하여사용자매뉴얼에기재된방법대로정량해분석하였다. 6) PGE 2 측정 RAW 264.7 세포를 RPMI 1640 배지를이용하여 1.5 10 5 cells/ml 로조절한후 24 well plate에접종하고, 5% CO 2 항온기에서 18시간전배양하였다. 이후배지를제거하고 MGH를각각 100, 250μg/ml 농도로 1시간동안처리한후, 그람-음성박테리아내독소인 LPS (200 ng/ml) 로 RAW 264.7 대식세포주를자극한후세포부유액을원심분리하여세포들을침전시켜상층액을수집하고, 상층액내 PGE 2 생성량을 EIA kit (R&D Systems Inc., Minneapolis, MN, USA) 를이용하여사용자매뉴얼에기재된방법대로정량해분석하였다. 7) RNA 분리및 RT-PCR MGH에의한 COX-2 와 inos 발현과의상관성을알아보기위하여 RT-PCR 로 mrna 발현을조사하였다. RNA 및 RT-PCR RNA 세포의준비는 6-well culture plate에 3 10 5 세포로분주한다음, 하루밤동안안정화시켰다. 이세포에 MGH 물추출물 (100, 250μg/ml) 을처리한후, LPS로자극하고 24시간후에세포를모아 PBS로세척하여이지블루 (Easy Blue, 인트론사 ) 1 ml를가하여실온에서교반하였다. chloroform 200μl를넣고다시교반하여 13,000 rpm, 4 o C에서 10분간원심분리한다음, 상등액 400μl에 isopropanol 을가하여다시원심분리하여 RNA pellet 을얻었다. 여기서얻어진 RNA에 MuLV역전사효소 (reverse transcriptase), 1 mm dntp 0.5μg을넣어 cdna 를만들었다. 여기에 COX-2, inos와 β-actin primer 를넣고유전자증폭기 (thermal cycler) 를이용하여증폭시켰다. 이때 inos는 94 o C에서 2분, 55 o C에서 30초, 72 o C에서 30초동안반응시켰으며, COX-2 와 β-actin은 94 o C에서 2분, 58 o C에서 30초, 72 o C에서 30초동안반응시켰다. 만들어진 RNA를 2% agarose gel에전기영동시켜 UV 검출기로확인하였다. 8) Western blot analysis 배양이끝난세포를수집하여 2~3회 PBS (phosphate buffered saline) 로세척한후 1 ml의 lysis buffer 을첨가 www.e-jkmr.org 29

김일현 최종환 이세원 송용선 하여 30분간 lysis 시킨후 12,000 rpm에서 20분간원심분리하여세포막성분등을제거하였다. 단백질농도는 BSA (bovine serum albumin) 를표준화하여 Bio-Rad Protein Assay Kit를사용하여정량하였다. 20~30μg 의 lysate를 8~12% mini gel SDS-PAGE 로변성분리하여, 이를 PVDF (polyvinylidene difluoride) membrane (BIO- RAD, Richmond, CA, USA) 에 200 ma로 2시간동안 transfer 하였다. 그리고 membrane의 blocking 은 5% skim milk가함유된 TBST (0.1% Tween20+TBS) 용액에서상온에서 2시간동안실시하였다. MAPKS 의발현양을검토하기위한항체로는 JNK, ERK anti-mouse (1:1,000) (Calbiochem, La Jolla, CA, USA) 를 TBST 용액에서희석하여상온에서 2시간반응시킨후 TBST로 3회세정하였다. 2차항체로는 HRP (horse radish peroxidase) 가결합된 anti-mouse IgG (Amersham Pharmacia Biotech, Little Chalfont, UK) 를 1:2,000으로희석하여상온에서 1시간반응시킨후, TBST로 3회세정하여 ECL 기질 (Amersham Biosciences, Piscataway, NJ, USA) 과 1~3 분간반응후 X-ray 필름에감광하였다. 9) 통계분석모든실험은 3회이상반복으로이루어졌으며, 통계분석은 SPSS 11.5를사용하였다. 실험결과는각항목에따라평균치 ± 표준편차 (SEM) 를구하여신뢰수준 *p<0.05, **p<0.005에서통계적유의성을검증하였다. 결과»»» 1. 세포생존율에대한 MGH 의영향 RAW 264.7 세포에서 MGH의세포독성을 MTS assay 방법으로조사하였다. 그결과 MGH를 50, 100, 250, 500 μg/ml의농도로처리하였을때 50~250μg/ml 의농도에서세포의생존율에영향을미치지않았다 (Fig. 1). 