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1 Original Article J Sasang Constitut Med 2014;26(2): 荊防敗毒散이 DNFB 로유발된생쥐의알레르기접촉피부염에미치는영향 박근희 임태형 박혜선 동신대학교한의과대학사상체질의학과 Abstract Effects of Hyeongbangpaedok-san (HBPDS) on Allergic Contact Dermatitis (ACD) induced by DNFB in Mice Geun-Hee Park, Tae-Hyung Lim, Hye-Sun Park Dept. of Sasang Contitutional Medicine, Graduate School of Dongshin University Objectives The present study was carried out to investigate effects of HBPDS on allergic contact (ACD) induced by 2,4-Dinitro-1-fluorobenzene (DNFB) in mice. Methods In this experiment, effects of HBPDS on body weights, skin thicknesses, skin weights, histopathological changes, clinical aspects, erythema index, melanin index, production levels of cytokines in ACD mice were investigated. In addition, effects on proliferation rates, release of β-hexosaminidase and histamine were also investigated in vitro. Results & Conclusions 1) HBPDS inhibited enlargement of skin thickness and weight significantly (P < 0.05). 2) HBPDS treatment prevented spongiosis, edema and immune cell infiltrations. 3) Erythema, desquamation and keratosis were diminished by oral administration of HBPDS. 4) Production levels of TNF-alpha and IFN-gamma in serum were decreased by HBPDS treatment in vivo. 5) More than 200 μg / ml of HBPDS treatment decreased β-hexosaminidase release and more than 400 μg / ml of HBPDS treatment also decreased histamine release in vitro. Key Words : Hyeongbangpaedok-san, Allergic Contact Dermatitis, DNFB, Sasang Contitutional Medicine Received May 07, 2014 Revised May 26, 2014 Accepted June 23, 2014 Corresponding Author Hye-Sun Park Dept. of Sasang Contitutional Medicine, Mokpo Oriental Hospital of Dongshin University, 313, Baengnyeon-daero, Mokpo-si, Jeollanam-do, , Republic of Korea Tel: Fax: medic73@hanmail.net C The Society of Sasang Constitutional Medicine. All rights reserved. This is an open acess article distributed under the terms of the Creative Commons attribution Non-commercial License ( creativecommons.org/licenses/by-nc/3.0/)

2 GH Park et al. 181 I. 緖論 접촉피부염이란외부물질과의접촉에서발생하는피부의염증반응으로발생기전에따라자극접촉피부염 (Irritant contact, ICD) 과알레르기접촉피부염 (Allergic contact, ACD) 으로세분된다. 이중알레르기접촉피부염은제 Ⅳ형과민반응에의한세포매개면역반응으로항원에대한감작이선행되어야하는알레르기질환으로임상상홍반, 구진, 수포를동반한습진의형태로나타나는데 1 한방에서는濕瘡의범위에속한다 2. 荊防敗毒散 (Hyeongbangpaedok-san, HBPDS) 은이제마가荊芥, 防風, 羌活, 獨活의降表陰작용과生地黃, 車前子, 柴胡, 地骨皮의淸裏熱작용을이용하여外寒包裏熱된少陽人表寒病을치료할목적으로龔信 古今醫鑑 의荊防敗毒散을加減하여少陽人의脾受寒表寒病에응용한처방이다. 少陽人의病證은脾受寒表寒病과胃受熱裏熱病으로구별되는데두病證은각각陰氣의升降장애와陰氣의부족으로유발되기때문에火熱위주의증상이나타나기쉽다 3. 알레르기접촉피부염의경우병변이국소적이고홍반, 구진, 수포등의습진형태로나타나며외부자극이있어야만질병이발현하므로 1 少陽人의경우外邪에의해유발되는脾受寒表寒病傷寒범주로인식하는것이타당하다고생각하였고성질상 熱多寒少之病 인것으로이해하였다. 이에알레르기접촉피부염의제반증상완화에降表陰淸裏熱하는荊防敗毒散이효과가있을가능성이있다고생각하여본연구를기획하였다. 본연구에서는荊防敗毒散의항피부염효과를살펴보기위하여생쥐에 2,4-Dinitro-1-fluorobenzene (DNFB) 을이용하여알레르기접촉피부염을유발하고荊防敗毒散을구강투여하여생쥐의체중, 병소에 발생하는피부염의정도, 홍반지수, 멜라닌지수, 피부조직의조직병리학적변화등을관찰하였다. 또한荊防敗毒散의작용기전을살펴보기위하여 in vitro 에서비만세포주 (mast cell) 에대한세포독성및 β -hexosaminidase 와 histamine 분비에미치는영향을관찰하였다. 위실험결과荊防敗毒散이알레르기접촉피부염에미치는영향에대한有意한결과를얻었으므로이에보고하는바이다. Ⅱ. 材料및硏究方法 1. 재료 1) 동물 6주령된수컷 Balb/c 생쥐를샘타코 (Incheon, Korea) 에서구입하여사용하였다. 모든실험동물은일주일이상실험실환경에적응시킨후온도와습도가조절되는환경 (24±3, 12-hr light-dark cycle) 에서고형사료와물을마음껏섭취하게하며실험에사용하였다. 2) 세포주흰쥐유래비만세포주 (mast cell line) 인 RBL-2H3 cell 은한국세포주은행 (Seoul, Korea) 으로부터냉동상태로구입하여사용하였다. 3) 약재본실험에사용된荊防敗毒散 (Hyeongbangpae dok-san, HBPDS) 은羌活, 獨活, 荊芥, 防風, 生地黃, 茯苓, 前胡, 車前子, 地骨皮, 柴胡로구성되며, 모든약재는동신대학교부속목포한방병원을통하여구입정제하여사용하였다. 荊防敗毒散 (HBPDS) 의처방구성은 東醫壽世保元 新定少陽人病應用要藥十七方 을따랐다 (Table 1).

