<C6EDC1FD2D33322D32C8A320BEC8C0CCBAF1C0CEC8C4B0FAC7D0C8B8C1F62E687770>

Size: px

Start display at page:

Download "<C6EDC1FD2D33322D32C8A320BEC8C0CCBAF1C0CEC8C4B0FAC7D0C8B8C1F62E687770>"

길수 좌
3 years ago
Views:

1 J Korean Med Ophthalmol Otolaryngol Dermatol 2019;32(2): pissn eissn Original Article / 원저 DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 손미주 1 이소민 1 박성환 1 김영은 2 정지연 1* 1 한국한의학연구원임상의학부 ( 선임연구원 ) 2 한국한의학연구원미래의학부 ( 선임연구원 ) The Effects of Orally Administrated Gwakhyangjeonggi-san on DNCB-induced Atopic Dermatitis like Mice Model Mi-Ju Son 1 So-Min Lee 1 Seong-Hwan Park 1 Young-Eun Kim 2 Jee-Youn Jung 1* 1 Clinical Medicine Division, Korea Institute of Oriental Medicine 2 Future Medicine Division, Korea Institute of Oriental Medicine Abstract Objectives : This study was conducted to investigate the effects of Gwakhyangjeonggi-san(GJS) on atopic dermatitis(ad) induced by 2,4-dinitrochlorobenzene(DNCB) in mice. Methods : The mice(balb/c mice) were divided into three groups; normal Balb/c mice with oil treatment(sham group), DNCB-induced AD mice(ad group), and GJS treated AD mice(gjs group). GJS group were orally administered GJS daily for 2 weeks. We observed changes of clinical skin severity score, the expression of thymic stromal lymphopoietin(tslp), interleukin(il)-4 and tumor necrosis factor(tnf)-α in skin and mast cell infiltration. Also, serum immunoglobuline(ige), IL-4, TNF-α and IL-6 were evaluated. Results : The clinical skin severity score of GJS group was decreased compared to AD group. In hematoxylin and eosin staining results, GJS group showed a significant reduction of epithelial skin thickness. In addition, expression of TSLP and mast cell infiltration in skin were also reduced by GJS treatment compared to those of AD group. Thus, we evaluated expression of IL-4, Th2-dependent cytokine, and TNF-α, pro-inflammatory cytokine in skin. GJS significantly reduced both IL-4 and TNF-α compared to AD mice. Moreover, levels of IgE, IL-4, TNF-α and IL-6 in plasma also significantly decreased by oral GJS treatment. Conclusion : The present study suggests that GJS can significantly reduced symptoms of AD, therefore it can be a promising candidate for anti-atopic dermatitis treatment. Key words : Atopic Dermatitis; Gwakhyangjeonggi-san; IL-4; in vivo; TNF-α c 2019 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. 94

2 손미주외 4 인 : DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 Ⅰ. 서론 아토피피부염은영유아기의흔한피부질환중하나로소양감과피부의홍반, 부종, 삼출, 태선화등의증상이특징적인만성재발성피부염이다 1). 2017년국민건강영양조사에따르면청소년의 25.1%, 성인의 4.1% 가의사로부터아토피피부염으로진단받은경험이있었으며 2), 성인의경우유병률이꾸준하게증가하는추세이다 3). 아토피피부염의원인은유전적소인과후천적소인으로나눌수있다. 특히성인의아토피피부염의증가추세는아토피피부염의발생과악화에있어서유전적소인뿐만아니라산업화, 주거행태및식습관의서구화등으로인한생활환경적요인과같은후천적소인이중요하게작용하고있음을시사한다 4). 최근위장질환과아토피질환과의관련성이보고되고있는데 5), 위장은음식의소화흡수로인체내영양분의원활한공급하는기능을할뿐만아니라면역시스템에서의중요한작용을하고있다 6). 한의학에서는胎中遺熱혹은先天稟賦不耐등으로표현되는유전적인소인과출생이후의부적절한생활환경습관관리또는飮食失節을아토피피부염의병인으로보고있다 7). 藿香正氣散은기능성소화불량, 설사등위장관기능저하증상뿐만아니라 8) 알레르기질환과밀접한과민성대장증후군환자에서도프로바이오틱스와의병용투여로효과적인것으로보고되었다 9). 또한이등 10) 은소음인아토피피부염환자에서藿香正氣散이효과가있었다는증례를보고한바있으며, 양등 11) 은藿香正氣散을포함한 3종의한약이아토피피부염의 SCORAD 수치를개선시키며, 혈액내 Corresponding author : Jee-Youn Jung, Clinical Medicine Division, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon, 34054, Republic of Korea. (Tel : , jjy0918@kiom.re.kr) Received 2019/4/19 Revised 2019/4/25 Accepted 2019/5/2 cytokine 의변화를일으킨다고보고한바있다. 이전연구로미루어볼때, 藿香正氣散은아토피피부염증상개선을시킬수있을것으로추측되나, 기존에연구된藿香正氣散은 東醫寶鑑 또는 東醫壽世保元 에기반한처방으로현재시판되는한약제제藿香正氣散과는그구성및용량에차이가있다. 본연구에서는藿香正氣散의아토피피부염치료효능을확인하고자, DNCB 로유발된아토피피부염동물모델에서육안피부상태, 표피와진피에서의 TSLP (thymic stromal lymphopoietin), mast cell 침윤을확인하고, 피부조직의 interleukin(il)-4, tumor necrosis factor(tnf)-α 및혈중 IL-4, IL-6, TNFα 수치변화를관찰하여藿香正氣散경구투여가아토피피부염에미치는영향을규명하고자하였다. II. 재료및방법 1. 재료 1) 약물 본실험에사용한藿香正氣散 (Gwakhyangjeonggisan, 이하 GJS) 은한풍제약에서제조한엑스과립을구입하여사용하였다 (Table 1). GJS는성인 1일 3 회, 3g 복용을권고하고있으며, 임상농도를마우스투여농도로환산하여사용하였다. 2) 동물雄性 6주령의수컷 Balb/c 마우스 (20.88±0.14g) 를 새론바이오에서공급받아실험당일까지고형사료와물을자유롭게섭식할수있도록공급하고실온 22±2, 상대습도 40-60%, 조도 200 lux(8시점등, 20시소등 ) 를계속유지하면서 1주일간실험실환경에적응시킨후실험에사용하였다. 본실험은한국한의학연구원실험동물윤리위원회 ( 승인번호 : ) 의규정을준수하여설계, 수행되었다. 95

