대한한의학회지제 39 권제 1 호 (2018 년 3 월 ) J Korean Med. 2018;39(1):1-12 pissn 1010-0695 eissn 2288-3339 Original Article 黃芪추출물의외용도포가자발성원형탈모생쥐에미치는영향 권혁제, 김미혜, 양웅모 경희대학교한의과대학 Effects of Topical application of Astragalus membranaceus in Spontaneous Alopecia Mice Model Hyeok Je Kwon, Mi Hye Kim, Woong Mo Yang Department of Convergence Korean Medical Science, College of Korean Medicine, Kyung Hee University Objectives: Astragalus membranaceus has been reported to inhibit immune responses, but its effect on hair loss is not clear. In this study, the effect of A. membranaceus extract (AM) on hair regrowth in C57BL/6 mice with natural hair loss in the telogen phase was investigated. Methods: Mice with natural hair loss were topically treated with 1% AM on the dorsal skin for 2 weeks. Dorsal skin samples were stained with hematoxylin and eosin and probed with an anti-mouse CD8a IgG. The mrna expression levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-4 were measured by reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. Results: AM treatment induced hair regrowth in hair loss mice, while control mice suffered continued hair loss. Tapering hair shafts and broken hair follicles were decreased as well as CD8+ T lymphocyte infiltration. In addition, the expressions of TNF-α, IFN-γ and IL-4 were reduced by AM treatment. Also, AM treatment significantly increased the KGF expressions in Hs68 fibroblast cells. Conclusion: These results suggest that topical application of A. membranaceus may be an alternative therapy for hair loss. Key Words : Astragalus membranaceus, hair loss, hair follicle, T lymphocyte, cytokine 서론모발은물리적충격, 자외선, 한랭자극으로부터신체를보호하는역할을담당하는피부의부속기관으로, 1~6년의성장기 (anagen), 1~3주의퇴행기 (catagen), 2~3개월의휴지기 (telogen) 등 3 단계의모주기를통하여성장과탈락을반복한다 1). 탈모는모낭의소형화, 휴지기모낭의증가, 모발의길이와 밀도의감소로인하여두피, 액와, 음부등에존재하는모발이탈락하는질환이다 2),3). 탈모는흔히반흔성탈모증과비반흔성탈모증으로구분되는데 4), 이중반흔성탈모증은홍반성루푸스, 박리모낭염, 여드름, 켈로이드등의원인으로인하여모낭이파괴되며, 모낭의섬유화로모구가폐색되어발생한다 5). 반면, 비반흔성탈모증은남성형탈모증, 원형탈모증, 휴지기탈모증, 생장기탈모증등으로구 Received:21 Nobember 2017 Revised:14 December 2017 Accepted:17 January 2018 Correspondence to: 양웅모 (Woong Mo Yang) 서울시동대문구회기동 1 번지경희대학교한의과대학 Tel:+82-2-961-2209, E-mail:wmyang@khu.ac.kr 1