따라서이후실험은 MGH의물추출물이세포독성이나타나지않는농도범위에서수행하였다. 2. Nitric oxide (NO) 생성에대한 MGH 의억제효과 NO는활성산소중하나이며, 최근염증유발에중요한역할을하는것으로알려져있다. 따라서 NO 생성에대한 MGH의효과를알아보기위해 Griess assay 방법을이용하여세포배양액중에존재하는 NO 2 의형태로측정하였다. MGH 물추출물은 100μg/ml, 250μg/ml의처리농도에서각각농도의존적이고유의적으로 ( 각각 p< 0.05, p<0.005) NO 생성이억제되는것으로나타났다 (Fig. 2). Fig. 1. Effect of MGH on cell viability in RAW 264.7 cells. The cell viability assessed using an MTS assay following incubation with different doses (50, 100, 250 and 500μg/ml) of MGH for 24 hours. Values are the mean±s.e. of duplicate determinations from three separate experiments. Fig. 2. Effect of MGH on LPS-induced NO production in RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated concentration of MGH for 30 minutes before being incubated with LPS (200 ng/ml) for 24 hours. The culture supernatant was subsequently isolated and analyzed for LPS treated group. Statistical significance: *p<0.05, **p<0.005, Significant differences between treated groups were determined using the Dunnett s t-test. Values are the mean±s.e. of duplicate determinations from three separate experiments. 30 J Korean Med Rehab 2013;23(3):27-35

3. inos mrna 발현에대한 MGH 의억제효과 5. COX-2 mrna 발현에대한 MGH 의억제효과 NO는 inos에의해생성되어염증상태에서혈관투과성, 부종등의염증반응을촉진시킬뿐만아니라염증매개체의생합성을촉진하여염증을심화시키는것으로알려져있다. 따라서 NO를생성하게하는 inos의 mrna 발현을알아보기위해 RT-PCR 을수행하였다. 그결과, LPS 처리에의해형성되는 inos mrna 발현모두 100, 250μg/ml에서현저한억제양상을보였다 (Fig. 3). MGH의 COX-2 mrna 발현에대한효과를알아보기위해 Western blot과 RT-PCR 을수행하였다. 그결과, LPS 처리에의해증가된 COX-2 mrna 발현은 100μg/ml, 250μg/ml 농도에서현저하게억제되는것을확인하였다 (Fig. 5). 6. IL-6 생성에대한 MGH의억제효과 4. PGE 2 생성에대한 MGH의억제효과대식세포매개 PGE 2 는 COX-1 과 COX-2 로부터생성되는데, 소량의경우생체대사에필수적이지만유도성 COX-2 의과발현에의한과량의 PGE 2 는강력한염증매개물질로작용하는것으로보고되어있다. 따라서본연구에서는 PGE 2 를대상으로 MGH의약리학적인효능평가를실시하였다. MGH를각각 100μg/ml, 250μg/ml 의농도로 30분동안처리한후, 그람-음성박테리아내독소인 LPS (200 ng/ml) 로 RAW 264.7 대식세포주를자극한후세포부유액을원심분리하여세포들을침전시켜상층액을수집하고, 상층액내 PGE 2 생성량을측정하였다. 그결과, control 에서는 PGE 2 생성량이매우낮게측정되었으며, LPS에의해현저히증가되었다. MGH는 100μg/ml, 250 μg/ml 의처리농도에서각각농도의존적으로 PGE 2 생성이유의성있게 (p<0.05) 억제되는것을확인할수있었다 (Fig. 4). IL-6는염증단계에서중추적역할을한다고알려져있다. 