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1 J Korean Med Ophthalmol Otolaryngol Dermatol 2020;33(2):83-99 pissn eissn Original Article / 원저감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 류지연 1 감은영 1 강은정 1 최정화 2 김종한 2 박수연 2 정민영 2 동신대학교한의과대학안이비인후피부과학교실 ( 1 수련의, 2 교수 ) Effects of Gammakdaejo-tang(GMD) on DNCB induced Atopic Dermatitis in Mice Ji-Yeon Ryu Eun-Young Kam Eun-Jeong Kang Jung-Hwa Choi Jong-Han Kim Soo-Yeon Park Min-Yeong Jung Department of Ophthalmology, Otolaryngology & Dermatology, College of Korean medicine, Dongshin University Abstract Objectives : Atopic dermatitis is a chronic inflammatory skin disease with frequent relapses. This study was to investigate the effects of Gammakdaejo-tang(GMD) in DNCB induced atopic dermatitis mice. Methods : The study was divided into five comparion groups. 2,4-dinitrochlorobenzene(DNCB) solution was applied to Nc/Nga mice to induce atopic dermatitis, followed by normal group, negative control group with distilled water, positive control group with Dexamethasione and GMD 200 mg / kg or 400 mg / kg. The control group was orally administered 200μl once daily for 4 weeks. Visual skin condition, Immunoglobulin E, Histamine, Cytokine, Immune cells, Tissue biomarkers were observed. Results : As a result of the dermatitis score evaluation, it was confirmed that the GMD-administered group improved symptoms compared to the negative control group. As a result of measuring IgE, the GMD-administered group significantly decreased compared to the negative control group. As a result of measuring Histamine, GMD group except 200mg / kg of GMD significantly decreased compared to negative control group. As a result of measuring cytokine, GMD 200mg / kg significantly reduced IL-1β, IL-6 and TNF-α compared to the negative control. 400mg / kg significantly reduced IL-1β, IL-4, IL-5, IL-6, IL-10, TNF-α and significantly increased IL-2, IFNγ. As a result of confirming the immune cells, all experimental groups showed no difference in basophil, GMD group significantly reduced monocyte and eosinophil compared to negative control group, and GMD 400mg / kg group significantly reduced white blood cell and neutrophil. And significantly increased lymphocytes. c 2020 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 ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 83

2 As a result of measuring the gene expression level, all GMD group significantly increased TGF-β1 compared with the negative control group, and filaggrin, VEGF and EGF were significantly increased in GMD 400mg / kg group. Epidermis, dermis thickness, and eosinophil infiltration were found to be decreased in all GMD groups compared with the negative control group. Conclusions : GMD is effective in atopic dermatitis by reducing imbalance of immune response of T cells (Th1 / Th2) and reducing skin tissue damage and inflammatory response. Keywords : Atopic dermatitis(ad), 2,4-dinitrochlorobenzene(DNCB), Gaammkdaejo-tang(GMD), Nc/Nga mice Ⅰ. 서론 아토피피부염 (AD, Atopic dermatitis) 은한의학적으로 奶廯, 胎斂瘡, 濕瘡, 浸淫瘡 등의범주에속하며소양감, 부종, 홍반, 삼출, 인설등의양상을나타내는습진의일종으로재발이잦아만성으로경과하는만성염증성질환이다 1). 한연구에따르면, 아토피피부염환자의 17.1% 정도가정신과진단을받은경험이있다고보고되어지므로아토피피부염은단순피부증상외에도수면장애, 정신적신체적스트레스등에영향을주어개인만의문제가아닌사회적문제로도이어질수있다 2). 현재까지아토피피부염에대한양방치료로는항히스타민제와염증을감소시켜주는국소스테로이드및항생제연고를사용하고, 증상이심한경우에는전신스테로이드제를사용하며, 기타치료법으로는광선치료, Cyclosporine 등이있다 3). 그러나스테로이드제의장기적사용은모세혈관확장과피부위축등의피부부작용과더불어쿠싱증후군, 성장지연등의전신적부작용을야기하는등다양한문제점들이제기되고있어한의학적치료에관심이높아지고있는추세이다 4). 甘麥大棗湯 (Gammakdaejo-tang, 이하 GMD) 은 金匱要略 에 婦人臟躁, 喜悲傷欲哭, 象如神靈所作, 數欠伸, 甘麥大棗湯主之 로기록되어있으며, 임상에서는不眠不 Corresponding author : Min-Yeong Jung, Department of Ophthalmology, Otolaryngology & Dermatology, College of Korean medicine, Dongshin University, 313 Baengnyeondaero, Mokpo-si, Jeollanam-do, 58665, Republic of Korea. (Tel : , mining32@naver.com) Received 2020/3/31 Revised 2020/5/5 Accepted 2020/5/12 安, 胃潰瘍, 筋痙攣, 精神病, 子宮痙攣, 神經衰弱및히스테리등에응용된다 5). 甘麥大棗湯의구성약물중君藥인甘草는生用하면解毒의효능이있으므로癰疽瘡瘍, 消化性潰瘍, 咽喉腫痛을치료하고 6), 주요성분인 isoliquiritigenin, liquiritin, liquiritigenin, glycyrrhizin 은항균작용이있는것으로알려져있어감염성피부질환치료에활용할수있다 7). 大棗는補脾和胃, 調營衛하는효능이있으며, 甘草와배합하면補脾生血, 益氣生津하는효과가증대되어지고 6), 비타민이풍부하며항산화물질인 polyphenol 성분이다량으로분포하여진정작용및항알러지작용에활용되어진다. 8) 小麥은益氣除熱止汗하는효능이있어自汗盜汗, 骨蒸勞熱에활용되어지며 9), 小麥의성분중하나인 arabinoxylan 은면역세포를활성화시켜주는작용이있다고알려져있다 10). 아토피피부염의가장두드러진증상은소양감으로볼수있다. 그러나소양감은개인마다매우주관적이며정신적스트레스등심리상태와도연관성이높은증상으로치료함에있어객관적인평가가어려운실정이다 11). 이와관련하여甘麥大棗湯이신체부동스트레스백서에미치는영향에관한연구 12) 에서는甘麥大棗湯이혈청스트레스호르몬으로알려진 corticosterone 의분비를억제하는효과가있음을보고하였고, 항우울및만성스트레스에대한효능에관한연구 13) 에서는만성스트레스를유발시킨생쥐에서 serotonine 의분비증가와스트레스를지속적으로받을시증가하는혈중 glucose 의함량을감소시킨다고보고하였다. 이외甘麥大棗湯에대한연구로는국소뇌혈관과심혈관계에미치는영향에관한연구 14), 알코올성간질 84

