J Korean Med Ophthalmol Otolaryngol Dermatol 2015;28(1):23-31 pissn 1738-6640 eissn 2234-4020 http://www.ood.or.kr http://dx.doi.org/10.6114/jkood.2015.28.1.023 Original Article / 원저加味潤燥湯이 LPS 로유도된 RAW 264.7 대식세포에서의항염효과연구 최종민 1 김용민 2 김희택 1 1 세명대학교한의과대학안이비인후피부과학교실 2 세명대학교한방화장품과학과 Anti-inflammatory Effects of Gamiyunjo-tang on Lipopolysaccharide Induced Inflammatory Responses in RAW 264.7 Cells Jong-Min Choi 1) Yong-Min Kim 2) Hee-Taek Kim 1) 1 Dept. of Oriental Ophthalmology, Otolaryngology & Dermatology, College of Korean medicine, Semyung University 2 Dept. of Oriental Medical and Herbal Cosmetic Sciences, Semyung University Abstract Objectives : Allergic disease has been well known as an IgE-dependent immunologic response. Recently, interest about the late inflammatory reaction has grown up as well as early allergic reaction characterized by IgE and mast cell. The purpose of this study was to find the anti-inflammatory effect of Gamiyunjo-tang(GMYJT) in allergic reaction. Methods : The experiment was performed using Raw 264.7 cells pretreated with GMYJT extracts. In this study, we observed the toxicity of cells by MTT analysis and measured the production of LPS-induced NO, PGE₂, IL-1β, IL-6 and TNF-α at a concentration of 50, 100, 200 and 400 μg / ml. Results : No toxicity of GMYJT (50, 100, 200, 400 μg / ml ) on RAW 264.7 cells was found after 24 hours incubation. LPS-induced NO production was reduced after treatment with GMYJT (100, 200, 400 μg / ml )(P<0.05). PGE₂was reduced after treatment with GMYJT (100, 200, 400 μg / ml )(P<0.05). IL-1β did not decrease at any dose. IL-6 decreased at 200, 400 μg / ml (P<0.05). TNF-α production decreased only at 400 μg / ml (P<0.05). c 2015 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. 23
