The authors conducted experiments using the same samples as PG extract, which had hair growth effect in our previous studies (27). The root of PG was obtained from Geumsan Ginseng Market (Geumsan-gun, Korea). The dried and crushed roots of PG (300 g) were extracted with 70% aqueous ethanol at 50°C for 8 h. The extracts were filtered and concentrated under reduced pressure at 60°C. The residue was dissolved with 100% ethanol and repeat filtration and vacuum distillation.

Minoxidil, MTT and dimethyl sulfoxide were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Human DKK-1 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Non-balding scalp specimens were obtained from patients undergoing hair transplantation surgery (IRB:DKUH 2013-08-012-001). The medical ethical committee of the Dankook Medical College (Department of Dermatology, Cheonan, Korea) approved all of the described studies, and informed written consent was obtained from the patients. HFs were isolated and cultured by the previously described method, with minor modifications (28). Cultured ORS keratinocytes of early passage were used for the experiments and were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Cell viability was determined using an MTT assay that was performed by a slight modification of the method described by Philpott et al (29). Briefly, ORS keratinocytes were seeded at a density of 2×10⁴ cells/well into 96-well plates and were cultured for 24 h. Prior to treatment, the cells were cultured for 24 h in a growth supplement-free medium. The cells were then treated with PG extract and DKK-1 for 24 h. The samples were assessed by measuring absorbance at 540 nm with a Synergy™ 2 Multi-Detection Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The cell viability rates were calculated from the optical density readings and are represented as percentages of the control value (untreated cells).

The total RNA was isolated using TRIzol™ reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 2 µg RNA was reverse-transcribed into cDNA using SuperScript® III Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). Quantitative real-time TaqMan PCR technology (TaqMan Universal PCR Master Mix, part no. 4304437) was used (Applied Biosystems; Thermo Fisher Scientific, Inc., Santa Clara, CA, USA). The cDNA samples were analyzed to determine the expression of the following: Hs00608023_m1 (Bcl-2), Hs00180269_m1 (Bax), and Hs02758991_g1 (GAPDH). Commercially available these probes were purchased and used in Thermo Fisher Scientific, Inc.

A TUNEL kit (In Situ Cell Death Detection kit, Fluorescein, Roche Diagnostics GmbH, Mannheim, Germany) was used according to the manufacturer's protocol to evaluate apoptotic cells. Briefly, ORS keratinocytes at 2×10⁴ cells/200 µl were seeded into eight-chamber slides (Nunc Lab-Tek; Thermo Fisher Scientific, Inc., Roskilde, Denmark), were serum-starved for 24 h, and were then treated with PG extract and DKK-1 for 24 h. These cells were then fixed in 4% paraformaldehyde for 10 min. After being washed with PBS, the cells were incubated with 0.1% Triton X-100 in 0.1% sodium citrate for 1 h at room temperature. After washing, the cells were treated with the TUNEL reaction mixture and then were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Representative images were taken with a fluorescence microscope (Olympus Corp., Tokyo, Japan) at ×100 magnification.

ORS keratinocytes at 2×10⁴ cells/200 µl were seeded into eight-chamber slides, and then treated with DKK-1 and PG extract for 24 h. These cells were then fixed in 4% paraformaldehyde for 10 min. After washing with Dulbecco's PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature and then blocked with 5% BSA in 0.05% Triton X-100 for 30 min at room temperature. The samples were incubated with Bcl-2 (1:200 dilution, sc-783; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and Bax antibody (1:200 dilution, sc-6236; Santa Cruz Biotechnology, Inc.) at 4°C overnight. They were then washed two times with PBS and four times with distilled water followed by incubation with a Alexa Fluor™ 488 anti-rabbit (1:200 dilution, A-11034; Thermo Fisher Scientific, Inc.) and Texas Red™-X anti-rabbit (1:200 dilution, T-6391; Thermo Fisher Scientific, Inc.) secondary antibodies in 5% BSA blocking solution for 2 h at room temperature. All samples were counterstained with DAPI to visualize the nuclei. Representative images were taken with a fluorescence microscope (Olympus Corp.) at ×100 magnification.

Anagen HFs from human scalp skin specimens were obtained from patients undergoing hair transplantation surgery. The medical ethical committee of the Dankook University Hospital (Cheonan, Korea) approved all of the described studies. A total of six HFs/well in 24-well plates were cultured in William's E medium at 37°C in a humidified atmosphere with 5% CO₂ in 500 µl basal medium supplemented with 10 µg/ml insulin, 10 ng/ml hydrocortisone, 2 mM L-glutamine, 0.1% Fungizone, 10 µg/ml streptomycin and 100 U/ml penicillin according to Philpott's method, as previous described (30). Each experimental group contained at least 30 anagen HFs derived from three different human donors/volunteers. DKK-1 (50 ng/ml) was determined to optimally induce catagen-like changes, shorten hair shaft length and hair bulbs with minimal other histological alterations to the HFs. Either the PG extract or MNX was added at the concentration, respectively, of 20 ppm or 50 µM. The incubation medium was renewed every 2 days. The HF elongation was measured directly at 2, 5 and 7 days of culture using a light stereo microscope (Olympus Corp.).

