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1 31 1 ( ) J Korean Oriental Med 2010;31(1): 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 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: , Fax: , omdchung@yahoo.com 138

2 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 ). 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) (21 23 ), (40 60%), (12 / ) 139

3 (140) 31 1 ( ) (, Korea), 1. 2) (Yikgeebohyul-tang(Yìqìbǔxuè-tāng), YBT) Table 1, g 3,000ml 2 rotary evaporator 156.7g mg/10g body weight ml 1 1, 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

4 4 : C57BL/6 Cytokine (141) ) 1, 4, 7, % isoflurane (EOS-10D, Canon, Japan). mouse. 3), NIH Image J software(ver. 1.36). (Hair Regrowth index, HRG index)., fig. 1-A , 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

5 (142) 31 1 ( ) 5) 0.05M Phosphate-Buffered Saline(PBS) 5 3, 1% H2O % 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 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, 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

6 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± ± ± ±15.2 Sample 0 4.2± ±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, , 10, 14 (Fig. 2).. 2. 육모지수의변화, 1 0%, 4 4.8±0.6%, ±4.9%, ±11.1%, ±15.2% %, 4 4.2±0.5%, ±5.6%, ±12.5%, ±10.8% 7 P<0.001, P<0.05 (Table 2, Fig. 2,3). 3. 발모상태의조직학적변화 14 H&E, Control Sample Fig

7 (144) 31 1 ( ) 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) Fig. 5. β β Fig

8 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, ±43.9pg/ml ±12.1pg/ml ±1.2pg/ml, ±25.3pg/ml, ±10.3pg/ml 7 P<0.05 IFNγ (Table 3). 7. 혈청 IL-4 의변화 IL ±1.2pg/ml, ± 1.5pg/ml, ±1.2pg/ml ±1.4pg/ml, ±1.6pg/ml, ±1.3pg/ml 7 P<0.05 IL-4 (Table 4). 8. 혈청 IL-10 의변화 IL ±5.3pg/ml, ±4.6pg/ml, ±4.2pg/ml Table 3. γ Groups 1st Days after hair removal 7th 14th Control 8.3± ± ±12.1 Sample 5.8± ±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 Sample 17.5± ±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

9 (146) 31 1 ( ) Table 5. Groups 1st Days after hair removal 7th 14th Control 53.5± ± ±4.2 Sample 45.7± ± ±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 ±4.1pg/ml, ±3.6pg/ml, ±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

10 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

11 (148) 31 1 ( ) 16)., 54),.,,,, 55). 54) 56).,,,,,,,, 57)., 19-25),.,. C57BL/6 mouse , melanocyte melanin 58,59). androgen 5 C57BL/6 1 6.,,, TGF-β1, EGF cytokine IFN-γ, IL-4, IL , 10, 14., P<0.001, P<0.05..,.,,. TGF-β1 148

12 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 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 , 14. cytokine. 결론 C57BL/6 14,, TGF-β1 EGF. IFN-γ, IL-4, IL ,. 3. TGF-β1. 4. EGF. 5. IFN-γ IL

