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31 1 (2010 1 ) J Korean Oriental Med 2010;31(1):138-152 Original article 益氣補血湯 育毛 홍정애 1, 송미연 1, 최인화 2, 손락원 3, 정석희 1 1 경희대학교한의과대학한방재활의학교실 2 경희대학교한의과대학안이비인후피부과학교실 3 경희대학교동서의학대학원신경과학및뇌질환전공 Effect of Yikgeebohyul-tang (Yìqìbǔxuè-tāng) on Hair Regrowth and Cytokine Changes on Hair-Removed C57BL/6 Mice Jung-Ae Hong 1, Mi-Yeon Song 1, In-Hwa Choi 2, Nak-Won Sohn 3, Seok-Hee Chung 1 1 Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Kyunghee University 2 Dept. of Oriental Ophthalmology & Otolaryngology & dermatology, College of Oriental Medicine, Kyunghee University 3 Dept. of Neuroscience and Brain Disease, Graduate School of East-West Medicine, Kyung-Hee University Objective: Yikgeebohyul-tang (Yìqìbǔxuè-tāng) is one of the representative prescriptions for invigoration of vitality and nourishing of the blood. This study was carried out to investigate the effects of Yikgeebohyul-tang (Yìqìbǔxuè-tāng) on hair regrowth and cytokine changes in a shaving model of C57BL6 mice. Method: Five-week-old mice were acclimated for 1 week at a temperature between 21-23 É, 40-60% relative humidity, and 12h of a light/dark cycle before beginning of the experiment. There were two groups including normal saline (control) and a positive control of oral intake of Yikgeebohyul-tang (Yìqìbǔxuè-tāng) extract (sample) in 18 female mice. The test compounds were topically treated once a day over 14 days. The hair regrowth was photographically and histologically determined during the experimental period of 14 days. Revelation of TGF-β1 and EGF were also determined using immunohistochemistry. In addition to that, IFN-γ, IL-4 and IL-10 were determined in serum. Results: Hair regrowth in the sample group was promoted earlier and faster than the control group, as shown by concentrations of hairs and thick-hair ratio in the sample group. TGF-β1 was not revealed in either control orsample group. EGF was strongly positive in out root sheath of some thick hair of the sample group. Serum IFN-γ was significantly decreased in the sample group compared with the control group at 7 experimental days. However, it was not significantly decreased at 14 experimental days. Serum IL-4 was significantly increased in the sample group compared with the control group at 7 experimental days. However, it was not significantly decreased at 14 experimental days. Serum IL-10 was decreased in the sample group compared with the control group, but with no real statistical significance. Conclusion: These results suggest that Yikgeebohyul-tang (Yìqìbǔxuè-tāng) has hair growth promoting activity and it can be used for treatment of alopecia. Also, these effects relate to EGF revelation of hair roots, a decrease in serum IFN-γ, and an increase of serum IL-4. Key Words : Alopecia, C57BL/6 mice, Yikgeebohyul-tang, hair regrowth. Received:27 August 2009 Revised:16 October 2009 Accepted:28 October 2009 Correspondence to: 정석희 (Seok-Hee Chung) 서울시동대문구회기동 1 번기경희의료원부속한방병원한방재활의학과교실 Tel:+82-2-958-9226, Fax:+82-2-963-4983, E-mail:omdchung@yahoo.com 138

4 : C57BL/6 Cytokine (139) 서론.. 1). 3-5, 1-2, 3-5 2) 2,3)., T 4). cytokine. cytokine EGF(Epidermal Growth Factor), TGF-α(Transforming Growth Factor-α), TGFβ (Transforming Growth Factor-β), KGF(Keratinocyte Growth Factor), IGF-1(Insulin-like Growth Factor-1), bfgf(basic Fibroblast Growth Factor), VEGF(Vascular Endothelial Growth Factor), HGF(Hepatocyte Growth Factor), IL-1(Interleukin-1), IFN-γ(Interferon-gamma), IL-4, IL-10, Il-16 5-7). EGF cytokine 8). TGF-β1 9,10). 11,12), 11,12), 13), 14), 12), 12), 15), 16) 17). 18),,., 19-25).. C57BL/6,, TGF-β1, EGF, cytokine IFN-γ, IL-4 IL-10. 실험 1. 재료 1) 5 C57BL/6 (, Korea) 36 1. (21 23 ), (40 60%), (12 / ) 139