3 182 Effects of Hyeongbangpaedok-san on Allergic Contact Dermatitis Table 1. The Composition of Prescription of Hyeongbangpaedok-san (HBPDS) Herb Pharmaceutical name Dose(g) 羌活 Osteric Radix 4.0 獨活 Angelicae Pubescentis Radix 4.0 荊芥 Schizonepetae Spica 4.0 防風 Saposhnikovia Radix 4.0 生地黃 Rehmanniae Radix 4.0 茯苓 Poria 4.0 前胡 Peucedani Radix 4.0 車前子 Plantaginis Semen 4.0 地骨皮 Lycii Radicis Cortex 4.0 柴胡 Bupleuri Radix 4.0 Total 방법 1) 약물의준비상기한내용과같이조제된荊防敗毒散 (HBPDS) 2첩 (80g) 을물 1,000ml와함께전기약탕기 ( 대웅, 한국 ) 로 3시간동안전탕한다음, 찌꺼기를제거하고상층액을여러번겹친거즈를이용하여여러차례여과하였다. 여과된추출액은감압농축기 (EYELA, Japan) 를이용하여감압농축한다음동결건조하였다. 최종적으로얻어진동결건조분말은 7.28g으로수득율은 9.1 % 였으며, 실험에사용될때까지냉동보관하였다. 2) 알레르기접촉피부염의유발알레르기접촉피부염유발을위하여 acetone과 olive oil을 4대 1로섞은용액에 2,4-Dinitro-1-fluorobenzene (DNFB) 을 0.1~2% 로희석하여사용하였다. 실험첫날부터 3일간 30μl의 0.1 % 2,4-Dinitro-1-fluorobenzene (DNFB) 용액을양측귀에 1일 1회씩도포해줌으로써감작 (Sensitization) 을시행하였고, 생체내의알레르기반응을위하여 3일동안방치하였다. 실험 7일째에 Animal clipper (Oster, USA) 를이용하여생쥐의등을가볍게제모하고제모제를이용하여잔털을깨끗이제거한다음상처치유를위하여 24시간방치하였다. 24시간이지난후 (day 8) 부터제모된등에 50μl의 0.2 % 2,4-Dinitro-1-fluorobenzene (DNFB) 용액을 2일간격으로총 4회도포하여알레르기접촉피부염을유발 (Challenge) 하였다 (Figure 1). 3) 약물투여및실험군분류상기한방법대로제조된荊防敗毒散 (HBPDS) 은실험시작 9일째 (day 9) 부터 1일 1회씩총 6일간 Oral zonde 를이용하여구강투여하였다 (Figure 1). 투여용량은성인의 1일복용량을기준으로 kg당 1일복용량을계산한다음생쥐의체중과비교하여저농도투여군의경우 10mg /day, 고농도투여군의경우 100mg /day의농도로 60kg 성인투여농도의 4배및 40배를투여하였다. 실험군은아래와같다. (1) 정상군 (NOR) : DNFB 대신 AOO 를도포하고, 荊防敗毒散 (HBPDS) 대신증류수를투여한군 (n=6). (2) 알레르기접촉피부염대조군 (CTL) : DNFB 로알레르기접촉피부염을유발하고증류수를투여한군 (n=8). (3) 荊防敗毒散 (HBPDS) 저농도군 (LOW) : DNFB 로알레르기접촉피부염을유발하고 10mg /day 의荊防敗毒散 (HBPDS) 을투여한군 (n=8). (4) 荊防敗毒散 (HBPDS) 고농도군 (HIGH) : DNFB 로알레르기접촉피부염을유발하고 100 mg /day 의荊防敗毒散 (HBPDS) 을투여한군 (n=8).

