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1 大韓本草學會誌제 31 권제 2 호 (2016 년 3 월 ) Kor. J. Herbol. 2016;31(2):63-69 ISSN (Print), ISSN (Online) 仙鶴草추출물의대식세포에서의 LPS- 유도염증반응에대한효능연구 황지혜 1#, 남주현 2, 김우경 3, 배효상 4* 1 : 동국대학교한방신약개발센터, 2 : 동국대학교의과대학생리학교실, 3 : 동국대학교의학전문대학원알레르기내과, 4 : 동국대학교한의과대학사상체질의학교실 Effects of Agrimoniae Herba 30% ethanol extract on LPS-induced inflammatory responses in RAW264.7 macrophage cells Ji Hye Hwang 1#, Joo Hyun Nam 2, Woo Kyung Kim 3, Hyo Sang Bae 4* 1 : Korean Medicine R&D Center, Dongguk University, 2 : Department of Physiology, College of Medicine, Dongguk University, 3 : Department of Internal Medicine, Graduate School of Medicine, Dongguk University, 4 : Department of Sasang Constitutional Medicine, college of Korean Medicine, Dongguk University ABSTRACT Objectives : The aerial parts of Agrimonia pilosa Ledeb (Agrimoniae Herba; AH) has been traditionally used as a Korean medicine to treatment of abdominal pain, sore throat, headaches, bloody discharge, parasitic infections and eczema. In this study, we investigated the effect of AH ethanol extract on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells. Methods : AH was extracted by 30% ethanol (AH-E). Raw264.7 cells were treated with AH-E extract at different concentrations for 30 min and then stimulated with LPS (1μg/ ml ) or without for indicated times. Cell viability was measured by MTT assay, and nitric oxide (NO) production was measured by Griess assay. The expression of inflammatory mediators, inos and COX-2 and inflammatory cytokines, TNF-α, IL-1β, and IL-6 was detected by RT-PCR, and the phosphorylation of ERK1/2, p38 and JNK MAP kinases (MAPKs) was analyzed by Western blot. Also, the expression of NF-κB in nuclear and cytosol was detected by Western blot. Results : AH-E extract significantly decreased LPS-induced NO production in RAW264.7 cells. AH-E extract inhibited the mrna expression of inos, COX-2, TNF-α, IL-1β, and IL-6 in LPS-stimulated cells with a dose-dependent manner. In addition, the phosphorylation of ERK, p38 and JNK MAPKs was also inhibited by AH-E extract. AP-E extract inhibited the nuclear translocation of NF-κB in LPS-stimulated cells. Conclusions : Our results suggest that AH-E extract has an anti-inflammatory activity in macrophages-mediated inflammation. Key words : Agrimonia pilosa Ledeb, 30% ethanol extract, anti-inflammation, RAW264.7 cells 서론 1) 아토피피부염 (Atopic dermatitis) 은주로유 소아에서발생하는만성재발성피부염으로알레르기반응이유전적으로발생하는아토피질환에속한다 1). 아토피피부염은대개생후 2-3개월부터 2세사이에발생하여일생동안지속적으로나타날수있는급 만성습진이며, 얼굴, 머리, 몸통, 팔다리등펴지는부위에주로발생하여태열과급성습진의양상을나타내게된다. 현재전세계적으로아토피피부염환자수가증가하는추세로우리나라에서도유병률이꾸준히증가하여 *Corresponding author : Hyo Sang Bae, Department of Sasang Constitutional Medicine, college of Korean Medicine, Dongguk University Tel : bjypapa@gmail.com #First author : Ji Hye Hwang, Korean Medicine R&D Center, Dongguk University Tel : jhbori@nate.com Received:21 February 2016 Revised:25 March 2016 Accepted:25 March 2016