(2) Journal of Korean Medicine 2018;39(1) 분할수있다. 남성형탈모증은연령증가로인한 dihydrotestosterone (DHT) 의영향으로두피의전두부와두정부의모낭의크기가축소되고, 성장기가단축되어발생하는질환이다 6),7). 원형탈모증은갑상선질환, 당뇨, 외상, 백신, 유전적, 정신적, 환경적요인등의원인이복합적으로작용하여성장기모낭에 T 림프구를매개한자가면역반응이유도되어발생한다 8),9). 현재 FDA 의승인을받은탈모치료제로는 Finasteride 와 Minoxidil 등이임상에서사용되고있다 10),11). 남성형탈모증치료제인 Finasteride는 testosterone에서 DHT로의전환을매개하는 5α-reductase를억제시키는약물로서그중진피유두부와전립샘및모낭에존재하는 type 2 5α-reductase를선택적으로억제한다 12),13). 또한두피내에존재하는 DHT를 41% 정도감소시키는것으로알려졌으나 14), 성욕감소, 무기력, 사정장애, 우울등의부작용이나타나는것으로보고되었다 15). Dutasteride는 Finasteride보다 type 2 5α -reductase는 3배, type 1 5α-reductase는 100배더강하게억제하며 16), 두피내 DHT를 51% 가량감소시키는것으로나타났으나 14) 사정장애, 성기능이상, 무기력, 발기장애, 여성형유방등의부작용이확인되어탈모치료에한계가있다 17). Minoxidil은 piperidinopyrimidine 계열로본래고혈압치료제였으나약물효과외의발모효과가관찰된이후, 치료용도를탈모치료제로변경하여현재까지사용되고있다 18),19). Minoxidil의정확한발모기전은아직까지밝혀진바없으나두피혈류의순환개선을통해모발성장을촉진한다는가설이가장유력하다 20),21). 그러나건조감, 소양감, 알러지성접촉성피부염등의다양한부작용이발생할우려가있어안전한탈모치료제개발이필요한실정이다 22). 그외에도원형탈모증등에는스테로이드 23), Cyclosporine 24), Diphenylcyclopropenone 25) 를이용한약물치료, Excimer lamp 26), 저강도광선요법 (Low-level light therapy) 27), 줄기세포치료 28), 표재성냉동치료 29), 자가혈소판농축혈장치료술 (Platelet -rich plasma) 30) 등다양한치료가시도되고있으나효과가미미하고치 료마다뚜렷한근거가부족한실정이다. 한의학문헌에서탈모는 黃帝內經 에서髮墮, 髮去, 毛拔, 髮落, 毛折으로기록되어있다 31),32). 晉代葛洪은 肘後備急方 33) 에서鬚鬢髮禿으로언급하였으며, 隋代巢元方은 諸病源候論 34) 에서탈모의원인에따라鬚髮禿落候, 鬼舐頭候, 毛髮不生候, 白禿候, 赤禿候, 火燒處, 髮不生候로구분하였다. 唐代孫思邈의 千金方 35) 에서는白禿, 赤禿, 髮落不生, 頭風白層으로, 明代陳實功의 外科正宗 36) 에서는油風으로, 淸代 醫宗金鑑 37) 에는鬼剃頭으로기재되었다. 이중현대의남성형탈모증와연관된병증은鬚髮禿落候이며변증으로는氣血兩虛, 肝脾濕熱, 肝腎陰虛 38) 39), 脾胃濕熱등이있다. 鬼舐頭候, 油風, 鬼剃頭는원형탈모증과유사하며, 변증으로血熱生風 40), 瘀血阻絡, 肝腎不足 41), 血瘀風燥, 氣滯血瘀 42) 및氣血虛弱 34) 이있다. 황기는콩과다년생본초인 Astragalus membranaceus Bunge의周皮를벗긴根으로性味가甘溫하며, 生用하면益衛固表, 利水消腫, 托毒生肌하며炙用하면補中益氣하고內傷勞倦을치료하는것으로알려져있다 43). 황기의성분은 isoflavonoid, saponin, polysaccharide 가포함되어있고, 그중 saponin인 astragaloside 44) 와 isoflavonoid인 formononetin, calycosin 45) 이황기의중요한생리활성작용과연관되어있다. 현재까지황기의약리작용으로는항산화 46), 간기능보호 47), 혈압강하 48), 항염증 49), 아토피증상완화 50) 등이보고되었다. 특히, 황기의주성분인 astragaloside IV와 formononetin의외용도포가탈모생쥐에서모낭내세포자멸사를억제하여발모효과를나타내는것으로보고되었다 51). 그러나황기추출물을이용한탈모생쥐에대한효능은아직까지연구되어있지않다. 따라서본연구에서는자연탈모가유발된 C57BL/6J mice에서황기추출물외용도포의발모효과를확인하고, 작용기전을확인하고자하였다. 1. 약물의제조및분석 실험재료및방법 2