3 류지연외 6 인 : 감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 환에대한치험 1례 5) 등이있으나아토피피부염에작용하는면역학적기전에관한연구는없었다. 이에저자는 GMD 가아토피피부염에효능이있는지알아보기위하여백서실험후육안적상태, IgE, Histamine, Cytokine, 면역세포, 조직내바이오마커등에대하여관찰및측정분석한결과유의성을얻었기에보고하는바이다. 1. 재료 1) 시료 Ⅱ. 재료및방법 실험에사용한甘麥大棗湯 ( 이하, GMD 으로표기 ) 의구성약재는대전한약국에서구입하여사용하였고甘草는충남금산, 小麥은전남구례, 大棗는경북경산에서채취한것을사용하였으며그내용및분량은다음과같다 (Table 1). Table 1. The Prescription of GMD Herbal medicine Scientific name Weight (g) name 甘草 Glycyrrhiza uralensis Fischer 40 小麥 Triticum aestivum Linne 48 大棗 Total amount 2) 실험동물및사육 Zizyphus jujuba Miller var. inermis Rehder 사용된 Nc/Nga mice ( 수컷, 5주령, 15-18g) 는 라온바이오 (Korea) 를통해공급받았으며실험당일까지고형사료와물을충분히공급하고습도 55±15%, 온도 22±2, 12시간씩 -12시간씩 (light-dark cycle) 의환경을조성하고 1주일간적응시켜실험에사용되었다. 위실험은대전대학교동물실험윤리위원회에서승인 ( 승인번호 DJUARB ) 을받았으며동물윤 리준칙에의거하여실험하였다. 3) 시약사용된시약은 DNCB (Sigma-Aldrich, USA), AccuPower CycleScript RT PreMix (Bioneer, Korea), dexamethasone (Sigma-Aldrich, USA), mouse magnetic luminex assay kit (R&D system, USA), total RNA prep kit (Intronbio, Korea), formalin (Merck, Germany), histamine ELISA kit (MyBioSource, U.S.A), mouse IgE single plex magnetic bead kit (Millipore, USA) 등을사용하였다. 4) 기기사용된기기는 rotary vacuum evaporator (EYELA Co., Japan), eep-freezer (Sanyo Co., Japan), reflux extractor (EYELA Co., Japan), freeze dryer (Ilshinbiobase Co., Korea), ICP-AES (Shimadzu, Co., Japan), HPLC (Shimadzu, Co., Japan), luminex (Millipore, U.S.A.), c d plate shaker (Lab-Line Co., U.S.A.), ice-maker (Vision scientific Co., Korea), nanodrop (Thermofisher, U.S.A.), autoclave (Sanyo Co., Japan), micro plate reader (Molecular Devices Co., U.S.A.), electrophoresis system (ADVANCE, Japan), lean bench (Vision scientific Co., Korea), alpha cycler 1 PCRmax (PCRmax, U.K.), centrifuge (Sigma Co., U.S.A.), 수은분석기 (TELEDYNE Leeman Labs, U.S.A.), vortex mixer (Vision scientific Co., Korea), real time PCR (Qiagen, Germany) 등을사용하였다. 2. 방법 1) 시료추출 GMD(102g) 에증류수 500ml을넣고환류추출을 3 시간동안하여얻은여과액을 rotary vacuum 85