3 Table 1. Prescription of Gwakhyangjeonggi-san 韓藥名 (Herbal Name) 白朮生藥名 (Scientific Name) Atractylodes macrocephala Koidzumi 用量 (Dose) (g) 1.00 半夏 Pinellia ternata Breitenbach 1.00 茯苓 Poria cocos Wolf 1.00 厚朴 Magnolia officinalis Rehd. et Wils 陳皮 Citrus unshiu Markovich 0.67 桔梗 Platycodon graucum Nakai 0.50 白芷 Angelica dahurica Bentham et Hooker 0.50 蘇葉 Perilla frutescens Britton var. acuta Kudo 0.33 藿香 Agastache rugosa(fisc.r et Mey.) Kuntze 0.33 大腹皮 Areca catechu L 大棗 Zizyphus jujuba Miller 0.67 乾薑 Zingiber officinale Roscoe 0.33 甘草 Glycyrrhiza uralensis Fischer 0.33 Total amount 방법 1) 아토피피부염유발약물및시료의투여 아토피피부염을유발하기위해 1-Chloro-2,4- dinitrobenzene( 이하 DNCB, sigma, USA) 를도포하였다. 병태모델유발을위해 olive oil과 acetone(sigma, USA) 을 4대 1로섞은혼합액에 DNCB 가혼합된용액을사용하였다. 먼저 Balb/c 마우스의등부위를깨끗이제모한후피부의미세상처가치유되도록 24시간방치하였다. 실험 1주일간 1% DNCB 용액 (acetone:olive oil=4:1) 를매일등부위 (100μl) 에도포하고 2주간일주일에 3번 0.5% DNCB용액 200μl를등부위 (100μl) 에도포하여염증을유발하였다. 정상군은 acetone:olive oil(4:1) 을같은용량과시기로도포하였다. 藿香正氣散의효능을관찰하기위하여실험군을정상군 (sham), 아토피피부염대조군 (AD), 그리고藿香正氣散군 (GJS) 으로총 3개군으로각각 5마리씩총 15마리로나누어실험을시행하였다. GJS군 (617mg/ kg, 임상농도를마우스투여농도로환산 ) 은실험 1주일후부터 2주간매일 0.1ml씩경구투여하였다. Sham군과 AD군은생리식염수를 GJS와같은용량, 용법으로투여하였다 (Fig. 1). Human 50 mg / kg = χ 0.081(mouse 상수 ) 12) Fig. 1. Schematic Representation of the Experiment 2) 피부손상정도측정 Balb/c 마우스의피부염은아토피피부염에서일반적으로사용되는임상적육안평가법을이용하였다. 육안평가결과는다음의 5가지항목을각각평가한점수의총합으로나타내었으며평가항목은홍반 (erythema), 가려움과건조피부 (pruritus & dry skin), 부종과혈종 (edema & excoriation), 짓무름 (erosion), 그리고태선화 (lichenification) 로나누어졌다. 이각각의항목은없음 (0), 약함 (1), 중등도 (2), 심함 (3) 으로나누어서채점하였다. 3) 혈액내면역글로불린 E(IgE) level 및 cytokine 측정 1 채혈및혈장분리각각의실험군의혈장내 IgE와 cytokine 에약물이미친영향을측정하기위해채혈후혈장을분리하였다. 이를위해서최종적으로실험이진행된마우스에 isoflurane(jwpharm, Korea) 와 O 2 혼합 gas로호흡마취를시킨후심장채혈을통해채취한혈액을 Lithium heparin 96

