23(6)17김상찬 hwp

Transcription

1 동의생리병리학회지제 23 권 6 호 Korean J. Oriental Physiology & Pathology 23(6): , 2009 회춘양격산물추출물의항염증효과 이태호 조미정 1 박숙자 1 이종록 백영두 김종열 2 권영규 3 김상찬 1 * 대구한의대학교한의과대학, 1 : 대구한의대학교한방신약개발팀 (BK21 Team), 2 : 한국한의학연구원, 3 : 부산대학교한의전문대학원 Inhibitory Effects of Hoechunyanggyeok-san on Inflammation in Vivo and in Vitro Tae Ho Lee, Mi Jeong Jo 1, Sook Jarh Park 1, Jong Rok Lee, Young Doo Back, Jong Reol Kim 2, Young Kyu Kwon 3, Sang Chan Kim 1 * College of Oriental Medicine, 1:BK21 Team, Daegu Haany University, 2:Korea Institute of Oriental Medicine, 3 : School of Oriental Medicine, Pusan National University Hoechunyanggyeok-san (HYS) is a traditional oriental herbal medicine widely used for treating inflammatory disorders. Although there are numerous clinical results of HYS reported in the literature of oriental hebal medicine, it has been rarely conducted to evaluate the immuno-biological activity. The present study was conducted to examine the anti-inflammatory effects of HYS extract (HYSE) in vivo and in vitro. To determine the cytotoxic concentration of HYSE, cell viability was tested by MTT assay. All four doses of HYSE (0.01, 0.03, 0.10 and 0.30 mg/ml) had no significant cytotoxicity during the entire experimental period. In order to measure NO levels in culture medium, the cells were treated with 1 µg/ml of LPS 1h before adding HYSE for 24 h and then culture medium were reacted with Griess reagent. Increased NO production and inos expression were detected in LPS-activated cells compared to control. However, these increases were dose-dependently attenuated by treatment with HYSE. LPS plays a key role in leading to the massive production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in macrophages. Thus, we next determined the levels of these cytokines. HYSE reduced the elevated production of TNF-α, IL-1β and IL-6 by LPS. Moreover, the effects of HYSE were in a dose-dependent manner. In vivo, histopathological study, HYSE effectively inhe efed the increases of hind paw skin thicknesses and inflammatory cell infiltrations induced by carrageenan treatment. It, therefore, considered that HYSE will be favorably inhe efed the acute edematous inanner. In s. These findings showed that HYSE could have anti-inflammatory effects through the reduction of NO and inflammatory cytokines in macrophage. Futhermore, the reduction of carrageenan-induced paw oedema by HYSE helps to understand its actions on inflammatory conditions. Key words : Hoechunyanggyeok-san, inflammation, cytokine, edema 서론 回春凉膈散은萬病回春에收載되어있는方劑로連翹, 黃芩, 梔子, 桔梗, 黃蓮, 薄荷, 當歸, 生地黃, 枳實, 赤芍藥, 甘草등의 11 種으로構成되어, 三焦火盛으로因한口舌生瘡을治療하는方劑이며, 淸熱解毒凉血散火의效能을갖고있다 1). 回春凉膈散에대한연구로는, 김등 2) 의 carrageenan 유도부 * 교신저자 : 김상찬, 대구시수성구상동 165 대구한의대학교한의과대학 sckim@dhu.ac.kr, Tel : 접수 : 2009/09/29 수정 : 2009/10/16 채택 : 2009/10/29 종의감소효과와, 강등 3) 의 LPS로활성화된 human monocyte의 IL-1β 생성억제등에대한보고만있으며, 回春凉膈散에대한연구는많은부분에서극히제한적인실정이다. 본연구에서는回春凉膈散이淸熱解毒凉血散火의효능이있음에근거하여, 回春凉膈散물추출물이 (HYSE; Hoechunyanggyeok-san extract) 이 LPS로활성화된 Raw cell에서나타나는염증매개물질들에미치는영향을평가하였으며, 또한回春凉膈散이동물모델에서도유효한항염증효과를가지는지를평가하기위하여 carrageenan으로유도된 rat의발부종모델에서부종의정도및조직학적평가를실시하였다