4 evaporator 를통해감압농축하였다. 농축된용액을 freeze dryer 로동결건조후 GMD 분말 14.6g( 수율 : 14.3%) 을얻었으며초저온냉동고 (-80 ) 에보관하며실험에필요한농도에맞춰증류수에희석하여사용하였다. 2) HPLC 분석 0.45μm membrane filter에 GMD 30mg을 80% 에탄올 1ml에녹인후여과하여 10μl를 HPLC 시료로사용하였다. water(a) 와 acetonitrile(b) 로 gradient elution system 을적용하여 0분-5분 (0% B), 5분-30 분 (40% B), 30분-40분 (80% B), 40분-55분 (100% B) 으로설정하였다. column 온도는 40 를유지하였으며유속은 1.0ml /min이었고, UV wavelength 는 210 nm로설정한후분석하였다. 3) 중금속검사납, 카드뮴, 비소를분석하기위해질산 10ml을넣고극초단파시료전처리장치전용용기에 GMD 0.5g 을넣은후후드안에용기를정치시켜발생가스를제거한후극초단파시료전처리장치를이용하여분해하였다. 분해가끝난후분해액을여과하여용량플라스크에넣고물을첨가하여표준액의농도범위에맞게희석후검액으로사용하였다. 극초단파시료전처리장치용기에질산 10ml를넣고검액조제와같은방법을이용하여공시험액으로사용하였다. 준비된공시험액, 표준약, 검액을가지고유도결합플라즈마분광계 (ICP) 를사용하여검량선을작성하였으며공시험액으로보정후검색을측정하였다. 수은분석의경우 GMD 50mg을수은분석기를이용하여특별한전처리과정없이측정하였다. 4) 아토피피부염유발및시료처리 1주동안안정기를가진뒤 6주령이된 Nc/Nga mice 의등표면을꼬리상단부에서귀하단부까지제모하고 24h 방치하였다. 그후 1% DNCB (dinitrochlorobenzen) 용액 ( 올리브오일 아세톤 = 1:3) 150 μl를 2주간격일로제모한부위에도포하였다. 2주도포후, 0.5% DNCB 용액 150μl를 1주에 3회씩도포하면서, 음성대조군은증류수를투여하고양성대조군은 dexamethasone 1mg / ml, GMD 200mg / kg, GMD 400 mg / kg으로 4주동안매일 1회 200 μl씩경구투여하였다. 비교군은 normal(non DNCB caused Nc/Nga mice), control(dncb-caused mice group was treated orally with D.W), positive control (DNCB-caused mice group was treated orally with dexamethasone 1mg / kg ), GMD 200mg / kg (DNCB-caused mice group was treated orally with GMD 200mg / kg ) GMD 및 GMD 400mg / kg (DNCB-caused mice group was treated orally with GMD 400 mg / kg ) 로총다섯군으로나누었으며각그룹별로 6마리의 Nc/Nga mice 가사용되었다. 5) 혈청분리및등조직채취실험이끝난후심장천자법을통해 1cc의전혈을얻었으며이중 0.25 cc의전혈을통해혈구분석을진행하였다. 나머지전혈을통해혈청을분리하여 30분동안상온에두어굳힌후원심분리 (3,000rpm, 15min) 하여 μl의혈청을획득하였다. 획득한혈청중 Millipore 사의 IgE kit 와 R&D system 사의 cytokine kit, Mybiosource 사의 histamine ELISA kit는혈청을각각 50μl사용하였다. 바이오마커측정용량에맞추어 ep tube 에넣고초저온냉동고 (-80 ) 에보관하여사용하였다. 혈액채취후발현된등부분을미세가위로근막전까지자르고질소로즉각냉각시킨다음 ep tube 에넣고초저온냉동고 (-80 ) 에보관하며사용하였다. 6) 육안적평가아토피피부염을유발시킨후시료를경구투여하는 6주차에사진촬영을진행하였으며사진을토대로아토 86

5 류지연외 6 인 : 감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 피성피부염에서일반적으로사용되어지는임상적육안평가법으로관능평가를실시하였다. 평가항목으로는건조피부 (dry skin), 홍반 (erythema), 짓무름 (excoriation), 부종과혈종 (edema & hematoma), 태선화 (lichenification) 등 5가지로나누었으며, 각각의항목에대한평가는없음 (0), 약함 (1), 중등 (2), 심함 (3) 으로채점하였다. 7) 혈액내바이오마커측정 1 Immunoglobulin E (IgE) 실험이끝난후획득한혈청을이용하여 Immunoglobulin E (IgE) 을측정하였다. 96 well plate에분리한혈청과 standard를 50μl, bead 를 25μl씩각 well 에넣었다. 상온에서 1h 반응시킨후, washing buffer 를사용하여 3회에걸쳐세척작업을진행하였다. 세척이끝난후 25 μl의 PE와 anti-mouse light chain 를넣고다시상온에서 15min 반응시켰다. 3회세척후 sheath fluid 200 μl씩넣고 luminex 를통해측정하였다. 2 Histamine 실험이끝난후획득한혈청을통해 histamine 을측정하였다. 96 well plate 를통해분리한혈청과 standard 를 50μl, biotin-labeled antibody 를 50μl씩각 well에넣은후, 37 에서 45min 반응시켰다. Washing buffer 를사용하여 3회에걸쳐세척작업을진행하였다. 세척후 HRP-streptavidin conjugate 100μl를넣은후 37 에서 30min 동안반응시킨다. 3회세척후 TMB substrate 90μl씩넣고 37 에서 15min 반응시킨후 stop solution 50μl씩넣었다. 흡광도는 micro plate reader 기를통해 450 nm에서측정하였다. 3 Cytokine 실험이끝난후획득한혈청을통해 TNF-α, IL-1β, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10 을 측정하였다. 96 well plate 에분리한혈청과 standard 를 50μl, microparticle cocktail를 50 μl씩각 well 에넣은후상온에서 2h 반응시켰다. washing buffer 를사용하여 3회에걸쳐세척작업을진행하였다. 세척후 biotin-antibody cocktail 50μl를넣고다시상온에서 1h 반응시켰다. 세척을 3회실시한뒤 streptavidin-pe 를 50μl씩넣고상온에서 30min 반응시킨후 washing buffer 를사용하여 3회세척작업을진행하였다. 세척한뒤 sheath fluid 150 μl를넣은후상온에서 2min 반응시키고 luminex 를통해측정하였다. 8) 면역세포분석실험이끝난후획득한혈액을통해면역세포 white blood cell, lymphocyte, monocyte, neutrophil, eosinophil, basophil 측정을대전대학교한방 Bio Fab Lab(Korea) 에의뢰하였다. 9) 조직내바이오마커측정실험이끝난후획득한등조직을통해 VEGF, filaggrin, TGF-β1 mrna 발현량을확인하였다. 조직 100 mg에 cell lysis buffer 1ml씩넣고미세가위로조직을자른후, chloroform 200 μl씩넣어다시혼합하였다. 이를원심분리 (13,000, 10분 ) 시킨후 400 μl의상층액을회수한다음 binding buffer 400 μl씩넣고나서혼합한후 column에주입하여원심분리하였다. column 에 washing buffer A 700 μl씩넣은후원심분리하였고 washing buffer B 700 μl씩넣고원심분리후 elution buffer 50μl을넣어원심분리하여 RNA 를추출하였다. 역전사 (reverse transcription) 반응은 RT premix (reverse transcriptase, dt20 primer, reaction buffer, stabilizer dntps) 에 total RNA 를 1μg첨가하여최종부피가 20μl가되도록 diethyl pyrocarbonate (DEPC) 처리된증류수를첨가하였다. 혼합액을섞어넣은후원심침강 (2,000, 5sec) 하 87