2 64 大韓本草學會誌 Vol. 31 No. 2, 2016 최근 20-30년사이어린이의 20% 이상에서발병하는것으로보고되고있다 2). 아토피피부염은나이에따라유아기, 소아기, 사춘기및성인기로구분하였으나최근알레르기유무에따라내인성과외인성으로구분하고있다. 아토피피부염은복합적요인으로발병하는다인자성질환으로발생원인은현재까지구체적으로밝혀지지않았으며가족력에따른유전적요인, 알레르겐이나자극물질, 감염, 기후, 스트레스등의환경적요인, 2형보조 (Th2) T세포면역반응의이상, 피부보호막의이상등의원인으로다양한아토피피부염증상이발현하게된다 1,3). 현재아토피피부염의치료는피부보습, 스테로이드연고, 국소면역조절제등을통한일반치료외항히스타민제, 피부감염치료, 감마리놀렌산등을이용한보조치료, 그리고광선치료, 전신스테로이드제, 면역억제제등의선택치료를사용하고있다. 이중국소스테로이드제는지난 50년이상아토피피부염의가장기본적이고효과적인치료제로사용되고있다 4). 그러나최근피부위축, 모세혈관확장증, 스테로이드성여드름, 자색반, 팽창선, 다모증, 자반, 녹내장등다양한부작용이알려지면서아토피피부염환자나보호자의 70% 가스테로이드제사용을꺼리는추세로민간요법에대한이용빈도가높고새로운치료요법이나치료제에대한관심도높은질환이다. 한의학에서아토피피부염은濕疹, 奶癬, 乳癬, 浸淫瘡, 胎熱, 血風瘡, 四彎風, 旋耳瘡등의범주로설명하고瘙痒感, 紅斑, 浮腫, 滲出物, 鱗屑, 痂皮등의증상이나타난다 5,6). 이들의발병원인을風熱, 血熱, 血虛, 脾胃運化機能失調에의한胎火濕熱, 風濕熱의侵入등으로본다 7). 한의학적기본치료방법은淸熱解毒이주가되며濕型과乾型으로나눌수있는데, 濕型의경우燥濕, 淸熱, 止痒의治法으로하고乾型의경우淸熱, 止痒, 佐以除濕의治法으로한다 8). 仙鶴草 (Agrimonia pilosa Ledeb; Agrimoniae Herba) 는장미과에속한다년생본초인짚신나물의全草로, 性은平하고味는苦澁하며收斂止血, 截瘧, 止痢, 解毒殺蟲의효능이있어喀血, 吐血, 崩漏下血, 血痢脫力勞傷등의出血症과瘧疾, 癰腫瘡毒, 陰痒帶下등에사용한다 9). 한방에서는지혈작용으로이질, 위궤양, 장염, 설사, 자궁출혈등에사용해왔고, 민간요법으로는폐암, 간암, 식도암, 종양, 통증제거, 지형, 지사, 토혈, 혈뇨, 결기등에활용되어왔다 10,11). 선학초는전초에서 agrimonin, agrimonolide, tomentic acid, ellagic acid 및잎과줄기에서 luteolin-7-glucoside이주요성분으로구성되어있고 12,13), 최근실험연구로서항여드름효과 14), 간보호효과 15), 혈관이완효과 16), 항산화활성및암세포성장저해활성 17), 폐암종양크기감소효과 18), 항알레르기효과 19), 항산화효소활성증진효과 12), 아토피피부염에대한화피 ( 樺皮 ), 만형자 ( 蔓荊子 ), 선학초외용제의임상효과 20) 및뇌출혈억제효과 21) 등이다양하게보고되었다. 본연구에서는선학초의항아토피효능중염증반응에대한효과를확인하기위해마우스대식세포에서의 LPS-유도염증반응에대한억제효과와기전을조사하였다. 1. 재료 재료및방법 1) 약재본실험에사용된선학초 (Agrimoniae Herba; AH) 는광명당제약 ( 울산, 한국 ) 으로부터표준약재를구입하여추출물제조에사용하였다. 2) 시약및기기실험에사용된시약으로 lipopolysaccharide(lps), MTTbased cell proliferation kit, Sulfanilamide, N-1- napthylethylenediamine dihydrochloride(ned) 는 Sigma- Aldrich사 (St Louice, CA, USA), TRIzol reagent, NE-PER nuclear and cytoplasmic extraction reagents 는 Thermo Fisher Scientific 사 (Grand Island, NY, USA), RIPA buffer, Quick Start Bradford Protein Assay 용액은 Bio-Rad Laboratories사 (Philadelphia, PA, USA), M-MLV reverse transcriptase는 Promega사 (Madison, WI, USA), Taq-based PCR enzyme은 Toyobo 사 (Osaka, Japan), ERK1/2, p38, JNK, NF-κB 에대한단클론항체 (mab) 는 Cell Signaling 사 (Denvers, MA, USA) 로부터구입하여사용하였다. 실험에사용된기기로회전식감압농축기 (rotary evaporator) 와동결건조기는선일아이라사 ( 경기도, 한국 ), Asys microplate reader는 Biochrom사 (Cambridge, UK), BioDoc-It 2 Imaging Systems은 UVP사 (Upland, CA, USA), Nano-Drop은 Thermo Fisher Scientific사제품을사용하였다. 2. 방법 1) 추출물제조선학초추출물은선학초 (Agrimoniae Herba; AH) 의전초 100 g을 30 % 에탄올로 95 에서 2시간추출하였으며 3겹거어즈및와트만거름종이 (Whatman No. 1) 로거른후회전식감압농축기를이용하여농축하였다. 