2 이태호 조미정 박숙자 이종록 백영두 김종열 권영규 김상찬 Carrageenan의주입은염증성근육통을유발하므로, 국소적염증과, 통각과민의연구에널리사용되고있으며 4), Raw cell은 murine 대식세포의 cell line으로서, 대식세포는여러종류의숙주반응에관여하여숙주의방어와숙주의항상성유지에관여하는것으로알려져있고, 염증반응시에는 interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) 및 interleukin-6 (IL-6) 와같은 cytokine을생산하여감염초기에생체방어에중요한역할을하는세포로알려져있다 5). Lipopolysaccharaide (LPS) 는인지질, 다당류및소량의단백질로구성되며, 염증반응을유발하는유력한인자로, 다양한 cytokine을생성시키므로염증반응을연구하는경우빈용하는실험모델 6) 로확립되어있다. 재료및방법 1. 回春凉膈散추출물 (HYSE) 의제조 回春凉膈散의재료는대원약업사 ( 대구, 한국 ) 에서구입하여관능평가를한후, < 방약합편 > 7) 의용량을근거로回春凉膈散 4 첩분량 g을물 2 L에넣고 3시간전탕한후추출물을거어즈로 1차여과하고 3000 g에서 3분간원심분리하였다. 원심분리후의상층액만을취하여 0.2 μm filter (Nalgene, New York, USA) 로여과하였다. 이여과액을 rotary evaporator (EYELA, Tokyo, Japan) 로동결건조하여 g을얻었으며, 사용때까지 -20 에서보관하였다. 回春凉膈散물추출물의수율은 % 였으며 in vitro처치시에는 DMEM에녹여사용하였으며, in vivo 실험에서는물에녹여사용하였다. 回春凉膈散의구성및용량은 Table 1과같다. Table 1. Composition of Hoechunyanggyeok-san 藥材名 生藥名 重量 (g) 連 翹 Forsythiae Fructus 4.50 g 黃 芩 Suctellariae Radix 2.62 g 梔 子 Gardeniae Fructus 2.62 g 桔 梗 Platycodi Rhizoma 2.62 g 黃 連 Coptidis Rhizoma 2.62 g 薄 荷 Menthae Folium 2.62 g 當 歸 Angelicae gigantis Radix 2.62 g 生地黃 Rehmanniae Radix 2.62 g 枳 殼 Aurantii Fructus 2.62 g 赤芍藥 Paeoniae Radicis rubra 2.62 g 甘 草 Glycyrrhizae Radix 2.62 g 合 計 g 2. 시약 LPS (Escherichia coli 026:B6) 와 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoleum (MTT) 은 Sigma (St. Louis, MO, USA) 에서구입하였고, fetal bovine serum (FBS) 과 antibiotics는 Gibco/BRL (Eggenstein, Germany) 로부터 구입하였으며, Antibody는 BD Bioscience (San Jose, CA, USA), Cayman (Ann Arbor, Mi, USA), Zymed (San Francisco, CA, USA) 에서구입하였고, NC paper는 Schleicher & Schuell (Dassel, Germany) 에서구입하였다. TNF-α, IL-1β와 IL-6의 ELISA Kit는 Pierce endogen (Rockford, IL, USA) 에서구입하였다. 3. 세포배양 Murine macrophage cell line인 Raw cells은한국세포주연구재단 (Seoul, Korea) 에서구입하였으며, Dulbecco s modified Eagle s medium (DMEM) 에 10% fetal bovine serum (FBS), 100 U/ml penicillin 및100 μg/ml streptomycin을혼합한배지를사용하여 37, 5% CO 2 incubator에서배양하였다. 실험과정의모든 cells은 80~90% 의 confluence에서실험하였고, 20 passages를넘기지않은 cell만사용하였다. 4. 세포생존율측정 Raw cell을 96-well plate에 cells/well로분주한다음回春凉膈散물추출물 (HYSE) 을농도별로처치하여세포의생존율을구하였다. 세포에 0.01, 0.03, 0.10, 0.30 mg/ml의농도로 HYSE를처치한후에 37, 5% CO 2 의환경이유지되는배양기에서배양하였다. 배양후생존세포에 MTT (0.1 mg/ml) 를 50 μl넣고 4시간배양한후배지를조심스럽게제거하고생성된 formazan crystals을 DMSO에녹여 Titertek Multiskan Automatic ELISA microplate reader (Model MCC/340, Huntsville, AL) 를사용하여 570 nm에서흡광도를측정하였다. 세포생존율은 control cell에대한백분율로나타내었다. i.e. viability(% control) = 100 /(absorbance of treated sample)/(absorbance of control) 5. NO생성량측정 Raw 세포주로부터생성된 nitric oxide (NO) 의양은세포배양액중에존재하는 NO 2-의형태로서 Griess 시약을이용하여측정하였다. 간략하게설명하면세포배양상등액 50 μl와 Griess시약 (1% sulfanilamide in 5% phosphoric acid + 1% α -naphthylamide in H 2O) 50 μl를 96 well plates에혼합하고암실에서 10분동안반응시킨후 540 nm에서 Titertek Multiskan Automatic ELISA microplate reader (Model MCC/340, Huntsville, AL) 로흡광도를측정하였다. NO 2-의농도는 sodium nitrate를희석하여흡광도를측정하여표준곡선을얻었다. 6. Immunoblot analysis 20 mm Tris Cl (ph 7.5), 1% Triton X-100, 137 mm sodium chloride, 10% glycerol, 2 mm EDTA, 1 mm sodium orthovanadate, 25 mm b-glycerophosphate, 2 mm sodium pyrophosphate, 1 mm phenylmethylsulfonylfluoride과 1 mg/ml leupeptin을함유하는 buffer를사용하여 cell을 lysis시켰다. Cell lysates를 10,000 g로 10분간원심분리하여 debris를제거하였다. 각 protein의발현은각각의 antibody를사용하여면역화학적방법으로분석하였으며, 2차 antibody는 alkaline phosphatase conjugated anti-rabbit을사용하였다. 각 protein의