4 GH Park et al. 183 Figure 1. Experimental design. All animals except normal group were received 0.1% of DNFB (30 μl in each ear) in AOO on day 1, 2, 3. Then CTL, HBPDS LOW and HBPDS HIGH group of mice received 0.2% of DNFB on day 8, 10, 12, 14. All animals were treated without HBPDS in AOO on 6 consecutive days (day 9-14). Mice were sacrificed on day 15. S : sacrifice, HBPDS : Hyeongbangpaedok-san 4) 체중측정실험시작일 (day 1) 측정한체중을기준으로 2주동안체중변화를관찰하였다. 체중측정은측정일오후 2시에전자저울 (Tanita, Japan) 을이용하여실시하였다. 5) 피부의두께및무게관찰실험마지막날, 생쥐로부터얻어진피부조직을버니어캘리퍼스 (Mitutoyo, Japan) 를이용하여두께를측정하고, 직경 5 mm 의동일한원형크기로잘라미량저울 (Sartorius, 한국 ) 을이용하여무게를측정하였다. 6) 피부조직의조직병리학적변화관찰실험마지막날, 생쥐로부터얻어진피부조직을 10% 포르말린에고정한후, 파라핀에포매하였다. 포매된조직을미세절단기 (Leica, Germany) 를이용하여 4 μm두께로잘라슬라이드글라스에부착하였다. 부착된조직으로부터파라핀을제거하고 hematoxylin 과 eosin을이용하여염색한후광학현미경 (Olympus, Japan) 으로관찰하였다 (x 100). 7) 피부표면의육안적관찰알레르기접촉피부염의유발정도를전반적으로관찰하기위하여실험마지막날생쥐를희생시킨다음, 알레르기접촉피부염이유발된등부위를 Digital camera (Olympus, Japan) 로촬영하여제시하였다. 8) 피부의홍반지수및멜라닌지수에미치는영향관찰실험마지막날, 생쥐의등에발생한병변부위에서홍반지수 (erythema index) 및멜라닌지수 (melanin index) 를 Color meter (Cortex tech., Denmark) 를이용하여측정하였다. 홍반지수및멜라닌지수는생쥐의등에서 3번씩각각서로다른부위를측정한후평균값을대푯값으로사용하였다. 9) Cytokine 분비에미치는영향관찰가장대표적인 Th1 skewing cytokine 인 TNF-alpha 와 IFN-gamma 의혈액내농도를측정하였다. 실험마지막날, 심장채혈로얻어진생쥐의혈액을원심분리기를이용하여 1500 rpm에서 10분간원심분리한후, 상층액을채취하여 cytokine 측정에사용하였다. 혈청중 TNF-alpha 와 IFN-gamma 의농도는각각의 ELISA kit (Raybiotech, USA) 를사용하여측정하였으며, 모든측정절차는제조사에서제시한가이드라인에따라진행되었다. 10) 세포주배양환경비만세포주 (mast cell line) 인 RBL-2H3 cell 의생육배지로는 DMEM (Dulbecco's odified Eagle Medium,

5 184 Effects of Hyeongbangpaedok-san on Allergic Contact Dermatitis high glucose, Gibco) 배지에 10% fetal bovine serum (Gibco, FBS) 과 penicillin-streptomycin (100 units/ ml, 100 μg / μl ) 을첨가하여사용하였고, 세포주의계대배양은 2일간격으로시행하였다. 부착세포의탈착을위해서 Trypsin-EDTA (Sigma) 를사용하였으며, 5% CO 2 가공여되는배양기속에서 37 를유지하며배양되었다. 11) 세포증식율에미치는영향측정세포증식율의측정은수용성 tetrazolium salt를이용하여살아있는세포를정량하는방법을이용한 Cell Counting Kit-8 (CCK-8) 을이용하여수행하였다. 먼저 96-well plate에측정하고자하는대상세포주를 well 당 5 x 10 3 개의분량으로분주하고, 37, 5% CO 2 가공여되는환경에서 24시간을배양하여부착및안정화를시행하였다. 24 시간의배양이끝난후, 상기한방법으로제조한荊防敗毒散 (HBPDS) 을인산완충액 (phosphate buffered saline, PBS) 에녹이고 0.45 μm크기의 syringe filter를이용하여거르는것으로부유된거대분자를제거한다음, 최종농도 800, 400, 200, 100, 0 μg / ml가되게배양액에희석하여부착및안정화된세포주에공급하고 24시간동안배양하였다. 24시간동안의배양이끝난후, 각 well 당 10μl의 CCK-8 용액을첨가하고 37, 5% CO 2 가공여되는환경에서 3시간동안방치하였다. 3시간후, Micro-plate reader (Bio-rad, CA) 를이용하여 450 nm파장에서흡광도를측정하였다. 정상대조군으로는약물을첨가하지않은 well 을사용하였고, 결과는정상대조군에대한백분율로환산하여나타내었다. 12) β-hexosaminidase 유리억제에미치는영향측정비만세포의탈과립에미치는영향을관찰하기위하여 β-hexosaminidase 유리능에미치는영향을관찰하였다. 먼저 96-well plate 에비만세포주를 well 당 개의분량으로분주하고, 37, 5% CO 2 가공여되는환경에서 24시간동안배양하여부착및안정화를 시행하였다. 24시간의배양이끝난후, 800, 400, 200, 100, 50 μg / ml농도의荊防敗毒散 (HBPDS) 을 1시간동안처리한이후배양액을깨끗이씻어내고, serum-free media 에 50 nm의 PMA 와 1 μm의 A23187을첨가하고 30분뒤상층액을취하였다. 얻어진상층액은 3000 rpm에서 20분동안원심분리하여유리된 β -hexosaminidase 농도측정에사용하였다. 세포내에존재하는 β-hexosaminidase 의총량측정을위하여원심분리가끝난 pellet에 triton X-100 을처리하여세포를파괴한다음세포내에존재하는 β-hexosaminidase 의총량을측정하였다. 상층액과 cell lysate에 1 mm 의 p-nag 를 0.1 M citrate buffer (ph 5) 에녹여처리한다음 37, 5% CO 2 가공여되는환경에서 1시간동안반응시키고난후, 200 μl의 carbonate buffer 를더하여반응을종료시켰다. β-hexosaminidase 의농도측정은 Micro-plate reader (Bio-rad, CA) 를이용하여 405 nm파장에서흡광도 (Opticald density, OD) 를측정하여다음과같은공식으로계산하였다. β-hexosaminidase release (%) = OD sup. /(OD sup. + OD pellet ) 100 OD sup. : 상층액에서얻어진흡광도 OD pellet : Cell pellet 을녹여서얻어진흡광도 13) Histamine 분비억제에미치는영향측정 Histamine 분비억제에미치는영향을측정하기위해먼저 24-well plate에비만세포주를 well 당 개의분량으로분주하고, 37, 5% CO 2 가공여되는환경에서 24시간동안배양하여부착및안정화를시행하였다. 24시간동안배양한후약물을처리하고, 비만세포를활성화한후배양액 500μl를취하여 0.1 M HCl 450 μl와 60% 과염소산용액 50μl를혼합한후 2000 rpm 에서 20분동안원심분리하여상층액을취하였다. 다시상층액 800μl를 5 M NaOH 용액 500μl, 증류수 3ml, n-butanol 10 ml, NaCl 1.2g 을혼합한시험관에넣고잘흔들어준뒤원심분리하였다. 원심분리한후, n-butanol 층 8ml를취해 0.1 M HCl 3ml, n-heptane