대식세포주인 RAW 264.7 세포로부터 IL-6의생성을 Fig. 4. Effect of MGH on LPS-induced PGE 2 production in RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated concentration of MGH for 30 minutes before being incubated with LPS (200 ng/ml) for 24 hours. The culture supernatant was subsequently isolated and analyzed for LPS treated group. *P<0.05, when compared to the LPS treated group. Significant differences between treated groups were determined using the Dunnett s t-test. Values are the mean±s.e. of duplicate determinations from three separate experiments. Fig. 3. Effect of MGH on LPS-induced inos mrna expression in RAW 264.7 cells. inos mrnas were assessed by RT-PCR in RAW 264.7 cells. Cells were pretreated with the indicated concentrations of MGH for 30 minutes before being incubated with LPS (200 ng/ml) for 24 hours. The β-actin mrna was assayed in parallel to confirm equivalency of the cdna preparation. The experiment was repeated three times, and similar results were obtained. Fig. 5. Effect of MGH on LPS-induced COX-2 mrna expression in RAW 264.7 cells. COX-2 mrnas were assessed by RT-PCR in RAW 264.7 cells. Cells were pretreated with the indicated concentrations of MGH for 30 minutes before being incubated with LPS (200 ng/ml) for 24 hours. The β-actin mrna was assayed in parallel to confirm equivalency of the cdna preparation. The experiment was repeated three times, and similar results were obtained. www.e-jkmr.org 31

김일현 최종환 이세원 송용선 Fig. 6. Effect of MGH on LPS-induced IL-6 production in RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated concentrations of MGH for 30 minutes before being incubated with LPS (200 ng/ml) for 24 hours. Production of IL-6 was measured by ELISA. Cells were pretreated with the indicated concentrations of MGH for 30 minutes before being incubated with LPS (200 ng/ml) for 6 hours. *p<0.05, when compared to the LPS treated group. Significant differences between treated groups were determined using the Dunnett s t-test. Values are the mean±s.e. of duplicate determinations from three separate experiments. ELISA를이용하여실험하였다. 그결과, LPS 자극원을처리하였을때 IL-6 생성이현저히증가하였고, MGH를처리하였을때 IL-6 생성억제효과가유의성있게 (p<0.05) 나타났다 (Fig. 6). 7. MAPKs 의인산화에대한 MGH 의억제효과 MAPKs 는세포의성장과분화및 cytokine 과 stress 제어에중요한역할을한다. 따라서 MGH의억제메커니즘이 MAPKs 를경유하는지알아보기위해 MAPKs 의인산화를 Western blot을통해확인하였다. 