4 손미주외 4 인 : DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 tube(bd, USA) 에수집한후원심분리기로 3,000 rpm으로 30분간원심분리하여상층의혈장을분리하였다. 2 혈장내 IgE 측정혈장 IgE량은실험종류후희생하여채취한혈장을이용하여 ELISA kit(bethyl, USA) 로측정하였다. 실험방법은먼저혈장샘플및 standards를 100μl씩 96well plate에넣고 1 시간동안실온에반응시킨다. 그후 anti-ige 를 well당 100μl씩넣고 1시간동안실온에반응한후 IgE를인지하는 2차항체를 well당 100 μl씩넣고 30분간실온에반응시킨다. Substrate 인 TMB(tetramethylbenzidine) solution을 well당 100μl씩넣고 30분간반응시킨후, 100 μl의 stop solution 을넣었다. 실험결과는 ELISA reader기의 450nm의 filter에서값을측정하였다. 3 혈장내 cytokine(il-4, TNF-α 및 IL-6) 측정혈장내의 cytokine 인 IL-4, TNF-α, 그리고 IL-6는 Bio-Plex Pro Mouse Cytokine Th1/Th2 Assay kit(bio-rad, USA) 를통해측정하였다. IL-4, TNF-α, 그리고 IL-6의 beads 를 50μl씩 96well plate 에넣고활성화시킨후혈장및 standards를 50μl씩넣어상온에서 30 분간 beads 와결합시킨다. antibody 를 25μl씩넣고상온 850rpm으로 30분간반응시킨후 SA-PE(streptavidin-phycoerythrin) 를 50μl씩넣고상온 850rpm으로 10분간반응시킨다. 실험결과는 Bio-Plex 200 System(Bio-Rad, USA) 으로측정하였다. 4) 피부조직의조직병리학적소견명확한조직병리학적관찰을위해등피부전체두께 ( μm ), 단위면적당등피부진피내축적염증세포와비만세포수 (cells/ mm2 ), 등피부진피및표피내 TSLP 세포수 (cells/ mm2 ) 를관찰하였다. 1 조직병리학적평가 GJS 투여로개선된생쥐피부의조직을관찰하기위하여 H&E(hematoxylin & Eosin stain) 염색과 TB 염색 (Toluidine blue stain) 을시행하였다. 마우스를희생시킨후피부조직을일정부분절제한후이를 10% 포르말린에고정한다음파라핀에포매하였다. 포매된조직을미세절단기 (Leica, Germany) 를사용하여 4 μm두께로박절하였다. 이후슬라이드에부착시킨후탈수와투명 (cleansing) 과정을거쳐흐르는물에침전시켰고파라핀을제거하는과정을거친조직절편은일반조직학적검사를위해 H&E 염색을실시하였고, 비만세포를관찰하기위해 TB 염색을실시하여광학현미경 (Nikon, Japan) 으로관찰하였다. 2 면역조직학적검사 10 % 포르말린으로고정하여파라핀으로포매한피부조직의표피와진피에서 TSLP 의면역반응을확인하였다. TSLP antibody(thermofisher, USA) 를이용하여 ABC(Avidin-Biotin Complex, Thermofisher) 기반면역조직화학적방법을통해실시하였고, 광학현미경 (Nikon) 으로관찰하였다. 5) 피부조직의단백추출및 Western blot 분석채취한마우스의피부조직을 RIPA buffer 를넣고분쇄기로균질화한후 4 에서 30 분간반응시킨후, 13,000rpm 으로 10 분간원심분리하여단백질을추출하였다. 단백질정량은 bicinchoninic acid (BCA, Thermofisher) 법을이용하여단백질의동량을 4-12% Bis-Tris Protein Gels(Thermofisher) 로전기영동하여분리한후 nitrocellulose membrane 으로 transfer하였다. 비특이적 antibody 결합을방지하기위해 membrane 을 5% skim milk가함유된 Tris-buffered saline(tbs) 용액에상온에서 1 시간동안 blocking하였다. 그후 1:1,000으로희석한각 97

