Article pubs.acs.org/JAFC
Stimulatory Effect of Brazilian Propolis on Hair Growth through Proliferation of Keratinocytes in Mice Shota Miyata, Yozo Oda, Chika Matsuo, Haruto Kumura, and Ken Kobayashi* Laboratory of Dairy Food Science, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan ABSTRACT: Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation. KEYWORDS: propolis, hair growth, keratinocyte, epidermis, anagen induction
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induction.20 Thus, the active proliferation of hair epithelial cells is required for hair bulb formation during the telogen−anagen transition and for hair shaft elongation during middle and late anagen. Propolis has been reported to facilitate reepithelialization during cutaneous wound healing by stimulating the proliferation of keratinocytes.21,22 These stimulatory effects of propolis on epithelial cell proliferation have also been observed in corneal and oral epithelium.23,24 Since hair growth requires epithelial cell proliferation, propolis may be able to stimulate hair growth; however, the influence of propolis on hair growth is yet unknown. In this study, we employed two types of in vivo mouse models: the gentle anagen induction model, which is useful for estimating the anagen induction ability of a given treatment,16,25 was prepared by shaving back skin during the telogen phase, and the forced anagen induction model was prepared by depilating back skin, using wax, during the telogen phase. Depilation immediately induces synchronized entry into the anagen phase.26 Therefore, depilated mice can be used to compare hair growth related activity, under different treatment conditions, at corresponding stages of the hair follicle cycle. We also prepared an organotypic culture model, using depilated skin at the anagen stage, to investigate the dose-dependent effects of propolis on the proliferation rate of matrix keratinocytes. These experiments enabled us to evaluate the potential of propolis as a treatment to promote hair growth.
INTRODUCTION Propolis is a natural honeybee hive product that is formed from resinous plant substances and honeybee secretions.1 Honeybees use propolis as a physical sealant for small, unwanted gaps in the hive that result from its waxy nature. Propolis is also an antibacterial agent that inhibits fungal and bacterial growth in the hive.2,3 Interestingly, ancient people used propolis for medicinal purposes, including as an antitumor therapy and as a treatment for inflammation and wounds.4,5 Propolis contains multiple physiologically active substances, which have various medical effects. For example, caffeic acid, chlorogenic acid, p-coumaric acid, kaempferide, and kaempferol exhibit anti-inflammatory activity.6−8 Drupanin, kaempferol, and baccharin are potent tumor-suppressive components of propolis.9−11 In addition, caffeic acid induces keratinocyte differentiation by activating PPAR-α.12 Caffeic acid phenethyl ester (CAPE) improves cutaneous wound healing by exerting anti-inflammatory and antioxidant effects.13,14 Furthermore, topical application of chlorogenic acid facilitates reepithelialization during cutaneous wound healing.15 These components of propolis may also affect the growth of hair, a keratinized, epithelial structure. Hair follicles (HFs) are tubular pouches in the skin in which active proliferation of hair epithelial cells (also known as matrix keratinocytes) gives rise to hair growth.16 HFs continuously cycle through anagen (growth), catagen (involution), and telogen (resting) stages.17 In early anagen, quiescent HFs initiate invagination into the subcutaneous fat layer, with actively proliferating hair epithelial cells forming hair bulbs and outer root sheath (ORS) structures.18 In middle and late anagen, hair matrix keratinocytes in the hair bulb undergo active proliferation and migration, as well as simultaneous differentiation into hair shafts.16 However, upon entry into the catagen stage, hair matrix keratinocytes stop proliferating, and hair bulb regression occurs.19 Finally, HFs enter telogen, a quiescent state without hair bulbs, until the next anagen © XXXX American Chemical Society