黃芪추출물의외용도포가자발성원형탈모생쥐에미치는영향 (3) 본실험에사용된황기는정도생약국 (Seoul, Korea) 에서건조된뿌리형태로구입한후경희대학교한의과대학융합한의과학교실에서형태학적및화학적으로동정하여사용하였다. 황기 (150 g) 에 30% 에탄올 (1.5 L) 을가하여상온에서 24시간추출한후여과하였다. 여과액은 55 ± 2 에서회전감압농축기 (rotary vacuum evaporator, EYELA, Tokyo, Japan) 로감압농축후, 동결건조하여 24.3g 파우더상태의황기에탄올추출물을얻었다 ( 수율 : 16.2%). 최종적으로얻어진황기 30% 에탄올추출물표본 (Voucher specimen: #AME030) 은본실험실에보관되었다. 황기추출물의성분분석은 High-performance liquid chromatography (HPLC) 를통하여확인하였다. HPLC system은 chromatographic pump (G1311A Quat pump; Agilent Technologies, Santa Clara, CA, USA), automated sample injector (ALS G1329A; Agilent Technologies) 와 thermostatted column compartment equipped with an evaporative light-scattering detector (1200 series; Agilent Technologies) 로이루어져있으며, output signal은 G1315D Diode Array Detector (DAD; Agilent Technologies) 로측정하였다. 분석 column은 Gemini RP C 18 column (5μm, 4.6 I.D. 250 mm ; Phenomenex, Torrance, CA, USA) 을사용하였으며이동상용매는증류수 (A) 와 acetonitrile (B) 를이용하였다. 유속은 0.5mL/min으로유지하였고 UV 277 nm에서검출하였다. 2. 실험동물실험동물은체중 20g 내외의암컷 7주령의 C57BL/ 6J 정상생쥐와자발적인원형탈모생쥐를이용하였다. 모든생쥐는 ( 주 ) 라온바이오 (Yongin, Korea) 로부터구매하여일주일동안의적응기를통해본실험에사용되었다. 각군의생쥐 (n = 7) 는정상적인모발주기를가진정상대조군 (Normal, normal control group), 자발적으로원형탈모가유발된음성대조군 (Control, negative control group), 자발적으로 원형탈모가유발된생쥐에황기추출물을처리한실험군 (AM, sample group) 으로나뉘어졌다. 정상대조군과음성대조군에는 vehicle( 증류수 ) 를처리하였으며, 실험군은황기추출물을 1% w/v 농도로 100 μl 씩원형탈모가일어난등부위에동일하게처리하였다. 매일 1회씩총 14일간반복하였으며, 15일째되는날실험목적에따라생쥐를희생하였다. 탈모부위의조직학적분석및각종분자학적지표분석을위하여병변부위의피부조직을적출하였다. 전실험기간동안모든생쥐들은표준화된고형사료와물을공급받았으며, 사육실의온도는 23±2 C, 습도 50±10%, 광주기와암주기 (light-dark cycle) 는 12시간-12시간으로유지되었다. 모든동물실험과정은 National Institutes of Health의실험동물관리규정 (Principle of Laboratory Animal Care) 에근거하여이루어졌으며, 경희대학교동물실험윤리위원회의승인 (Permit Number: KHUASP(SE)-13-046) 을받아동물윤리준칙에의거하여수행되었다. 3. 육안적발모소견자발적으로원형탈모가유발된생쥐에서황기추출물의발모효능을관찰하기위하여실험시작 15 일째되는날생쥐를희생하기전졸레틸과럼푼이 1:1로혼합된마취제를희석하여복강투여하였다. 마취된생쥐의등부위를카메라 (Sony, Tokyo, Japan) 로촬영한후정상대조군및음성대조군과비교관찰하였다. 4. 조직학적평가적출한등피부조직을 24시간동안 4% 포르말린에고정한후, 조직의탈수를위해순서대로저농도의 70% 에탄올부터 80% 에탄올, 90% 에탄올, 95% 에탄올및고농도 100% 알코올까지탈수하고, 자일렌에투명과정을거친다음파라핀블록을제작하였다. 제작된블록은 4 μm 두께로박절하여탈파라핀및 100% 에탄올에서 70% 에탄올을거쳐함수시켰다. 증류수에수세시킨조직슬라이드를 hematoxylin & eosin (H&E) 로각각염색하여 permount로봉입하 3