Conclusions : These data suggest that GMYJT has anti-inflammatory effects in late allergic reaction. Key words : Anti-inflammation; NO; PGE 2; IL-1β; IL-6; TNF-α; Gamiyunjo-tang; Ⅰ. 서론 하고자한다. 알레르기초기면역반응에서는항원에노출된후 비만세포의탈과립이일어나면서알레르기증상을유 발시키는 histamine, prostaglandin, leukotriene 과 같은다양한매개물질들이분비된다. 그리고후기반 응은 T 세포, 호염기구, 호산구로이루어진염증세포 들의유입과이로인해특징지어지는복합적인염증 반응에의해이루어진다 1,2). 알레르기질환의지속적인염증반응에있어서는상 피세포, 비만세포와후기반응의주된역할을하는호 산구, 호염기구, T 세포와같은염증세포모두가관여 하여알레르기염증반응을유발하고지속시킨다. 이 러한일련의과정에서대식세포는 T 세포에항원을제 공하는기능을하여면역반응을개시하는데중요한 역할을하고, 비특이적인면역반응을담당하게된다. 그중 NO 는주로대식세포에서생성되는중간물질 3) 로 염증반응의정도를가늠하는척도가된다 4,5). 활성화 된대식세포는 interleukin-1 beta(il-1β), IL-6, tumor necrosis factor-α(tnf-α) 와같은 pre- inflammatory cytokine 과 prostaglandin E 2 (PGE 2 ) 등을생산하고, 이를통해염증반응을촉진시킴으로 써다양한염증관련질환의발달에기여한다 6,7). 본연구에사용한加味潤燥湯 8) 은全身瘙痒血燥風 毒性症에다용되며피부질환의급성기보다는만성기 에사용할수있는처방이다. 이에저자는 LPS 로유 도된 RAW 264.7 대식세포에서 NO 생성과염증반 응에관여하는사이토카인인 IL-1β, IL-6, TNF-α 에 대한실험을진행하여유의한결과를얻었기에보고 Corresponding Author : Hee-Taek Kim, Semyung University Oriental Medicine Hospital, 66, Semyeong-ro, Jecheon-si, Chungcheongbuk-do, Korea(Tel:043-649-1817, E-mail:kht8725c@naver.com) Recieved 2015/1/19 Revised 2015/2/2 Accepted 2015/2/9 1. 실험재료 Ⅱ. 실험방법 1) 처방구성및시료제조 加味潤燥湯 (Gamiyunjo-tang, 이하는 GMYJT) 의 구성약물을 HMAX( 제천, 한국 ) 에서구입하여사 용하였다 (Table 1). 가미윤조탕 2 첩분량에해당하는 112 g 을 3 차증류수 2 L 와혼합하여 100 로 4 시간 동안열수추출하였으며, 여과지로여과한추출액을 rotary evaporator 를이용하여 100 ml까지농축하고 -80 로동결하였다. 농축한동결액을 freezing dryer system (Labconco, USA) 을이용하여 7 일간동결건 조하였다 ( 수율약 18%). Table 1. The Amount and Composition of GMYJT Herbal Name Scientific Name Dose(g) 熟地黃 Rehmanniae Radix Preparat 6 生地黃 Rehmanniae Radix 6 白芍藥 Paeoniae Radix Alba 6 知母 Anemarrhenae rhizoma 6 黃芩 Scutellariae Radix 6 秦艽 Gentianae Macrophyllae Radix 6 玄蔘 Scrophulariae Radix 6 黑芝麻 Sesami Semen Nigrum 4 防風 Saposhnikovia Radix 4 浮萍 Spirodelae Herba 4 甘草 Glycyrrhizae Radix 2 Total 56 24
최종민외 4 인 : 加味潤燥湯이 LPS 로유도된 RAW 264.7 대식세포에서의항염효과연구 2) 세포배양실험에사용된 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 (100 U/ ml ) 및 streptomycin (100 μg / ml ) 등이포함된 DMEM 배지로배양되었다. 배양세포들은 75 cm 2 flask (Falcon, USA) 에서충분히증식된후배양 3일간격으로배양세포표면을 PBS 용액으로씻어준뒤 50 ml flask 당 1 ml의 0.25% trypsin-edta 용액을넣고실온에서 1분간처리한다음 trypsin 을버리고 37 에서 5분간보관하여세포를탈착하여계대배양하였다. 탈착된세포는 10% FBS가첨가된 DMEM 배양액 10 ml에부유시킨다음새로운배양용기 (50 ml culture flask) 에옮겨 1:2 split ratio로 CO 2 배양기에서배양하였다. 2. 