3 회춘양격산물추출물의항염증효과 band는 ECL western blotting detection reagents (Amersham) 를사용하여 manufacturer's instruction에따라발색하였다. 발색후단백질의발현량을평가하기위하여 image analyzing system (Ultra-Violet Products Ltd., Upland, CA, USA) 을이용하여 Densitometric analysis를실시하였다. 7. Cytokine 측정 Cytokine을측정하기위하여 6-well plate에 cells ( /ml) 을분주하고 HYSE를농도별로처치한다음, 1시간후에 LPS를처치하였다. LPS 처치후각 cytokine마다특정시간에배지를수거하여 cytokine을측정하였다. 수거된배지는바로측정하거나, 측정전까지 -70 에서보관하였다. TNF-α, IL-1β와 IL-6는 ELISA Kit (Pierce endogen, Rockford, IL, USA) 를사용하여측정하였으며, 실험의방법은 manufacturer's instruction에따랐다. Kim, et al 8) 의방법을변형시켜, 발등및발바닥피부 ( 상피에서진피 ) 의두께를 mm 단위로 40배현미경시야에서자동영상분석장치 (DMI-300 Image Processing; DMI, Korea) 를이용하여각각측정하였으며, 각각 1 mm 2 의발등및발바닥피부에침윤된염증세포의수역시자동영상분석장치를이용하여, 200배현미경시야에서측정하였다. 12. 통계적검증실험결과는 mean ± S.D. 로나타내었으며, 처치군간의유의성은 one way analysis of varience (ANOVA) 로검정한후 Newman-Kleuls test로검정하였다. 통계적유의성검정은 p<0.05 또는 p<0.01로하였다. 8. 실험동물및처치실험동물은 4주령된 Sprague-Dawley계수컷흰쥐 ( g) 를 1주일동안환경에적응시킨후실험에사용하였으며, 사육실환경은온도 20-23, 습도 60%, 12시간 light/dark cycle을유지하고, 사료 (Nestle Purina Petcare Korea, Seoul, Korea) 와음료는자유롭게섭취하도록하였다. 실험은아무런처치를하지않은군을 Normal군으로하고기염제인 carrageenan (Sigma Chemical Co., St. Louis, USA; 100 μl/rat) 만을피하주사한군을 carrageenan군으로하였으며, carrageenan과 dexamethasone (1 mg/kg, P.O) 을투여한 dexamethasone군, carrageenan과 0.3 g/kg의回春凉膈散을투여한 0.3 g/kg HYSE군, carrageenan과 1 g/kg의回春凉膈散을투여한 1 g/kg HYSE군으로나누었으며, 각군당 n수는 6마리로하였다. dexamethasone과 HYSE는 4 일동안매일 1회투여하였으며, 마지막약물투여 1시간후 carrageenan을 100 μl/rat로 rat의오른쪽발바닥에주입하였다. 9. Paw edema의유도및측정 4일째약물을투여하고 1시간후 carrageenan을투여하여 Paw edema를유발시켰다. Paw edema의측정은 carrageenan을주입후시간별로 (0, 1, 2, 3, 4시간 ) 부종측정기 (Plethysmometer, LE 7500; LETICA Scientific Instruments, Spain) 를이용하여부종정도를측정하였다. Fig. 1. Schedule of the in vivo study. 결과 1. 回春凉膈散이 LPS로유도된 Raw cell의 NO production 에미치는영향 Raw cell에서 HYSE의 NO 생성억제정도를관찰하기위하여 HYSE를 0.01, 0.03, 0.10, 0.30 mg/ml의농도로세포에처리하여생성되는 NO양을측정하였다. LPS군에서는 control군에비교하여 NO의생성량이 LPS처치후 18 h, 24 h에서각각 2.5 배, 5배정도로유의하게증가하였으며, HYSE를처치한실험군에서는 18 h, 24 h처치에서전농도가유의한 NO억제를나타내었다 (Fig. 2). 10. 조직처리 Paw edema의유발과회복여부를살펴보기위해, carrageenan으로염증이유발된 rat의오른쪽하지의발목아래를절단하여조직을채취하여, 後肢의발등 (dorsum pedis) 및발바닥 (ventrum pedis) 의피부실질조직을분리하여 10% 중성포르말린에 6시간이상고정시킨 (d 탈수및파라핀포매를실시하고, 3~4 μm의 longitudinal 절편을제작하여 Hematoxylin-eosin 염색을실시하고, 광학현미경 (Nikon, Japan) 하에서관찰하였다. 11. 조직학적평가 Fig. 2. Effects of HYSE on the production of NO by LPS. Raw cells were treated with 0.01, 0.03, 0.1, 0.3 mg/ml of HYSE dissolved in media for 1 h prior to the addition of LPS (1 μg/ml), and the cells were further incubated for 24 h. The concentrations of nitrite and nitrate in culture medium were monitored as described in the materials and methods section. Data represent the mean ± S.D. with eight separate experiments. (*, significant as compared to control. **P<0.01; #, significant as compared to LPS alone, ##P<0.01)

