PHYTOTHERAPY RESEARCH Phytother. Res. 28: 1088–1095 (2014) Published online 26 December 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5101

Ginsenoside Rg3 Up-regulates the Expression of Vascular Endothelial Growth Factor in Human Dermal Papilla Cells and Mouse Hair Follicles Dae Hyun Shin,1 Youn Jeong Cha,1 Kyeong Eun Yang,2 Ik-Soon Jang,2 Chang-Gue Son,3 Bo Hyeon Kim1* and Jung Min Kim3* 1

R&D Center, Somang Cosmetics Corporation, 687–14 Kozan-dong, Namdong-gu, Incheon 405-310, Republic of Korea Division of Life Science, Korea Basic Science Institute, 113 Gwahangno, Yusung-Gu, Daejeon 305-333, Republic of Korea Liver and Immunology Research Center & NAR Center, Inc., Daejeon Oriental Hospital of Daejeon University, 22–5 Daeheung-dong, Jung-gu, Daejeon 301-724, Republic of Korea 2 3

Crude Panax ginseng has been documented to possess hair growth activity and is widely used to treat alopecia, but the effects of ginsenoside Rg3 on hair growth have not to our knowledge been determined. The aim of the current study was to identify the molecules through which Rg3 stimulates hair growth. The thymidine incorporation for measuring cell proliferation was determined. We used DNA microarray analysis to measure gene expression levels in dermal papilla (DP) cells upon treatment with Rg3. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in human DP cells were measured by real-time polymerase chain reaction and immunohistochemistry, respectively. We also used immunohistochemistry assays to detect in vivo changes in VEGF and 3-stemness marker expressions in mouse hair follicles. Reverse transcription polymerase chain reaction showed dose-dependent increases in VEGF mRNA levels on treatment with Rg3. Immunohistochemical analysis showed that expression of VEGF was significantly up-regulated by Rg3 in a dose-dependent manner in human DP cells and in mouse hair follicles. In addition, the CD8 and CD34 were also up-regulated by Rg3 in the mouse hair follicles. It may be concluded that Rg3 might increase hair growth through stimulation of hair follicle stem cells and it has the potential to be used in hair growth products. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Rg3; hair growth; DP cell; gene expression profiles; VEGF; stemness marker. Abbreviations: DP, dermal papilla; H&E, hematoxylin-eosin; IHC, immunohistochemistry; SD, standard deviation; VEGF, vascular endothelial growth factor

INTRODUCTION The roots of Panax ginseng C.A. Meyer, commonly known as ginseng, have been a valuable and important folk medicine in East Asian countries (Yun, 2001). Ginseng has been documented to possess hair growth activity and is widely used to treat alopecia (Matsuda et al., 2003; Park et al., 2011). Ginsenoside Rg3 (Rg3), a product formed in the preparation of red ginseng from the crude root of P. ginseng (Kitagawa et al., 1983, 1989; Kasai et al., 1983), has been reported to promote hair growth (Matsuda et al., 2003), to stimulate blood circulation (Matsuda et al., 2000), and to inhibit both blood aggregation (Yamamoto et al., 1988) and the metastasis of tumor cells (Sato et al., 1994).

* Correspondence to: Jung Min Kim, PhD, NAR Center, Inc. & Liver and Immunology Research Center, Daejeon Oriental Hospital of Daejeon University, 22-5 Daeheung-dong, Jung-gu, Daejeon 301-724, Korea; Bo Hyeon Kim, BS, R&D Center, Somang Cosmetics Corporation, 687-14 Kozan-dong, Namdong-gu, Incheon 405-310, Korea. E-mail: [email protected] (Jung Min Kim); [email protected] (Bo Hyeon Kim)

Copyright © 2013 John Wiley & Sons, Ltd.