6 GH Park et al. 185 Figure 2. Effects of HBPDS on changes in body weights in ACD mice. Body weights were measured once in a week. Changes in body weights were represented as average weights, which were expressed as percentages of weight on day 1. NOR : naive group, CTL : ACD group, LOW : 10 mg /day of HBPDS treated ACD group, HIGH : 100 mg /day of HBPDS treated ACD group. Values are represented as mean±sd (n=8). 10ml를더하여잘흔들어준후재차원심분리하였다. 여기서얻어진수층 2ml에 1M NaOH 400μl, 1% o-phthaldialdehyde 용액 100μl를가하여혼합하고 2분동안방치한다음 emission 438 nm, excitation 353 nm에서형광강도 (fluorescence intensity, FI) 를측정하였다. 측정된형광강도값은 PMA 와 A23187을이용하여활성화시킨군에대한백분율로나타내었다. 3. 통계처리 실험자료에대한통계적분석은통계패키지인 SAS (The SAS System for Windows, ver. 6.12, SAS Institute, U.S.A.) 를이용하였다. 실험성적은평균 ± 표준편차로나타내었으며, 각실험군간평균차이를검정할때에는 Mann-Whitny test 로검정하여 P값이 0.05 미만일때유의한차이가있는것으로판정하였다. Ⅲ. 結果 Figure 3. Effects of HBPDS on skin thickness changes in ACD mice. Skin thicknesses were measured using vernier calipers on day 15. NOR : naive group, CTL : ACD group, LOW : 10mg /day of HBPDS treated ACD group, HIGH : 100mg /day of HBPDS treated ACD group. Values are represented as mean±sd. ## P < 0.01 vs. non-treated naive (NOR) group, * P < 0.05 vs. ACD (CTL) group, (n=8). 1. 체중변화에미치는영향 2 주동안체중변화를관찰한결과정상군 (NOR) 은 12.1% 의체중증가를보인반면, 대조군 (CTL) 은 5.0%, 荊防敗毒散 (HBPDS) 저농도군 (LOW) 은 6.1%, 荊防敗毒散 (HBPDS) 고농도군 (HIGH) 은 6.8% 의체중증가를보였다. 알레르기접촉피부염이유발된모든실험군에서정상군 (NOR) 에비해체중증가율이감소되는경향을보였으나통계적유의성은없었다 (Figure 2). 2. 피부의두께변화에미치는영향 실험마지막날, 생쥐로부터피부조직을적출하여피부두께를관찰한결과 ACD를유발한대조군의평균피부두께는정상군에비하여유의한수준으로증가하였다 (P < 0.01). 고농도 (HIGH) 의荊防敗毒散 (HBPDS) 투여는이러한피부두께의증가를유의한수준으로감소시켰으며 (P < 0.05), 저농도군 (LOW) 은감소하는경향을보였으나통계적유의성은없었다 (Figure 3).

7 186 Effects of Hyeongbangpaedok-san on Allergic Contact Dermatitis Figure 4. Effects of HBPDS on skin weight changes in ACD mice. Skin weights were measured using micro balance on day 15. NOR : naive group, CTL : ACD group, LOW : 10 mg /day of HBPDS treated ACD group, HIGH : 100 mg /day of HBPDS treated ACD group. Values are represented as mean±sd. ## P < 0.01 vs. non-treated naive (NOR) group, * P < 0.05 vs. ACD (CTL) group, (n=8). Figure 5. Effects of HBPDS on histopathological changes in ACD tissue. Skin tissues were stained with hematoxylin and eosin, then observed using microscope. (A) NOR, (B) CTL, (C) LOW, (D) HIGH. Filled arrows mean infiltration site of immune cells. Blue bars mean range of spongiosis (x100). 3. 피부의무게변화에미치는영향 실험마지막날, 생쥐로부터피부조직을적출하여피부무게를관찰한결과알레르기접촉피부염을유발한대조군 (CTL) 의평균피부무게는정상군 (NOR) 에비하여유의한수준으로증가하였다 (P < 0.01). 저농도또는고농도의荊防敗毒散 (HBPDS) 투여는이러한피부무게의증가를유의한수준으로감소시켰다 (P < 0.05), (Figure 4). 4. 피부조직의조직병리학적변화에미치는영향 Vehicle 만을처리한정상군 (NOR) 의피부조직은특별한이상소견이발견되지않은반면 (Figure 5A), 대조군 (CTL) 의피부조직에서는비후된스폰지조직 (spongiosis) 과부종 (edema), 면역세포의침윤이관찰되었다 (Figure 5B). 저농도와고농도의荊防敗毒散 (HBPDS) 투여군에서는스폰지조직 (spongiosis) 과면역세포침윤이줄어드는경향을보였다 (Figure 5C, D). Figureig 6. Effects of HBPDS on clinical aspect in ACD mice. Clinical aspects of skin lesions were observed using digital camera on day 15. (A) NOR, (B) CTL, (C) LOW, (D) HIGH. 5. 피부의증상변화에미치는영향 Vehicle 만을처리한정상군 (NOR) 의피부에서는특별한변화를관찰할수없었고 (Figure 6A), 반복되는 DNFB 접촉이이루어진대조군의피부에서는홍반, 낙설등중등도이상의알레르기접촉피부염증상이관찰되었다 (Figure 6B). 저농도와고농도의荊防敗毒散 (HBPDS) 처리는상기한알레르기접촉피부염