이를냉동시킨후동결건조기로건조하였으며이때수율은 18.4 % 였다. 건조된추출물은가루상태로마쇄한후일정용기에담아냉동보관하였으며, 1x PBS에일정농도로완전용해시킨후 0.22 μm주사기필터 (syringe filter) 로필터한후시험약물 (AH-E extract) 로사용하였다. 2) 세포배양 RAW264.7 세포는 ATCC사 (Manassas, VA, USA) 로부터구입하여 10 % FBS와 1 % penicillin- streptomycin이함유된 Dulbecco's Modified Eagle's Medium(DMEM) 을기본배지로하여배양하였다. 3) 세포독성평가 RAW264.7 세포에서선학초추출물의독성농도를선정하기위해 MTT-based proliferation assay를수행하였다. 즉, RAW264.7 세포 ( / ml ) 를 24-well culture plate에분주하여하룻밤배양한후선학초추출물을 0.05, 0.1, 0.2, 0.5 mg / ml농도로처리한후 24시간배양하였다. 여기기에 10 μl MTT(5 mg / ml ) 용액을넣고 37 CO 2 incubator에서 2시간이상반응시킨후상층액을모두제거하고 100 μl DMSO를첨가하여교반기위에서 15분간용해시켰다. 반응

3 仙鶴草추출물의대식세포에서의 LPS- 유도염증반응에대한효능연구 65 액의흡광도를 microplate reader기를이용하여 590 nm에서측정하였다. 세포생존도 (%) 는정상세포의생존률 100 % 를기준으로선학초추출물처리군의생존율을계산하였다. 4) Nitric oxide(no) 측정세포배양액내 NO 농도를측정하기위해 Griess assay를수행하였다. 먼저 RAW264.7 세포 ( / ml ) 를 24-well culture plate에분주하여하룻밤배양한후선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하여 30분간배양하였다. 여기에 LPS(1 μg / ml ) 를처리혹은처리하지않고 24시간배양한후세포배양액을수거하였다. 세포배양액 100 μl를 96- well plate에넣고 1 % sulfanyamide(in 5 % phosphoric acid) 용액 50 μl와 0.1 % NED 용액 (in water) 50 μl를각각넣은후실온에서 10분간정치하였다. 반응액의흡광도를 microplate reader 기를이용하여 540 nm에서측정하였으며, 0.1 M sodium nitrite(in water) 표준용액의흡광도곡선 (reference curve) 을기준으로 NO의농도를계산하였다. 5) RT-PCR 세포내유전자발현변화를확인하기위해 RT-PCR을수행하였다. 즉, RAW264.7 세포 ( / ml ) 를 6-well culture plate에분주하여하룻밤배양한후선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하여 30분간배양하였다. 여기에 LPS(1 μg / ml ) 를처리혹은처리하지않고 5시간배양한후세포를수거하고 Trizol reagent 를이용하여 total RNA를분리하였다. 분리된 RNA는 DEPC water에녹인후 Nano- Drop을이용하여정량하였다. 5 μg RNA에 M-MLV reverse transcriptase를넣고 25 10분, 42 60분, 72 15분조건으로반응시켜 cdna를합성하였으며, cdna에 Taqbased PCR enzyme 과 primer(table 1) 를넣어 94 30초, 58~61 30초, 72 60초조건을 30회반복하는 PCR을수행하였다. PCR 반응산물은 EtBr이함유된 1 % agarose gel에서 100V 조건으로 20분간전기영동한후 BioDoc-It 2 Imaging Systems 을이용하여확인하였다. 각밴드는 Image J program 을이용하여 density 를측정하고 GAPDH 발현에대한비율로계산하여반복실험에대한결과를 Histogram 으로나타내었다. Table 1. Primer sequences for PCR Name inos Sequence (5' 3') CCCGAAGTTTCTGGCAGC GGCTGTCAGAGCCTCGTGGCTT 6) Western blot 세포내단백질발현변화를확인하기위해 Western blot 을수행하였다. 즉, RAW264.7 세포 ( / ml ) 를 6-well culture plate 에분주하여하룻밤배양한후선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하여 30 분간배양하였다. 여기에 LPS(1 μg / ml ) 를처리혹은처리하지않고 15 분 (MAPK), 2 시간 (NF-κB) 배양한후세포를수거하고 RIPA buffer 를이용하여세포질을분리하였다. 한편 NF-κB 의핵에서의발현확인을위해서 NE-PER nuclear and cytosol isolation kit 를이용하여핵과세포질을각각분리하였다. 세포질내단백질의양을 Bradford protein assay 용액으로측정한후 30 μg단백질을 SDS-PAGE 하고, nitrocellulose membrane 에 transfer 하였다. Membrane 은 3 % bovine serum albumin(bsa) 용액으로실온에서 1 시간 block 한후각단백질에대한 primary antibody 를넣고 4 에서 O/N 반응시켰다. Membrane 을 Trisbuffered saline with 0.1 % Tween 20(TBST) buffer(ph 7.5) 로 5 회세척한후 HRP- conjugated secondary antibody 용액과실온에서 1 시간반응시켰다. 