The DP is a discrete population of specialized dermal fibroblasts found at the base of the hair follicle (Paus and Cotsarelis, 1999), and plays a central role in hair follicle morphogenesis (Jahoda and Reynolds, 2000; Reynolds et al., 1999) and mesenchymal-epithelial interactions (Richardson et al., 2005). DP cells have been the focus of much interest because the DP not only regulates hair follicle development and growth, but is also thought to be a reservoir of multipotent stem cells (Driskell et al., 2011). On the other hand, the Rg3 stimulates hair growth, yet our understanding of its mechanism of action on human hair follicle DP cells is very limited. DNA microarray technology is a very effective tool to measure genome-wide changes in transcript levels and detect hair growth-related genes (Ishimatsu-Tsuji et al., 2010). The VEGF is not only a potent stimulator of vasodilation, microvascular hyperpermeability, and angiogenesis, but it also serves as a multifunctional growth factor for a variety of cells. Previous studies have reported that DP cells express VEGF and that VEGF acts on DP cells as an autocrine growth factor. These reports also showed that minoxidil stimulates the production of VEGF in cultured DP cells, and this led to the conclusion that minoxidil promotes hair growth through the induction of VEGF (Lachgar et al., 1998; Li et al., 2001). Received 22 October 2013 Revised 11 November 2013 Accepted 18 November 2013

GINSENOSIDE RG3 UP-REGULATES VEGF EXPRESSION

The aim of the current study was to identify the molecules through which Rg3 stimulates hair growth. Little is known about which genes are regulated by Rg3 in DP cells and genome-wide large-scale screening of Rg3’s regulatory effects has not been reported. We used DNA microarray analysis to measure gene expression levels in DP cells upon treatment with Rg3 and found significant changes in expression levels of genes involved in signal transduction pathways such as the VEGF pathway, immune defense, cell cycle control, cell structure, and motility. We focused on the changes in the mRNA and protein expression levels of VEGF in human DP cells using real-time polymerase chain reaction (PCR) and IHC, respectively. We also used IHC assays to detect in vivo changes in VEGF expression in mouse hair follicles. Finally, we investigated the expression of the stemness markers CD8, Cd34, and Ki-67 in mouse hair follicles that contain DP cells. Our results suggest that Rg3 promotes hair growth in vitro and in vivo by increasing VEGF levels and by activating CD8 and Cd34.

MATERIALS AND METHODS Materials. Ginsenoside Rg3 (95.7% purity) was purchased from ChromaDex Inc. (Santa Ana, CA, USA). Minoxidil was purchased from Sigma (St. Louis, MO, USA). Rg3 and minoxidil were dissolved in DMSO and stored at 20 °C. Culture of human dermal papillae cells. Human DP cells were cultured as described previously. Briefly, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) containing 2 mM L-glutamine, 1000 μg/mL streptomycin sulfate, 1000 units/mL penicillin G sodium, 2.5 μg/ mL amphotericin B, and 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) at 37 °C in a 5% CO2 incubator. Fourth-passage DP cells were used in all experiments. Thymidine incorporation assay. The thymidine incorporation assay for measuring cell proliferation has been previously described. Briefly, DP cells were plated in 6-well plates and grown for 12–24 h in DMEM supplemented with 10% FBS. After washing twice with phosphate-buffered saline (PBS), the cells were placed in FBS-free DMEM together with 1 μCi of 3H thymidine and varying concentrations of Rg3. Following incubation for 48 h, the cells were washed twice with PBS and once with 5% cold trichloroacetic acid. The cells were then lysed in 0.1 N NaOH and 1% SDS, and the levels of radioactivity were measured in a liquid scintillation counter (Beckman Coulter, Fullerton, CA, USA). RNA isolation. Total RNA from each sample was extracted using TRIZOL reagent (GibcoBRL, Rockville, MD, USA) according to the manufacturer’s instructions. RNA was treated with RNase-free DNase I (Promega, Madison, WI, USA) to reduce DNA Copyright © 2013 John Wiley & Sons, Ltd.