실험방법 1) MTT assay 세포독성유발효과를알아보기위하여 3-(4,5- dimrthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 방법을사용하여측정하였다. 96 well plate에 1 10 5 cells/well 의 cell을 100 μl씩넣고 37, 5% CO₂가공급되는배양기에서 24시간동안배양한후배지를버리고배양세포표면을 1 PBS 용액으로씻어주었다. 같은양의배지와 PBS 에녹인시료를농도별로각 well에처리하고 24시간배양하였다. 배양이끝난후 PBS에녹인 1 μg / ml MTT (Sigma, USA) 를 100 μl씩각 well에처리하여알루미늄호일로차광시킨뒤 2시간동안같은조건에서배양하였다. 배양액을모두제거한후 DMSO 를 100 μl처리하고 37 에서 2시간방치한다음 microplate reader 를이용하여 490 nm에서흡광도를측정하였다. 세포생존율은다음과같은공식으로계산되었다. Viability(%) = 100 AT/AC AT : absorbance of tested extract solution AC : absorbance of control 2) NO assay 96 well plate 에 1 10 5 cells/well 의 cell을 100 μl씩넣고 37, 5% CO₂가공급되는배양기에서 24 시간동안배양하여세포를안정화시켰다. 안정화시킨세포에 lipopolysacharride (LPS) 1 μg / ml와가미윤조탕추출물을농도별로처리하고 24시간동안 3 7, 5% CO₂가공급되는배양기에서배양한후세포배양상등액 60 μl을채취하여여기에 Griess 시약 100 μl을혼합하여 15분동안반응시킨뒤 microplate reader 를이용하여 540 nm에서흡광도를측정하였다. 3) PGE2 생성측정 PGE 2 의측정은 commercial competitive enzyme immunoassay kit를 R&D systems (Minneapolis, USA) 에서구입하여사용하였다. RAW 264.7 세포에가미윤조탕과 1 μg / ml의 LPS를처리하여 24시간배양한후세포배양상층액을수거하여 PGE 2 측정에사용하였다. 배양액을 goat anti-mouse 로 coating 된 96 well plate에각각의배양액을 100 μl씩 loading 하고여기에 primary antibody solution 50 μl와 PGE 2 conjugate 50 μl씩첨가하여 4 에서 overnight 시켰다. 기질용액을 200 μl씩처리하여 5~20 분간반응시킨후, 50 μl의 stop solution 을처리하고 450 nm에서흡광도를측정하였다. 4) Cytokine 생성측정 96 well plate 에 1 10 5 cells/well 의 cell을 100 μl씩넣고 37, 5% CO₂가공급되는배양기에서 24 시간동안배양하여세포를안정화시켰다. 안정화시킨세포에 LPS 1 μg / ml와가미윤조탕추출물을농도별로처리하고 24시간동안배양하였다. 배양이끝나 25
면상등액을채취하여 Bio-Plex Suspension Assay System 을이용, Quantitative Multiplexed Cytokine/ Chemokine Assay 를실시하여 IL-1β, IL-6 및 TNFα 생성량을측정하였다. 5) 통계분석 실험결과는 SPSS Window program(ver. 12.0) 을 이용하였으며, 모든측정값은평균값 ± 표준편차 (Mean ± SD) 로나타내었고, 대조군과각실험군과의 평균차이는 Student's t-test 로분석하여 p-value 가 0.05 미만일때통계학적으로유의한차이가있는것 으로판정하였다. Ⅲ. 실험성적 1. 세포독성에미치는영향 가미윤조탕의농도가세포내에서독성을일으키는 지확인하기위해 MTT assay 를이용하여측정하였 다. 