(4) Journal of Korean Medicine 2018;39(1) 였고. 염색된조직은광학현미경 (Leica Microsystems Inc., IL, USA) 에서 200 및 400 배율로관찰하였다. 5. 면역조직화학적염색파라핀슬라이드의탈파라핀및수세과정은조직학적평가에사용했던방법과동일하게진행되었다. 3% 과산화수소처리로인하여 endogenous peroxidase 활성이억제된조직슬라이드에 normal goat serum이포함된 1차항체 goat anti-mouse CD8a (1:200) 을처리하여실온에서 4시간동안반응시켰다. 그후 2차항체는 biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) 를 1:400으로희석하여실온에서 1시간반응시킨후 Avidin/ Biotinylated enzyme Complex kit (Vector Laboratories) 용액에담가실온에서 1시간가량반응시킨후 3-3 diaminobenzidine으로발색시켜통상적인방법에따라탈수와투명화를거쳐 permount로봉입하였다. 염색된조직은광학현미경 (Leica Microsystems Inc., IL, USA) 에서 400 배율로관찰하였다. 6. Reverse transcription polymerase chain reaction (RT-PCR) 과 Real-time PCR (qpcr) 적출한등피부조직에서 mrna을추출하기위하여 Trizol 방법을사용하였다. 100 mg의피부조직에 Trizol 1 ml을섞어상온에서 30분정도반응시킨후 homogenizer로분쇄하였다. 클로로포름과이소부탄올을이용하여추출된 1μg RNA을 cdna synthesis kits (Invitrogen Corp., Carlsbad, CA, USA) 을이용하여 45 C에서 60분, 95 C에서 5분동안 cdna로합성하였다. 합성된 cdna 증폭은 GoTaq (Promegar M712B) 을이용하였고 TNF-α, IL-4와 IFN-γ primer와함께 thermal cycler (Perkin Elmer 2400, USA) 에서 45 cycles 증폭되었다. 사용된 primer는 RT-PCR과 qpcr 모두사용가능한제품으로주문하였다. 각 cycle은 94 C에서 20초 denaturation, 60 C에서 30초 annealing 및 72 C에서 2분 extension시켰다. PCR에사용된 TNF-α sequence 는 5 -GGT GCA ATG CAG AGC CTT CC-3 (forward) and 5 -CAG TGA TGT AGC GAC AGC CTG G-3 (reverse), IL-4 sequence는 5 -ATG GGT CTC AAC CCC CAG C-3 (forward) and 5 -GCT CTT TAC GCT TTC CAG GAA GTC-3 (reverse), IFN-γ sequence는 5 -AGC GGC TGA CTG AAC TCA GAT TGT AG-3 (forward) and 5 -GCT CTT TAC GCT TTC CAG GAA GTC-3 (reverse) 이며, GAPDH sequence 는 5 -CCA TCA CCA TCT TCC AGG AG 3 (forward) and 5 -CCT GCT TCA CCA CCT TCT TG-3 (reverse) 였다. 증폭된생성물은 1% agarose gel에전기영동후 Gel Doc (Bio-Rad, USA) 에서관찰하였다. 7. 면역형광검사 Hs68 섬유아세포의배양을위하여 Dulbecco s modified Eagle s medium (DMEM) 과 fetal bovine serum (FBS), 1% penicillin-streptomycin 을혼합하여사용하였다. 6 well chamber에서세포가 confluent한상태가될때까지배양한후황기추출물을농도별로 1, 10, 100 μg/ml 처리한후 24시간째에배지를제거하고 4% 포르말린용액을처리하여세포를고정시켰다. 1차항체로 goat anti-rabbit keratinocyte growth factor (KGF) 를이용하여실온에서 4시간동안반응시켰다. 2차항체로 Alexa fluor 488 rabbit anti-goat를반응시키고, 핵은 DAPI로염색하였다. 염색된세포는형광현미경으로관찰하였다. 8. Western blotting 상기기재된동일한방법으로황기추출물을섬유아세포에처리후, 배지를제거하고 trypsin-edta 시약을첨가하여세포를걷어내었다. Protease inhibitors cocktail이포함된 protein extraction buffer (0 mm Tris HCl, 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mm EDTA) 를 pipetting을통하여분쇄하였다. 정량된단백질 30 μg을 SDS 12% polyacrylamide gel에전기영동하여분리한후 PVDF membrane으로이동시켰다. 1차항 4