1089

contamination. Total RNA concentration and purity were determined spectrophotometrically with a 260:280 nm absorbance ratio. The integrity of RNA samples was confirmed by the appearance of distinct 28S and 18S bands of ribosomal RNA using a Bioanalyzer 2100 system (Agilent Technology, Santa Clara, CA, USA). Microarray analysis. The DP cells (1 × 106 cells) were seeded into 100-mm diameter cell culture dishes. After 24 h, the cells were treated with 10 μM Rg3 or 10 μM minoxidil for 6 h. For control and test RNAs, the synthesis of target cRNA and the hybridizations were performed using the Low RNA Input Linear Amplification Kit PLUS (Agilent Technology, Santa Clara, CA, USA) according to the manufacturer’s instructions, as described previously (Shin et al., 2013.). Briefly, each 0.5 μg sample of total RNA was mixed with the diluted spike mix and T7 promoter primer mix and incubated at 65 °C for 10 min. The cDNA master mix (5 × first-strand buffer, 0.1 M DTT, 10 mM dNTP mix, RNase-Out, and MMLV-RT) was prepared and added to the reaction mixture. The samples were incubated at 40 °C for 2 h and the reaction was stopped by heating at 65 °C for 15 min. The transcription master mix was prepared as per the manufacturer’s protocol (4 × transcription buffer, 0.1 M DTT, NTP mix, 6.4 μL of 50% PEG, RNase-Out, inorganic pyrophosphatase, T7-RNA polymerase, and cyanine 3/5-CTP). Transcription of dsDNA was performed by adding the transcription master mix to the dsDNA reaction samples and incubating the mixture at 40 °C for 2 h. Amplified and labeled cRNA was purified on RNase Mini Spin Columns (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The labeled cRNA target was quantified using an ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). After checking labeling efficiency, each 750 ng of the cyanine three-labeled and cyanine five-labeled cRNA targets were mixed, and the cRNA was fragmented by adding 10× blocking agent and 25× fragmentation buffer (Agilent Technology) and incubating at 60 °C for 30 min. The fragmented cRNA was resuspended in 2× hybridization buffer (Agilent Technology) and pipetted directly onto an assembled Agilent Whole Human Genome Oligo Microarray. The arrays were hybridized at 65 °C for 17 h in an Agilent hybridization oven. The hybridized microarrays were washed as per the manufacturer’s protocol (Agilent Technology), and the hybridization images were analyzed with an Agilent DNA Microarray Scanner. The average fluorescence intensity for each spot was calculated and local background was subtracted with the Agilent Feature Extraction software package. All data normalization and selection of up-regulated and down-regulated genes was performed using GeneSpring GX 7.3 (Agilent Technology). Intensity-dependent normalization was performed in which the ratio was reduced to the residual of the Lowess fit of the intensity versus ratio curve. The averages of normalized ratios were calculated by dividing the average of the signal channel intensity by the average of the control channel intensity. Functional annotation of genes was performed according to the Gene OntologyTM Consortium (http://www.geneontology.org/ Phytother. Res. 28: 1088–1095 (2014)