8 GH Park et al. 187 Figure 7. Effects of HBPDS on erythema index in ACD mice. Erythema index were measured using color meter on day 15. NOR : naive group, CTL : ACD group, LOW : 10mg /day of HBPDS treated ACD group, HIGH : 100 mg /day of HBPDS treated ACD group. Values are represented as mean±sd. ### P < vs. non-treated naive (NOR) group, * P < 0.05 vs. ACD (CTL) group, (n=8). Figure 9. Effects of HBPDS on serum level of TNF-alpha in ACD mice. Level of TNF-alpha in serum was measured using ELISA method. NOR : naive group, CTL : ACD group, LOW : 10 mg /day of HBPDS treated ACD group, HIGH : 100 mg /day of HBPDS treated ACD group. Values are represented as mean±sd. ### P < vs. non-treated naive (NOR) group, * P < 0.05, ** P < 0.01 vs. ACD (CTL) group, (n=8). 지수가유의한수준으로상승하였고 (P < 0.001), 고농도군 (HIGH) 의고농도荊防敗毒散 (HBPDS) 투여는이러한홍반지수상승을유의한수준으로억제하였다 (P < 0.05). 저농도군 (LOW) 은홍반지수가하강하는경향을보였으나통계적유의성은없었다 (Figure 7). 7. 피부의멜라닌지수에미치는영향 Figure 8. Effects of HBPDS on melanin index in ACD mice. Melanin index were measured using color meter on day 15. NOR : naive group, CTL : ACD group, LOW : 10 mg /day of HBPDS treated ACD group, HIGH : 100 mg /day of HBPDS treated ACD group. Values are represented as mean±sd. ### P < vs. non-treated naive (NOR) group, * P < 0.05 vs. ACD (CTL) group, (n=8). 증상을감소시키는경향을보였다 (Figure 6C, D). 실험마지막날, 생쥐의피부병소로부터멜라닌지수 (melanin index) 를측정한결과대조군 (CTL) 에서멜라닌지수가유의한수준으로상승하였고 (P < 0.001), 고농도군 (HIGH) 의고농도荊防敗毒散 (HBPDS) 투여는이러한멜라닌지수상승을유의한수준으로억제하였다 (P < 0.05). 저농도군 (LOW) 은멜라닌지수가하강하는경향을보였으나통계적유의성은없었다 (Figure 8). 6. 피부의홍반지수에미치는영향 실험마지막날, 생쥐의피부병소로부터홍반지수 (erythema index) 를측정한결과대조군 (CTL) 에서홍반 8. 혈중 TNF-alpha 함량에미치는영향실험마지막날, 심장채혈로얻은혈청에서 TNF-alpha의농도를분석한결과대조군 (CTL) 에서 TNF-alpha의농도가유의한수준으로증가하였으며

9 188 Effects of Hyeongbangpaedok-san on Allergic Contact Dermatitis Figure 10. Effects of HBPDS on serum level of IFN-gamma in ACD mice. Level of IFN-gamma in serum was measured using ELISA method. NOR : naive group, CTL : ACD group, LOW : 10mg /day of HBPDS treated ACD group, HIGH : 100mg /day of HBPDS treated ACD group. Values are represented as mean±sd. ## P < 0.01 vs. non-treated naive (NOR) group, * P < 0.05 vs. ACD (CTL) group, (n=8). Figure 11. Effects of HBPDS on proliferation rates of RBL-2H3 cells in vitro. Proliferation rates were measured using highly water-soluble tetrazolium salt in various concentrations. Values were represented as mean±sd. (P < 0.001), 荊防敗毒散 (HBPDS) 투여는이러한증가를농도의존적으로감소시켰다 (LOW, P < 0.05; HIGH, P < 0.01), (Figure 9). 9. 혈중 IFN-gamma 함량에미치는영향 실험마지막날, 심장채혈로얻은혈청에서 IFN-gamma 의농도를분석한결과대조군 (CTL) 에서 Figure 12. Effects of HBPDS on β-hexosaminidase release of RBL-2H3 cells in vitro. Cells were pre-treated with HBPDS in indicated concentrations, then activated using 50 nm of PMA and 1 μm of A β-hexosaminidase release was measured using spectrophotometric method in various concentrations. Rest : non-treated (resting) RBL-2H3 cells, Active : 50 nm of PMA and 1 μm of A23187 treated (activated) RBL-2H3 cells (control). Values were represented as mean±sd. ### P < vs. non-treated naive (Rest) group, * P < 0.05 vs. non-treated control (Active) group of three independent experiments. IFN-gamma 의농도가유의한수준으로증가하였으며 (P < 0.01), 고농도군 (HIGH) 의고농도荊防敗毒散 (HBPDS) 투여는이러한증가를유의한수준으로감소시켰다 (P < 0.05). 