(140) 31 1 (2010 1 ) (, Korea), 1. 2) (Yikgeebohyul-tang(Yìqìbǔxuè-tāng), YBT) Table 1, 10 804g 3,000ml 2 rotary evaporator 156.7g. 10 26.1mg/10g body weight 1. 1 0.2ml 1 1, 2. 2. 방법 1) 6 C57BL/6 animal hair clipper (, Korea). C57BL/6 (Control), (Sample). 18, Table 1. Herbs Dosage(g) 黃耆 ( 密炙 ) Astragali Radix(stir-bake with honey) 6.0 白朮 Atractylodis Rhizoma Alba 6.0 山査肉 Crataegi Fructus 4.0 香附子 Cyperi Rhizoma 4.0 半夏 Pinelliae Tuber 4.0 陳皮 Citri Percarpium 4.0 白茯苓 Hoelen 4.0 神麯 ( 炒 ) Massa Medicata Fermentata(stir-bake) 4.0 麥芽 ( 炒 ) Hordei Germinatus Fructus(stir-bake) 4.0 甘草 Glycyrrhizae Radix 4.0 厚朴 Magnoliae Cortex 3.2 砂仁 Amomi Xanthioides Fructus 3.2 當歸身 Angelicae Gigantis Radix 2.8 白芍藥 Paeoniae Radix 2.8 乾地黃 Rehmanniae Recens Radix 2.8 人蔘 Ginseng Radix 2.4 白茯神 Poria 2.4 麥門冬 Ophiopogonis Radix 2.4 遠志 Polygalae Radix 2.4 川芎 Cnidii Rhizoma 2.0 木香 Saussureae Radix 2.0 生薑 Zingiberis Rhizoma Recens 4.0 大棗 Zipyphi Fructus 4.0 Total 80.4 140

4 : C57BL/6 Cytokine (141) 10 1 2. 2) 1, 4, 7, 10 14 2% isoflurane (EOS-10D, Canon, Japan). mouse. 3), NIH Image J software(ver. 1.36). (Hair Regrowth index, HRG index)., fig. 1-A 4 0 3. 4 0, 3 1, 2 2, 1 3., (Fig. 1). 0% 300%. 4) 14 sodium pentobarbital dry ice-isophentane, 10% neutral formalin. 8 Hematoxyline & Eosin (H&E). A B score 0 score 1 score 2 score 3 HRG Index = (area% of A 1) + (area% of B 2) + (area% of C 3) = (35% 1) + (55% 2) + (10% 3) = 175% Fig. 1. 141

(142) 31 1 (2010 1 ) 5) 0.05M Phosphate-Buffered Saline(PBS) 5 3, 1% H2O2 10-15 3 10% normal horse serum(vectastain, USA) bovine serum albumin(sigma, USA) PBS blocking solution. PBS 3, primary antibody. Primary antibody anti-tgf-β1(sc-146, Santa Cruz, USA) anti-egf(mab236, R&D Systems, USA) PBS Triton X-100 4 12. PBS, abidin-biotin immunoperoxidase(abc Vectastain Kit, USA). NiCl2 H2O(Sigma, USA) diaminobenzidine tetrachloride(sigma, USA) 5-10, poly-l-lysine 2-3,. 6) cytokine 1, 7 14 6 sodium pentobarbital 1,000g 15. IFN-γ, IL-4 IL-10 colorimetric Sandwich ELISA ki (Quantikine, R&D Systems, USA). 96 well plate well standard 100μl 2 wash buffer 400μl 4, conjugate 200μl 2 wash butter 400μl 4. well substrate 200μl 30 stop solution 50μl. 30 ELISA reader(bio-tek Instruments, USA) 450nm. 3. 통계분석 p<0.05 Mann-Whitney U test. Mean ± Standard error. 18, 12 6 (n=18, 12, 6). 성적 1. 발모상태의육안적변화 4, 7 1st day 4st day 7st day 10st day 14st day Control Sample Fig. 2. 142