6 여 45 heating block 에서 60min 동안반응시킨후 first-strand cdna 를합성하였다. 이를다시 95 에서 5min 동안반응시키고 M-MLV RT를불활성화시킨다. 그후합성이완료된 cdna 를 PCR 에사용하였다. 합성이완료된 cdna 를증폭시키기위해 real-time PCR을진행하였다. cdna 1μl, 각 primer 2μl, DEPC-DW 5μl, SYBR Green 10μl를 real-time 전용 tube 에넣은후다음과같이진행하였다. 95 2min 반응시킨후 95 5초, 초를 1 cycle 로정하고총 40 cycle 을시행하였다. 그후유전자발현량은대조군과비교하여계산을하였고, 사용된 primer 의 sequence 와 size는다음과같다 (Table 2). 10) 조직학적검사실험종료후등피부를채취하여 10% 포르말린에조직을고정시킨다. 이후조직을실험검사기관인 KPNT (Korea) 에조직염색 (M&T 염색, H&E) 을의뢰하였고, 염색된조직은광학현미경을통해호산구침윤변화와피부두께를비교분석하였다. 3. 통계처리실험결과는 ANOVA 와 SPSS 18.0 의 unpaired student's T-test 를이용하여통계처리하였다. p < 0.001, p < 0.01 and p < 0.05 수준에서유의성을검정하였다. 1. HPLC 분석 Ⅲ. 실험결과 GMD의 HPLC 분석을한결과, 2min, 4min, 23min 대에피크가확인되었다 (Fig. 1). Fig. 1. HPLC analysis of GMD *GMD were analysis with HPLC chromatogram. HPLC : high performance liquid chromatography 2. 중금속함량 중금속함량을측정한결과, GMD 는비소가기준치 (3mg/ kg ) 이하로검출되었고납, 카드뮴, 수은은검출되지않았다 (Table 3). Table 3. Content of Pb, As, Cd and Hg in GMD Pb As Cd Hg permissible concentration GMD N.D 0.1 N.D N.D *Unit : mg / kg Table 2. The Sequences of Primers for Real-Time PCR Primer F/R* Sequences F TACTGGTGCTAAAGDTGGCA Filaggrin R CCTTTATAATCAGTTTTACT F AGGAGACGGAATACAGGGCT VEGF R GGTCCTCCCCGACGTTTGGCTG F CGAGCCGCGAGGAACCGAAG TGF-β1 R GAGCCCAGAAGTTCGGACGAA F TGCTGCATACGAGCATCCAT GAPDH R TGTCCTCAAAGTGAACCGCA * F : forward, R : reverse Size (bp)

7 류지연외 6 인 : 감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 3. 육안적평가 DNCB 처리로인한정상군을제외한대조군, 실험군그룹에서짓무름과심한상처에의한피부염발현현상을확인하였고, 6주차에육안으로각그룹의피부상태를관찰한결과, 대조군은시간이경과할수록각질의탈락과각화의현상이심한반면, GMD 투여군은아토피피부염증상이현저하게감소되는것을육안으로관찰할수있었다 (Fig. 2). Fig. 3. The effects of GMD on IgE level in the DNCB-caused AD mice. *The level of IgE was examined by luminex multiplex assays. Each data represent the mean ± SD. (***p < 0.001, *p < 0.05 compared with the control group.) 2) Histamine Histamine level 을측정한결과, 정상군은 12.0± 3.7ng/ ml, 양성대조군은 32.8±3.1ng/ ml, 음성대조군은 62.6±3.1ng/ ml로나타난반면, GMD 200, 400mg / kg투여군은 58.9±3.3ng/ ml, 47.6±2.2ng/ ml GMD 200 mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 4). Fig. 2. Dermatitis score in the DNCB-caused AD mice. *Clinical skin severity scores were monitored once in the last week. Values express the mean ± SD of the total score from 6 mice for each group. (***p < compared with the normal group and ***p < compared with the control group). 4. 혈액내바이오마커측정 1) Immunoglobulin E (IgE) IgE level 을측정한결과, 정상군은 245.6±50.4ng/ ml, 양성대조군은 ±400.9ng/ ml, 음성대조군은 ±282.0ng/ ml로나타난반면, GMD 200, 400 mg / kg투여군은 ±298.3 ng/ ml, ± 419.2ng/ ml로나타나모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, * p < 0.05 ) 감소가나타났다 (Fig. 3). Fig. 4. The effects of GMD on histamine level in the DNCB-caused AD mice. *The level of histamine was examined by ELSIA. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 3) Cytokine 1 IL-1β : IL-1β level 을측정한결과, 정상군은 1.4±1.3pg/ ml, 양성대조군은 17.1±2.3pg/ ml음성대조군은 40.3±3.7pg/ ml로나타난반면, GMD 200, 400mg / kg투여군은 33.1±3.0pg/ ml, 24.8±2.9pg/ ml로나타나모든투여군은음성대 89