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MATERIALS AND METHODS
Propolis. Brazilian propolis ethanol extract was obtained from Yamada Bee Company, Inc. (catalog #110711, Okayama, Japan). The analysis of the ethanol extract of Brazilian green propolis was Received: April 8, 2014 Revised: October 28, 2014 Accepted: November 22, 2014
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dx.doi.org/10.1021/jf503184s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
conducted using high-performance liquid chromatography (HPLC), carried out with a Sunniest-C18 column (150 × 4.6 mm, 5 μm; ChromaNik, Osaka, Japan). Analytes were dissolved in a methanol (CH3OH) gradient solvent system (5 min hold at 15%, 5−50 min, linear gradient of 15−40%, and 50−160 min, linear gradient of 40− 80%) in 0.1% trifluoroacetic acid in water. The flow rate was 1.0 mL/ min, and analytes were detected at 275 nm. All analyses by HPLC were carried out at the Institute for Bee Products & Health Science, Yamada Bee Company, Inc. Shaving and Depilation of Mice. Female C57BL/6N mice were purchased from Japan SLC (Shizuoka, Japan) and were maintained under conventional conditions at 22−25 °C. At 8 weeks of age, when the back skin was at the telogen stage, mice were anesthetized by intraperitoneal administration of sodium pentobarbital and were then shaved or depilated. For the gentle anagen induction experiments, the back skin of mice (n = 10), under anesthesia, was shaved with an electric shaver, and ethanol-extracted propolis, diluted 10-fold in 99.5% ethanol, was topically applied to the shaved skin at 100 μL/d for 3 weeks. Control mice (n = 10) were similarly shaved, and 99.5% ethanol was used in place of diluted propolis. The treated skin regions of all 20 mice were excised and fixed in 4% formaldehyde in phosphate buffered saline (PBS). For the forced anagen induction experiments, the back hair of anesthetized mice was shortened with an electric shaver, and then club hairs were removed from the back skin using depilatory wax. Eight days after depilation, 10% ethanol-extracted propolis was topically applied to the dorsal skin at 100 μL/d for 3 d (n = 6). Control mice (n = 5) were depilated and then treated with ethanol only. The treated skin regions were excised and fixed in 4% formaldehyde in PBS. All experimental procedures in this study were approved by the Animal Resource Committee of Hokkaido University (approval number: #09-0125) and were conducted in accordance with Hokkaido University guidelines for the care and use of laboratory animals. Identification of Anagen Induction in Shaved Mice. In shaved mice, the hair cycle progresses from the caudal end toward the cranial end of the back skin, and melanin granules are produced in the hair bulb during middle and late anagen.16,27 Therefore, we visually identified middle anagen induction based on the degree of melanization of the caudal regions of the back skin in shaved mice. Antibodies. The following antibodies were used as primary antibodies for immunofluorescence staining: mouse antibodies against pan-keratin (Sigma-Aldrich, St. Louis, MO, U.S.A.), cytokeratin 10 (CK10; Progen, Heidelberg, Germany), cytokeratin 14 (CK14; SigmaAldrich), cytokeratin 15 (CK15; Thermo Scientific, Fremont, CA, U.S.A.), and 5-bromo-2′-deoxyuridine (BrdU; BioLegend, San Diego, CA, U.S.A.). Similarly, rabbit antibodies against perilipin (Cell Signaling Technology, Danvers, MA, U.S.A.), laminin (LSL, Tokyo, Japan), and zonula occludens-1 (ZO-1; Life Technologies, Gaithersburg, MD, U.S.A.), a rat antibody against Ki-67 (DakoCytomation, Glostrup, Denmark), and a goat antibody against versican (R&D Systems, Minneapolis, MN, U.S.A.) were also used as primary antibodies. The following antibodies were purchased from Cell Signaling Technology and were used as secondary antibodies: Alexa Fluor 488conjugated goat anti-rabbit, Alexa Fluor 488-conjugated and Alexa Fluor 546-conjugated goat anti-mouse, Alexa Fluor 488-conjugated donkey anti-mouse, Alexa Fluor 546-conjugated donkey anti-goat, and Alexa Fluor 488-conjugated and Alexa Fluor 546-conjugated goat antirat antibodies. Immunofluorescence Staining. After fixation (as described above), surgically excised regions of skin were embedded in paraffin and sliced into 5 μm sections. The sections were deparaffinized and rehydrated. After they were heated in a microwave oven in antigen retrieval buffer consisting of 10 mM Tris-HCl (pH 9.0) and 0.5 mM ethylene glycol tetraacetic acid (Sigma-Aldrich), the sections were incubated in blocking solution (5% bovine serum albumin in PBS) to preclude nonspecific interactions. The sections were then incubated overnight at 4 °C in various primary antibodies diluted in blocking solution. After the sections were washed with PBS containing 0.05%