D. H. SHIN ET AL.

Real-time PCR. The DP cells (1 × 106 cells) were seeded into 100-mm diameter cell culture dishes. After 24 h, the cells were treated with 5 μM and 10 μM Rg3 or minoxidil for 6 h. The forward primer sequence for VEGF (NM_003376) was 5′-C AACATCACCATGCAGATTATGC-3′, and the reverse primer sequence was 5′-GCTTTCGTTTTTGCCCCTTTC-3′. The forward primer sequence for glyceraldehyde-3-phosphate dehydrogenase (NM_002046) was 5′-GGTCTCCTCTGAC TTCAACA-3′, and the reverse primer sequence was 5′-AG CCAAATTCGTTGTCATAC-3′. Total RNA (1 μg) was reverse-transcribed to cDNA according to the manufacturer’s instructions (M-MLV Reverse Transcriptase; Invitrogen, Carlsbad, CA, USA). Reactions were carried out on an ABI Prism® 7900HT Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA), and relative transcript levels were determined using glyceraldehyde-3-phosphate dehydrogenase as the endogenous control gene. Each reaction was performed in triplicate. Thermal cycling conditions were 95 °C for 10 min followed by 45 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. The data were analyzed using the Sequence Detection System software (SDS version 2.0, PE Applied Biosystems). Animals and skin harvesting. The study was approved by the regional ethics committee for animal care (KBSI-AEC1107). Syngenic female C57BL/6 mice 6–8 weeks of age and weighing 15–18 g in the telogen stage of the hair cycle were purchased from Japan SLC Inc. (Shizuoka, Japan; http://www.jslc.co. jp/). The mice were housed in community cages with 12 h light periods and had free access to food and water. Immunohistochemistry. Cultured human DP cells were placed on a standard microscope slide and incubated with 4% (w/v) paraformaldehyde in PBS (pH 7.4). Slides were quenched in hydrogen peroxide (0.3–3%) to block endogenous peroxidase activity and then washed in an automation buffer (Biomeda, Foster City, CA, USA). Slides were blocked in 5% normal serum for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibody antiVEGF (1:200 dilution; Abcam, Cambridge, MA, USA). Slides were then probed with horseradish peroxidase-conjugated secondary antibodies for 30 min. Anti-rabbit secondary antibodies from the DakoCytomation EnVision detection system (Dako, Carpinteria, CA, USA) and the immune complexes were detected by incubation with the horseradish peroxidase substrate 3,3′-diaminobenzidine. Slides were dehydrated sequentially in ethanol, rinsed with xylene, and mounted with Permount. For IHC studies on mouse skin tissue, the mice were processed performed as described below. The backs of the mice were shaved at 7 weeks of age, and a sterile glass filter (GF-C Whatman) was surgically placed under the skin to protect the skin from the mechanical trauma of daily treatments and to ensure even spread Copyright © 2013 John Wiley & Sons, Ltd.

of the applied solutions. Rg3 and minoxidil were diluted in physiological saline to concentrations of 100, 500, and 1000 μM, and a 100-μL aliquot was applied daily onto the skin of one side of animals that had been anesthetized with avert in injections. The opposite side received 100 μL of saline solution as a control. The animals were killed by intracardiac perfusion of 4% (w/v) paraformaldehyde in PBS (pH 7.4). A 5-mm thick section across the dorsoventral diameter of the skin was cut and fixed in ice-cold 10% paraformalin overnight and embedded in paraffin. Serial sections (4 μm thick) were cut and processed for H&E and immunohistological staining. Paraffin sections (4 μm thick) on the microslides were deparaffinized in xylene and rehydrated sequentially in ethanol. Antigen retrieval was achieved by incubation with 10 mM citrate buffer (pH 6.0) in a domestic microwave oven at 700 W for 30 min. Slides were quenched in hydrogen peroxide (3%) to block endogenous peroxidase activity and then washed in tris-buffered saline buffer (0.05 M, pH 7.6). Slides were blocked with 3% bovine serum albumin in PBS for 1 h at room temperature and incubated overnight at 4 °C with primary antibody diluted in serum-free protein blocking buffer. Primary antibodies were directed against the following antigens (using the following dilutions): VEGFA (1:200; Abcam, Cambridge, MA, USA), CD8 (1:50; GenWay Biotech Inc., San Diego, CA, USA), CD34 (1:50; AnaSpec Inc., San Jose, CA, USA), and Ki-67 (1:50; Dako, Glostrup, Denmark). The Labeled Streptavidin Biotin kit was used for the avidin-biotin peroxidase complex method, and slides were counterstained with hematoxylin. Slides were dehydrated sequentially in ethanol, rinsed with xylene, and mounted with Permount.

Image acquisition and protein quantification. Samples were analyzed with a Zeiss AxioImagerZ1 microscope system with a charge-coupled device camera and the TissueFAXSTM automated acquisition system (TissueGnostics, Vienna, Austria). The percentages

*p