5 타겟단백질인 1차항체 IL-4, TNF-α, 그리고 β -actin(santacruz, USA) 를 12 시간이상처리하였다. 그후 TBS-Tween 20으로 3회세척한다음 1:2,000으로희석시킨 2차 antibody(enzo, USA) 를상온에서 1 시간반응시키고 TBS-tween 20으로 3회세척한후 ECL solution(bio-rad) 을반응시켜발현정도를 Chemidoc image analyzer(bio-rad) 을사용하여측정후정량하였다. 6) 통계분석통계학적유의성검정은 one-way ANOVA (analysis of variance) test 후 Tukey s post hoc test를시행하였으며, 유의수준 p<0.05 의범위내에서그결과들은평균 ± 표준오차로나타내었다. DNCB 로피부염을유발한아토피동물모델에서 GJS 2주투여후홍반, 가려움과피부건조, 부종과혈종, 짓무름, 태선화정도를 3점척도로점수를부여하여 Atopic dermatitis index(adi) 를측정하였다 (Fig. 2). AD군은홍반, 가려움과피부건조, 부종과혈종, 짓무름, 태선화등의증상이 2주에가장심해서 ADI 평균점수가 8.0±0.41 이었으며 3주의 ADI는 2주에비하여약간감소하는경향을보였다 (6.6± 0.27, Fig. 3). GJS군의경우 2주부터 AD군에비해유의하게아토피증상이감소하였으며 (p<0.05, Fig. 3), 치료가끝난 3주까지 AD군에비하여 ADI 평균점수가유의하게감소하였다 (p<0.01, Fig. 3). Fig. 2. Effects of Gwakhyangjeonggi-san(GJS) on Clinical Aspect in AD Induced Mice Sham, Normal mice with oil treatment; AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. Ⅲ. 결과 1. 藿香正氣散이아토피피부증상에미치는영향 Fig. 3. Change of Atopic Dermatitis Index after Gwakhyangjeonggi-san(GJS) Treatment AD-like skin lesions were evaluated by visual observation. Atopic dermatitis index were defined as the sum of scores for five symptoms: erythema, pruritus & dry skin, edema & excoriation, erosion, and lichenification. Data expressed as mean ± SEM. *, p<0.05 vs. Sham; **, p<0.01 vs. Sham; ***, p<0.001 vs. Sham; #, p<0.05 vs. AD; ##, p<0.01 vs. AD. Sham, Normal mice with oil treatment; AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. 2. 피부조직두께미치는영향 H&E 염색법을이용하여피부조직을분석한결과, Sham군의귀조직에서는표피, 진피등이정상적으로관찰되었으나, AD군은표피의각질화가발생되었으며 GJS군에서는표피의각질화도심하지않은것으로관찰되었다 (Fig. 4A). 더불어 GJS의투여가생쥐의 98

손미주외 4 인 : DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 Fig. 4. Histologic Features of Skin Lesion and Epithelial Thickness (A) H&E staining and (B) Change of epithelial thickness after GJS treatment.

6 손미주외 4 인 : DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 Fig. 4. Histologic Features of Skin Lesion and Epithelial Thickness (A) H&E staining and (B) Change of epithelial thickness after GJS treatment. Data expressed as mean ± SEM. **, p<0.01 vs. Sham; ***, p<0.001 vs. Sham; ###, p<0.001 vs. AD. Sham, Normal mice with oil treatment; AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. DE, dermis; EP, epidermis; HF, hair follicle. 게증가하기는하였으나 (p<0.05), AD군에비해서는유의하게두께의감소가있음을확인할수있었다 (p<0.001, Fig. 4B). 3. IgE 발현에미치는영향 Fig. 5. Change of IgE Levels after Gwakhyangjeonggi-san(GJS) Treatment Data expressed as mean ± SEM. **, p<0.01 vs. Sham; ***, p<0.001 vs. Sham; ##, p<0.01 vs. AD. Sham, Normal mice with oil treatment; AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. 표피두께에미친영향을 H&E 염색을통해서분석한결과, Sham군에비해서 AD군에서유의하게두꺼워져있는것을확인하였다 (p<0.001, Fig. 4B). 특히아토피유발후 GJS 투여군은 Sham군에비해서유의하 GJS 2주투여종료후혈액내 IgE를측정하였다. Sham군에비하여 AD군은유의하게 IgE수치가증가하였으며 (3126±791.8, p<0.001, Fig. 5), GJS군은 Sham군에비하여서는유의하게증가하였으나 (p<0.01, Fig. 5), AD군에비해서는유의하게감소하고있음을관찰하였다 (p<0.01, Fig. 5). 4. 피부조직의 TSLP 발현에미치는영향피부조직의 TSLP 발현을살펴본결과, 표피와진피에서 TSLP가 AD군에서증가하였다가 GJS군에서비교적감소하는것을확인하였다. 특히단위면적당진피의 TSLP의발현량은 AD군에서 Sham군에비하여유의하게증가하고있음을확인하였다 (p<0.001, Fig. 99