이를다시 TBST buffer 로 5 회세척한후 X-ray film 에감광시켜밴드유무를확인하였다. 각밴드는 Image J program 을이용하여 density 를측정하고 β-actin 발현에대한비율로계산하여반복실험에대한결과를 Histogram 으로나타내었다. 7) 통계학적검정모든실험결과는최소 3 회반복실험에대한평균과표준편차 (mean ± SD) 로나타내었으며, 통계학적유의성은 GraphPad Prism 5.0 분석프로그램 (GraphPad Software, La Jolla, CA, USA) 에서 one-way ANOVA 와 Tukey's test 를이용하여검정하였고, p 값이 0.05 이하인경우를유의성있는것으로판정하였다. 1. 세포독성평가 결과 RAW264.7 세포에서선학초추출물 (AH-E extract) 의독성정도를평가하기위해 MTT assay 를수행하였다. 그결과, 선학초추출물 0.05, 0.1, 0.2 mg / ml처리농도에서는세포만배양한경우와유사한생존도를나타내었으며, 0.5 mg / ml처리농도에서는독성이있는것으로나타났다 (Fig. 1). 따라서이후실험에서는독성이나타나지않은 0.5 mg / ml이하농도에서수행하였다. 150 COX-2 TNF-α IL-1β IL-6 GAPDH GGAGAGACTATCAAGATAGTGATC ATGTCAGTAGACTTTTACAGCTC ATAGCTCCCAGAAAAGCAAGC CACCCCGAAGTTCAGTAGACA GCCTTGGGCCTCAAAGGAAAGAATC GGAAGACACAGATTCCATGGTGAAG TGGAGTCACAGAAGGAGTGGCTAAG TCTGACCACAGTGAGGAATGTCCAC GACATCATACTTGGCAGGTT CTCGTGGAGTCTACTGGTGT Cell viability (%) a***, b*** 0 AH-E extract (mg/ml) LPS (1 ug/ml) Fig. 1. Effect of Agrimoniae Herba exthanol extract on cell viability in RAW264.7 cells. Cells were treated with Agrimoniae Herba 30 % ethanol (AH-E) extract at 0.05, 0.1, 0.2, and 0.5 mg / ml for 30 min, and then stimulated with or without LPS (1 μg / ml ) for 24 hr. Cell viability was measured by MTT assay. Data represents mean ± SD of three independent experiments. *** P < vs. normal (a) or LPS alone (b).

66 大韓本草學會誌 Vol. 31 No. 2, 2016 2. NO 생성에대한효과활성화된대식세포로부터분비되는대표적염증물질인 Nitric oxide(no) 에대한선학초추출물 (AH-E extract) 의억제효과를확인하기위해세포배양액내 NO 농도를 Griess assay 방법으로측정하였다.

NO ( M) 100 80 a*** 60 a*** a*** 40 은 LPS 자극에의해증가되었으며, 선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하였을때처리농도에의존적으로감소하였다 (Fig. 4). 한편선학초추출물단독처리군에서는 COX-2의유전자발현이낮은것으로나타났다. Fig. 4. Effect of Agrimoniae Herba exthanol extract on LPSinduced COX-2 expression in RAW264.

4 66 大韓本草學會誌 Vol. 31 No. 2, NO 생성에대한효과활성화된대식세포로부터분비되는대표적염증물질인 Nitric oxide(no) 에대한선학초추출물 (AH-E extract) 의억제효과를확인하기위해세포배양액내 NO 농도를 Griess assay 방법으로측정하였다. 그결과, NO의농도는 LPS 자극에의해현저히증가되었으며, 선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하였을때 LPS 처리군에비해유의적으로또한처리농도에의존적으로감소하는것으로나타났다 (Fig. 2). 한편선학초추출물을단독으로처리한군에서는정상군과마찬가지로 NO의농도증가가나타나지않았다. NO ( M) a*** 60 a*** a*** 40 은 LPS 자극에의해증가되었으며, 선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하였을때처리농도에의존적으로감소하였다 (Fig. 4). 한편선학초추출물단독처리군에서는 COX-2의유전자발현이낮은것으로나타났다. Fig. 4. Effect of Agrimoniae Herba exthanol extract on LPSinduced COX-2 expression in RAW264.7 cells. Cells were treated with Agrimoniae Herba 30 % ethanol (AH-E) extract at 0.05, 0.1, 0.2, and 0.5 mg / ml for 30 min, and then stimulated with or without LPS (1 μg / ml ) for 5 hr. (A) The expression of COX-2 mrna was determined by RT-PCR. (B) COX-2 band was analyzed per a GAPDH as an internal control, and represents histogram with mean ± SD of three independent