정상군의생존율을 100 ± 3.96% 로계산하였을 때모든농도에서세포독성이없음을확인하였다 (Table 2). Table 2. The Cytotoxic Effect of Gamiyunjo-tang (GMYJT) Water-extract on RAW 264.7 Macrophage Cells by MTT assay Cell Viability Normal 100 ± 3.96 50 94.95 ± 5.51 100 98.75 ± 4.49 200 95.56 ± 5.64 400 94.94 ± 5.51 Values are the mean ± SD of the three independent experiments. Normal : not treated with GMYJT 50 : Treated with GMYJT (50 μg / ml ) 100 : Treated with GMYJT (100 μg / ml ) 200 : Treated with GMYJT (200 μg / ml ) 400 : Treated with GMYJT (400 μg / ml ) 2. NO 생성에미치는영향 가미윤조탕이마우스대식세포의 NO 생성에미치 는영향을관찰하였다. LPS 로유도된 NO 의생성율을 100 ± 1.86% 로계산하였을때가미윤조탕 100 μg / ml, 200 μg / ml및 400 μg / ml농도로처리한군에서 LPS 단독처리한군에비하여통계학적으로유의한 감소가나타났다 (Table 3). Table 3. The Effect of Gamiyunjo-tang (GMYJT) Water-extract on NO Production of RAW 264.7 Macrophage Cells NO production Control 100 ± 1.86 LPS + 50 98.50 ± 1.28 LPS + 100 85.33 ± 0.47* LPS + 200 82.57 ± 1.34* LPS + 400 77.81 ± 0.16* Values are the mean ± SD of the three independent experiments. Table 4. The Effect of Gamiyunjo-tang (GMYJT) Water-extract on PGE2 Production of RAW 264.7 Macrophage Cells PGE 2 production Control 100 ± 2.10 LPS + 50 98.41 ± 1.92 LPS + 100 90.29 ± 1.80* LPS + 200 79.30 ± 1.78* LPS + 400 74.20 ± 1.72* Values are the mean ± SD of the three independent experiments. 26
최종민외 4 인 : 加味潤燥湯이 LPS 로유도된 RAW 264.7 대식세포에서의항염효과연구 3. Prostaglandin E2 생성에미치는영향 가미윤조탕이마우스대식세포의 Prostaglandin E 2 생성에미치는영향을관찰하였다. LPS 로유도된 PEG 2 의생성율을 100 ± 1.65% 로계산하였을때가 미윤조탕 100 μg / ml, 200 μg / ml및 400 μg / ml농도로 처리한군에서 LPS 단독처리한군에비하여통계학적 으로유의한감소가나타났다 (Table 4). 4. IL-1β 생성에미치는영향 가미윤조탕이마우스대식세포의 IL-1β 생성에미 치는영향을관찰하였다. LPS 로유도된 IL-1β 의생 성율을 100 ± 2.71% 로계산하였을때가미윤조탕을 처리한모든군에서 LPS 단독처리한군에비하여통 계학적으로유의한감소가나타나지않았다 (Table 5). Table 5. The Effect of Gamiyunjo-tang (GMYJT) Water-extract on Interleukin-1β Production of RAW 264.7 Macrophage Cells IL-1β production Control 100 ± 2.71 LPS + 50 101.54 ± 2.52 LPS + 100 101.28 ± 1.90 LPS + 200 97.01 ± 1.55 LPS + 400 93.20 ± 2.42 Values are the mean ± SD of the three independent experiments. 