4 이태호 조미정 박숙자 이종록 백영두 김종열 권영규 김상찬 2. 回春凉膈散이 Raw cell의생존율에미치는영향 HYSE가 0.01, 0.03, 0.10, 0.30 mg/ml의농도에서 LPS로유도된 Raw264.7 cell의 NO의생성을감소시킨것이, HYSE의세포독성으로인한것인지를관찰하기위하여, HYSE를 0.01, 0.03, 0.10, 0.30 mg/ml로처리하고 24시간후에 MTT assay를실시하여세포생존율을측정하였다. 측정결과 12 h에서는모든경우유의한세포독성이나타나지않았으나, LPS단독처리 24 h에서는 Control에비교하여약 70% 정도의세포독성을나타내었다. 그러나, LPS와 HYSE (0.01, 0.03, 0.10, 0.30 mg/ml) 를처리한군에서는 LPS 단독처치군에비교하여유의한세포독성을나타내지않았으며, 오히려 HYSE는농도의존적으로유의한세포독성을억제함이관찰되었다 (Fig. 3). 4. 回春凉膈散이 LPS로유도된 Raw cell의 cytokine에미치는영향염증반응에있어서중요한역할을하는 TNF-α는 LPS반응의주요매개체로서내재면역에있어서도중요한역할을한다 9,10). 본연구에서 LPS는 TNF-α의분비를유의성있게증가시켰으며, HYSE는 0.10 및 0.30 mg/ml의농도에서 TNF-α의생성량을유의하게감소시켰다 (Fig. 5A). IL-1β는 NK cell의활성, T-cell의활성화, B-cell의성숙을활성화하는 10) cytokine으로, 본연구에서도 LPS의자극에의하여 Raw264.7 cell의 IL-1β의분비가유의성있게증가하였으며, HYSE는 0.10 및 0.30 mg/ml의농도에서유의하게 IL-1β의생성량을줄였다 (Fig. 5B) IL-6는 B-cell이활성화되어항체를생산하는 plasma세포로분화되도록촉진하고, 항체의분비를자극하는 cytokine으로 B-cell 분화단계의후기에주로작용하여 imunoglobulin의생성을유도하고, T-cell의증식에도관련되어있다 11). 본실험에서 LPS는 IL-6의생성을유의성있게증가시켰으며, HYSE는 0.03, 0.10, 0.30 mg/ml의농도에서 LPS 로유도된 IL-6를유의성있게감소시켰다 (Fig. 5C). A) Fig. 3. Effects of HYSE on the cell viability in LPS stimulated Raw264.7 cells. Raw264.7 cells were treated with 0.01, 0.03, 0.10, 0.30 mg/ml of HYSE dissolved in media for 1 h prior to the addition of LPS (1 μg/ml), and the cells were further incubated for 24 h. Data represent the mean ± S.D. with eight separate experiments. (*, significant as compared to control. **P<0.01) A) B) B) C) Fig. 4. Effect of HYSE on the induction of inos by LPS. The levels of inos and actin protein were monitored 18h after treatment of cells with LPS (1μg/ml) with or without HYSE (0.01, 0.03, 0.10 and 0.30 mg/ml) pretreatment (i.e. 1h before LPS). Equal amounts of total protein were resolved by SDS-PAGE. Expressions of inos protein were determined by immunoblot analysis using inos specific antibodies. The actin was used as a loading control (A). The relative density levels of protein bands were measured by scanning densitometry (B). The data represent the mean ± SD of three separate experiments.(*: significant compared with the control, **P<0.01, #: significant compared with the LPS alone, ##P<0.01). Fig. 5. The Effect of HYSE on LPS-stimulated cytokine production. Production of cytokine was measured in the medium of Raw264.7 cells cultured with LPS (1 μg/ml) in the presence or absence of HYSE for 24 h. The amount of cytokine was measured by immunoassay as described in materials and methods. Data represent the mean ± S.D. with three separate experiments. (*, significant as compared to control. **P<0.01; #, significant as compared to LPS alone, #P<0.05, ##P<0.01)

5 회춘양격산물추출물의항염증효과 5. 回春凉膈散이 carageenan으로유도된 Rat의 paw edema에미치는영향부종측정기를이용하여 paw edema를측정한결과, carrageenan을주입한군에서는 0, 1, 2, 3, 4 시간에각각 1.00 ± 0.08, 1.68 ± 0.14, 2.21 ± 0.22, 3.02 ± 0.45, 3.02 ± 0.37을나타내어, 유의한발부종이유발되었다. 그러나, 발부종유도후 dexamethasone을처치한군에서는 1.03 ± 0.08, 1.02 ± 0.14, 1.01 ± 0.17, 1.09 ± 0.16, 1.05 ± 0.17을나타내어실험시간동안유의한발부종억제를나타내었다. HYSE 0.3 g/kg를투여한군에서는 0, 1, 2, 3, 4 시간에 1.12 ± 0.11, 1.22 ± 0.09, 1.74 ± 0.10, 2.02 ± 0.24, 1.90 ± 0.15를나타내어 1, 3, 4 시간에유의한발부종억제를나타내었으며, HYSE 1.0 g/kg를투여한군에서도 1.12 ± 0.12, 1.27 ± 0.12, 1.90 ± 0.23, 2.03 ± 0.09, 1.69 ± 0.15로유의한억제를나타내었다 (Fig. 6). 