7 6A). 더불어, GJS는 Sham 군에비하여서는유의하게증가하였으나 (p<0.05, Fig. 6B), AD군에비하여서는유의하게감소하고있었다 (p<0.001, Fig. 6B). 5. Mast cell 침윤에미치는영향피부조직의 mast cell 침윤을확인하기위하여 toludine blue(tb) 염색을시행한결과, Sham 군에비하여아토피유발군에서확연한염증세포의침윤정도가증가함을확인할수있었다 (Fig. 7A). 특히단위면적당 mast cell 수치또한 AD군에서 Sham군에비하여증가하였으며 (p<0.001, Fig. 7B), GJS군에서는침윤정도가유의하게감소하였다 (p<0.001, Fig. 7b). 6. 피부조직의 IL-4, TNF-α 발현변화 TSLP의발현에따른 Th1면역관련한 IL-4 발현을측정하였다. AD군은 Sham에비하여유의하게증가한 IL-4 발현을보였으며 (p<0.05, Fig. 8A), GJS에서 는 AD군에비하여유의하게감소하였다 (p<0.05, Fig. 8A). 더불어 pro-inflammatory cytokine인 TNF-α 의발현또한 AD군은 Sham에비하여유의하게증가하였으나 (p<0.05, Fig. 8B), GJS에서는 AD군에비하여유의하게감소하였다 (p<0.05, Fig. 8B). 7. 혈중 IL-4, TNF-α, IL-6 분비변화 GJS의아토피피부염효과가피부조직의 cytokine 분비뿐만아니라혈중의 cytokine 에도영향이있는지살펴보기위하여 T helper type 2(Th2) cytokine인 IL-4와 proinflammatory cytokine 인 TNF-α, IL-6 수치를분석하였다. AD군은 Sham군대비 IL-4(p<0.001, Fig. 9A), TNF-α (p<0.001, Fig. 9B), IL-6(p<0.001, Fig. 9C) 가모두유의하게증가하였으며, GJS는 AD군대비혈중 IL-4(p<0.01, Fig. 9A), TNF-α(p<0.05, Fig. 9B), IL-6(p<0.05, Fig. 9C) 수치또한유의하게감소하였다. Fig. 6. Change of TSLP expression after Gwakhyangjeonggi-san(GJS) treatment Data expressed as mean ± SEM. *, p<0.05 vs. Sham; ***, p<0.001 vs. Sham; ###, p<0.001 vs. AD. Sham, Normal mice with oil treatment AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. 100

8 손미주외 4 인 : DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 Fig. 7. Change of Mast Cell Infiltration after Gwakhyangjeonggi-san(GJS) Treatment. (A) Toludine blue (TB) staining (B) and count of mast cell in dermis. Data expressed as mean ± SEM. ***, p<0.001 vs. Sham; ###, p<0.001 vs. AD. Sham, Normal mice with oil treatment AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. Fig. 8. Change of Expression of IL-4 and TNF-α after Gwakhyangjeonggi-san(GJS) Treatment Data expressed as mean ± SEM. *, p<0.5 vs. Sham; #, p<0.05 vs. AD. Sham, Normal mice with oil treatment AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. 101

