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Lab. Anim. Res. 2010: 26(1), 55-62 Effect of Sodium Silicate on Hair Growth in C57BL/6 Mice Jin-Joo Hue 1, Bum-Ki Jo 1, Bong Su Kang 1, Jun-hyeong Kim 1, Sang Yoon Nam 1, Young Won Yun 1, Jong Soo Kim 1, Jae-Hwang Jeong 2, Sang-Hwa Lee 3, Jun Bae Ahn 4 and Beom Jun Lee 1 * 1 College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, Korea 2 Department of Biotechnology and Biomedicine, Chungbuk Province College, Okcheon, Koea 3 Department of Food and Nutrition, Seowon University, Chungju, Korea 4 Department of Food Service Industry, Seowon University, Chungju, Korea A promoting effect of on hair regrowth was investigated using an animal model of C57BL/6 mice. There were four experimental groups including distilled water (DW, a negative control), 5% minoxidil (MXD, a positive control), 50%, and 100% solution. The animals were shaved with an electric clipper and then test solutions applied daily with a volume of 0.2 ml per to the dorsal skin of mice for 3 weeks. Body weight and food and water consumption were measured weekly. Photographs of hair regrowth were taken at experimental day 0, 4, 7, 10, 14, 17, and 21. Activities of alkaline phosphatase and γ-glutamyl transpeptidase as well as expressions of growth factors were also determined in the dorsal skin of mice. The animal body weight were not significantly changed among the experimental groups. The MXD and accelerated hair regrowth compared with DW. The elongation of hair follicles were evidently observed in MXD and 50 or 100% groups. MXD significantly increased γ-glutamyl transpeptidase at day 14, compared with DW (P<0.05). But the activities of alkaline phosphatase and γ-glutamyl transpeptidase were not significantly increased in groups, compared with DW. The expression of epidermal growth factor was significantly increased in MXD and groups, compared with DW (P<0.05). The expression of vascular endothelial growth factor was not significantly changed by MXD or treatments. The expression of transforming growth factor (TGF)-β1 was clearly decreased in MXD and groups, compared with DW. These results indicate that may have a hair growth-promoting activity and it can be used for treatment of alopecia or boldness in humans. Key words:, hair growth, ALP, γ-gt, EGF, VEGF, minoxidil (Received 24 February 2010; Revised version received 10 March 2010; Accepted 13 March 2010) yw, w wš, j ƒ w wwš. û û k ƒ ƒw w š. û yw t w ƒ š. k p, ³x, y w w. l (hair follicle) x» yw,»(anagen),»(catagen), {»(talogen), kv» (exogen) 4 ù.» *Corresponding author: Beom Jun Lee, Department of Veterinary Public Health, College of Veterinary Medicine, Chungbuk National University, 48 Gaeshin-dong, Heungduk-gu, Cheongju 361-763, Korea Tel: +82-43-261-3357 Fax: +82-43-271-3246 E-mail: beomjun@cbu.ac.kr l s (apoptosis) ƒ w š, {» ƒ l j»ƒ (Burton and Marshall, 1979; Stenn, 1991). v ƒ k xk, k, {» k ƒ ƒ k (Kaufman, 1996). k» z û w ƒ w x k û k û x k š š x k (Muller-Rover et al, 2001). k š, e ƒ ƒ š e x p ƒ š (Kaufman, 1996; Muller, 2001). û x k ƒ w w l m l ù, x l m l w l m l ƒ 55