저농도군 (LOW) 의 IFN-gamma 농도는감소하는경향을보였으나통계적유의성은없었다 (Figure 10). 10. 비만세포주의증식율에미치는영향 비만세포주인 RBL-2H3 cell 에荊防敗毒散 (HBPDS) 을최종농도 800, 400, 200, 100, 0 μg / ml로처리하고 24시간동안배양한후, 세포증식율에미치는영향을관찰한결과荊防敗毒散 (HBPDS) 은투여한모든농도에서 RBL-2H3 cell 의증식에특별한영향을미치지않았다 (Figure 11). 11. β-hexosaminidase 유리억제에미치는영향

10 GH Park et al. 189 Ⅳ. 考察 Figure 13. Effects of HBPDS on histamine release of RBL-2H3 cells in vitro. Cells were pre-treated with HBPDS in indicated concentrations, then activated using 50 nm of PMA and 1 μm of A histamine release was measured using fluorescence detecting method in various concentrations. Rest : non-treated (resting) RBL-2H3 cells, Active : 50 nm of PMA and 1 μm of A23187 treated (activated) RBL-2H3 cells (control). Values were represented as mean±sd. ### P < vs. non-treated naive (Rest) group, * P < 0.05 vs. non-treated control (Active) group of three independent experiments. 비만세포주의활성화에의하여세포내에저장된 β-hexosaminidase 의유리가유의한수준으로증가하였고 (P < 0.001), RBL-2H3 cell 에荊防敗毒散 (HBPDS) 을농도별로처리하고 β-hexosaminidase 유리에미치는영향을관찰한결과, 400 μg / ml이상의荊防敗毒散 (HBPDS) 처리에의하여 β-hexosaminidase 유리가유의한수준으로감소하였다 (P < 0.05) (Figure 12). 12. Histamine 유리억제에미치는영향 비만세포주의활성화에의하여정상군 (NOR) 에비하여 10배이상증가된 histamine 유리를보였고 (P < 0.001), RBL-2H3 cell 에荊防敗毒散 (HBPDS) 을농도별로처리하고 histamine 유리에미치는영향을관찰한결과 200 μg / ml이상의荊防敗毒散 (HBPDS) 처리에의하여 histamine 유리가유의한수준으로감소하였다 (P < 0.05) (Figure 13). 접촉피부염의경우알레르기접촉피부염 (ACD) 의발생빈도는약 20% 로추측되나접촉피부염의대부분을차지하는자극접촉피부염 (ICD) 의경우대개일과성으로경미한경우가많아치료현장에서는상대적으로알레르기접촉피부염을접할기회가많은것으로보인다 1. 양방에서는알레르기접촉피부염의치료를위해면역억제제와스테로이드제를사용하는데국소적인경우외용스테로이드제를국소도포하고전신적인경우에는스테로이드제를전신투여한다 4. 하지만스테로이드제복용으로도완치가안되는경우다른처치가불가능하고 3주이상의스테로이드제복용은중단후최고 1년까지뇌하수체- 부신피질억제를나타낼수도있기때문에 1 기존치료를대체할수있는새로운치료법이필요한실정이다. 少陽人은脾大腎小한특징을지닌체질로脾局의熱氣가太過하기쉽다. 脾受寒表寒病의경우脾局의熱氣太過로中上焦의陰氣가下焦로하강하지못하고背表에囚滯되어초기에는惡寒, 發熱, 關節痛등의寒性증상이나타나지만險危證으로진행되면便閉, 發狂, 譫語, 動風등의熱性증상이나타나게된다. 이는少陽人發病의근본원인이과도한裏熱이기때문에表寒病이더라도겉으로는熱性증상이뚜렷하게표현되는것이다 3. 이제마가 甲午本 에서裏熱病으로인식한陽毒發斑證을 辛丑本 에서는表寒病 熱多寒少之病 으로改抄한것 5 과도一脈相通한다. 알레르기접촉피부염증상의경우항원자극에의해국소적인습진형태로나타나며외부항원자극이있어야만발현되므로少陽人의경우裏熱病보다表寒病 熱多寒少之病 으로인식하는것이적합하다고생각하였다. 荊防敗毒散을이용한최근연구로는이 6 와민 7 의노화관련연구가있었고폐렴에응용한한 8 의증례보고가있었다. 또한알레르기접촉피부염에대해서는단일약재 9-16 와처방 을이용한다양한연구가있었지만체질적으로접근한연구로는알레르기접촉피부

11 190 Effects of Hyeongbangpaedok-san on Allergic Contact Dermatitis 염을熱性病證으로인식하고접근한조 30 와김 31 의연구가있을뿐이었다. 본연구에서는 T세포의제4형과민반응유발을위해다용되는 32 2,4-Dinitro-1-fluorobenzene (DNFB) 을 Balb/c 생쥐에반복적으로도포하여중등도이상의피부염소견이유발된알레르기접촉피부염동물모델을만들었다. 이러한반복적인 DNFB 의처리는피부조직의두께와무게를유의한수준으로증가시켰는데이는알레르기접촉피부염에서볼수있는염증반응에의한조직내부종 (edema), 면역세포침윤, 조직내부종의흔적인스폰지조직형성 (spongiosis) 및각질화세포 (keratinocyte) 와섬유아세포 (fibroblast) 의과다증식으로조직비후가일어난것으로 33 荊防敗毒散의투여가피부조직비후를유의하게억제시켰음을알수있다 (Figure 3, 4). 또한荊防敗毒散 (HBPDS) 의투여로알레르기접촉피부염발생조직의스폰지조직형성 (spongiosis) 및면역세포의침윤이감소되는경향을보였다 (Figure 5). 대조군의피부조직에서는스폰지조직의비후소견과헤마톡실린에의하여푸른점상으로염색된단핵구들이증가하는소견이관찰되었으나 (Figure 5B), 荊防敗毒散 (HBPDS) 투여군에서는스폰지조직및부종이줄어드는경향을보였으며, 염증조직내로침윤된단핵구도감소하는경향을보였다 (Figure 5C, D). 저농도荊防敗毒散 (HBPDS) 투여군에서의스폰지조직은대조군에비해감소하였으나단핵구침윤은상당부분진행되어있었다 (Figure 5C). 