가는 dexamethasone 및 HYSE 0.3, 1.0 g/kg의처치에의해 7.20 ± 2.28, ± 2.61, 7.00 ± 2.70 (cells/1 mm 2 ) 을나타내어유의하게감소되었다. 부종발의발바닥조직에서침윤염증세포의수 (cells/1 mm 2 ) 는정상군이 6.40 ± 2.70개이었으며, carrageenan군은 ± 개로유의한증가를나타내었다. 이러한염증세포침윤의증가는 dexamethasone 및 HYSE 0.3, 1.0 g/kg의처치에의해 ± 64.10, ± , ± (cells/1 mm 2 ) 로유의하게염증세포의침윤을억제하였다 (Table 2, Fig. 7, 8). Table 2. Changes of histomorphometrical measurements in the present study Groups Dorsum pedis skin Thickness (mm) Infiltrated inflammatory cells (cells/1 mm 2 ) Ventrum pedis skin Thickness (mm) Infiltrated inflammatory cells (cells/1 mm 2 ) Normal 0.87± ± ± ±2.70 Carrageenan 1.95±0.35** 28.40±4.56** 1.91±0.24** ±124.99** Dexamethasone 0.75±0.21 ## 7.20±2.28 ## 0.84±0.35 ## ±64.10 ## HYSE 0.3 g/ml 1.17±0.33 ## 10.60±2.61 ## 1.37±0.13 ## ± ## HYSE 1.0 g/ml 1.08±0.21 ## 7.00±2.70 ## 1.23±0.31 ## ±80.10 ## Values are expressed as mean ± SD of 5 histological fields ** p<0.01 as compared with Normal control ## p<0.01 as compared with Carrageenan control. Fig. 6. Inhibition of Carrageenan-induced paw edema by HYSE. HYSE was administered to rats at an oral dose of 0.3, 1.0 g/kg/day for 4 days before the induction of paw edema. Paw edema was induced by subcutaneously injecting 1% solution of carrageenan dissolved in saline (0.1 ml per animal) into the right hind paw. The swelling of the paw was measured 0~4 h after carrageenan injection. Dexamethasone (1 mg/kg p.o.) was used as a positive control. Data represent the mean ± S.D. of six animals. (## P<0.01, significant compared with carrageenan alone) 6. 回春凉膈散이 carageenan으로유도된 Rat의 paw의조직변화에미치는영향 HYSE의 carrageenan 유도발부종에대한조직학적영향을평가하기위하여 4 시간째의발부종의측정을완료한후실험동물을희생하여부종발의발등부분과발바닥부분에서피부두께및침윤염증세포의수를측정하였다. 본실험의결과, 발등피부의두께는정상군에서 0.87 ± 0.11 mm, carrageenan군이 1.95 ± 0.35 mm로유의성있게증가하였으며, 이러한피부의두께의증가는 dexamethasone 및 HYSE 0.3, 1.0 g/kg를처치한실험군에서 0.75 ± 0.21, 1.17 ± 0.33, 1.08 ± 0.21 (mm) 로유의성있게감소하였다. 발바닥부위의피부두께에있어서도정상군은 0.70 ± 0.11 mm이었으나, carrageenan군에있어서는 1.91 ± 0.24 mm로유의성있게증가하였으며, 이러한발바닥의피부두께의증가역시 dexamethasone 및 HYSE 0.3, 1.0 g/kg를처치한실험군에서 0.84 ± 0.35, 1.37 ± ± 0.31 (mm) 으로유의성있게감소하였다. 또한, 부종발의발등조직에서침윤염증세포의수 (cells/1 mm 2 ) 는정상군에서는 6.40 ± 2.30개였으며, carrageenan군은 ± 4.56개로유의성있게증가하였다. 이러한염증세포의증 Fig. 7. Changes on histological profiles of the Dorsum Pedis skin in normal control (a, b), Carrageenan control (c, d), Dexamethasone (e, f), HYSE 0.3 g/kg (g, h) and HYSE 1.0 g/kg (i, j) treated groups. Note that marked increases of skin thicknesses due to edematous changes were detected by carrageenan treatment with increases of inflammatory cell infiltrations. However, these increases of skin thicknesses and inflammatory cell infiltrations were effectively inhibited by treatment of dexamethasone and two different dosages of HYSE, respectively. Arrow indicated total thicknesses measured. All HE stain; Scale bars = 80 μm