experiments AH-E extract (mg/ml) LPS (1 ug/ml) Fig. 2. Effect of Agrimoniae Herba exthanol extract on LPSinduced NO production in RAW264.7 cells. Cells were treated with Agrimoniae Herba 30% ethanol (AH-E) extract at 0.05, 0.1, 0.2, and 0.5 mg / ml for 30min, and then stimulated with or without LPS (1 μg / ml ) for 24 hr. NO levels were measured in culture medium by Griess assay. Data represents mean ± SD of three independent experiments. *** P < vs. LPS alone (a). 3. inos 발현에대한효과 NO 합성효소인 inos 발현에대한선학초추출물 (AH-E extract) 의억제효과를확인하기위해 RT-PCR을수행하였다. 그결과, inos의유전자발현은 LPS 자극에의해증가되었으며, 선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하였을때처리농도에의존적인감소를나타내었다 (Fig. 3). 한편선학초추출물을단독처리군에서는정상군과유사하게증가하지않는것으로나타났다. 5. 염증성사이토카인발현에대한효과대표적염증성사이토카인인 TNF-α, IL-1β, IL-6 발현에대한선학초추출물 (AH-E extract) 의억제효과를확인하기위해 RT-PCR을수행하였다. 그결과, TNF-α, IL-1β, IL-6의유전자발현은모두 LPS 자극에의해증가되었고, 선학초추출물을 0.05, 0.1, 0.2 mg / ml농도로처리하였을때농도의존적인감소를나타내었다 (Fig. 5). 한편선학초추출물을단독처리군에서는정상군과마찬가지로 TNF-α, IL-1 β, IL-6 유전자발현에영향을주지않는것으로나타났다. Fig. 3. Effect of Agrimoniae Herba exthanol extract on LPS-induced inos expression in RAW264.7 cells. Cells were treated with Agrimoniae Herba 30 % ethanol (AH-E) extract at 0.05, 0.1, 0.2, and 0.5 mg / ml for 30 min, and then stimulated with or without LPS (1 μg / ml ) for 5 hr. (A) The expression of inos mrna was determined by RT-PCR. (B) inos band was analyzed per a GAPDH as an internal control, and represents histogram with mean ± SD of three independent experiments. * P < 0.05 vs. LPS alone (a). 4. COX-2 발현에대한효과 inos와마찬가지로대표적염증물질인 COX-2 발현에대한선학초추출물 (AH-E extract) 의억제효과를확인하기위해 RT-PCR을수행하였다. 그결과, COX-2의유전자발현 Fig. 5. Effect of Agrimoniae Herba exthanol extract on LPSinduced expression of inflammatory cytokines in RAW264.7 cells. Cells were treated with Agrimoniae Herba 30% ethanol (AH-E) extract at 0.05, 0.1, 0.2, and 0.5 mg / ml for 30 min, and then stimulated with or without LPS (1 μg / ml ) for 5 hr. (A) The mrna expression of TNF-α, IL-1β, IL-6 was determined by RT-PCR. (B) Each band was analyzed per a GAPDH as an internal control, and represents histogram with mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01 vs. LPS alone (a). 6. 염증신호분자발현에대한효과 활성화된대식세포에서염증물질의합성과분비를조절하는염증신호전달분자들의활성화에대한선학초추출물 (AH-E extract) 의억제효과를확인하기위해 ERK1/2, p38, JNK MAPKs와 NF-κB 에대한 Western blot을수행하였다. 그결과, ERK1/2, p38, JNK MAPK 분자들은모두 LPS 자극에의해인산화가증가되었고, 선학초추출물 0.1 mg / ml과 0.2 mg / ml농도에서감소하는것으로나타났다 (Fig. 6A). 한편선학초추출물을단독처리군에서는정상군과마찬가지로

仙鶴草추출물의대식세포에서의 LPS- 유도염증반응에대한효능연구 67 ERK1/2, p38, JNK MAPK 분자들의인산화에영향을주지않는것으로나타났다. 또한핵과세포질에서의 NF-κB 발현변화를조사한결과, LPS 자극후 NF-κB 의핵으로의이동에의한발현증가가관찰되었으며, 선학초추출물처리에의해감소하는것으로나타났다 (Fig. 6B). Fig. 6. Effect of Agrimoniae Herba exthanol extract on LPSinduced expression of MAP kinases and NF-κB in RAW264.