4 : C57BL/6 Cytokine (143) Table 2. Groups 1st (n=18) 4th (n=12) Day after hair removal 7th (n=12) 10th 14th Control 0 4.8±0.6 27.6±4.9 130.8±11.1 208.9±15.2 Sample 0 4.2±0.5 69.2±5.6*** 168.2±12.5* 253.5±10.8* Data present Mean ± Standard error. YBT; Yikgeebohyul-tang(Yìqìbǔxuè-tāng). Control; group treated without any treatment after the hair removal. Sample; group treated with Yikgeebohyul-tang(Yìqìbǔxuè-tāng) water-extract administration after the hair removal. Statistical significance was verified with Mann-Whitney U test between the control and sample group(*, P<0.05; ***, P<0.001)., 10, 14. 7 7, 10, 14 (Fig. 2).. 2. 육모지수의변화, 1 0%, 4 4.8±0.6%, 7 27.6±4.9%, 10 130.8±11.1%, 14 208.9±15.2% 1 7 7 14. 1 0%, 4 4.2±0.5%, 7 69.2±5.6%, 10 167.2±12.5%, 14 243.5±10.8% 7 P<0.001, 10 14 P<0.05 (Table 2, Fig. 2,3). 3. 발모상태의조직학적변화 14 H&E, Control Sample Fig. 3. 143

(144) 31 1 (2010 1 ) 1 2 3 4 Fig. 4. (Fig. 4-1),, (Fig. 4-2). (Fig. 4-3), (Fig. 4-4). 4. TGF-β1 발현의변화 14, TGF-β1,, TGF-β1 (Fig. 5-1). TGFβ1 (Fig. 5-2). 1 1 2 2 Fig. 5. β β Fig. 6. 144

4 : C57BL/6 Cytokine (145) 5. EGF 발현의변화 14, EGF, (± +), (-) (Fig. 6-1). EGF (+) (++), (+++) EGF (Fig. 6-2). 6. 혈청 IFN-γ 의변화 IFNγ 1 8.3±1.5pg/ml, 7 218.3 ±43.9pg/ml 14 53.2±12.1pg/ml. 1 5.8±1.2pg/ml, 7 105.6±25.3pg/ml, 14 48.5±10.3pg/ml 7 P<0.05 IFNγ (Table 3). 7. 혈청 IL-4 의변화 IL-4 1 18.3±1.2pg/ml, 7 10.3± 1.5pg/ml, 14 13.5±1.2pg/ml. 1 17.5±1.4pg/ml, 7 15.2±1.6pg/ml, 14 14.8±1.3pg/ml 7 P<0.05 IL-4 (Table 4). 8. 혈청 IL-10 의변화 IL- 10 1 53.5±5.3pg/ml, 7 43.7 ±4.6pg/ml, 14 46.2±4.2pg/ml Table 3. γ Groups 1st Days after hair removal 7th 14th Control 8.3±1.5 218.3±43.9 53.2±12.1 Sample 5.8±1.2 105.6±25.3 * 48.5±10.3 Data present Mean ± Standard error. YBT; Yikgeebohyul-tang(Yìqìbǔxuè-tāng). Control; group treated without any treatment after the hair removal. Sample; group treated with Yikgeebohyul-tang(Yìqìbǔxuè-tāng) water-extract administration after the hair removal. Statistical significance was verified with Mann-Whitney U test between the control and sample group(*, P<0.05). Table 4. Groups 1st Days after hair removal 7th 14th Control 18.3±1.2 10.3±1.5 13.5±1.2 Sample 17.5±1.4 15.2±1.6 * 14.8±1.3 Data present Mean ± Standard error. YBT; Yikgeebohyul-tang(Yìqìbǔxuè-tāng). Control; group treated without any treatment after the hair removal. Sample; group treated with Yikgeebohyul-tang(Yìqìbǔxuè-tāng) water-extract administration after the hair removal. Statistical significance was verified with Mann-Whitney U test between the control and sample group(*, P<0.05). 145