5. IL-6 생성에미치는영향 가미윤조탕이마우스대식세포의 IL-6 생성에미치 는영향을관찰하였다. LPS 로유도된 IL-6 의생성율 을 100 ± 1.23% 로계산하였을때가미윤조탕 200 μg / ml및 400 μg / ml농도로처리한군에서 LPS 단독처 리한군에비하여통계학적으로유의한감소가나타 났다 (Table 6). Table 6. The Effect of Gamiyunjo-tang (GMYJT) Water-extract on Interleukin 6 Production of RAW 264.7 Macrophage Cells IL-6 production Control 100 ± 1.23 LPS + 50 99.70 ± 1.04 LPS + 100 93.09 ± 1.01 LPS + 200 85.86 ± 1.89* LPS + 400 81.77 ± 1.83* Valuse are the mean ± SD of the three independent experiments. Table 7. The Effect of Gamiyunjotang (GMYJT) Water-extract on Tumor Necrosis Factor-α (TNF-α) Production of RAW 264.7 Macrophage Cells TNF-α production (% of control) Control 100 ± 5.91 LPS + 50 97.31 ± 4.23 LPS + 100 89.74 ± 3.03 LPS + 200 84.17 ± 3.89 LPS + 400 79.40 ± 4.73* Values are the mean ± SD of the three independent experiments. 27
6. TNF-α 생성에미치는영향가미윤조탕이마우스대식세포의 TNF-α 생성에미치는영향을관찰하였다. LPS로유도된 TNF-α 의생성율을 100 ± 5.91% 로계산하였을때가미윤조탕 400 μg / ml처리한군에서 LPS 단독처리한군에비하여통계학적으로유의한감소가나타났다 (Table 7). Ⅳ. 고찰알레르기질환은일반적으로정상인사람들에게별로해가없는외부항원에대한불필요한면역반응, 즉과민반응을보이는것으로항원의반복적인노출에따른변화된상태, 즉이상반응이라는광범위한의미에서부터자극적이거나해로운작용을일으키는면역반응으로해석되고있다 9,10). 알레르기면역반응은항원에대해 IgE에의해매개되는면역반응으로초기수분내에일어나는즉각적초기반응과 4~8시간이후에일어나는후기반응으로나눌수있다. 초기반응은지속적으로항원에노출될경우 B세포로부터 IgE 항체가생성되어비만세포 (mast cell) 를활성화시키고탈과립으로유리되는다양한화학매개물질 (histamin, tryptase, chymase, bradykinin, heparin) 에의해혈관확장, 점막부종, 기관지평활근수축, 점액분비증가, 콧물생성등이유발되게된다 11-13). 후기반응은조기반응후 T세포, 호염기구, 호산구로이루어진염증세포들의유입에의한염증반응으로이루어지며, 이들염증세포에서분비되는매개물질에의해그증상이지속되게된다. 즉후기반응이알레르기질환의지속적인염증반응과연관이있다. 이러한만성적인염증반응으로생성되는결과물중 NO는주로대식세포에서생성되는작고불안정한무기가스로, 급만성염증에핵심적인역할을하며 Th1의감소, 사이토카인의생성에중요한역할을한다. 그리고만성알레르기비염과천식환자의점막과혈정, 날숨 등에서 NO의농도가높게측정되었으며, 이를이용하여알레르기비염과천식등의진단에이용할수있다고주장하는연구가있다 5,14). 활성산소종인 NO는주로대식세포에서 L-arginine 으로부터 NO 합성경로 (NO synthase pathway ; NOS) 를통해 inos 에의해합성되는작은분자량의자유라디칼로서세포내항상성유지, 신경전달물질운반, 항암작용및세포독성등에관여하는신호전달자로서, 특히 LPS나 IFN-γ, β-amyloid 등의자극으로활성화된대식세포로부터과도하게생성되어세포독성과염증반응을유발하는것으로알려져있다 15-17). 따라서염증반응이진행되는동안유의적으로증가하는 NO 생성을효과적으로억제하는억제제개발에대한연구가최근이루어지고있으며다양한염증질환의유용한치료방법으로여겨지고있다 18-20). PGE₂ 는 prostaglandin endoperoxide synthase 인 COX-2 에의해합성되는염증물질로서 NO와마찬가지로다양한염증질환의병태생리에기여하며 21,22), 최근 COX-2 저해제가각종염증질환개선에효능이있는것이알려지면서치료제개발연구가활발히진행되고있다. Bacterial endotoxin 인 LPS는염증을유발하는주요 inflammagen 이며, 종양괴사인자 (tumor necrosis factor, TNF) 와관련하여강력한염증반응을유도하는물질로서 TNF-ɑ, IL-1β 를포함한다양한사이토카인분비를통해신체각부위의면역반응을담당하게된다 23). 특히 TNF-ɑ, IL-1β 는 LPS에의해활성화된대식세포로부터다량분비되어각종염증질환에서염증유발에핵심적인역할을한다. 따라서최근에는대식세포에서 LPS 인식기전의이해및염증유발관련유전자의발굴, 나아가이들의조절을통한퇴행성뇌질환치료에대한연구가활발히이루어지고있다 24). LPS는또한대식세포를활성화시킴으로써 NO, TNF-α, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1α (MIP-1α), IL-1β, IL-6, IL-8, IL-18, macrophage 28