그결과 LPS에의해활성화된 RAW 264.7 대식세포에 MGH를 100, 250μg/ml 을처리한경우 JNK1/2 와 ERK1/2 인산화를억제하는것을확인하였다 (Fig. 7). 그러나 p38의인산화에는영향이없는것을확인할수있었다. 고찰»»» 戊己丸은宋代 太平惠民和劑局方 8) 에처음기록된처방으로黃連, 吳茱萸, 白芍藥으로구성되어脾受濕氣泄痢 9) 不止米穀遲化臍腹刺痛을치료한다고하였다. 또한許는 東醫寶鑑 에痢疾의한종류인濕痢를치료한다하 Fig. 7. Effect of MGH on the phosphorylation (P-) of MAPKs in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with the indicated concentrations of MGH for 30 minutes before being incubated with LPS (200 ng/ml) for 30 minutes. Whole-cell lysates were analyzed by western blot analysis. The experiment was repeated three times, and similar results were obtained. 였다. 戊己丸의구성약재의본초학적효능으로黃連은大苦大寒하면서質이燥하여淸熱燥濕의효능이매우강력할뿐아니라解毒작용을겸하고있기때문에胃腸의濕熱로인한嘔吐, 瀉痢, 痔瘡등의병증에응용하며, 吳茱萸는性味가辛苦熱하면서燥하여溫散開鬱의효능이있으며, 熱證에속한병증에도황련과같은寒冷藥物을배합하여응용한다. 또한白芍藥은濕熱下痢증으로인한腹痛, 裏急後重의병증에黃芩, 黃連, 大黃, 木香, 甘草등의調和氣血, 淸熱解毒, 止痢의효능이있는약물을배합하여응용할수있다 18). 이러한약재들의효능과배합으로볼때戊己丸은이질과같은증상뿐만아니라염증성질환을치료하는데사용할수있을것으로생각되어, 본연구에서는설치류대식세포주인 RAW 264.7 세포를이용하여 LPS로유도된염증반응에서戊己丸이어떤작용기전을통해항염증효과를나타내는지연구하였다. 염증은자극에대한생체조직의방어반응의하나로서감염에의한외인적인요인및조직의 stress와기능부전에의한내인적인요인에의해유도되며 19) 손상된조직을수복하거나재생하려는기전이다 20). 그러나지속적인염증반응은점막손상을촉진시켜결과적으로통증, 부종, 32 J Korean Med Rehab 2013;23(3):27-35

발적, 발열등을일으켜기능장애를유발하며당뇨병, 동맥경화증, 관절염및암등의발생과깊은연관을갖고있다 21,22). 선천면역을담당하는대식세포는능동및수동면역반응에서매우중요한역할을한다. 또한, 초기염증반응에관여하는대표적인면역세포로서여러자극이나면역세포들이분비하는 cytokine 등에의해활성화되어 TNFα, IL-1, IL-6와같은 pro-inflammatory cytokine을비롯하여 eicosanoids, ROS, NO, PGE2, superoxide (O 2 ) 등의다양한염증매개물질들을생성함으로써통증, 부종, 열등의염증반응을유발하고, 염증부위로면역세포의이동을촉진시킨다 23-26). 최근, LPS에의한대식세포의활성은 toll-like receptor (TLR) 4의발현을조절한다고알려져있다. LPS와 TLR4가결합하여활성화되면세포질조절단백질인 MyD88 이모이게되고여러신호전달기전을통하여전사인자인 nuclear factor-κb (NF-κB) 가세포질내로이동하여활성화된다고보고되고있다 27). 이러한 NF-κB 전사인자의활성화는염증매개물질들인 NO, prostaglandins (PGs) 그리고 pro-inflammatory cytokine 등을조절한다 28). NO는반응성이높은물질로 NO synthase (NOS) 에의해 L-arginine 으로부터생성되며, 신경전달과혈관확장등생리적인기능을조절하는중요한역할을하지만, 과발현된 NO는혈관투과성, 부종, 염증을심화시켜조직을손상시키고암으로의진행을촉진한다 29,30). NOS는 constitutive NOS와 inducible NOS (inos) 로나누어지는데, 특히 inos는외부자극이나 pro-inflammatory cytokine 등에의해자극받게되면 hepatocytes, smooth muscle cells, bone marrow cells, monocytes, macrophages 등다양한세포에서발현되어다량의 NO를생성한다고보고되고있다 31). 염증반응의중요한매개체인 prostaglandin (PG) E 2 는 COX에의해생성되는데, COX에는 COX-1 과 COX-2 두종류가있으며, COX-1 은생체내의대부분의조직에존재하며일정하게발현되는반면, COX-2 는염증자극원에의해 macrophage 와같은염증성세포에서유도되며, PGE 2 와같은다량의 PGs를생성함으로써염증관련질병을유발하는것으로밝혀져있다 32). 