6 이태호 조미정 박숙자 이종록 백영두 김종열 권영규 김상찬 Fig. 8. Changes on histological profiles of the Ventrum Pedis skin in normal control (a, b), Carrageenan control (c, d), Dexamethasone (e, f), HYSE 0.3 g/kg (g, h) and HYSE 1.0 g/kg (i, j) treated groups. Note that marked increases of skin thicknesses due to edematous changes were detected by carrageenan treatment with marked increases of inflammatory cell infiltrations. However, these increases of skin thicknesses and inflammatory cell infiltrations were effectively inhibited by treatment of dexamethasone and two different dosages of HYSE similar to that of dorsum pedis, respectively. M, muscle layers; Arrow indicated total thicknesses measured. All HE stain; Scale bars = 80 μm. 고 찰 回春凉膈散은太平惠民和劑局方의凉膈散에서大黃, 芒硝, 竹葉, 蜂蜜을제거하고, 黃連, 桔梗, 枳殼, 生地黃, 赤芍藥, 當歸를加한方劑이다 1). 凉膈散의구성은크게胸膈부위의邪熱을제거하는黃芩, 梔子, 連翹, 薄荷, 邪熱을大小便으로下하는大黃, 芒硝, 竹葉그리고淸熱潤燥, 調和諸藥하는蜂蜜, 甘草의 3가지로구성되어있다. 回春凉膈散은胸膈의邪熱을제거하기위하여凉膈散의連翹, 黃芩, 梔子, 薄荷에黃連을加하여淸熱의의미를강화하였고, 여기에桔梗, 枳殼 ( 桔梗枳殼湯 ) 을배합하여, 理氣의의미를강화하였고, 또한生地黃, 赤芍藥, 當歸, 甘草등을加하여邪熱로인한진액의손상을고려하고있다. 回春凉膈散에대한연구로김등 2) 은 mg/20 g의회춘양격산추출물을투여하여 carrageenan으로유도된 paw edema 를 21% 정도억제함을보고하였고, 강등 3) 은 human monocyte에회춘양격산추출물을 , 0.001, 0.01% 처리하여 IL-1β가 8, 28, 43% 가감소함을보고하였을뿐回春凉膈散에대한연구는많은 부분에서극히제한적인실정이다. 이러한까닭에본연구에서는回春凉膈散물추출물의 NO 억제기전및염증관련 cytokine의발현에대한연구를수행하기위하여열수추출된回春凉膈散 (HYSE) 이 LPS로활성화된 Raw cell에서나타나는염증매개물질들에미치는영향을평가하였으며, 또한 carrageenan 유발발부종에서의부종의정도및염증관련지표에대한回春凉膈散의효과를평가하였다. 산화질소는 arginine과 O 2 로부터 nitric oxide synthase를경유하여생성되는 radical로, 세포내에서혈관조절, 숙주면역, 방어, 신호전달등에서중요한역할을한다. septic shock, 뇌경색, 당뇨, 퇴행성신경질환등이 NO의과량생산에연관되어있다 12). 대식세포가이물질에대응할때분비되는 IL-1β, TNF-α 및 nitric oxide (NO) 는숙주에치명적인결과를초래할수있는것으로보고되고있으므로 13-15), NO 생성저해제에대하여연구가활발하게이루어지고있으며, 최근에는靑黛, 苦楝皮, 當歸, 香附 16-21) 子, 牡丹皮등의한약에서이러한조절제를찾기위해많은연구가진행되고있다. Raw cell에서 HYSE의 NO 생성억제정도를관찰하기위하여 HYSE를 0.01, 0.03, 0.10, 0.30 mg/ml의농도로세포에처리하여생성되는 NO양을측정하였다. LPS군에서는 control군에비교하여 NO의생성량이유의성있게증가하였으며, HYSE 0.01 ~ 0.30 mg/ml을처치한실험군에서는농도의존적으로유의성있는 NO의생성억제를나타내었다. 또한, HYSE는 12 h 및 24 h 에서세포독성을나타내지않았으며, 오히려농도의존적으로 LPS에의한세포독성을억제하였다. Sharifi 등 22) 의연구결과에의하면, 납 (Pb) 이 PC-12 cell에서유발하는독성은, 납에의한 NO의과다생성으로인해세포독성이유발됨을밝혔으며, 박등 23) 은감초성분중의하나인 liquiritigenin이납에의해생성되는 NO의생성을억제하여세포독성을억제함을보고한바있다. HYSE가 LPS로유도되는세포독성을억제한것이 NO의독성을억제함에기인한것인지는좀더연구가진행되어야할것으로생각된다. NO는 inos를경유하여생성되는것이므로 24), HYSE와 inos단백질의관련성을조사하기위하여 inos단백질의발현량을조사하였다. LPS처치시에는 inos 단백질이유의하게발현이증가되었으나, LPS에 HYSE 0.10, 0.30 mg/ml을처치한실험군에서는 inos의량이농도의존적으로유의하게감소하였다. 이러한결과는 HYSE가 inos의발현을억제함으로서 NO의생성을억제함을의미한다. TNF-α는 pro-inflammatory cytokine으로서 macrophages, mast cells, endothelial cells 등으로부터분비된다. 근래 TNF-α는많은자가면역질환에있어서염증의개시및유지에핵심적역할을하는것으로알려져있다 9,10). 본연구에서도 LPS는 Raw264.7 cell에서 TNF-α의분비를유의하게촉진시켰으며, HYSE는 0.1 0및 0.30 mg/ml의농도에서 TNF-α의생성량을유의하게감소시켰다. 이러한결과는 HYSE가여러종류의면역게재염증성질환을유의하게개선시킬수있음을의미한다

7 회춘양격산물추출물의항염증효과 염증관련 cytokine 중, IL-1β는 TNF-α, IL-2, IL-6와함께 pro-inflammatory cytokine으로서여러면역학적작용들과연관되어있다. 특히 IL-1β는 T-cell의활성화, B-cell의성숙, NK cell 의 activity를활성화한다 10). 본연구에서는 LPS의자극에의하여 IL-1β의분비가유의성있게증가하였으며, HYSE는 0.10, 0.30 mg/ml의농도에서모두유의하게 IL-1β의생성량을줄였다. 단핵구나대식세포에서분비되는 IL-6는, 림프구를활성화시켜항체생산을증가시키는것으로 10), 본실험에서 LPS는 IL-6의분비를유의성있게증가시켰으며, HYSE 0.10, 0.30 mg/ml은 LPS로유도된 IL-6를유의성있게감소시켰다. Carrageenan의국소적용은염증세포의침윤을동반한급성부종을초래하므로, 현재여러가지항염증물질의급성염증에대한효력평가에널리이용되고있는동물모델중하나로 25-27), 조직학적으로투여부위에국소적인염증세포침윤을동반한피부조직의부종이관찰되는것으로알려져있다 28-31). 