56 Jin-Joo Hue et al. w, p w m l ƒ k ùkû (Hamada and Suzuki, 1996). x w (minoxidil, MXD) vù l (finasteride)ƒ t t (FDA) t. šx e w x y ù š š, z w» x ¾ y x x y m w œ ƒ e (Potassium channel opening) z w ƒ š. w vù l û y w z 5-α-reductase y j (Hattori and Ogawa, 1983; Hamada and Suzuki, 1996)., k x x w in vivo sƒ l s w in vitro sƒ š. ù in vitro w sƒ l ü s y l v y, š x y w e w š. ƒ yw sƒ mw k z w» w ¾ x w z sƒ x š. w yw, w x w ƒ ù,» k ƒ y w š (Schwarz, 1978; Meister and Anderson, 1983; Nielsen, 1991; Seaborn and Nielsen, 2002). yw z γ-glutamyl transpeptidase (γ-gt) s w z ke (glutathione) s mw rp, w p»(anagen)» ƒ t š (Schwarz, 1978). w, transforming growth factor β (TGF-β) s,, y s xk w w. l» w TGF-β1 ƒw l w š šw z l» nw», {» ƒ l» w w w, w w š š. w l k negative regulator, catagen talogen phase ú hair regression w k. ³ ü» w 1-10 ppm ƒ, ƒy v lë (hair cuticle) d vjpj(epicuticle)d (Smith, 1993; Wickett et al., 2007). ³ v w x w w w ùkþ š w (Smith, 1993; Wickett et al., 2007). w ü w x w» w, y e w š (Barel et al., 2005; Wickett et al., 2007). x ³ Sodium Metasilicate ( ) silica (SiO 2 ) w k» xk y t Silicateƒ w z» silica w. w k pw x 13-18%, GRAS (generally regarded as safe) t ƒ» w (Elmore, 2005). ƒ š w ù w w k C57BL/6 w z minoxidil w sƒ w. l w œ x (D.W.) 5% MXD w. 5% MXD w û ew x t w w. x DW sy k 100% 50% w. lò 6 ƒ ù f C57BL/6 w 1 x k z, 7 x w. (50±10%) (23±3 o C) w f 5 w. w. ƒƒ 18 72 x w. f ( ) Ÿ f w. x w x z(iacuc) w š ww. x ¾ w ú l 1 1z d. x s l d w x ¾ w ( z2 ) d w w.

Ðg y» {» w» w»» w l w. v ƒw, ù kü. z 2 1z 21 w. l ¾ (0.2 ml) x w z w š s g., 5% (MXD), š x sw. ¼j x z 0, 4, 7, 10, 17, 14, 21 l k y w» w l w z w, l k q w.» Nikon D70 (Tokyo, Japan) w. ƒ z ƒ ƒ w 0-19% ( ), 20-39% (±), 40-59% (+), 60-79% (++), 80-100% (+++) q w. j x21 s w z 10% s w š w. g k w q v s w z, j m (HM-315 model, Microm Co., Germany) w 4 µm r w g q v w. x m w Ÿ wx (Olympus, Tokyo, Japan) l w y w. n m Ð x21 v w þ g. (1 g) v w 4 0.1 M š ³ y g. 12,000 rpm 20 w z w z y d w. Alkaline phosphatase (ALP) y z d (GSCC) š γ- glutamyl transpeptidase (γ-gt)y p-nitrophenolphosphate (PNPP)-DEAV w d w. d yw» Hitachi-747 w w Daichi w,»k w x» w. Effects of sodium silicate on hair growth in mice 57 š k z 2 z wš z w w ƒƒ well. 2 z w z q w š ƒƒ well d w. VEGF EGF d ƒƒ Mouse VEGF immunoassay kit Mouse EGF immunoassay kit (R&D SYSTEMS, USA) w w. œw w w. 5('β8FTUFSOCMPUBOBMZTJT v protein prep (intron Biotechnology, Seongnam, Korea) ³ y w Bradford protein assay (Bradford MM, 1976) w. 30 µg loading w z, 12% SDS PAGE» w z, PVDF membrane (Hoefer Inc., San Francisco, USA) transfer w. 5% BSA blockingwš, TGF-β1 antibody (1:500, Santa Cruz Biotechnology, Inc., California, USA) β-actin (1:2000, Cell Signalling Technology Inc., Boston, USA) 1 antibody 3% BSA w 4C o overnight w. z, 2 antibody anti-rabbit horseradish peroxidase-conjugated antibody (1:4000, Santa Cruz Biotechnology, Inc., California, USA) anti-mouse horseradish peroxidase-conjugated antibody (1:4000, Santa Cruz Biotechnology, Inc., California, USA) k shaker 1 k z, ECL w, Western x x w. j l s³±t t wš, x sƒ one-way analysis of variance(anova), Turkey HSD test n P<0.05 level sƒw. m C57BL/6 x» ƒw x n (Figure 1). y x ƒ. Vesicular epidermal growth factor (VEGF) Epidermal growth factor (EGF) ƒƒ py w ww z d w. x ƒƒ well m 1 z l ƒƒ sw. x 7 ¾ l. 10 z