5 仙鶴草추출물의대식세포에서의 LPS- 유도염증반응에대한효능연구 67 ERK1/2, p38, JNK MAPK 분자들의인산화에영향을주지않는것으로나타났다. 또한핵과세포질에서의 NF-κB 발현변화를조사한결과, LPS 자극후 NF-κB 의핵으로의이동에의한발현증가가관찰되었으며, 선학초추출물처리에의해감소하는것으로나타났다 (Fig. 6B). Fig. 6. Effect of Agrimoniae Herba exthanol extract on LPSinduced expression of MAP kinases and NF-κB in RAW264.7 cells. Cells were treated with Agrimoniae Herba 30% ethanol (AH-E) extract at 0.05, 0.1, 0.2, and 0.5 mg / ml for 30 min, and then stimulated with or without LPS (1 μg / ml ) for 15 min (for MAPKs) and 2 hr (for NFκB). (A) The phosphorylation of ERK1/2, p38, and JNK MAPKs was determined in cytosol by Western blot. (B) The expression of NFκB p65 was determined in nuclear by Western blot. 고찰 선학초는薔薇果에속한다년생본초인짚신나물의全草로, 큰등골짚신나물 (Agrimonia eupatoria L.) 외에짚신나물 (Agrimonia pilosa L.), 산짚신나물 (Agrimonia coreana Pilosella Satake) 등이알려져있으며, 이들을통틀어용아, 용아초, 황아초, 황용초, 지선초등으로부르기도한다 9,10). 선학초는다양한추출물형태에서효능보고가이루어져있는데, 메탄올추출물의여드름유발균증식억제효과 14), 물추출물의흰쥐에서의 CCL 4-유도급성손상에대한간보호효과 15), 부탄올추출물의흰쥐대동맥혈관에서의이완효과 16), 다양한분획물들의항산화활성및대장암, 위암, 자궁경부암세포에서의성장저해활성 17), 폐암유발마우스에서의종양크기감소효과 18), 메탄올추출물의난알부민-유도폐염증유발마우스에서의알레르기성염증억제효과 19), 물추출물의항산화효소활성증진효과 12), 아토피피부염에대한화피 ( 樺皮 ), 만형자 ( 蔓荊子 ), 선학초외용제의임상효과 20) 및흰쥐에서의뇌선조체내출혈억제효과 21) 등이보고되었다. 본연구와유사하게메탄올추출물이 NO, PGE 2, IL-1β, IL-6 분비와 inos, COX-2 발현을억제한다는보고가있으나 ERK, JNK, p38 MAPK 의인산화에대해효과가없는것으로보고 19) 되어차이를나타내었다. 이는추출물유형차이로인해활성에차이가나타난것으로해석할수있으나이에대해서는추후좀더연구되어야할것으로사료된다. 본연구자는아토피피부염에서나타나는소양감, 피부손상, 염증반응중에서선학초는염증을억제하는효능이있다는것에착안하여아토피피부염에대한효능을검증하기위한연구중하나로본연구에서와같이 RAW264.7 대식세포에서의 LPS-유도염증반응에대한억제효과를조사하였다. 아토피피부염은심한소양감을동반하는만성재발성피부염증이기때문에염증억제는중요한치료목표이며스테로이드제제에비해부작용이적은천연물제제에대한관심이높아지고있다 22-24). 이러한변화에맞추어염증과알레르기에관련된세포내분자들을대상으로직접적인항염증인자및간접적으로병리적유관성을가진인자들에대한다양한연구가이루어지고있다 25-27). 한편초기염증반응에서는항원을인지한대식세포가항원을처리하면서면역세포에항원을제공함과동시에염증물질인 NO를생성하고, TNF-α, IL-6, IL-1β와같은사이토카인들을분비하게된다 28,29). TNF-α 는염증초기에감염부위의모세혈관투과성을증가시켜해당부위로호중구를유도하며, 급성염증반응을일으키는주요인자이고 30), IL-6는백혈구내의 B 림프구의분화와성장을증가시키며, 항체를생성하게되면서알레르기성염증을만성적인단계로진행시키게된다 31). 일반적으로면역세포내염증반응이일어나게되면 NO가다량합성되는것으로알려져있는데, NO는 inos에의해합성되고면역반응에중요한요소로서특히대식세포에서만들어지는고농도의 NO는산화적스트레스를통해염증유발및조직손상과관련이있는것으로알려져있고, 다양한염증질환에서의증상유발과질병악화에기여하는것으로알려져있다 32,33). 본연구에서선학초의 30 % 에탄올추출물은 RAW264.7 세포에서 LPS 자극에의한 NO의분비를농도의존적으로감소시킴으로써염증반응을억제할수있는것으로나타났다. COX-2는염증물질인 PGE 2 를합성하는효소단백질로 PGE 2 합성을통해혈관확장, 통증, 발열작용등을통한염증유발과면역반응을과도하게촉진시키는작용이있어염증조절에중요한인자로알려져있다 34). 따라서 inos와 COX-2 의발현이나 NO, PGE 2 의정량평가는약물의항염증효능을평가하는데유용한지표로사용되고있다. 본연구에서선학초의 30 % 에탄올추출물은 LPS 자극으로부터발현이증가되는 inos와 COX-2의전사단계조절을통해염증물질의생성을감소시킬수있는것으로나타났다. LPS는대식세포에서 toll-like receptor(tlr) 4와결합함으로써활성화되어각종염증물질의합성 분비를통해염증의개시및유지에중요한작용을한다. 이러한염증반응은 ERK, p38, JNK MAPK와같은염증신호분자들의인산화와염증전사인자인 NF-κB pathway를통해각종염증성사이토카인들이나염증물질의합성과분비로급성염증반응뿐아니라만성염증반응으로의이행을유도하게된다 35). MAPK들은대식세포에서염증반응을유발하도록세포밖신호를핵내로전달하게하는대표적인염증신호전달분자로, LPS와같은자극원으로부터인산화되어염증전사인자인 NF-κB 의핵내로의이동을촉진시킴으로써대식세포내염증반응개시를유도하게되고, 이러한염증기전가동에의해 NO, inos, COX-2, TNF-α, IL-1β, IL-6와같은염증물질들이합성, 분비됨으로써염증이발달하게된다 36,37). 본연구에서선학초의 30 % 에탄올추출물은 LPS 자극으로부터활성화된대식세포에서