기염제로는김등 31) 과같이 carrageenan을사용하였으며, HYSE는 0.3, 1.0 g/kg의용량으로투여하였다. 김등 2) 은 HYSE 를 mouse에 2.55 g/kg를투여하였으나, 본연구에서는 HYSE의수율이 % 인점을고려하여 0.3 g/kg ( 건조약재로서 1.7 g/kg), 1.0 g/kg ( 건조약재로서 5.7 g/kg) 로투여하였다. 본실험의결과에서도 carrageenan은발의부종을유도하였으며, 0.3, 1.0 g/kg HYSE는유의하게발부종을억제하였다. 또한 carrageenan은정상대조군에비해현저한침윤염증세포의수적증가및부종성변화에의한발등및발바닥피부두께의증가가관찰되었다. 이러한 carrageenan 유발급성부종성염증소견은 HYSE의투여에의해유의성있게억제되었다. 그러나, 본연구는連翹, 黃芩, 梔子, 桔梗, 黃連, 薄荷, 當歸, 生地黃, 枳殼, 赤芍藥, 甘草의복합추출물로서, 이들약물개별의효과및약물간의상호관계, 각약물의비율등에대해서는아직많은부분이밝혀져있지않으므로, 향후이들의효과및상호관련성에대하여추가적연구가필요할것으로생각된다. 결론 回春凉膈散물추출물 (HYSE) 의항염증효능을평가하기위하여, Raw cell을 LPS로활성화시킨후 nitric oxide의생성량, inducible nitric oxide synthase의발현및 interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) 등에미치는영향및 carageenan으로유도된 rat의 paw edema에미치는영향을살펴본바다음과같은결론을얻었다. HYSE는 LPS에의해증가된 NO를농도의존적으로유의하게억제하였다. 또한 HYSE처치군은 LPS단독처리군에비교하여유의한세포독성을나타내지않았다. HYSE는 LPS로증가된 inos의발현을유의하게감소시켰다. HYSE는 LPS에의해증가된 TNF-α, IL-6, IL-1β를유의성있게감소시켰다. HYSE는 carrageenan 으로유도된 rat의 paw edema에대하여급성부종성염증소견을유의성있게억제하였다. 감사의글 본연구는지식경제부지역혁신센터사업 ( 대구한의대학교한방생명자원연구센터 ) 의지원에의하여이루어진것입니다. 참고문헌 1. 허준. 동의보감. 남산당, 서울, p 252, 420, 김경준, 김중호, 채병윤. 회춘양격산이항알레르기및소염진통해열효과에미치는영향. 대한외관과학회지 7: 1-13, 강승원, 노석선. 回春凉膈散과龍石散이抗炎作用에미치는影響. 대한외관과학회지 12(1):47-78, 한유진, 이용태, 장경전. Carrageenan 유발염증에대한 15Hz 전침의효과에대한연구. 대한침구학회지 20(3): , Higuchi, M., Higashi, N., Taki, H., Osawa, T. Cytolytic mechanism of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanism acts as synergistically as the major cytolytic mechanism of activated macrophages. J Immunol. 144: , 국윤범. 황련해독탕이자발적고혈압백서의혈압및신장기능에미치는영향. 대한한의방제학회지 10(1): , 黃道淵原著. 南山堂編輯局飜譯. 對譯證脈方藥合編. 南山堂. 서울, pp , Kim, H.D., Cho, H.R., Moon, S.B., Shin, H.D., Yang, K.J., Park, B.R., Jang, H.J., Lim, L.S., Lee, H.S., Ku, S.K. Effect of exopolymers from Aureobasidum pullulans on formalin-induced chronic paw inflammation in mice. J Microbiol Biotechnol. 16: , Lee, A.K., Sung, S.H., Kim, Y.C., Kim, S.G. Inhibition of lipopolysaccharide-inducible nitric oxide synthase, TNF-α and COX-2 expression by sauchinone effects on I-κBα phosphorylation, C/EBP and AP-1 activation. British journal of pharmacology 139: 11-20, Delgado, A.V., McManus, A.T., Chambers, J.P. Production of tumor necrosis factor-alpha, interleukin 1-beta, interleukin 2, and interleukin 6 by rat leukocyte subpopulations after exposure to substance P. Neuropeptides. 37(6): , Jirik, F.R., Podor, T.J., Hirano, T., Kishimoto, T., Loskutoff, D.J., Carson, D.A., Lotz, M. Bacterial lipopolysaccharide and inflammatory mediators augment IL-6 secretion by human endothelial cells. J Immunol. 142(1): , Mori, M. Regulation of nitric oxide synthesis and apoptosis by arginase and arginine recycling. J Nutr. 137: 1616S-1620S, McDaniel, M.L., Kwon, G., Hill, J.R., Marshall, C.A. and Corbett, J.A. Cytokines and nitric oxides in islet inflammation and diabetes. Proc. Soc. Exp. Biol. Med. 211: 24-32,