8 조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 5). GMD 200, 400mg / kg투여군은 65.4±8.0pg/ ml, 59.6±6.3pg/ ml로나타나 GMD 200mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 7). Fig. 5. The effects of GMD on IL-1β level in the DNCB-caused AD mice. *The level of IL-1β was examined by luminex multiplex assays. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 2 IL-2 : IL-2 level 을측정한결과, 정상군은 45.7±3.2pg/ ml, 양성대조군은 40.4±2.6pg/ ml, 음성대조군은 19.0±2.4pg/ ml로나타난반면, GMD 200, 400mg / kg투여군은 22.9±4.1pg/ ml, 29.6±2.8pg/ ml로나타나 GMD 200mg / kg투여군을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 증가가나타났다 (Fig. 6). Fig. 7. The effects of GMD on IL-4 level in the DNCB-caused AD mice. *The level of IL-4 was examined by luminex multiplex assays. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 4IL-5 : IL-5 level을측정한결과, 정상군은 1.3±1.9pg/ ml, 양성대조군은 18.9±3.5pg/ ml, 음성대조군은 33.4±4.4pg/ ml로나타난반면, GMD 200, 400mg / kg투여군은 28.4±1.7pg/ ml, 25.4±2.5pg/ ml로나타나 GMD 200mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 8). Fig. 6. The effects of GMD on IL-2 level in the DNCB-caused AD mice. *The level of IL-2 was examined by luminex multiplex assays. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 3 IL-4 : IL-4 level 을측정한결과, 정상군은 5.1±2.2pg/ ml, 양성대조군은 34.4±5.9pg/ ml, 음성대조군은 72.7±7.1pg/ ml로나타난반면, Fig. 8. The effects of GMD on IL-5 level in the DNCB-caused AD mice. *The level of IL-5 was examined by luminex multiplex assays. Each data express the mean ± SD. (**p < 0.01, *p < 0.05 compared with the control group). 5IL-6 : IL-6 level 을측정한결과, 정상군은 90

9 류지연외 6 인 : 감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 17.1±11.6pg/ ml, 양성대조군은 135.8± 16.4pg/ ml, 음성대조군은 270.9±12.4pg/ ml로나타난반면, GMD 200, 400 mg / kg투여군은 226.3±28.0pg/ ml, 176.4±19.8pg/ ml로나타나모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 9). 7 TNF-α : TNF-α level 을측정한결과, 정상군은 94.9±68.5pg/ ml, 양성대조군은 570.2± 79.3pg/ ml, 음성대조군은 982.4±54.1pg/ ml로나타난반면, GMD 200, 400 mg / kg투여군은 848.7±61.3pg/ ml, 712.7±79.3pg/ ml로나타나모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 11). Fig. 9. The effects of GMD on IL-6 level in the DNCB-caused AD mice. *The level of IL-6 was examined by luminex multiplex assays. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 6 IL-10 : IL-10 level 을측정한결과, 정상군은 1.5±2.0pg/ ml, 양성대조군은 25.7±3.1pg/ ml, 음성대조군은 59.7±4.6pg/ ml로나타난반면, GMD 200, 400mg / kg투여군은 56.1±5.4pg/ ml, 46.5±4.6pg/ ml로나타나 GMD 200mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 10). Fig. 11. The effects of GMD on TNF-α level in the DNCB-caused AD mice. *The level of TNF-α was examined by luminex multiplex assays. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 8 IFN-γ : IFN-γ level을측정한결과, 정상군은 70.4±5.0pg/ ml, 양성대조군은 62.8±7.0pg/ ml, 음성대조군은 41.2±6.4pg/ ml로나타난반면, GMD 200, 400mg / kg투여군은 44.5±5.2pg/ ml, 58.8±6.4pg/ ml로나타나 GMD 200mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( ** p < 0.01) 증가가나타났다 (Fig. 12). Fig. 10. The effects of GMD on IL-10 level in the DNCB-caused AD mice. *The level of IL-10 was examined by luminex multiplex assays. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). Fig. 12. The effects of GMD on IFN-γ level in the DNCB-caused AD mice. *The level of IFN-γ was examined by luminex multiplex assays. Each data express the mean ± SD. (**p < 0.01 compared with the control group). 91

10 5. 면역세포분석 1) White blood cell White blood cell level 을측정한결과, 정상군은 1.1± cells/ μl, 양성대조군은 1.5± cells/ μl, 음성대조군은 3.3± cells/ μl로나타난반면, GMD 200, 400mg / kg투여군은 3.2± cells/ μl, 2.3± cells/ μl로나타나 GMD 200 mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01) 감소가나타났다 (Fig. 13). 3) Neutrophil Neutrophil level을측정한결과, 정상군은 5.7±0.6%, 양성대조군은 7.5±0.2% 로나타난반면, 음성대조군은 10.9±0.4% 로나타난반면, GMD 200, 400mg / kg투여군은 10.6±0.4%, 8.3±0.4% 로나타나 GMD 200 mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01 ) 감소가나타났다 (Fig. 15). Fig. 13. The effects of GMD on white blood cell level in the DNCB-caused AD mice. *The level of white blood cell data express the mean ± SD. (***p < 0.001, **p < 0.01 compared with the control group). 2) Monocyte Monocyte level 을측정한결과, 정상군은 7.4±1.1%, 양성대조군은 17.4±1.0%, 음성대조군은 25.0±1.9% 로나타난반면, GMD 200, 400mg / kg투여군은 21.5±2.2%, 19.3±1.0% 로나타나모든투여군은음성대조군에비해유의성있는 ( ** p < 0.01, * p < 0.05 ) 감소가나타났다 (Fig. 14). Fig. 15. The effects of GMD on neutrophil level in the DNCB-caused AD mice. *The level of neutrophil data express the mean ± SD. (***p < 0.001, **p < 0.01 compared with the control group). 4) Eosinophil Eosinophil level 을측정한결과, 정상군은 5.4±0.3%, 양성대조군은 6.7±0.4%, 음성대조군은 10.8±0.4% 로나타난반면, GMD 200, 400mg / kg투여군은 9.8±0.2%, 9.6±0.4% 로나타나모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 감소가나타났다 (Fig. 16). Fig. 14. The effects of GMD on monocyte level in the DNCB-caused AD mice. *The level of monocyte data express the mean ± SD. (**p < 0.01, *p < 0.05 compared with the control group). Fig. 16. The effects of GMD on eosinophil level in the DNCB-caused AD mice. *The level of eosinophil data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 92