黃芪추출물의외용도포가자발성원형탈모생쥐에미치는영향 (5) 체로 goat anti-rabbit KGF를반응시킨후, HRP goat anti-rabbit을처리하였다. 발현된단백질밴드는 enhanced chemiluminescence assay kit를이용하여 LAS Image Gauge 프로그램에서측정하였다. 하여 HPLC를이용하여 formononetin 표준물질 (Fig. 1A) 과황기추출물 (Fig. 1B) 을비교분석하였다. formononetin 표준물질과황기추출물의 peak는 31.7 min으로동일한것을확인하였다. 9. 통계처리실험결과는평균 ± 표준오차 (mean ± S.E.M) 값으로표시하였으며, 집단간평균치차이는각실험결과를통하여 ANOVA (analysis of variance) 를구한후 Duncan s multiple range test를이용하여 p < 0.05 수준에서검증하였다. 결과 1. HPLC를이용한황기추출물의품질분석황기추출물내 formononetin 함량을확인하기위 2. 황기추출물의육안적인발모효능자발적으로원형탈모가유발된대조군생쥐의경우, 모발주기가정상인정상군생쥐에비하여등부위에부분적으로원형탈모가일어난것을확인할수있었다. 자발적원형탈모유발생쥐의병변부위피부는 C57BL/6J 생쥐의모발생성주기중퇴행기를나타내는회색으로변한상태였다. 황기추출물을원형탈모부위에 14일동안외용도포한결과병변부위전체에서육안적인발모현상과육모현상이관찰되었다 (Fig. 2). 뿐만아니라, 황기추출물로인하여모발의밀도가대조군에비하여증가된것을확인할수있었다. Fig. 1. Quality evaluation of Astragalus membranaceus. HPLC chromatograms of standard formononetin (A) and A. membranaceus extract (B). It takes 31.7 min for the retention times of formononetin itself and formononetin in A. membranaceus extract. 5
(6) Journal of Korean Medicine 2018;39(1) Fig. 2. A. membranaceus induces hair regrowth in hair loss mice. Morphological fingdings on the back of the C57BL/6J mice. The mice of A. membranaceus group were treated with 100 μ L of 1% (w/v) A. membranaceus for 14 days. Following challenge for 15 days, photographs were taken and mice were sacrificed. Normal, non-treated normal control mice; Control, vehicle-treated alopecia mice; AM, AM-treated alopecia mice. 3. 황기 추출물의 모낭 성장 효능 H&E 염색으로 병변 부위 조직을 확인한 결과, 정 Fig. 3. A. membranaceus recovers the dystrophy of hair follicles. Histological findings by hematoxylin and eosin (H&E) staining of dorsal skin sections (n = 7). The magnifications were 100 (upper panel) and 200 (lower panel). Arrow indicates swarm of bees. Arrowhead indicates tapering hair shaft. Normal, non-treated normal control mice; Control, vehicle-treated alopecia mice; AM, AM-treated alopecia mice. 상 생쥐의 모낭은 손상 없이 털이 표피 밖으로 빠져 나와 있는 반면, 자발적 원형탈모를 유발한 대조군 생쥐의 병변 부위 조직의 모낭은 이어져 있지 않고 불규칙하게 끊어져 있었다. 그리고 모낭의 크기가 축 소되어 모낭 안쪽의 모발 섬유가 없이 비어있는 것 을 확인할 수 있었다. 또한 모낭 주위에 염증세포가 무리를 지어 군집을 형성한 swarm of bees 현상을 나타내었다. 황기 추출물을 외용 도포한 생쥐의 경우 손상되었던 모낭이 회복되어 진피층 뿐만 아니라 피 하층까지 성장한 것을 확인할 수 있었다(Fig. 3). 