Tween 20, they were exposed to secondary antibodies diluted in blocking solution for 1 h at room temperature. Controls for autofluorescence and the unspecific binding of the secondary antibody were treated in the same manner, but without primary antibodies. Images of the stained sections were obtained using a confocal laserscanning microscope (TCS SP5; Leica, Mannheim, Germany). Measurement of Matrix Cell Migration Distance. In middle and late anagen, hair matrix keratinocytes actively proliferate and then migrate toward the upper part of the HFs, leading to hair shaft elongation.19 Therefore, to evaluate hair growth activity, proliferating matrix keratinocytes were labeled by intraperitoneal injection of BrdU into mice (1 mg/mouse in 100 μL of PBS, Sigma-Aldrich). Mice were sacrificed 24 h after injection, and skin samples were excised and fixed (as described above). The migration distance between the bottom of the hair bulb and the furthermost BrdU-positive cells in the hair shaft was measured in 19 sagittally sectioned HFs from 6 propolis-treated and 5 vehicle-treated (control) mice. Skin Organ Culture. Organ culture was performed using depilated, middle anagen stage back skin to investigate the influence of propolis on the proliferation of hair matrix keratinocytes. The back skin was harvested from mice 5 d after depilation and washed with RPMI-1640 medium containing penicillin and streptomycin. Each skin sample was placed on a cell culture insert with a pore size of 0.2 μm and was then cultured for 3 d in RPMI-1640 containing 10 μg/mL insulin, 10 ng/mL hydrocortisone, and either a low concentration (0.005%) of propolis (n = 6), a high concentration (0.05%) of propolis (n = 6), or ethanol as vehicle (n = 6). After 3 d in culture, skin samples were fixed in 4% formaldehyde in PBS, and paraffin sections were prepared. To measure the proliferation rate of hair matrix keratinocytes, paraffin sections were assayed by immunofluorescence with anti-Ki-67 antibodies and stained with 4′,6-diamidino-2-phenylindole (DAPI). The Ki-67-positive cells were counted, and the percentages of proliferating cells were calculated by dividing this count by the total number of DAPI-stained matrix cells. Keratinocyte Isolation and Culture. Keratinocytes were isolated from the back skin of neonatal C57BL/6N mice 1 d after birth, as follows. Excised regions of back skin were immersed in 0.1% collagenase type III (Worthington Biochemical, Freehold, NJ, U.S.A.), and the epidermis and dermis were separated with fine forceps. Keratinocytes in the separated epidermis were collected by washing with keratinocyte growth medium (CELLnTEC Advanced Cell Systems, Bern, Switzerland), and the cell suspension was passed through a 40 μm cell strainer (BD Biosciences, San Jose, CA, U.S.A.). The cell suspension was then centrifuged at 500g for 5 min, and the epidermal keratinocytes obtained were seeded in 96-well plates at 1 × 103 cells/well and cultured for 1 d. The medium was then replaced with new medium that lacked growth factors but contained either propolis or vehicle (ethanol). After 2 d of culture, the proliferation activity of these keratinocytes was evaluated using a WST-8 Cell Counting Kit (Dojindo, Osaka, Japan). Statistical Analysis. Statistical analysis was performed using the Analyze-It add-in (Analyze-it Software, Ltd., Leeds, United Kingdom) for Microsoft Excel (Microsoft Corporation, Redmond, WA, U.S.A.). In the case of in vitro experiments, differences between propolis and vehicle groups were analyzed using the unpaired Student’s t-test. Differences were considered significant at a p-value of