6 68 大韓本草學會誌 Vol. 31 No. 2, 2016 MAPK 분자들의인산화억제와 NF-κB pathway 차단을통해 NO 분비및 inos, COX-2의발현억제및 TNF-α, IL-1β, IL-6 등염증성사이토카인들의전사단계조절을통해염증반응을제어할수있는것으로나타났다. 결론 본연구에서는 RAW264.7 대식세포에서선학초 30% 에탄올추출물의염증억제효과를확인하여다음과같은결론을얻었다. 1. 선학초추출물 (0.05, 0.1, 0.2 mg / ml ) 은 RAW264.7 세포에서 LPS-유도 nitric oxide의생성을억제하였다. 2. 선학초추출물 (0.05, 0.1, 0.2 mg / ml ) 은 RAW264.7 세포에서 LPS-유도 inos와 COX-2의유전자발현을억제하였다. 3. 선학초추출물 (0.05, 0.1, 0.2 mg / ml ) 은 RAW264.7 세포에서 LPS-유도 TNF-α, IL-1β, IL-6의유전자발현을억제하였다. 4. 선학초추출물 (0.1, 0.2 mg / ml ) 은 RAW264.7 세포에서 LPS-유도 ERK1/2, p38, JNK MAPK의인산화를억제하였다. 5. 선학초추출물 (0.1, 0.2 mg / ml ) 은 RAW264.7 세포에서 LPS-유도 NF-κB 의핵에서의발현증가를억제하였다. 따라서선학초 30% 에탄올추출물은대식세포활성화에따른염증반응억제효과가있는것으로확인되었으며, 아토피피부염에서의피부조직염증억제에효과가있을것으로사료되었다. 감사의글 본연구는한국보건산업진흥원을통해보건복지부 " 양 한방융합기반기술개발사업 " 의재정지원을받아수행된연구입니다 (HI15C0256). References 1. Boguniewicz M, Leung DYM. Atopic dermatitis. J Allergy Clin Immunol ; 117(Suppl) : Kapoor R, Menon C, Hoffstad O, Bilker W, Leclerc P, Margolis DJ. The prevalence of atopic dermatitis in children with physician-confirmed atopic dermatitis. J Am Acd Dermatol ; 58 : Buske-Kirschbaum A, Gierens A, Hollig H, Hellhammer DH. Stress-induced immunomodulation is altered in patients with atopic dermatitis. J Neuroimmunol ; 129 : Charman CR, Williams HC. The use of corticosteroid and corticosteroid phobia in atopic dermatitis. Clin in Dermatol ; 21 : Hong CE. Pediatrics. Seoul : Daehan Publisher : Ha YT. A Study on Combination of Topical Jachobokhabbang (JBB) and Internal Hwangtogamibang (HTGMB) for the Treatment of Atopic Dermatitis. Daejeon Univ Park SN. Effect of Sopungsan in BALB/c Mouse Model of Atopic Dermatitis. Kyunghee Univ Cha KB, Kim YS, Seol IC. Literatual study on Atopic dermatitis. J Orient Med Res Center Daejeon Univ ; 14(2) : The National College of Oriental Medicine Herbology Classroom. Herbology. Seoul : Youngrimsa : 190-1, 427-8, Kang BS. Medical herbs. Seoul : Youngrimsa : Lee Y, Kim M, Chung D. Effect of extract Agrimonia pilosa L. on biological activity in rats. Korean J Medicinal Crop Sci ; 10(3) : 167.\ 12. Yoon JY, Lee SY, Lee JY. Anti-aging effects of solvent fraction from Agrimonia pilosa L. extracts. J Appl Biol Chem ; 55(1) : Pei YH, Li X, Zhu TR. Studies on the structure of a new ellagic acid glycoside from the root sprouts of Agrimonia pilosa. Yao Xue Xue Bae ; 25 : Kim HJ, Lim HW, Kim BH, Choi SW, Yoon CS. Studies on the Anti-acne Effect of Agrimonia pilosa Ledeb. J Soc Cosmet Scientists Korea 2006 ; 32(1) : Kang SC, Lee CM, Koo HJ, Ahn DH, Choi H, Lee JH, Bak JP, Lee MH, Choung ES, Kawk JH. Hepatoprotective effects of aqueous extract from aerial part of Agrimmony. Kor J Pharmacogn ; 37(1) : Hua CL, Lee JK, Cho KH, Kang DG, Kwon TO, Kwon JW, Kim JS, Sohn EJ, Lee HS. Mechanism for the vascular relaxation induced by butanol extract of Agrimonia pilosa. Kor J Pharmacogn ; 37(2) : Min KJ, Song JW, Cha CG. The antioxidative and antitumor activity of extracts of Agrimonia pilosa. J Fd Hyg Safety ; 23(2) : Choi SJ, Baik JW, Park JH, Jun CY, Choi YK, Ko SG. Mechanism of apoptosis & tumor growth inhibition of Agrimonia pilosa Ladebour (APL) in vitro and in vivo. Korean J Orient Int Med ; 30(2) :