또한 IL-6, IL-1β, TNF-α는염증반응을촉진하며면역계를자극하는염증유발인자로종양의침습에도관련이있다고알려져있 다 33). 이러한사실에기초하여 MGH가 RAW 264.7 세포에서 LPS에의해유도된 NO의생성을저해함을확인하였다. 또한 Western blot으로분석한결과 MGH에의한 inos 의발현억제는 NO 형성억제와유사한경향을나타냄으로써 NO 형성억제는 inos의발현저해를경유한것임을알수있었다. MGH가 LPS에의해유도된 inos 단백질및 mrna 발현을저해되는것으로보아 MGH가 LPS 에의해유도된 inos의전사와번역모두를저해한다고추정된다. 다수의항염증약물들의작용기전은 prostaglandin 합성을억제하며이는 COX-2 의생성및효소활성저해에의한것이다. 따라서 COX-2 에의한 prostaglandin 의합성은염증반응을매개하는것으로여겨지고있다고보고 MGH가 LPS에의해형성되는 PGE 2 를유의성있게감소시키며이는 COX-2 단백질과 mrna 의발현저해에의한것임을확인할수있었다. 염증매개물질인 IL-6는 in vivo 및 in vitro 모두에서염증반응을조절하는것으로알려져있다. 이러한 cytokine 들은서로상호작용이있는것으로알려져있으며 LPS등의염증자극물질에의해생성이유도된다고보고되었다 34). 따라서본연구에서 LPS에의해유도된 IL-6의생성에대한 MGH의억제효과를실험하였다. 그결과, IL-6의생성을 ELISA로분석하고, 이러한 COX-2, inos 그리고 pro-inflammatory cytokines 의발현에대해조사하였다. 또한, 현재까지비교적잘알려진염증반응의분자신호전달기전은 MAPK superfamily에속하는세가지효소들로 extracellular signal-regulated kinase (ERK), c-jun NH2-protein kinase (JNK)/stress-activated protein kinase (SAPK), serine/threonine protein kinase인 p38 MAPK 등을들수있다. 이들 MAPKs 는모두다양한세포외자극에반응해 upstream MAPK kinase (MEK) 에의해 tyrosine과 threonine에서인산화가일어남으로써활성화된다. 위의 MAPKs 의활성형은그후에다른 kinase 나전사인자를인산화, 활성화시키고, 결국표적유전자의발현을변화시킨다. 어떤보고에의하면 RAW 264.7 세포에서 LPS로유도된 COX-2 발현이 MAPK 저해제들인 PD98059와 SB203580에의해 ERK1/2와 p38 MAPK 활성화가각각차단됨으로써부분적으로억제된다는것을증명했다 35). 유사하게마우스대식세포 J774에서 phospholipase A2 저해제인 methyl arachidonyl fluorophospho- www.e-jkmr.org 33

김일현 최종환 이세원 송용선 nate에의한 COX-2 유도는 PD98059 와 SB203580 을포함한 MAPK 저해제에의해억제되었다 36). 마찬가지로 LPS 는단핵세포에서 ERK2와 p38 MAPK 의인산화와활성화처럼 COX-2 단백질과그것의 mrna 전사물이발현을유도하였다는보고가있다 37). 따라서본연구에서도 MGH가 NO와 COX-2, IL-6와같은염증매개체들을어떠한신호전달을통해억제하는지알아보기위해 MAPKs 의인산화를억제하는지알아보았다. 그결과 MGH가 MAPKs 의인산화에서는 JNK1/2 와 ERK1/2 의인산화는억제하는반면, p38 MAPK 의인산화에는아무런영향을주지못했다. 이는 LPS로유도한대식세포의염증반응은 JNK1/2와 ERK1/2의신호전달경로를경유하는반면 p38의경로와는다르다는것을보여준다. 본실험결과를요약하면, MGH는 RAW 264.7 세포에서 LPS에의해유도되는 inos, COX-2, 그리고 IL-6 유전자의발현, PGE2 생성을효과적으로저해한다. 그러므로이러한결과들은한의학에서 MGH의항염증기전을밝힘으로써그과학적근거를제시할수있을것으로생각된다. 결론»»» 설치류의대식세포주인 RAW 264.7 세포를 LPS로자극하였을때戊己丸의항염증효과를실험하여다음과같은결론을얻었다. 1. 戊己丸은 LPS로유도된대식세포에서 NO 생성을농도의존적으로유의성있게 (100μg/ml, 250μg/ml의처리농도에서각각 p<0.05, p<0.005) 억제하였다. 2. 戊己丸은 LPS로유도된대식세포에서 PGE 2 생성을농도의존적으로유의성있게 (p<0.05) 억제하였다. 