58 Jin-Joo Hue et al. γ-gt z y d w Figure 5 ùkü. γ- GT z y MXD e 2 z w ƒw (P<0.05), x ³ e ù, 2 z ƒw w (Figure 5). Figure 1. Change in body weight of C57BL/6 mice for 3 weeks. The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. D.W: distilled water, MXD: 5% minoxidil. Date represent mean±sd (n=5). ƒ ƒw w (Figure 2). w e 10 z l l» w. x 14 z MXD 80% l w. x 14 l z (Figure 2). w z w Table 1 ùkü. j m x v l l (hair root) w e z w» w sw z w z w w. x s w l j»ƒ š ƒw. ù»k w (Figure 3). w MXD l ¼ x (Figure 3). "-1n m m ALP z hair growth cycle x x t, v z y d w. x n 1-3 z ALP z y d w Figure 4 ùkü. ALP z y SiO 1-2 z w ƒw ù (Figure 4). MXD 1-2 z ALPz y ƒ w ù y ùkù (Figure 4). γ(5n m m γ-gt z» x x t, v z y d w. x n 1-3 z &QJEFSNBMHSPXUIGBDUPS &('m Epidermal growth factor v w. Figure 6 w n ³ n ƒ (P<0.05). 7FTJDVMBSFQJEFSNBMHSPXUIGBDUPS 7&('m VEGF v x w. ³ n VEGF y ƒw. ù w y w m (Figure 7). 5('β l TGF-β1 x w, w 5% MXD x x ƒ w ƒ, TGF-β1 x MXD w x n» ù, w y w (Figure 8). y ù ³x y k ù š û eƒ ƒwš. j k w ù j e w. m 100,000 l, ƒ l y s³ 20 l w (Rebora, 2004). s³ k w 100ƒ. k ù w w š, k šm w û ƒ ƒw y., k wš j e wš, w š. x, z ƒ t w š w C57BL/6 w z, 50% 100% x ( ) 5% minoxidil z w. C57BL/6 ƒ š w ù w w k (Muller-

Effects of sodium silicate on hair growth in mice 59 Figure 2. Photograph of hair regrowth in C57BL/6 mice. The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. The photographs were taken day 0, 4, 7, 10, 14, 17, and 21. DW: distilled water, MXD: 5% minoxidil. Rover et al, 2001). z 7 f C57BL/6 w, w z {» k y w z x w. 3 x sw, x 10 z 5% MXD, 50%, 100% DW w z. x 2 z 80% z š, ù z. x 3 z MXD w z., jš, z MXD z w ùkù. x 5% MXD ƒ ùkùù,. x z w yw x mw z š w. H&E, 5% MXD, 50%, 100% w l ƒ ƒ wš, l ¾ w ƒw y w. x MXDƒ {» k l

60 Jin-Joo Hue et al. Table 1. Comparison of hair regrowth in C57BL/6 mice for 3 weeks Groups Weeks 50% 100% D.W. MXD 1 - - - - 2 - ++ Û + 3 + +++ +++ +++ The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. Hair growth was evaluated by the scoring index: 0-19% (-), 20-39% (±), 40-59% (+), 60-79% (++), 80-100% (+++). D.W: distilled water, MXD: 5% minoxidil.» j z ƒ y w, MXD ƒ {» l» j z y w. ³ (Si) ü w, m w. w ü w x w» w (Barel et al., 2005; Wickett et al., 2007). y v ƒ w ù colloidal silicic acid n e z m k v Ì w š, v ̃ É y w (Lassus, 1993). ³ xk ch-osa (Choline-stabilized orthosilicic acid) ³ xk OSA (orthosilicic acid) g», w ch-osa n w, 20 z v p m x w z ùkü (Barel et al., 2005). x z mw z w. ALP z y MXD 1-2 z w ƒw. ALP x w z, anagen» v ƒ É ALP ƒƒ ùkù š w (Hamada and Suzuki, 1996). z v x œ w yw w z x w y ƒ ùký š (Meister and Anderson, 1983). v (pilosebaceous unit) w ALPy ùk üš, xk ALPy yƒ ùkù š w (Handjisiki et al., 1994). x z γ-gt z y MXD 2 z w ƒw, ƒw w. γ-gt» x x t»» y ùkü š, {» j w. v γ-gt z l l (hair root) ül (hair root) Figure 3. Histological change of hair follicles in the dorsal skin of C57BL/6 mice (H&E, 40). The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. D.W: distilled water, MXD: 5% minoxidil.