8 이태호 조미정 박숙자 이종록 백영두 김종열 권영규 김상찬 14. Corbett, J.A. and Mac, Daniel, M.L. Intraislet release of interleukin-1 inhibits beta cell function by inducing beta cell expression of inducible nitric oxide syntheses. J. Exp. Med. 181: , Cetkovic Cvrlje, M. and Eizirik, D.L. TNF and IFNγ potentiate the deleterious effects of IL-1β on mouse pancreatic islets mainly via generation of nitric oxide. Cytokine. 6: , Kawamata, H., Ochiai, H., Mantani, N., Terasawa, K. Enhanced expression of inducible nitric oxide synthase by Juzen-taiho-to in LPS-activated RAW264.7 cells, a murine macrophage cell line. Am J Chin Med. 28: , Lee, B.G., Kim, S.H., Zee, O.P., Lee, K.R., Lee, H.Y., Han, J.W., Lee, H.W. Suppression of inducible nitric oxide synthase expression in RAW macrophages by two-carboline alkaloids extracted from Melia azedarach. Eur J Pharmacol. 406: , 장선일, 김형진, 황기명, 배현옥, 윤용갑, 정헌택, 김윤철. 활성화된설치류 RAW 대식세포에서당귀에탄올추출물의항염증효과. 대한한의학방제학회지 10(2): , Seo, W.G., Pae, H.O., Oh, G.S., K.Y. Chai, Kwon, T.O., Y.G. Yun, N.Y. Kim, H.T. Chung. Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide production by murine macrophage cell line, RAW cells. J Ethnopharmacol. 76: 59-64, 이영선, 한옥경, 신상우, 박종현, 권영규. 향부자열수추출물의 Nitric oxide 생성및 inos 유전자발현에미치는영향. 동의생리병리학회지 17(3): , Chun, S.C., Jee, S.Y., Lee, S.G., Park, S.J., Lee, J.R., Kim, S.C. Anti-Inflammatory Activity of the Methanol Extract of Moutan Cortex in LPS-Activated Raw264.7 Cells. Evid Based Complement Alternat Med. 4(3): , Sharifi, A.M., Mousavi, S.H., Bakhshayesh, M., Tehrani, F.K., Mahmoudian, M., Oryan, S. Study of correlation between lead-induced cytotoxicity and nitric oxide production in PC12 cells. Toxicol Lett. 160: 43-48, 박은영, 박숙자, 이종록, 지선영, 변성희, 김상찬. PC-12 cell에서감초성분의 Liquiritigenin이납에의해유도된세포독성과 nitric oxide production에미치는영향, 본초학회지 22(2):17-24, Palmer, R.M., Ashton, D.S., Moncada, S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 333: , Lee, J.H., Choi, Y.H., Choi, B.T. The anti-inflammatory effects of 2 Hz electroacupuncture with different intensities on acute carrageenan-induced inflammation in the rat paw. Int J Mol Med. 16: , Gupta, M., Mazumder, U.K., Gomathi, P., Selvan, V.T. Antiinflammatory evaluation of leaves of Plumeria acuminata. BMC Complement Altern Med. 6: 36, Rao, C.V., Verma, A.R., Gupta, P.K., Vijayakumar, M. Anti-inflammatory and anti-nociceptive activities of Fumaria indica whole plant extract in experimental animals. Acta Pharm. 57: , Holt, S., Comelli, F., Costa, B., Fowler, C.J. Inhibitors of fatty acid amide hydrolase reduce carrageenan-induced hind paw inflammation in pentobarbital-treated mice: comparison with indomethacin and possible involvement of cannabinoid receptors. Br J Pharmacol. 146: , Liu, J., Zhang, W., Zhou, L., Wang, X., Lian, Q. Anti-inflammatory effect and mechanism of osthole in rats. Zhong Yao Cai. 28: , Beloeil, H., Ababneh, Z., Chung, R., Zurakowski, D., Mulkern, R.V., Berde, C.B. Effects of bupivacaine and tetrodotoxin on carrageenan-induced hind paw inflammation in rats (Part 1): hyperalgesia, edema, and systemic cytokines. Anesthesiology. 105: , Kim, Y.W., Zhao, R.J., Park, S.J., Lee, J.R., Cho, I.J., Yang, C.H., Kim, S.G., Kim, S.C. Anti-inflammatory effects of liquiritigenin as a consequence of the inhibition of NF-kappaB-dependent inos and proinflammatory cytokines production. Br J Pharmacol. 154(1): ,