그러나고농도荊防敗毒散 (HBPDS) 투여군에서는스폰지조직감소효과뿐만아니라면역세포침윤억제효과도있는것으로나타났다 (Figure 5D). 위의결과를종합하여보면, 荊防敗毒散 (HBPDS) 이조직내에서면역세포침윤, 부종, 스폰지조직비후등을억제시켜피부조직의비후를감소시킨것을알수있었다. DNFB 처리로유발된낙설과홍반, 각질화된가피 (Figure 6B) 등은荊防敗毒散 (HBPDS) 의투여로피부병변이감소되는경향을보였다 (Figure 6C, D). 이는荊防敗毒散 (HBPDS) 에의한조직병리학적변화와 도서로통하는결과로부종, 해면화, 면역세포침윤등을억제함으로써피부표면에나타나는증상도완화시켰음을알수있었다. 육안적관찰을객관적인수치로분석하기위하여 color meter를이용하여피부의홍반지수와멜라닌지수를측정한결과저농도荊防敗毒散 (HBPDS) 투여군에서는홍반지수와멜라닌지수가감소하는경향을보였으나통계적유의성은없었고, 고농도荊防敗毒散 (HBPDS) 투여군에서는홍반지수와멜라닌지수가유의한수준으로감소하였다 (Figure 7, 8). 또한, 홍반지수는급성염증상태및출혈반등에기인하여증가하고, 멜라닌지수는만성피부염의특징중에하나인태선화 (lichenification) 과정에의한멜라닌색소침착으로증가함 34 을감안하면, 荊防敗毒散 (HBPDS) 이두가지과정을모두억제할수있다는가설을세울수있었다. TNF-alpha 와 IFN-gamma 는대표적인 Th1 skewing reaction에관여하는염증성 cytokine 으로 DNFB 에의한피부염모델에서유의한수준으로증가됨이알려져있다 35. 연구에사용한동물모델의피부병변에서는출혈반이거의발생하지않았으므로 (Figure 6B) 본연구에서의홍반지수증가는염증반응과관련될가능성이높은것으로해석될수있었다. 이를확인하기위하여대표적인염증성 cytokine 인 TNF-alpha 와 IFN-gamma 의혈액내함량을조사하였고, 그결과荊防敗毒散 (HBPDS) 이효율적으로 TNF-alpha 와 IFN-gamma 의혈액내농도를낮춘것을확인할수있었으며 (Figure 9, 10), 荊防敗毒散 (HBPDS) 이 Th1 계열과관련된염증반응을억제하는작용이있음을알수있었다. 비만세포는다양한자극에의하여탈과립이일어나피부염증반응에서가려움증을유발하며 36,37, 염증성 cytokine인 TNF-alpha 를분비하여피부염증상의악화에관여하는것으로알려져있다 38. 본연구에서비만세포주에荊防敗毒散 (HBPDS) 을최고농도 800 μg / ml까지처리한결과비만세포주의세포증식율에특별한변화는보이지않았다 (Figure 11). 또한최고농도 800 μg / ml범위내에서荊防敗毒散

12 GH Park et al. 191 (HBPDS) 이비만세포의탈과립작용에미치는영향을 β-hexosaminidase 와 histamine 유리능에미치는영향을통하여살펴본결과 β-hexosaminidase 유리는 400 μg / ml이상에서, histamine 유리는 200 μg / ml이상농도에서유의한수준으로억제되었으며반응은농도의존적이었다 (Figure 12, 13). 이러한결과는荊防敗毒散 (HBPDS) 이비만세포의탈과립작용을억제함으로써조직의염증반응및피부증상을완화시킨것으로해석된다. 이상을정리하면荊防敗毒散 (HBPDS) 이피부조직비후와알레르기접촉피부염의주요증상인홍반, 낙설, 각질화등을효과적으로완화하였으며, TNF-alpha와 IFN-gamma 와같은염증성 cytokine의생성을억제하여 Th1 계열의염증반응을감소시켰고, 비만세포의탈과립억제를통해병변의염증반응을억제하여가려움을방지하였음을알수있었다. Ⅴ. 結論 荊防敗毒散 (HBPDS) 열수추출물이 DNFB 에의하여유발된알레르기접촉피부염에미치는영향및비만세포주의탈과립에미치는영향을관찰한결과다음과같은결론을얻었다. 1. 알레르기접촉피부염유발에의하여발생한피부의무게증가와두께증가를유의하게감소시켰다. 2. 피부조직에서발생한스폰지조직의비후와부종및면역세포침윤을감소시키는경향을보였다. 3. 알레르기접촉피부염유발에의하여발생한홍반, 낙설, 각화등의피부증상을감소시키는경향을보였다. 4. 알레르기접촉피부염유발에의하여증가된홍반지수와멜라닌지수를유의하게감소시켰다. 5. Th1 반응의대표적인 cytokine인 TNF-alpha와 IFN-gamma 의혈액내함량을대조군과비교하여유의하게감소시켰다 μg / ml이상에서비만세포주의 β-hexosaminidase 유리를유의한수준으로감소시켰다 μg / ml이상에서비만세포주의 histamine 유리를유의한수준으로감소시켰다. 이상의결과로부터少陽人脾受寒表寒病에응용되는荊防敗毒散 (HBPDS) 이알레르기접촉피부염의치료에도움을줄수있음을알수있었다. Ⅵ. References 1. Jung JY, Han KH. Allergic Contant Dermatitis in Korea. Seoul:MD world publisher. 2008:20-26, 28-34, (Korean) 2. National federation of department of dermatologic surgery, colleges of oriental medicine. Dermatologic surgery. Pusan:Sunwoo publisher. 2007: (Korean) 3. National federation of department of sasang constitutional medicine. Sasang constitutional medicine. Seoul:Jipmoondang. 2008: , 253, 391. (Korean) 4. Professors of medical college in Korea, New Today's theraphy Seoul:Daeyoung publisher. 2007: (Korean) 5. Kim SH, Kim YH, Jang HS, Lee JH, Ko BH. A study of Soyangin's Delirious speaking bad disease in exterior cold disease induced from spleen affected by cold. J Sasang Constitut Med. 2008;20(3): (Korean) 6. Lee SY, Ahn TW. Effects of Hyeongbangpaedok-san and Dokhwaljihwang-tang that Get Weight, Hematology, Biochemistry Change by Wistar rat's aging. J Sasang Constitut Med. 2005;17(3): (Korean) 7. Min KH, Ahn TW. Anti-Oxidative Effect of Soyangin Hyeongbangpaedok-san Decoction in Kidney and