일 Fig. 4. A. membranaceus reduces the infiltration of CD8+ + cells in skin tissues. Immunohistochemical CD8 + cell staining. Arrow indicates brown-stained CD8 cells. Normal, non-treated normal control mice; Control, vehicle -treated alopecia mice; AM, AM-treated alopecia mice. 부 모낭들에서는 황기 추출물 처리로 인하여 모낭 안쪽의 모발 섬유가 자라났으며 대조군 생쥐에 비하 + 조직에서는 표지된 CD8 T 림프구를 거의 관찰할 여 황기 추출물을 외용 도포한 생쥐에서는 모낭의 + 수 없어 황기 추출물이 모낭 내 CD8 T 림프구의 수가 증가되는 현상을 나타내었다. 침윤을 감소시키는 것을 확인할 수 있었다(Fig. 4). 4. 황기 추출물의 모낭 내 CD8+ T 림프구 저해 5. 황기 추출물의 피부 조직 내 싸이토카인 mrna 발현 조절 효능 효능 자발적으로 원형탈모가 유발된 대조군에서는 CD8+ 탈모 병변 부위의 피부 조직에서 탈모에 관련된 T 림프구가 침윤되어 모낭 주위에서 강하게 면역반 싸이토카인 (TNF-α, IL-4와 IFN-γ) mrna 발현량을 응을 나타내었다. 반면 황기 추출물 처리군의 피부 RT-PCR과 qpcr 모두에서 측정한 결과, 대조군 생 6
黃芪추출물의외용도포가자발성원형탈모생쥐에미치는영향 (7) Fig. 5. A. membranaceus decreases the mrna expressions of cytokines in skin tissues. Expression of TNF-α, IL-4 and IFN-γ mrna levels by RT-PCR (A) and qpcr (B). Results are presented as mean ± S.E.M. ## and ### indicates the mean differs significantly between Normal group and Control group (p < 0.01 and p < 0.001, respectively). *** indicates that the mean differs significantly between Control and AM group (p < 0.001). Fig. 6. A. membranaceus increases the KGF expression in Hs68 fibroblast cells. Expression of KGF fluorescence by immunofluorescence staining (A) and protein level by Western blotting (B) in Hs68 fibroblast cells. Green, KGF. Blue, DAPI. Results are presented as mean ± S.E.M. *** indicates that the mean differs significantly between non-treated cells and AM-treated cells (p < 0.001). 쥐의피부조직에서 TNF-α, IL-4와 IFN-γ mrna 발현량이유의성있게증가한것으로확인되었다. TNF-α 발현은정상군에비하여대조군의피부조직에서약 22배증가하였고, IL-4 발현은약 59배증가, IFN-γ 발현은약 25배증가하였다. 반면탈모된병변부위에황기추출물을외용도포한피부조직에서 TNF-α, IL-4와 IFN-γ mrna 발현량이모두정상군의발현량과비슷한수준으로감소되었다 (Fig. 5, p < 0.001). KGF를녹색형광으로염색한결과피부모유두세포에서황기추출물은농도의존적으로 KGF 발현을증가시켰다 (Fig. 6A). 황기를처리했을때 DAPI로염색된세포의핵의수는그대로유지되었기때문에황기가피부모유두세포에서 KGF의발현을증가시키는효능을확인하였다. 뿐만아니라 Western blotting으로확인한 KGF 단백질발현량또한황기추출물에의하여농도의존적으로유의성있게늘어난것을확인하였다 (Fig. 6B). 6. 황기추출물의피부모유두세포내 KGF 발현증가효능피부모유두세포인 Hs68 세포에서황기추출물을농도별로처리하여 KGF 발현량을확인하였다. 고찰 탈모로인하여손상된모낭은크기가축소되어있거나심한경우모구가폐색되어모낭이파괴되어 7