11 류지연외 6 인 : 감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 5) Basophill Basophil level 을 측정한 결과, 정상군은 10.6±0.1%, 양성대조군은 9.9±0.2%, 음성대조군은 10.7±0.1% 로나타난반면, GMD 200, 400mg / kg투 여군은 10.6±0.2%, 11.5±0.3% 로나타나모든실험 군차이를나타내지않았다 (Fig. 17). 6. 조직바이오마커측정 1) Filaggrin Filaggrin mrna 발현량을측정한결과, 정상군은 2.2±0.1, 양성대조군은 1.7±0.1, 음성대조군은 1.0±0.1로나타난반면, GMD 200, 400mg / kg투여군은 1.2±0.2, 1.3±0.1, 로나타나 GMD 200mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 증가가나타났다 (Fig. 19). Fig. 17. The effects of GMD on basophil level in the DNCB-caused AD mice. *The level of basophil data express the mean ± SD. 6) Lymphocyte Lymphocyte level 을측정한결과, 정상군은 70.6±2.4%, 양성대조군은 57.8±6.2%, 음성대조군은 41.7±5.1% 로나타난반면, GMD 200, 400mg / kg투여군은 46.8±6.5%, 51.3±1.6% 로나타나 GMD 200 mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( ** p < 0.01, * p < 0.05 ) 증가가나타났다 (Fig. 18). Fig. 19. The effects of GMD on filaggrin mrna level in the DNCB-caused AD mice. *The mrna level of filaggrin was examined by a real-time PCR. Each data express the mean ± SD. (***p < 0.001, **p < 0.01, *p < 0.05 compared with the control group). 2) VEGF VEGF mrna 발현량을측정한결과, 정상군은 1.7±0.1, 양성대조군은 1.5±0.1, 음성대조군은 1.0± 0.1로나타난반면, GMD 200, 400 mg / kg투여군은 1.1±0.1, 1.4±0.1로나타나 GMD 200 mg / kg을제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01, * p < 0.05) 증가가나타났다 (Fig. 20). Fig. 18. The effects of GMD on lymphocyte level in the DNCB-caused AD mice. *The level of lymphocyte data express the mean ± SD. (**p < 0.01, *p < 0.05 compared with the control group). Fig. 20. The effects of GMD and DCL on VEGF mrna level in the DNCB-caused AD mice. *The mrna level of VEGF was examined by a real-time PCR. Each data express the mean ± SD. ***p < 0.001, (**p < 0.01, *p < 0.05 compared with the control group). 93

12 3) TGF-β1 TGF-β1 mrna 발현량을측정한결과, 정상군은 2.2±0.1, 양성대조군은 1.6±0.1, 음성대조군은 1.0±0.2 로나타난반면, GMD 200, 400mg / kg투여군은 1.3±0.1, 1.5±0.1로나타나모든투여군은음성대조군에비해유의성있는 ( ** p < 0.01, * p < 0.05) 증가가나타났다 (Fig. 21). 7. 조직학적검사 1) 피부두께피부두께변화를측정한결과, 음성대조군은정상군에비해 epidermis (E; black arrow) 와 dermis (D; red arrow) 두께가현저하게증가하였고, 모든투여군은음성대조군에비해 epidermis와 dermis의두께가감소하였다 (Fig. 23). Fig. 21. The effects of GMD on TGF-β1 mrna level in the DNCB-caused AD mice. *The mrna level of TGF-β1 was examined by a real-time PCR. Each data express the mean ± SD. (**p < 0.01, *p < 0.05 compared with the control group). 4) EGF EGF mrna 발현량을측정한결과, 정상군은 2.3±0.0, 양성대조군은 1.7±0.1, 음성대조군은 1.0±0.1 로나타난반면, GMD 200, 400mg / kg투여군은 1.1±0.3, 1.4±0.2 로나타나 GMD 200mg / kg제외한모든투여군은음성대조군에비해유의성있는 ( *** p < 0.001, ** p < 0.01) 증가가나타났다 (Fig. 22). Fig. 23. The effects of GMD on skin thickness in the DNCB-caused AD mice. *The skin thicknesse were examined by M&T staining of skin sections (magnification : 50, scale bar : 200 μm ). 2) 호산구침윤 호산구의침윤변화를측정한결과, 음성대조군은정상군에비해호산구의침윤이현저하게증가하였고, 모든투여군은음성대조군에비해침윤이감소하였다 (Fig. 24). Fig. 22. The effects of GMD on EGF mrna level in the DNCB-caused AD mice. *The mrna level of EGF was examined by a real-time PCR. Each data express the mean ± SD. ***p < 0.001, (**p < 0.01 compared with the control group). Fig. 24. The effects of GMD on osinophil infiltration in the NCB-caused AD mice. *The eosinophil infiltration was examined by H&E staining of skin sections (magnification : 200, scale bar : 50 μm ). 94