13 192 Effects of Hyeongbangpaedok-san on Allergic Contact Dermatitis Spleen cells of Aged Rats. J Sasang Constitut Med. 2010;22(1): (Korean) 8. Han DY, Shim KH, Kim JY, Kim DL. A clinical study of the treatment prescribed Hyeongbangpaedok-San for Aspiration Pneumonia concurred in Soyangin with Stroke. J Sasang Constitut Med. 2003;15(2): (Korean) 9. Son MY, Nam SH. Effect of Eucommia ulmoides Extracts on Allergic Contract Dermatitis and Oxidative Damage induced by repeat elicitation of DNCB. J of The Korean Society of Food Science and Nutrition. 2007;36(12): (Korean) 10. Kim SC, Lee JR, Choi KI, Park SJ, Kwon YK, Byun SH. Effect of Lonicerae Flos-Skin on contact hypersensitivity induced by repeat elicitation of DNCB. J of The Korea Association of Herbology. 2006;21(1): (Korean) 11. Kim YH, Park YS. Effect of Acanthopanacis cortex water extract on antioxidative activity, lipid profile and epidermal thickness in DNCB-induced Allergic Contact Dermatitis animal model. J of Korean Society of Food Science and Technology. 2006;38(5): (Korean) 12. Son MY. Effect on Onion(Allium cepa L.) extracts on Allergic Contact Dermatitis and oxidative damage induced by repeat elicitation og DNCB. J of Korean Society of Food Science and Technology. 2007;12(2): (Korean) 13. Song JS, Lee JC, Chol JH, Kim JH, Park SY. Effects of Scrophulariae Radix(SR) on Allergic contact Dermatitis(ACD) induced by DNCB in mice. J of The Korean Oriental Ophthalmology & Otorhinolaryngology & Dermatology. 2011;24(3):1-16. (Korean) 14. Kim CJ, Kim YB, Koo YH, Nam HJ. The effects of Sophorae Radix and Coptidis Rhizoma-Iontophoresis in Allergic Contact Dermatitis. J of The Korean Oriental Ophthalmology & Otorhinolaryngology & Dermatology. 2005;18(1): (Korean) 15. Shin KS, Son JS, Kim DE, Jeon WJ, Jung SH, Shin KJ, et al. Effect of Rhemanniae Radix extract on Allergic Contact Dermatitis on mice induced by DNCB. J of The Korean Academy of Oriental Medicine Prescription. 2000;8(1): (Korean) 16. Kim YH, Park YS. Effect of Scutellaria baicalensis water extract on antioxidative activity and epidermal thickness in DNCB-induced Allergic Contact Dermatitis animal model. J of The Korean Society of Food Science and Nutrition. 2006;35(5): (Korean) 17. Hwang SY, Lee KY, Lee SH, Kim HJ. The ameliorative effects of Gagam-Gongjin-dan(WSY-1075) in contact -induced animal model. J of The Korean Academy of Oriental Medicine Prescription. 2013;21(1): (Korean) 18. Park EH, Yoon MY, Kim SB, Kim DH. Effects of GP extract on oxidative stress and contact in NC/Nga mice induced by DNCB. Dept. of Physiology, College of Korean Medicine, Daejeon University. 2007;16(2): (Korean) 19. Bae EY, Kim DH. Effect of Geopungjeseupbang on contact induced by DNCB. Korean Journal of Oriental Physiology & Pathology. 2007;21(6): (Korean) 20. Kim SH, Kim HT. The effects og Gyejijakyakjimo- Tang on the Allergic Contact Dermatitis induced by DNCB. J of The Korean Oriental Ophthalmology & Otorhinolaryngology & Dermatology. 2010;23(1): (Korean) 21. Ahn JY, Lim IL, Kim JH, Shin MG, Kwon SW, Kim DK, et al. Inhibitory effect of Gamigunggui- Tang on Allergic Contact Dermatitis. Korean Journal of Oriental Physiology & Pathology. 2010;24(2): (Korean) 22. Byun SH, Lee BW, Kim SC. Effect of Samyotongan-

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