최종민외 4 인 : 加味潤燥湯이 LPS 로유도된 RAW 264.7 대식세포에서의항염효과연구 colony-stimulating factor(m-csf) 등의염증반응물질을분비하도록자극함으로써이를통해조직내염증반응을촉진시킴으로써다양한염증관련질환의발달에기여하게된다 25). TNF-ɑ 와 IL-1β 는주로단핵구나대식세포에서생성되는데, 특히기관지천식환자의경우 TNF-ɑ 가알레르기성염증반응과관련이있고실제환자의혈청과기관지세척액에서 TNF-ɑ 의농도가증가되어있으며 mrna 와단백질발현이증명되어 TNF-ɑ 는기관지천식이나만성폐질환등의병인기전에중요한역할을하는사이토카인으로인식되고있다 26,27). 加味潤燥湯은韓方臨床 40 年 8) 에수록되어全身瘙痒血燥風毒性症을치료하는처방으로熟地黃, 生地黃, 白芍藥, 知母, 黃芩, 秦艽, 玄蔘, 胡麻子, 防風, 浮萍草, 甘草로구성되어있다. 임상에서는陰氣를보충해주고祛風淸熱, 潤燥除濕하는효능으로피부질환의급성기보다는만성기에사용되고있지만이에대한연구로는이 28) 의아토피성피부염환자에대한임상연구외에는처방의기전에대한연구는없었다. 이에본연구에서는가미윤조탕이알레르기후기반응에관련한염증억제효과를확인하기위하여가미윤조탕이 LPS로유도된 RAW 264.7 대식세포에서 NO생성, PGE₂ 생성, 사이토카인인 IL-1β, IL-6, TNF-α 등의생성에미치는영향을관찰하여항염증에유의한결과를얻었다. 실험에사용된가미윤조탕추출물 ( 이하 GMYGT water-extract) 의세포독성을평가하기위해시행한 MTT assay 결과모든처리농도에서세포독성은보이지않았다 (Table 2). GMYGT water-extract 의항염작용을알아보기위해 100, 200 및 400 μg / ml농도에서시행한 NO assay 에서모든군에서 LPS 단독처리한군에비하여통계학적으로유의한감소가나타났으며 (Table 3), Prostaglandin E 2 생성에미치는영향을실험한결과에서도 100, 200 및 400 μg / ml농도로처리한군에서 LPS 단독처리한군에비하여통계학적으로유의한감 소가나타났다 (Table 4). IL-1β 생성에미치는영향에대한실험에서는모든농도에서 LPS 단독처리한군에비하여통계학적으로유의한감소가나타나지않았다 (Table 5). IL-6 생성에미치는영향에대한실험에서는 200 및 400 μg / ml농도처리한군에서 LPS 단독처리한군에비하여통계학적으로유의한감소가나타났으며 (Table 6), TNF-α 생성에미치는영향에대한실험에서는 400 μg / ml처리한군에서 LPS 단독처리한군에비하여통계학적으로유의한감소가나타났다 (Table 7). 본연구를통해 GMYGT water-extract 가만성염증반응에중요한역할을하는염증물질인 NO, PGE 2, IL-6, TNF-ɑ 생성의억제효과를확인할수있었지만향후이에대한기전연구등후속연구가이루어져야할것으로생각된다. Ⅴ. 결론알레르기후기염증반응에있어가미윤조탕의염증억제기전을확인하기위하여 LPS를처리하여염증을유도한 RAW 264.7 대식세포에가미윤조탕을처리하여 NO생성정도, Prostaglandin E 2, IL-1β, IL-6, TNF-α 의생성에미치는영향을관찰하여다음과같은결과를얻었다. 1. 가미윤조탕추출물은모든농도에서세포독성이없었다. 2. 가미윤조탕추출물은 100, 200 및 400 μg / ml농도에서농도비례하여 NO의생성을억제하였다. 3. 가미윤조탕추출물은 100, 200 및 400 μg / ml농도에서농도비례하여 PGE₂ 생성을감소시켰다. 4. 가미윤조탕추출물은실험한모든농도에서 IL-1 β 생성을감소시키지못했다. 5. 가미윤조탕추출물은 200 및 400 μg / ml농도에서농도비례하여 IL-6 생성을감소시켰다. 29
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