13 류지연외 6 인 : 감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 Ⅳ. 고찰 아토피피부염 (Atopic dermatitis, AD) 은피부발적, 소양감, 태선화등의병변을특징으로하는만성염증성피부질환으로겉으로드러나는피부증상이외에도수면장애, 감정적, 신체적스트레스, 사회생활등에제한을주고삶의질을떨어뜨리므로개인및가족의문제만이아니라사회공동체의문제이기도하다. AD의발병기전에관해서는확실히알려진바가없으나, 환경인자, 유전적소인, 면역학적변화등복잡하고다양한인자들로인해발생되어지는것으로보고있다 15,16). AD의양방학적치료로는국소치료, 전신치료, 항원특이면역치료, 기타치료등으로이루어진다. 국소치료로는국소스테로이드, 습포밀폐요법, 국소칼린뉴린억제제, 국소, 선택적 PDE-4 억제제치료, 전신치료로는항히스타민제, 항균제, 항염증치료제, 기타치료로는광선치료, 항원특이면역치료등이사용되어지고있으며최근에는염증세포나매개체에특이적으로표적작용하는생물학적제제치료에대한연구들이지속적으로이루어지고있다 16). 그러나 AD의치료제로사용어지는항히스타민은아토피피부염환자에게서가려움증을억제하기에충분하지못하며, 스테로이드의장기적적용은피부의위축이나소아환자에있어성장지연가능성등각종부작용을유발할수있으므로안전하고우수한효과를가진새로운치료법에대한연구가지속되고 있다 17,18). 한의학에서아토피피부염은 ' 浸淫 ', ' 浸淫瘡 ', ' 奶癬 ', ' 胎斂瘡 ', ' 乳癬 ' 등으로불리며 19), 諸病源候論, 小兒雜病諸侯. 癬候 에 小兒面上, 癬皮如甲錯起乾燥, 謂之乳癬 이라정의하여최초로기록되어있고, 外科正宗 에서는 奶癬因兒在胎中母食五辛父餐炙煿遺熱與兒. 라하여아토피피부염의원인에대하여기록하고있다. 또한, 醫宗金鑑 에는胎斂瘡이라하여건조하고가려워白屑이생기는乾斂, 소양감이극심하여潢水가전신으로퍼져나가는濕斂으로나누어변증함으로써현대의아토피피부염과가장유사하게설명하고있다 19,20). 甘麥大棗湯은 金匱要略 에 婦人臟躁, 喜悲傷欲哭, 象如神靈所作, 數欠伸, 甘麥大棗湯主之 로기록되어있으며, 養心安神하고和中緩急시키기위해사용되어왔다. 임상에서는不眠不安, 胃潰瘍, 筋痙攣, 精神病, 子宮痙攣, 神經衰弱및히스테리등에응용된다 5). 甘麥大棗湯의구성약물인甘草는和中緩急, 解毒, 潤肺하는효능이있어生用하면解毒의효능이있으므로소화성궤양, 癰疽瘡瘍, 咽喉腫痛을치료하고, 灸用하면성질이溫하게변하므로脾胃虛弱, 食少, 腹痛便溏, 心悸등에활용된다 6). 또한, 감초의주요성분인 isoliquiritigenin, liquiritin, liquiritigenin, glycyrrhizin 은항균작용이있는것으로알려져있다 7). 아토피피부염환자 90% 정도에서감염균 Staphylococcus aureus 가발견되는것으로보아항균작용이있는甘草를감염성피부질환치료에활용할수있다 21). 大棗는補脾和胃, 調營衛하는효능이있으며, 甘草와배합하면補脾生血, 益氣生津하는효과가증대되어脾胃虛弱과營血不足으로인한心悸怔忡, 婦人臟燥症등에활용되어지고 6), 비타민이풍부하며항산화물질인 polyphenol 성분이다량으로분포하여항암작용, 진정작용및항알러지작용에활용된다 8). 小麥은益氣除熱止汗하는효능이있어自汗盜汗, 骨蒸勞熱에활용되어지고養心安身작용으로부인의히스테리나虛煩證에이용되어지며 9), 소맥의성분중하나인 arabinoxylan 은면역세포를활성화시켜주는작용이있다고알려져있다 10). 甘麥大棗湯에대한연구로는알코올성간질환에대한치험 1례 5), 항우울및만성스트레스에대한효능에관한연구 13), 국소뇌혈관과심혈관계에미치는영향에관한연구 14) 등이있으나피부질환과관련된연구는없었다. 이에저자는 GMD 가아토피피부염에효능이있는지알아보기위하여 DNCB 용액을 Nc/Nga mice 에도포하여접촉성피부염을일으켜아토피피부염과같은모델로유발시킨후 normal군, 증류수투여한 Negative control 군, Dexamethasione 투여한 Positive control 군, 甘麥大棗湯저농도 (200 mg / kg ) 및고농도 95