13 (150) 31 1 ( ) IL-10. 참고문헌 1. Botchkarev VA, Kishimoto J. Molecular control of epithelial-mesenchymal interactions during hair follicle cycling. J Investig Dermatol Symp Proc. 2003;8(1): Stenn KS, Paus R. Controls of hair follicle cycling. Physiol Rev. 2001;81(1): Soma T, Ogo M, Suzuki J, Takahasi T, Hibino T. Analysis of apoptotic cell death in human hair follicles. J Invest Dermatol 1998;11: Kalish RS, Gilhar A. Alopecia areata: autoimmunitythe evidence is compelling. J Invest Dermatol Symp Proc. 2003;8: Peus D, Pittelkow MR. Growth factors in hair organ development and the hair growth cycle. Dermatol Clin. 1996;14(4): Jindo T, Tsuboi R, Imai R, Takamori K, Rubin JS, Ogawa H. The effect of hepatocyte growth factor/scatter factor on human hair follicle growth. J Dermatol Sci. 1995;10(3): Mitsui S, Ohuchi A, Hotta M, Tsuboi R, Ogawa H. Gene for a range of growth factors and cyclin-dependent kinase inhibitors are expressed by isolated human hair follicle. Br. J Dermatol. 1997;137: Mak KK, Chan SY. Epidermal growth factor as a biologic switch in hair growth cycle. J Biol Chem. 2003;278(28): Inui S, Fukuzato Y, Nakajima T, Yoshikawa K, Itami S. Androgen-inducible TGF-beta1 from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of androgen on human hair growth. FASEB J. 2002;16(14): Soma T, Dohrmann CE, Hibino T, Raftery LA. Profile of transforming growth factor-beta responses during the murine hair cycle. J Invest Dermatol. 2003:121(5): Lee hs, Song JY, Yun Km, Jae YM. Hair growth effect of Mori Cortex Mixture on the hair of rat. Korean J. Seric. Sci. 2000;42(2): Choi W, Choi JH, Kim JH. Effect on Hair Growth by various herb medicine. The Journal of Oriental Ophthalmology & Otolaryngology & dermatology. 2002;15(2): Shin YS. Studies on the effect of five herbal extracts on hair growth activity. Dept. of Oriental MedicineGraduate School Daejeon University Kim MY, An BJ. A Study of Hair Loss Prevention and Hair Growth Promotion by Korean Traditional Fomular. Korean journal of herbology. 2004:19 (2); Park WS, Sung DS, Kim DK, Cho WH, Lee HK, Lee CH, et al. The Effect of Hair Essence (HHRHG ) Containing Five Herbal Extracts on Hair Growth and the Prevention of Alopecia in vitro& vivo. J Korean Oriental Med. 2004;25 (1): Kwon GJ. Experimental Studies on Hair Growth Effect of Samultang-gamibang in Spontaneous Alopecia Areata Mice. Professional Graduate School of Oriental Medicine Wonkwang University KIm PS, Kim HT, Roh SS, Whang CY. Effect of DanGuiBoHyulTangGami-Bang on the Alopecia and Hair Growth Stimulation. The Journal of Oriental Ophthalmology & Otolaryngology & dermatology. 2004;17(3): Kim JJ. Dongyangeuihak Jinryoyogam. Seoul: The Institute of Oriental medicine. 2007; Hwang HJ, Kim JD. A Case Study on the Iron Deficiency Anemia. Korean J. Orient. Int. Med. 2000;21(4):

14 4 : C57BL/6 Cytokine (151) 20. Lee G. Dongyuashizhongyishu. Seoul:Daeseong medicine publishing company. 1983;35-7, Youn GY. Dongeuiyimsangbangjehak. Seoul: Myungbo publishing company. 1987;254-63, Lee HY, Baek TH. Effect of Jaeumgeonbi-tang Extract on Indomethacin-Induced Gastric Mucosal Lesions. J Korean Oriental Med. 2004;25(3): Lim KM, Jeong HW. Mechanism of Jaeumgeonbitang adding Aurantii Fructus Gastrogae Rhizoma on the Improvement and Changes of Cerabral Hemodynamics. Korean Journal of Oriental Physiology and Pathology. 2003;17(1): Hong DS. The Effect of Jaeumgunbitang on The Brain Catecholamine Contents and Body Weight of Rats in Immobilization Stress. Graduate School Kyung Hee University Kim JH, Lee KS. Experimental Study on the Effects of Jaeumgeonbi-tang on Sedative Action and Hematosis. J Korean Oriental Med. 1988;9 (1): Randall VA, Sundberg JP, Philpott MP. Animal and in vitro models for the study of hair follicles. J Investig Dermatol Symp Proc. 2003;8(1): Powell B, Crocker L, Rogers G. Hair follicle differentiation: expression, structure and evolutionary conservation of the hair type II keratin intermediate filament gene family. Development. 1992;114(2): Rogers GE. Hair follicle differentiation and regulation. Int J Dev Biol. 2004;48(2-3): Kreplak L, Mérigoux C, Briki F, Flot D, Doucet J. Investigation of human hair cuticle structure by microdiffraction: direct observation of cell membrane complex swelling. Biochim Biophys Acta. 2001;1547(2): Hirobe T. Structure and function of melanocytes: microscopic morphology and cell biology of mouse melanocytes in the epidermis and hair follicle. Histol Histopathol. 1995;10(1): Li J, Tzu J, Chen Y, Zhang YP, Nguyen NT, Gao J, et al. Laminin-10 is crucial for hair morphogenesis. EMBO J. 2003;22(10): Peters EM, Hansen MG, Overall RW, Nakamura M, Pertile P, Klapp BF, et al. Control of human hair growth by neurotrophins: brain-derived neurotrophic factor inhibits hair shaft elongation, induces catagen, and stimulates follicular transforming growth factor beta2 expression. J Invest Dermatol. 2005;124(4): Danilenko DM, Ring BD, Yanagihara D, Benson W, Wiemann B, Starnes CO, et al. Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development, and differentiation. Normalization of the nu/nu follicular differentiation defect and amelioration of chemotherapy-induced alopecia Am J Pathol. 1995;147(1): Lindner G, Botchkarev VA, Botchkareva NV, Ling G, van der Veen C, Paus R. Analysis of apoptosis during hair follicle regression (catagen). Am J Pathol. 1997;151(6): Paus R, Christoph T, Muller-Rover S. Immunology of the hair follicle: a short journey into terra incognita. J Invest Dermatol Symp Proc. 1999;4 (3): Christoph T, Muller-Rover S, Audring H, Tobin DJ, Hermes B, Cotsarelis G, et al. The human hair follicle immune system: cellular composition and immune privilege. Br J Dermatol. 2000;142 (5): Paus R, Hofmann U, Eichmuller S, Czarnetzki BM. Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle. Br J Dermatol. 1994;130(3): Ito T, Ito N, Saathoff M, Bettermann A, Takigawa M, Paus R. Interferon-gamma is a potent inducer of catagen-like changes in cultured human anagen hair follicles. Br J Dermatol. 2005;152 (4):