(8) Journal of Korean Medicine 2018;39(1) 있다. 특히, 손상된모낭주위에림프구가침윤되는 swarm of bees 현상이일어나모낭에염증을일으킨다 9). 침윤된림프구는 CD4 + T 림프구와 CD8 + T 림프구로구성되어있는데, CD8 + T 림프구와, CD4 + T 림프구를매개한자가면역반응으로모낭이훼손되면모발이정상적으로기능을수행할수없다. 본연구에서는자발적원형탈모가유발된동물모델을통해황기추출물의탈모개선효과를실험하였다. 배측병변부위에황기추출물을도포한생쥐의병변부위모발은육안상으로대조군에비하여비병변부위의모발의길이가회복되었으며, 모발의밀도도높아지는등의경향성이관찰되었다. 조직학적평가에서대조군에비해황기추출물처치군에서는염증세포의침윤이감소하였으며, 면적당모낭수가증가했고모낭내모발의정상성장을확인하였다. 면역조직화학적염색으로 CD8 + T 림프구의발현을확인한결과, 황기추출물의외용도포로인하여 CD8 + T 림프구가감소하였다. 스트레스, 피부미세손상, 감염인자외설명할수없는여러발병원인들로인하여모낭및모낭주위세포들이 interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) 등의싸이토카인을분비한다 52). TNF-α 는여러감염과염증에연관되어있으며 IFN-γ와같은 Th1 계통의싸이토카인이다. 염증매개작용과함께세포성장과분화등비면역반응에도영향을미치는 TNF-α는 53),54) 최근연구에따르면 in vitro에서 IL-1α, IL-1β와함께모구 (hair bulb) 에있는기질의공포화, 크기감소, 모낭내 melanocyte의붕괴, precortical cell과내모근초 (inner root sheath) 의비정상적분화와각화를일으키는작용을하는것으로보고되었다 55). 뿐만아니라 TNF-α 는모발을퇴행기의모발과유사한형태인곤봉형태로변화시킨다고알려져있다 56). 본연구에서는정상생쥐에비하여탈모가유발된병변부위에서 TNF-α mrna 레벨이유의하게증가된것을확인할수있었다. 탈모병변부위의증가된 TNF-α 발현은황기추출물외용도포로인하여유의성있게감소되었다. IFN-γ은모낭내 MHC class I, II의발현을유도하여성장기모낭의면역특권을소실하는것으로보고되어있다 57),58). IFN-γ가결핍된생쥐는모낭주위의 MHC I, II의이상발현이정상대조군에비해서적어원형탈모증을유발하지않았다 59). 이는원형탈모증에서모낭내 MHC class I, II의발현에대한 IFN-γ의역할을나타낸다. 본실험에서황기추출물도포군은탈모가유발된생쥐에서 IFN-γ의발현을유의하게감소시켰다. 따라서황기는 IFN-γ의발현을억제하여모낭내 MHC class I, II의발현을낮추어 CD8 + T 림프구의모낭파괴를감소시킨것으로유추할수있다. 또한 IL-4는 Th2 계열의싸이토카인중하나로, 원형탈모증에이환된환자에서높게나타나는것으로밝혀져있다 60),61). 본연구에서는황기추출물을처리한생쥐에서 Th2 싸이토카인인 IL-4가대조군에비해유의하게감소한것을확인하였다. 이상의결과를종합하면, 황기추출물외용도포는 TNF-α, IFN-γ 및 IL-4 등의염증성싸이토카인의발현감소를통해기질, melanocyte, 근초등모낭의기능정상화에기여하였을것으로사료된다. 염증성싸이토카인외에도 IGF-1, KGF, fibroblast growth factor-1 (FGF-1), FGF-2, FGF-5, hepatocyte growth factor, vascular endothelial growth factor 등모발성장을유도하는여러가지인자가있다. 그중 KGF는섬유모세포성장인자의하나인 FGF family 에속하는인자이다 62). KGF는모낭의성장에필수적인인자이며, 표피의기저층과외모근초 (outer root sheath) 에존재하는 KGF 수용체를억제할경우, 모낭의형태가비정상적으로변화되고모낭개수가감소되는것을관찰할수있었다. 그외에도피부, 위장관, 유선등의상피세포의분열과분화가 KGF에의해촉진되는것으로알려져있다 62),63). KGF는피부내기저층과모낭및피지선안의 keratinocyte를자극하여성장을촉진시키고모발의성장을유도한다 64). 황기추출물의처리는 Hs68 섬유아세포의 KGF의발현을농도의존적으로증가시켰다. 이러한 KGF의발현증가를통해황기추출물이모발성장을유도할수있을것으로판단된다. 8
黃芪추출물의외용도포가자발성원형탈모생쥐에미치는영향 (9) 이상과같이황기추출물처리는자발적원형탈모가유발된 C57BL/6J mice의병변부위에서의모발의성장을유도하고, 염증성싸이토카인을감소시켰다. 또한섬유아세포에서 KGF 발현을증가시키는것을확인할수있었다. 이를통해황기의외용도포제로써탈모치료에응용할가치가있다고사료된다. 추후황기의탈모효과개선에대한기전규명을위한추가적인연구가필요할것이다. 결론본연구는황기추출물의자발적원형탈모에대한개선효과를객관적으로검증하기위하여황기의외용도포가염증세포의침윤, CD8 + T 림프구침윤, IL-4, IFN-γ, TNF-α의발현감소및 KGF의발현증가를확인하였다. 