14 (400 mg / kg ) 투여한군총 5개의비교군으로나누어연구를진행하였다. 비교군에는각각의시료들을 4주동안매일 1회 200 μl씩경구투여한후, 육안적인피부상태, Immunoglobulin E, Histamine, Cytokine(IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, IFN-γ), 면역세포, 조직내바이오마커등에대해관찰하였다. HPLC 를통해 GMD의유효성분을분석결과, 2분, 4 분, 23분대에피크가확인되었다 (Fig. 1). GMD 의중금속함량을측정한결과, 비소가기준치이하로검출되었고납, 카드뮴, 수은은검출되지않았다 (Table 3). DNCB 는혈청내 IgE, 호염구, 호산구 Th2 를증가시켜아토피피부염과유사한상태를나타내므로본연구에서아토피피부염을발현시키기위한목적으로사용하였으며 22), DNCB 처리에의하여정상군을제외한대조군, 실험군그룹에서짓무름과심한상처에의한피부염유발현상을확인하였다. 6주차에육안으로각그룹의피부상태를관찰한결과 GMD 투여군은아토피피부염증상이현저하게감소되는것을관찰할수있었다 (Fig. 2). 아토피피부염은여러면역관련세포중 T세포의활성화와큰관련성을가지고있다 23). T세포는각각의 Cytokine 에의해 Th1과 Th2로분류되어지며아토피피부염은 Th2 반응과연관성을가지게된다. Th2 세포는 IL-4, IL-5 등에의해 B세포에서 IgE를생산시키고호산구를활성화시켜혈액내에 IgE와 Histamine 등의독성물질들의함량을높이고면역세포를피부로침윤시켜아토피피부염을더악화시킨다. 반면, Th1 세포에의해생성되어진 IL-12 와 IFN-γ 등은 Th2 로인하여생성되는 IL-4, IL-5 등을억제하는역할을한다 24-27). 본연구에서 GMD 경구투여가 IgE와 Histamine 에영향을주는것을알아보기위해실험한결과 IgE level 은음성대조군에비해모든투여군에서유의한감소가나타났으며 Hitamine level 은고농도 (400mg / kg ) 투여군에서유의한감소가나타났다 (Fig. 3, 4). GMD 경구투여가각종 Cytokine 들에영향을주는것을알아보기위해실험한결과, 모든투여군에서 IL-1β, IL-6 및 TNF-α 는모두감소하였고 (Fig. 5, 9, 11), GMD 400 mg / kg투여군에서 IL-4, IL-5, IL-10 는감소하였으며 (Fig. 7, 8, 10) IL-2, IFN-γ 는증가하였다 (Fig. 6, 12). 위결과를토대로 GMD는 Th1 Cytokine 은활성화시키고 Th2 Cytokine 은억제하여아토피피부염의면역학적불균형을완화시켜준다는것을확인할수있다. GMD 경구투여가혈액내면역세포에영향을주는것을알아보기위해실험한결과, 모든투여군에서 Basophil은차이가없었고 (Fig. 17), Monocyte 와 Eosinophil 는모든투여군에서유의하게감소하였으며 (Fig, 14, 16) GMD 400mg / kg투여군에서 White blood cell, Neutrophil 는유의한감소를 (Fig. 13, 15) Lymphocyte 는유의한증가를보였다 (Fig. 18). Basophil에는유의미한차이는없었으나 Monocyte, Eosinophil, White blood cell, Neutrophil 는감소하고 Lymphocyte 는유의한증가를보이는것을토대로 GMD 가혈액내면역세포에작용하여염증반응을경감시켜주는것을확인할수있다. 상처의재생및치유를위해서는혈관손상의회복이가장중요한데이과정은 vascular endothelial call growth factor(vegf), transforming growth factor beta 1(TGF-β1), epidermal growth factor(egf) 등의 Cytokine 에의해이루어진다 28). 또한, filaggrin 은피부장벽을형성하고보습하는기능을가진단백질로, filaggrin 유전자기능상실변이가일어나면손상된피부장벽을통해항원이쉽게투입되어 Th2 면역반응, 선천면역활성화등각종면역반응을일으켜아토피피부염을유발한다 29). GMD가피부재생에효과가있는지알아보기위해실험한결과, filaggrin 은 GMD 400 mg / kg투여군에서유의하게증가하여피부장벽형성에도움을주는것을확인할수있고 (Fig. 19) VEGF 와 EGF 또한 GMD 400 mg / kg투여군에서유의하게증가하였으며 (Fig. 20, 22) TGF-β1 은모든투여군에서유의한증가 (Fig. 21) 를보여 GMD 가손상받은피부조직의재생및치료에도움 96

15 류지연외 6 인 : 감맥대조탕이 DNCB 로유발된생쥐의아토피피부염에미치는영향 을주는것으로나타났다. 실험이끝난후, 쥐의등피부조직을채취하여피부두께와호산구침윤의변화를측정한결과, 모든투여군은음성대조군에비해 epidermis 와 dermis 의두께가감소 (Fig. 23) 하였으며호산구침윤또한감소 (Fig. 24) 한것을확인할수있었다. 이상의결과들을종합하여볼때, GMD는아토피피부염에있어피부재생및면역, 항염증과관련하여효과가있을것이라판단되어진다. GMD 는항염증작용뿐만아니라피부장벽형성에도도움을주는것으로보아외용제로서의효과또한충분히고려되므로아토피피부염치료제로서 GMD 가다양한제제로활용가능한연구들이추후필요하리라사료된다. 또한, GMD는항스트레스및불면등정신과적질환에다용되는처방으로피부증상이외에도수면장애, 감정적, 신체적스트레스, 사회생활등에제한을주는아토피피부염환자들의삶의질을개선시키는데도움이되리라사료되며본연구결과를바탕으로다양한임상실험및효능보강이이루어진다면아토피피부염치료를동반한피부조직의회복및재생에대한한의학적처방으로써활용이가능할것으로사료된다. Ⅴ. 결론 甘麥大棗湯 (GMD) 추출물의아토피피부염에대한효과를검증하기위해 DNCB 를통해아토피를유발시킨 Nc/Nga mice 로실험한결과다음과같은결론을얻었다. 1. 육안적평가결과, GMD 투여군은음성대조군에비해증상이유의성있게호전되었다. 2. IgE을측정한결과, GMD 투여군은음성대조군에비해유의성있게감소시켰다. 3. Histamine 을측정한결과, GMD 200mg / kg을제외한 GMD 투여군은음성대조군에비해유의성있게감소시켰다. 4. Cytokine 을측정한결과, GMD 200mg / kg은 TNFα, IL-1β, IL-6 를음성대조군에비해유의성있게감소시켰다. GMD 400mg / kg은 IL-1β, IL-4, IL-5, IL-6, IL-10, TNF-α 를유의성있게감소시켰고 IL-2, IFN-γ 를유의성있게증가시켰다. 5. 면역세포를확인한결과, 모든실험군은 basophil 의차이를나타내지않았으며, 모든 GMD 투여군은 monocyte와 eosinophil을음성대조군에비해유의성있게감소시켰으며, GMD 400 mg / kg투여군만 white blood cell, neutrophil 을유의성있게감소시키고 lymphocyte 를유의성있게증가시켰다. 6. 유전자발현량을측정한결과, 모든 GMD 투여군은 TGF-β1 을음성대조군에비해유의성있게증가시켰으며, GMD 400 mg / kg투여군에서 filaggrin 과 VEGF, EGF 가유의성있게증가시켰다. 7. 호산구침윤변화와피부조직내의표피와진피의두께를확인한결과, 모든 GMD 투여군에서음성대조군에비해감소하였다. ORCID Ji-Yeon Ryu ( Eun-Young Kam ( Eun-Jeong Kang ( Jung-Hwa Choi ( Jong-Han Kim ( Soo-Yeon Park ( Min-Yeong Jung ( 97

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