15 (152) 31 1 ( ) 39. Bodemer C, Peuchmaur M, Fraitaig S, Chatenoud L, Brousse N, De Prost Y. Role of cytotoxic T cells in chronic alopecia areata. J Invest Dermatol. 2000;114(1): Neville LF, Mathiak G, Bagasra O. The immunobiology of interferon-gamma inducible protein 10 kd (IP-10): a novel, pleiotropic member of the C-X-C chemokine superfamily. Cytokine Growth Factor Rev. 1997;8(3): Miceli MC, Parnes JR. Role of CD4 and CD8 in T cell activation and differentiation. Adv Immunol. 1993;53: Meng X, Sawamura D, Tamai K, Hanada K, Ishida H, Hashimoto I. Keratinocyte gene therapy for systemic diseases. Circulating interleukin-10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin. J Clin Invest. 1998; 101(6): Gilhar A, Landau M, Assy B, Ullmann Y, Shalaginov R, Serafimovich S, et al. Transfer of alopecia areata in the human scalp graft/ Prkdc(scid) (SCID) mouse system is characterized by a TH1 response. Clin Immunol. 2003;106(3): Tanyasiri K, Hira K, Mitsuishi K, Ueki R, Sekigawa I, Ogawa H. Interleukin-16 in patients with alopecia areata. J Dermatol Sci. 2005;37 (1): Kim JH, Lee TH. Effects of Bojungikgitang on Starvation stress in Mice. The Korean Journal of Oriental Medical Prescription. 2006;14(1): Kim SM, Sim SY, Byun HS, Kim KJ. The Effect of Bojungikgi-tang in mouse model of Allergic rhinitis. The Journal of Oriental Ophthalmology & Otolaryngology & dermatology. 2005;18(3): Baek TH, Jee HC. Study on effect of Bojungikgitang affecting lactate tolerance and recovery rate. Journal of Oriental Rehabilitation Medicine. 1998;8(1): Park SD, Seo GS, Park WH. Effects of Bojungikgitang, Soeumin Bojungikgi-tang and its component groups on Antioxidant Activities in alloxaninduced diadetic rats. Kor. J. Herbology. 2001;16 (2): Kim JS, Sim SW, U YS, Nam HJ, Kim KJ. The Effect of Bojungikgi-tang on the NAsal tissue in allergic rhinitis of ovalbimin-inhalation Rats. Journal of the oriental medical surgery, ophthalmology & otolaryngology. 2004;17(1): Eun JS, Choi H, Song JM. Effect of Bojungikgitang on the Specific immune Response in BALB/c Mice. Korean J. Oriental Physiology & Pathology. 2002;16(5): Gong TX. Wanbinghuichun. Seoul:Haenglim publishing company. 1972; Heo J. Dongeuibogam. Seoul:Namsandang ;202-3, Chen SW. Taipinghuiminhejijufang. Beijing:People s Medical Publishing House. 1985;179-80, Yang YG. Huangdineijingsuwenyijie. Seoul:Seongbosa 1980;5-10,93-4,337, Yang YG. Huangdineijinglingshuyikjie. Seoul: Seongbosa. 1980;104, Chao YF. Zhubingyuanhoulun. Beijing:People s Medical Publishing House. 1983; Lim SBN, Choi KD, Kim SK. The Oriental and Western Medical Study of Alopecia. The Journal of Jeahan Oruental Medical Academy. 1999;4 (1): Otomo S. new hair growth drugs and their nonclinical evaluation methods. Hyangjanghwoiji. 1997;21: Kagoura M, Morohashi M, Ogita Z. Animal models for the study of alopecia. Hyangjanghwoiji. 1997;21:

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