7 仙鶴草추출물의대식세포에서의 LPS- 유도염증반응에대한효능연구 Kim JJ, Jiang J, Shim SW, Kwon SC, Kim TJ, Ye SK, Kim MK, Shin YK, Koppula S, Kang TB, Choi DK. Anti-inflammatory and anti-allergic effects of Agrimonia pilosa Ledeb extract on murine cell lines and OVA-induced airway inflammation. J Ethnopharmacol ; 140 : Hong JH, Jung HA. Clinical efficacy of external application containing Betula platyphyllae Cortex, Viticis Fructus, Agrimoniae Herba in atopic dermatitis. J Korean Med Ophthalmol Otolaryngol Dermatol ; 27(1) : Kim YS, Choi YS. Neuroprotective Effects of Agrimoniae Herba against Intrastriatal Hemorrhage in Rats. Kor J Herbol ; 25(4) : Hoffman ES, Smith RE, Renaud BC Jr. From the analyst's couch: TLR-targeted therapeutics. Nat rev drug discov ; 4(11) : Kim TY. Treatment of Atopic Dermatitis : an Update and Review of the Literature. Pediatr Allergy Respir Dis(Korea) ; 19(3) : Lee EH, Lee SE, Lee SH. Current Trends of Diagnosis and Treatment of Atopic Dermatitis. Korean Soc Skin Barrier Res ; 12(1) : Kang RY, Park BK, Gim SH, Choi HJ, Kim DH. The effects of HYT on various immunological factors related to pathogenesis of allergic dermatitis in NC/Nga mice induced by Biostir AD. J Orient Med Res Center Daejeon Univ ; 18(2) : Kim MW. The cure effect of Chengsimyenjatang on atopic dermatitis by regulation of Th2 cell differentiation and anti-inflammatory mechanism. Dongguk Univ Kim HG. Inhibitory Effect of Galgeunhaegi-Tang on Compound48/80 stimulated Allergic Reaction. Daegu Univ Yun KJ, Kim JY, Kim JB, Lee KW, Jeong SY, Park HJ, Jung HJ, Cho YW, Yun KJ, Lee KT. Inhibition of LPS-induced NO and PGE 2 production by asiatic acid via NF-κB inactivation in Raw macrophages: Possible involvement of the IKK and MAPK pathways. Int Immunophar ; 8 : Baugh JA, Bucala R. Mechanisms for modulating TNF-αin immune and inflammatory disease. Curr Opin drug discov devel ; 4(5) : Zhang Y, Ramos BF, Jakschik BA. Neutrophil recruitment by tumor necrosis factor from mast cells in immune complex peritonitis. Science ; 258 : Gabay C. Interleukin-6 and chronic inflammation. Arthritis Res Ther ; 8 : Chae SC, Jung HT. NO and the immune response. Allergy Asthma Respiratory disease ; 22(3) : Kwon YH, Joo KB, Bae SC, Kim SY. Nitric oxide(no) in inflammatory arthritis. Hanyang Medical Reviews ; 16(2) : Yang Y, Kim SC, Yu T, Yi YS, Rhee MH, Sung GH, Yoo BC, Cho JY. Functional roles of p38 mitogen-activated protein kinase in macrophagemediated inflammatory responses. Mediators Inflamm ; 2014 : Limtrakul P, Yodkeeree S, Pitchakarn P, Punfa W.Suppression of Inflammatory Responses by Black Rice Extract in RAW Macrophage Cells via Downregulation of NF-kB and AP-1 Signaling Pathways. Asian Pac J Cancer Prev ; 16(10) : Ivashkiv LB. Inflammatory signaling in macrophages: transitions from acute to tolerant and alternative activation states. Eur J Immunol. 2011; 41(9) : Aggarwal BB, Shishodia S, Takada Y, Jackson- Bernitsas D, Ahn KS, Sethi G, Ichikawa H. TNF blockade: an inflammatory issue. Ernst Schering Res Found Workshop ; (56) :

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386-390.hwp 386 HANYANG MEDICAL REVIEWS Vol. 29 No. 4, 2009 우리나라 미숙아의 통계와 의료비용 Statistics and Medical Cost of Preterm in Korea 윤혜선 을지대학교 노원을지병원 소아청소년과학교실 Hye Sun Yoon, M.D., Ph.D., Department of Pediatrics, Nowon