3. 戊己丸은 LPS로유도된 inos와 COX-2 의 mrna 발현을억제하였다. 4. 戊己丸은 LPS로유도된전염증성사이토카인 IL-6발현을유의성있게 (p<0.05) 억제하였다. 5. 戊己丸은 LPS로유도된 MAPKs JNK 1/2, ERK1/2 의인산화를억제하였으나 p38 인산화는억제하지못하였다. 이와같은결과로보아戊己丸은대식세포에작용하여 MAPKs 의인산화활성의저해를통해 NO, PGE 2, IL-6의생성과 inos, COX-2 발현을억제함으로써항염증효과가있음을알수있다. 참고문헌»»» 1. Morson BC. Pathology of inflammatory bowel disease. Gastroenterol Jpn. 1980;15(2):184-7. 2. Cline MJ. Leukocyte function in inflammation: the ingestion, killing, and digestion of microorganisms. Ser Haematol. 1970;3(2):3-16. 3. Bosca L, Zeini M, Traves PG, Hortelano S. Nitric oxide and cell viability in inflammatory cells: a role for NO in macrophage function and fate. Toxicology. 2005;208(2): 249-58. 4. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992;6(12):3051-64. 5. Turini ME, DuBois RN. Cyclooxygenase-2: a therapeutic target. Annu Rev Med. 2002;53:35-57. 6. Wei W, Li XY, Zhang HQ, Wu SG. Antiinflammatory and immuno pharma cology. 1st ed. Beijing:Renmin weisheng chubanshe. 2004:10-7. 7. Andreakos E, Foxwell B, Feldmann M. Is targeting Toll-like receptors and their signaling pathway a useful therapeutic approach to modulating cytokine-driven inflammation. Immunol Rev. 2004;202:250-65. 8. 宋太平惠民和劑局. 太平惠民和劑局方. 中國中醫藥出版社. 1996:158. 9. 許俊. 東醫寶鑑. 서울 : 동의보감출판사. 2007:406. 10. Bae EA, Han MJ, KIM NJ, Kim DH. Anti-Helicobacter pylori activity of herbal medicines. Biol Pharm Bull. 1998;21(9):990-2. 11. 정효원, 박용기. 황련추출물의분획화및 BV2 microglial cells 에서 LPS 에의해유도되는 nitric oxide 생성억제효과검정. 대한본초학회지. 2007;22(2):73-8. 12. Kuo CL, Chi CW, Liu TY. The anti-inflammatory potential of berberine in vitro and in vivo. Cancer Lett. 20. 2004;203(2):127-37. 13. Takada Y, Kobayashi Y, Aggarwal BB. Evodiamine abolishes constitutive and inducible NF-kappaB activation by inhibiting IkappaBalpha kinase activation, thereby suppressing NF-kappaB-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion. J Biol Chem. 29. 2005;280(17):17203-12. 14. Kang SS, Kim JS, Kim EM, Yun-Choi HS. Platelet anti-aggregation of Paeony root. Kor J Pharmacogn. 1991;22: 215-8. 15. Ro HS, Ko WK, Yang HO, Park KK, Cho YH, Park HS. 34 J Korean Med Rehab 2013;23(3):27-35

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