Effects of sodium silicate on hair growth in mice 61 Figure 4. Alkaline phosphatase (ALP) changes in the dorsal tissue of C57BL/6mice. The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. D.W.: Distilled water, MXD: Minoxidil. Data represent mean±sd (n=5). Figure 6. Activities of Endothelial growth factor (EGF) in the dorsal tissue of C57BL/6 mice. The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. D.W.: Distilled water, MXD: 5% Minoxidil. Data represent mean±sd (n=5). *Significantly different from the D.W. group (P<0.05). Figure 5. γ-glutamyl transpeptidase (γ-gt) changes in the dorsal tissue of C57BL/6 mice. The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. DW: Distilled water, MXD: Minoxidil. Data represent mean±sd (n=5). *Significantly different from the D.W. group (P<0.05). ùkùš v l x w š, v l x ùkù š w (Kawabe et al., 1994). Figure 7. Activities of vascular endothelial growth factor (VEGF) in the dorsal tissue of C57BL/6 mice. The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. D.W.: Distilled water, MXD: 5% Minoxidil. Data represent mean±sd (n=5). EGF VEGF x s w d e ùkþ. EGF l v w e v w e v w Figure 8. Expression of TGF-β in the dorsal skin of C57BL/6 mice. The animals were shaved with electric clipper and the test compounds applied with 0.2 ml per to the dorsal skin of mice for 3 weeks. D.W.: Distilled water, MXD: 5% Minoxidil.

62 Jin-Joo Hue et al. e (Chapman and Hardy, 1998). x EGF y w MXD n x n ƒ. x ³ k. VEGF v x w. x VEGF y w MXD n x n ùkû. x ƒ VEGF y ƒ g z ùký. w TGF-β1 ƒ ƒ y catagen talogen» ƒ» x ƒ MXD w, y ƒ ù. v negative regulator TGF-β1 x y w, x w v x ƒ û y w., ³ MXD C57BL/6 w öe γ-gt, ALP, VEGF, EGF ƒ jš, negative regulator TGF-β1 x mw» l j w š. q x l(ric) v y w ey t l w. k Barel, A., Calomme, M., Timchenko, A., De Paepe K., Demeester, N., Rogiers, V., Clarys, P. and Vanden Berghe, D. (2005) Effect of oral intake of choline stabilized orthosilicic acid on skin, nails and hair in women with photodamaged skin. Arch. Dermatol. Res. 297, 147-153. Burton, J.L. and Marshall, A. (1979) Hypertrichosis due to minoxidil, Br. J. Dermatol. 101, 593-595. Carlberg, I. and Mannervik, B. (1975) Purification and characterization of the flavoenzyme glutathione reductase from rat liver. Bio. Chem. 250, 5475-5480. Chapman, R.E. and Hardy, M.H. (1988) Effects of intradermally injected and topically applied mouse epidermal growth factor on wool growth, skin and wool follicles of merino sheep. Aust. J. Biol. Sci. 41, 261-268. Chase, H.B., Rauch, H. and Smith, V.W. (1951) Critical stages of hair development and pigmentation in mouse. Phyiol. Zool. 24, 1-8. Deyoung, L.M., Richards, W.L., Bonzelet, W., Tsai, L.L. and Boutwell, R.K. (1978) Localization and significance of γ- glutamyltranspeptidase in normal and neoplastic mouse skin. Cancer. Res. 38, 3697-3701. Elmore, A.R. (2005) Final report on the safety assessment of potassium silicate, sodium metasilicate, and sodium silicate. Int. J. Toxicol. 