Pulmonary gastrointestinal and urogenital pharmacology
The inductive effect of ginsenoside F2 on hair growth by altering the WNT signal pathway in telogen mouse skin

https://doi.org/10.1016/j.ejphar.2014.02.024Get rights and content

Abstract

This study was conducted to confirm the possibility of using minor ginseng saponin F2 by oral administration on hair anagen induction effects. The signaling pathway and anagen induction effect of ginsenoside F2 were investigated and compared with finasteride on the effect of hair growth induction. The cell-based MTT assay results indicated that the proliferation rates of HHDPC and HaCaT treated with F2 significantly increased by 30% compared with the finasteride-treated group. A western blot study showed that the expression of β-catenin Lef-1 and DKK-1 increased by 140, 200% and decreased by 40% in the F2-treated group, respectively compared to that of finasteride-treated group. C57BL/6 mice were subjected to the same treatments. The hair growth promotion rates were compared with groups treated with finasteride, which was 20% higher in the F2-treated group. Tissue histological analysis results showed the number of hair follicles, thickness of the epidermis, and follicles of the anagen phase which increased in the F2-treated group, compared with the finasteride-treated groups. Moreover, the effect of F2 on hair growth was confirmed through the immunofluorescence (IF) methods indicating the expression aspect of Wnt signal pathway-related factors in the tissue of C57BL/6 mouse. Our results considered the expression increase in β-catenin, Lef-1 which was suggested as a major factor related to the development and growth of hair follicle and the decrease in DKK-1 when entering catagen by F2. As the data showed, F2 might be a potential new therapeutic source for anagen induction and hair growth through the Wnt signal pathway.

Introduction

Hair growth appears through the compositive interaction of the epithelial cell and dermal cell of the hair follicle. Based on that interaction, hair growth cycle is composed of three stages: a growing phase (anagen), a regressing phase (catagen), and a resting phase (telogen) (Buhl et al., 1990, Paus and Foitzik, 2004). Many studies have been conducted about the mechanism and substance of the hair cycle as well as the development of medicine for the treatment of hair growth and anagen induction (Stenn and Paus, 2001).

The cellular signaling system of the hair follicle cell known for hair growth and anagen induction is related to Wnt signaling and many growth factors are secreted in the hair follicle cell (Krause and Foitzik, 2006). Wnt signaling takes a significant part in anagen phase with regard to prosperous cell proliferation (Schmidt-Ullrich and Paus, 2005). β-catenin is the transcription factor of Wnt signaling expression in the hair stem cell-related cell cycle and promoting hair growth (Närhi et al., 2008). While β-catenin moves inside the nucleus, it would be combined with the lymphoid enhancer binding factor (Lef)/T-cell factor (Tcf) and especially combined with Lef-1 to activate Wnt signaling pathway (Wang et al., 2012). The combined β-catenin and Lef-1 would affect keratinocyte, morphogenesis of the hair follicle and the hair cycling (Niemann et al., 2002).

On the contrary, the Dickkopf-1 (DKK-1) is known as the antagonist of Wnt/β-catenin signaling pathway (Bafico et al., 2001). Opposed to Wnt signaling, DKK-1 was expressed during the catagen stage and related to transition anagen to catagen of the hair cycle (Kwack et al., 2012). Thus, the Wnt signaling pathway study is accompanied by the study about the induction anagen of hair cycle and the mechanism effect.

Finasteride, which was developed by Merck, is a substance that inhibits the activation of 5α-reductase affecting androgen metabolism (Drake et al., 1999). 5α-reductase prevents the conversion of testosterone to dihydrotestosterone (DHT), which is active in metabolite (Chen et al., 1996). Also, finasteride was known that it had hair loss preventive activity by inhibiting 5α-reductase activity. Recently, there were studies finasteride has the effects of hair growth by increasing anagen follicle in vivo and clinical study (Dhanotia et al., 2011, Sawaya et al., 2002, Van Neste et al., 2000).

The pharmacological effects of Panax ginseng were very diverse, like anti-aging, antioxidant effect (Liu et al., 2003), hair growth effect of Rb1, Rd (Cai et al., 2009), and 5α-reductase inhibiting effect of Ro (Kurata et al., 2012), anti-cancer effect (Wang et al., 2006), immune regulatory function (Lim et al., 2004), anti-inflammatory effect (Yim et al., 2005) have been reported. Also there were reports, ginsenoside Rg1 had estrogen‐like effects and Ro had anti-androgen effects, which was related to the hair re‐growth of androgenic alopecia (Chan et al., 2002).

The most important pharmaceutical active in Panax ginseng revealed a structure divided into about 40 species of saponin. Major saponin Rb1 is hydrolyzed by intestinal microorganism in the body and changed to minor saponin F2 (Ko et al., 2007). The study on the effect of F2 was mainly conducted for anti-cancer activity (Mai et al., 2012, Shin et al., 2012) while the study about hair growth was not conducted.

C57BL/6 mouse is a widely used model because its genetic characteristics are well known (Seiberg et al., 1997). The method used to evaluate hair growth effect involves determining the degree of transition from telogen follicle to anagen follicle. The regular hair period of the C57BL/6 mouse, contrary to that of humans and telogen, would hold out from 7 weeks to 16 weeks. This means longer telogen compared to other animal models. Thus, it is appropriate to observe the effect of transition from telogen follicle to anagen follicle (Paus et al., 1989).

This study was conducted to confirm the possibility of oral administration of F2, a minor saponin of ginseng for hair growth. F2 was compared to finasteride on the effect of hair growth induction. The signaling pathway and anagen induction effect of ginsenoside F2 were investigated by hair cell, C57BL/6 model, and the study mechanism including the expression of β-catenin Lef-1 and DKK-1.

Section snippets

Materials

The mesenchymal stem cell (MSC) medium kit came from Sciencell (Carlsbad, USA). Fetal Bovine Serum (FBS) was obtained from Gibco-BRL (Gaithersburg, MD, USA). RPMI-1640, antibiotics and trypsin were purchased from Cambrex (Walkersville, MD, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RIPA lysis buffer, carboxymethylcellulose (CMC), dimethyl sulfoxide (DMSO), Finasteride, Hematoxylin and Eosin stain and reagents without indication were purchased from Sigma-Aldrich

The effect of hair-related cell proliferation by ginsenoside F2

The rate of cell proliferation was confirmed to determine the effect of hair-related cell proliferation of F2 using HHDPC and HaCaT through MTT assay.

HHDPC, which had been cultured for 24 h without any treatment of sample, was set as a control group. After F2 was treated in concentrations of 0.01, 0.1, 1, and 10 μM, the 0.2 μM of finasteride was treated as a comparative control group cultured for 24 h and the rate of cell proliferation through MTT assay. When F2 was treated with the concentration

Discussion

This study verified the effect of ginsenoside F2 on hair anagen induction and hair growth by altering the WNT signal pathway via comparison with finasteride.

The cell-based MTT assay results indicated that the proliferation rates of HHDPC and HaCaT treated with F2 significantly increased by 30% compared with the finasteride-treated group (Fig. 1). For the mechanism, which studied the effect of F2 on anagen induction and hair growth, the expression aspect of β-catenin, Lef-1 and DKK-1, which are

Acknowledgments

This work was carried out with the support of Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET, 810006033SB110), Republic of Korea.

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