(146) 31 1 (2010 1 ) Table 5. Groups 1st Days after hair removal 7th 14th Control 53.5±5.3 43.7±4.6 46.2±4.2 Sample 45.7±4.1 40.3±3.6 41.2±4.4 Data present Mean ± Standard error. YBT; Yikgeebohyul-tang(Yìqìbǔxuè-tāng). Control; group treated without any treatment after the hair removal. Sample; group treated with Yikgeebohyul-tang(Yìqìbǔxuè-tāng) water-extract administration after the hair removal. Statistical significance was verified with Mann-Whitney U test between the control and sample group.. 1 45.7±4.1pg/ml, 7 40.3±3.6pg/ml, 14 41.2±4.4pg/ml (Table 5). 고찰 26)., 27-29)., keratin keratin fibril 29). 1,2,30). 31). cytokine 5). TGF-α, TGF-β, afgf (acidic FGF), FGF5, HER-3 (Human Epidermal Growth Factor Receptor-3), TGF-β, EGFR (EGF Receptor), FGFR2 (Fibroblast GFR2), KGFR (Keratinocyte GFR) TGF-β, KGF, HGF (Hepatocyte Growth Factor), VEGF (Vascular EGF), FGFR1 (Fibroblast GFR1). VEGF. KGF, HGF EGF, TGF-α, EGFR, KGFR 6,8,9,32,33). EGF cytokine 8), TGF-β1 9,10)., 34)., MHC 146

4 : C57BL/6 Cytokine (147) 35,36). MHC 35,37). IFN-γ MHC MHC 38). MHC 36,38,39). MHC, 40). 41). cytokine IFN-γ CD4+ T Th1 Th1 cytokine IFN-γ 38,40,41). IL-4 CD4+ T Th2 Th2 cytokine Th1 IFN-γ 41). IL-10 cytokine 42) 39,43) T 41). IL-16 IL-16 cytokines 44). 18),,,,.,,,,,,,,,,,,,,,,,,,,,,,,. 19),,. 20) 21).,, 45-48) cytokine IFN-γ 49,50). 51) 52),.,,, 22-25). 53),, 21). 147

(148) 31 1 (2010 1 ) 16)., 54),.,,,, 55). 54) 56).,,,,,,,, 57)., 19-25),.,. C57BL/6 mouse 3 2 6 4., melanocyte melanin 58,59). androgen 5 C57BL/6 1 6.,,, TGF-β1, EGF cytokine IFN-γ, IL-4, IL-10. 4 7 10 14. 7 7, 10, 14., 1 7 7 14 7 P<0.001, 10 14 P<0.05..,.,,. TGF-β1 148

4 : C57BL/6 Cytokine (149) TGF-β1. TGF-β1,. EGF,, EGF, EGF EGF. cytokine IFN-γ, 7 14, 7 P<0.05 IFN-γ 14. Th1. IL-4 7, 14, 7 P<0.05 IL-4 14. Th2 Th1 IFN-γ. IL-10 7, 14. EGF IFN-γ IL-4.. IFN-γ IL-4, 7 IFN-γ 14 7, IL-4 7 IL-4 14 7., 14. cytokine. 결론 C57BL/6 14,, TGF-β1 EGF. IFN-γ, IL-4, IL-10. 1.. 2.,. 3. TGF-β1. 4. EGF. 5. IFN-γ 7 14. 6. IL-4 7 149

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