24, 103-117. Hamada, K. and Suzuki, K. (1996) Evaluation of biochemical indices as a hair cycle marker in C3H mice. Exp. Anim. 45, 251-256. Handjisiki, B.K., Eichmuller, S., Hofmann, U., Czrnetzki, B.M. and Paus, R. (1994) Alkaline phosphatase activity and localization during the murine hair cycle. Br. J. Dermatol. 131, 303-310. Hattori, M. and Ogawa, H. (1983) Biochemical analysis of hair growth from the aspects of aging and enzyme activities. J. Invest. Dermatol. 10, 45-54. Kaufman, K.D. (1996) Clinical studies on the effects of oral finasteride, a type II 5α-reductase inhibitor on scalp hair in men with male pattern baldness. In Hair Research for the Next Millenium (Neste, D and Randall, V.A. ed.), pp. 363-364, Elsevier Science, Amsterdam. Lassus, A. (1993) Colloidal silicic acid for oral and topical treatment of aged skin, fragile hair and brittle nails in females. J. Int. Med. Res. 21, 209Æ215. Meister, A. and Anderson, M.E. (1983) Glutatione. Annu. Rev. Biochem. 52, 711-760. Muller-Rover, S., Handjiski, B., van der Veen, C., Eichmuller, S., Foitzik, K., McKay I.A., Stenn, K.S. and Paus, R. (2001) Comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J. Invest. Dermatol. 117, 3-15. Nielsen, F.H. (1991) Nutritional requirements for boron, silicon, vanadium, nickel and arsenic: current knowledge and speculation. Faseb. J. 5, 2661-2667. Rebora, A. (2004) Pathogenesis of androgenetic alopecia. J. Am. Acad. Dermatol. 50, 777-779. Schwarz, K. (1978) Significance and functions of silicon in warmblooded animals. Review and outlook. Biochemistry of silicon and related problems. (Bendz, G. and Lindquist. I. ed.), pp. 270-330, Plenum Press, NewYork. Seaborn, C.D. and Nielsen, F.H. (2002) Silicon deprivation decreases collagen formation in wounds and bone, and ornithine transaminase enzyme activity in liver. Biol. Trace. Elem. Res. 89, 251-261. Smith, BL. (1993) Analysis of hair element levels by age, sex, race, and hair color. In Trace elements in man and animals- TEMA8 (Anke, M., Meissner, D. and Mills, C.F. ed.), pp 1091-1093, Kluwer, NewYork. Stenn, K.S. (1991) The molecular and structural biology of hair: introduction. Ann. NY. Acad. Sci. 642, 11-13. Sundberg, J.P., Cordy, W.R. and King. L.E.Jr. (1994) Alopecia areata in aging C3H/HeJ mice. J. Invest. Dermatol. 102, 847-856. Wickett, R.R., Kossmann, E., Barel, A., Demeester, N., Clarys, P., Vanden Berghe, D. and Calomme, M. (2007) Effect of oral intake of choline-stabilized orthosilicic acid on hair tensile strength and morphology in women with fine hair. Arch. Dermatol. Res. 299, 499-505.