9 Fig. 9. Change of Expression of (A) IL-4, (B) TNF-α and (C) IL-6 after Gwakhyangjeonggi-san(GJS) Treatment Data expressed as mean ± SEM. ***, p < vs. Sham; #, p < 0.05 vs. AD; ##, p < 0.01 vs. AD; ###, p < vs. AD. Sham, Normal mice with oil treatment AD, DNCB-induced atopic dermatitis model; GJS, Orally GJS treated DNCB-induced atopic dermatitis model. Ⅳ. 고찰 아토피피부염은다양한원인으로인해호전과악화를반복하는만성염증성피부질환으로, 병태생리에대하여다양한연구들이수행중이나아직명확한원인이밝혀지지않고있다. 다만, 유전적요인, 환경적요인등이복합적으로작용하는것으로보고되고있으며, 13) 부모에게아토피피부염의기왕력이있으면자녀에게서아토피피부염이발생하는경우가부모에게아토피피부염의기왕력이없는경우보다많았으며, 피부장벽을구성하는핵심단백질인필라그린 (filaggrin) 의돌연변이가대표적인유전적요인으로는보고되고있다 14). 또한유전적요인을가지고있어도아토피피부염이반드시나타나는것은아니기때문에 David Strachan 은위생환경이매우좋아짐에따라세균에대한면역반응이줄어들고알레르기를일으키는면역반응이증가하고있다고제시한바있다 15). 이러한알레르기를일으키는원인으로는집먼지진드기, 음식물, 애완동물의털, 바퀴벌레, 꽃가루등이꼽히고있으며 16), 치료가되었더라도외부항원노출시재발되는질병으로적극적인치료와예방관리가중요하다. 아토피피부염의한약치료효과에대해확인한체 계적문헌고찰연구결과, 아토피피부염에서한약치료는양약치료 (conventional medicines) 에비해전반적인유효율 (RR 1.43, 95% CI ) 이더높았으며, 가려움증은감소하는것 (SMD 0.83, 95% CI ) 으로보고된바있으며 17), 消風散, 補中益氣湯이아토피치료에효과적인것으로확인된바있다 18,19). 藿香正氣散은 東醫寶鑑 에傷寒陰證으로머리가아프고몸이아픈것을치료하는것으로제시되어있는처방으로 20), 정등 21) 은藿香正氣散은 LPS (Lipopolysaccharide) 로염증반응을유도한 RAW 세포주에서 proinflammatory cytokine인 TNF-α, IL-6, Prostaglandin E2의생성을억제시킨다는사실을보고한바있으며, 또한 Han 등 22) 의연구에서藿香正氣散은 DNFB 로유도한알레르기피부염동물모델에서귀부종및비만세포탈과립지수에서대조군에비해효과적으로억제하는것으로확인되었다. 이와같이藿香正氣散은항염증, 항알레르기효능이있어아토피피부염에효과가있을것으로추정되나, 본연구에서사용한藿香正氣散한약제제에대한유효성연구는보고되지않았다. 본연구에서는藿香正氣散의효능을관찰하기위해 Th2에특화되어면역학분야에광범위하게활용되는 102

10 손미주외 4 인 : DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 종인 Balb/c mouse에 hapten 자극으로 DNCB 를이용하여피부염을유발하여아토피피부염증상을유발하였다 23). 매일 1차례 1% DNCB 를 1주일간도포하여피부염을유발하고, DNCB 도포 7일째부터藿香正氣散을매일 1회경구투여한후 2주후육안피부상태, 표피와진피에서의 TSLP 발현및 mast cell 침윤을확인하였고, 피부조직의 IL-4, TNF-α 및혈중 IL-4, IL-6, TNF-α 변화를관찰하였다. 연구결과, 홍반, 가려움및건조, 부종및혈종, 짓무름, 태선화상태를측정하여점수를구성한 ADI 24) 수치부분에있어서, GJS군은 AD군대비 ADI 수치를유의하게감소시키는것을알수있었다. 특히, 그효과는치료 1주후부터유의했으며, 2주까지지속되었다. 이를통해藿香正氣散은아토피피부염의피부임상양상을개선시키는것을알수있었다. 긁기, 미생물, 알레르겐등의자극은 keratinocyte 로하여금 TSLP를유발시키고, 생성된 TSLP는 naive T세포를 Th2 세포분화시키며, mast cell 생산을촉진하여아토피피부염을유발및악화시키는데있어서중요한작용을하는것으로알려져있다 25-27). 본연구에서피부조직의 H&E 염색결과, AD 군은 Sham군에비해상피조직이두꺼워졌고 GJS군에서는 AD군에비해감소하였다. 더불어 AD군의피부조직에서 TSLP 발현및 mast cell 침윤이증가되어있었으며, 이를藿香正氣散이유의하게감소시켰다. 특히, TSLP 가유도하는 mast cell의활성화는 Th2 면역에중요한 cytokine 등을활성화시켜, 상피가두꺼워지거나, IgE 수치가높아지는등의면역반응을유발시킨다. 이에본연구에서 TSLP 및 mast cell 침윤증가와연관하여 IL-4와 IgE 수치를분석한결과, 피부조직과혈액에서모두 IL-4의수치가藿香正氣散에의해서감소되었으며, IgE 수치또한유의한감소를보였다. IL-4는 Th2 면역관련 cytokine 중 keratinocyte 의분화과정에서유도된유전자의발현을억제하며, fibronectin 생산을감소시켜피부의상처치유반응을손상시키는것으로알려져있으며 28), IgE는 mast cell과결합하여알레르기반응을유발하여피부에 leukocyte 를응집시키고, 피부염증, 알레르기반응을지속시키는작용을하는데 29), 藿香正氣散이이기전에작용하여, 아토피피부염의알레르기반응을감소시키는것으로추정된다. AD군에서는 sham군에비해피부조직의 TNF-α 수치가증가하였다가, GJS군에서는감소하였다. TNF-α 는염증반응의개시와관련되어있는 proinflammatory cytokine 으로, TNF-α 단독또는 Th2 cytokine 과의공동으로작용하여아토피피부염피부의지질장벽기능을조절하며, TSLP 의증식에도관여한다 30,31). 이에藿香正氣散은피부조직의 TNFα 조절을통해피부조직의염증반응을개선시키는것으로추측된다. 또한피부뿐만아니라혈액내의 TNF-α, IL-6 수치가藿香正氣散에의하여감소하였다. 아토피피부염환자에서혈중 pro-inflammatory cytokine 인 TNF-α, IL-6 수치가증가되어있다는사실이여러연구에서밝혀진바있다 31,32). 경구투여한藿香正氣散은장에서흡수되어혈액내 TNF-α, IL-6 수치를조절하며, 전신의면역반응을조절할것으로파악된다. 종합하면, 藿香正氣散은체내에흡수되어아토피피부염에서 Th2 cytokine 인 IL-4와 proinflammatory cytokine 인 TNF-α, IL-6를감소시키며, 혈중 IgE 수치를감소시킨다. 더불어피부조직에서 IL-4, TNF-α 의발현을감소시키고, TSLP 및비만세포침윤을억제하여아토피피부염임상증상을개선시키는것으로확인되었다. Ⅴ. 결론 藿香正氣散의항아토피효능을검증하기위하여 DNCB 로유발된아토피피부염동물모델을대상으로연구한결과, 다음과같은결론을얻었다. 1. 藿香正氣散경구투여는피부증상발현을효과적으로억제하였으며, ADI 수치또한통계적으로 103