이러한결과를통해황기외용도포는자발적원형탈모병변부위에모발생장을보였으며, 향후자발적원형탈모에대한치료소재로활용가능할것으로사료된다. References 1. Stenn KS, Paus R. Controls of Hair Follicle Cycling. Physiological Reviews. 2001;81(1): 449-94. 2. Min BG. Diagnosis and treatment of hair & scalp disorders. Hanmi Book. 2005:21-30. 3. Zoe DD. Cosmetics in dermatology. 2nd. London: Churchill Livingstone. 1995:179-91. 4. Ahn SG, Ji HG, Hwang SM, Jung J, Jang KH. Common Skin Disease. Pacific publisher. 2003. 437. 5. Korean Dermatology Association. Textbook of Dermatology. 6Th. Seoul:Med book. 2014:551-7. 6. Lee SH, Lee JR. Association of Diffuse Hair loss of Adult Male on Stress, Self-confidence, and Depression. Journal of the Society of Cosmetic Scientists of Korea. 2010;16(4):1171-9. 7. Fitzpatrick TB, Wolff K. Fitzpatrickʼs dermatology in general medicine. 7Th. NewYork:McGraw -Hill. 2008:766-9. 8. Moustafa AET, Hassan I, Essam AN, Mai SAD. Platelets rich plasma versus minoxidil 5% in treatment of alopecia areata: A trichoscopic evaluation. Dermatologic Therapy. 2017;30(1): e12437. 9. Amin SS, Sachdeva S. Alopecia areata: A review, Saudi Society of Dermatology and Dermatologic Surgery. 2013;17:37-45. 10. Blumeyer A, Tosti A, Messenger A, Reygagne P, Del Marmol V, Spuls PI, et al. Evidence-based (S3) guideline for the treatment of androgenetic alopecia in women and in men. Journal der Deutschen Dermatologischen Gesellschaft. 2011;9(6):51-7. 11. Varothai S, Bergfeld WF. Androgenetic Alopecia: An Evidence-Based Treatment Update. American Journal of Clinical Dermatology. 2014;15(3): 217-30. 12. Levy LL, Emer JJ. Female pattern alopecia: current perspectives. International Journal of Women's Health. 2013;5:541-56. 13. Yamana K, Labrie F, Luu-The V. Human type 3 5α-reductase is expressed in peripheral tissues at higher levels than types 1 and 2 and its activity is potently inhibited by finasteride and Dutasteride. Hormone Molecular Biology and Clinical Investigation. 2010;2:293-9. 14. Lee SY, Chun SW, Kim JB, No BI. A Case of Combination Therapy with Finasteride and Low Dose Dutasteride in the Treatment of Androgenetic Alopecia. Korean Journal of Dermatology. 2017;55(2):147-8. 15. Mounsey AL, Reed SW. Diagnosing and treating hair loss. American Family Physician. 2009;80(4):356-62. 9
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