11 유의하게감소하였다. 2. 藿香正氣散경구투여는피부조직비후, TSLP 발현및 mast cell 침윤을유의하게감소시켰다. 3. 藿香正氣散경구투여는혈액내 IgE 수치를유의한수준으로감소시켰다. 4. 藿香正氣散경구투여는피부조직에서의 IL-4 및 TNF-α 발현을유의하게감소시켰다. 5. 藿香正氣散경구투여는혈액내 IL-4, IL-6, TNF-α 함량을유의한수준으로감소시켰다. 감사의글 본연구는한국한의학연구원주요사업 장내미생물-면역-대사융합분석을통한아토피피부염한의치료기술의근거구축 (KSN ) 의지원을받아수행되었습니다. ORCID Mi-Ju Son ( So-Min Lee ( Seong-Hwan Park ( Young-Eun Kim ( Jee-Youn Jung ( References 1. Eichenfield LF, Tom WL, Chamlin SL, Feldman SR, Hanifin JM, Simpson EL, et al. Guidelines of care for the management of atopic dermatitis: section 1. Diagnosis and assessment of atopic dermatitis. Journal of the American Academy of Dermatology. 2014;70(2): Ministry of Health and Welfare. Korea youth risk behavior survey [Internet]. Korean Statistical information service Available from: URL: kosis.kr/stathtml/stathtml.do?orgid=117&t blid=dt_117_12_y094&conn_path=i2. 3. Ministry of Health and Welfare. Korea National Health and Nutrition Examination Survey [Internet] Available from: URL: stathtml/stathtml.do?orgid=117&tblid=dt_ 11702_N114&conn_path=I2. 4. Krakowski AC, Eichenfield LF, Dohil MA. Management of atopic dermatitis in the pediatric population. Pediatrics. 2008;122 (4): Jones MP, Walker MM, Ford AC, Talley NJ. The overlap of atopy and functional gastrointestinal disorders among 23,471 patients in primary care. Alimentary Pharmacology and Therapeutics. 2014;40 (4): Walker MM, Powell N, Talley NJ. Atopy and the gastrointestinal tract - a review of a common association in unexplained gastrointestinal disease. Expert Review of Gastroenterology and Hepatology. 2014;8 (3): Korea Institute of Oriental Medicine. Atopic dermatitis Korean Medicine Clinical Practice Guideline. Seoul: Elsevier Korea. 2015:

12 손미주외 4 인 : DNCB 로유발된아토피피부염동물모델에대한藿香正氣散경구투여의효과 8.Yuan Z, Cao Z, Guo Q, Yang Z. The Clinical Efficacy Observation of Huoxiang Zhengqi Caplsule to Functional Dyspepsia. Liaoning zhong yi zazhi. 2011; 38(3): Ko SJ, Han G, Kim SK, Seo JG, Chung WS, Ryu B, et al. Effect of Korean Herbal Medicine Combined with a Probiotic Mixture on Diarrhea-Dominant Irritable Bowel Syndrome: A Double-Blind, Randomized, Placebo-Controlled Trial. Evidence-based Complementary and Alternative Medicine. 2013;2013: Lee SY, Bae HS, Park SS. A Case Report of Soeumin Atopic Dermatitis Patient Improved by Gwakhyangjeonggi-san. Journal of Sasang Constitutional Medicine. 2010;22(4): Yang SW, Kim YB, Kim SY, Choi H, Sohn YJ, Park SK, et al. Effect of several herbal medicines on atopic dermatitis patients SCORAD and cytokin levels. The Korean Journal of Oriental Medical Prescription. 2008;16(1): Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. Journal of Basic and Clinical Pharmacy. 2016;7(2): Rha YH, Choi SH. Risk factors associated with development of atopic dermatitis. Allergy Asthma & Respiratory Diseases. 2009;19(2): Palmer CN, Irvine AD, Terron- Kwiatkowski A, Zhao Y, Liao H, Lee SP, et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nature Genetics. 2006; 38(4): Strachan DP. Hay fever, hygiene, and household size. British Medical Journal. 1989;299(6710): Nutten S. Atopic Dermatitis: Global Epidemiology and Risk Factors. Annals of Nutrition and Metabolism. 2015;66(suppl 1): Gu S, Yang AW, Xue CC, Li CG, Pang C, Zhang W, et al. Chinese herbal medicine for atopic eczema. Cochrane Database of Systematic Reviews. 2013;(9): Kobayashi H, Ishii M, Takeuchi S, Tanaka Y, Shintani T, Yamatodani A, et al. Efficacy and Safety of a Traditional Herbal Medicine, Hochu-ekki-to in the Long-term Management of Kikyo(Delicate Constitution) Patients with Atopic Dermatitis: A 6-month, Multicenter, Double-blind, Randomized, Placebo-controlled Study. Evidence-based Complementary and Alternative Medicine. 2010;7(3): Cheng HM, Chiang LC, Jan YM, Chen GW, Li TC. The efficacy and safety of a Chinese herbal product(xiao-feng-san) for the treatment of refractory atopic dermatitis: a randomized, double-blind, placebo-controlled trial. International Archives of Allergy and Immunology. 2011;155(2): Korea Institute of Oriental Medicine. Mediclassics [Internet]. Korea Institute of Oriental Medicine [cited 19 Apr 2019]. Available from: URL: 105

13 mediclassics.kr/books/8/volume/10#conten t_ Jeong SJ, Kim OS, Yoo SR, Seo CS, Kim Y, Shin HK. Anti-inflammatory and antioxidant activity of the traditional herbal formula Gwakhyangjeonggisan via enhancement of heme oxygenase1 expression in RAW264.7 macrophages. Molecular Medicine Reports. 2016;13 (5): Han Y. Experimental study on Huoxiangzhengqi decoction and it s drug-serum against allergic reaction. Kunming: Kunming medical university; Martel BC, Lovato P, Baumer W, Olivry T. Translational Animal Models of Atopic Dermatitis for Preclinical Studies. Yale Journal of Biology and Medicine. 2017; 90(3): Yamaguchi T, Maekawa T, Nishikawa Y, Nojima H, Kaneko M, Kawakita T, et al. Characterization of itch-associated responses of NC mice with mite-induced chronic dermatitis. Journal of Dermatological Science. 2001;25(1): Indra AK. Epidermal TSLP: a trigger factor for pathogenesis of atopic dermatitis. Expert Review of Proteomics. 2013;10(4): Soumelis V, Reche PA, Kanzler H, Yuan W, Edward G, Homey B, et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nature Immunology. 2002;3(7): Cianferoni A, Spergel J. The importance of TSLP in allergic disease and its role as a potential therapeutic target. Expert Review of Clinical Immunology. 2014;10 (11): Serezani APM, Bozdogan G, Sehra S, Walsh D, Krishnamurthy P, Sierra Potchanant EA, et al. IL-4 impairs wound healing potential in the skin by repressing fibronectin expression. The Journal of Allergy and Clinical Immunology. 2017;139(1): e Leung DYM. Role of IgE in atopic dermatitis. Current Opinion in Immunology. 1993;5(6): Danso MO, van Drongelen V, Mulder A, van Esch J, Scott H, van Smeden J, et al. TNF-α and Th2 Cytokines Induce Atopic Dermatitis-Like Features on Epidermal Differentiation Proteins and Stratum Corneum Lipids in Human Skin Equivalents. Current Opinion in Immunology. 2014;134(7): Sumimoto S, Kawai M, Kasajima Y, Hamamoto T. Increased plasma tumour necrosis factor-alpha concentration in atopic dermatitis. Archives of Disease in Childhood. 1992;67(3): Toshitani A, Ansel JC, Chan SC, Li SH, Hanifin JM. Increased Interleukin 6 Production by T Cells Derived from Patients with Atopic Dermatitis. Journal of Investigative Dermatology. 1993;100 (3):

( )Kju269.hwp

( )Kju269.hwp 만성세균성전립선염모델흰쥐에서 의항염효과 Anti-inflammatory Effect of Lycopene on Chronic Bacterial Prostatitis Rat Model Cho Hwan Yang, Dong Wan Sohn, Yong-Hyun Cho From the Department of Urology, The Catholic University