The results are expressed as mean ± standard deviation. The data was analyzed using a Student's t-test, and the two-tailed value of P<0.05 was considered to indicate a statistically significant difference. The data was processed by SPSS software for Windows, version 22.0 (SPSS Inc., Chicago, IL, USA).

To investigate the potential role of PG extract on the proliferation and inhibition of apoptosis in ORS keratinocytes, the authors performed an MTT assay one day after treatment in the presence or absence of DKK-1 and PG extract. The PG extract and DKK-1 concentrations were determined by a previous study of the authors (data not shown). The results indicated that the PG extract enhanced the proliferation of ORS keratinocytes (Fig. 1) compared to untreated negative controls and enhanced ORS growth cultured positive controls with growth supplement medium. Treatment with DKK-1 (50 ng/ml) significantly inhibited the viability of ORS keratinocytes compared to the untreated negative control. PG extract significantly counteracted the inhibitory effect of apoptosis by DKK-1 on ORS keratinocyte (Fig. 1) viability.

To confirm the inhibitory effect of apoptosis by the PG extract in ORS keratinocytes, a TUNEL assay was performed. TUNEL-positive cells undergoing apoptosis significantly increased when ORS keratinocytes (Fig. 2A and B) were treated with 50 ng/ml of DKK-1. The PG extract decreased TUNEL-positive cells despite co-treatment with DKK-1.

Previously, it was found that there are many kinds of ginsenosides in total extract by high-performance liquid chromatography analysis (27). In order to determine which of these ginsenosides is most effective, we selected three representative ginsenosides of PG extract. (Rb1, Re, Rg1) and conducted further experiments. As a result (Fig. 2C and Table I), it was demonstrated that each ginsenoside inhibited apoptosis induced by DKK-1 to some extent. However, each effect was found to be insufficient for total extract.

To further investigate the relevance of the anti-apoptotic effects of PG extract on DKK-1, changes in the expression of apoptosis-related genes were examined by RT-qPCR. ORS keratinocytes were treated with PG extract and/or DKK-1 for 24 h. DKK-1-induced apoptosis was accompanied by Bcl-2/Bax expression in many cells, including HF cells (12,13). In ORS keratinocytes, DKK-1 treatment significantly decreased anti-apoptotic factor Bcl-2 expression. PG extract alone increased Bcl-2 expression four times more than the untreated control, and it reversed the DKK-1-induced inhibition of Bcl-2 expression. DKK-1 also induced the expression of the pro-apoptotic factor Bax. PG extract significantly inhibited Bax expression in ORS keratinocytes (Fig. 3A) despite the presence of DKK-1. In other words, PG extract promotes ORS keratinocyte survival and increases the ratio of Bcl-2/Bax to further inhibit the cell death (Fig. 3B). Increased protein level of Bcl-2 and decreased protein of Bax were confirmed by immunocytochemistry (Fig. 3C). These results indicated that the effect of PG extract was mediated through Bcl-2/Bax expression on ORS keratinocytes.

In order to examine the effect of PG extract in the presence or absence of DKK-1 at the organ level, the authors performed an ex vivo culture of whole human scalp HFs. Minoxidil (MNX) and vehicle served as positive and negative controls, respectively. HFs treated with PG extract grew longer than the negative control HFs at 5 days, which was similar to the growth of HFs treated with MNX. This result is consistent with the authors' previous study (27). A low dose of DKK-1 (<50 ng/ml) produced no significant impairment of hair shaft elongation compared to the vehicle (data not shown), but a dose of 50 ng/ml DKK-1 significantly inhibited hair shaft elongation. The authors observed a narrower hair bulb in HFs treated with DKK-1 at the 50 ng/ml dose, which is reminiscent of catagen-like regressive changes. They also measured the anagen/catagen ratio (Fig. 4A), and DKK-1 treatment resulted in anagen-to-catagen changes in the HF organ culture. Thus, DKK-1 treatment at the dose of 50 ng/ml was used to establish an ex vivo model of HF catagen induction. With co-incubation of the PG extract and DKK-1, the PG extract significantly abrogated DKK-1-induced growth inhibition of cultured HFs ex vivo (Fig. 4B).

There is evidence to suggest that several factors such as cytokine, growth factor, and apoptosis-related factor are involved in the hair growth cycle (31). In the above results, the authors already confirmed that PG extract regulates hair growth-related factors at the in vitro level. To confirm the inhibitory effect of apoptosis by PG extract at the ex vivo level, changes in the gene expression were examined by RT-qPCR using HFs treated with PG extract and/or DKK-1 for 2, 5 and 7 days. The DKK-1 treatment significantly decreased anti-apoptotic factor Bcl-2 expression at 2 days. At the same time point, the PG extract completely abolished the effect of DKK-1 on Bcl-2 expression in HFs. On the other hand, DKK-1 treatment significantly increased Bax expression in HFs, whereas the PG extract strongly inhibited this induction of Bax for 5- and 7-day HFs (Fig. 5). Of note, the PG extract affected only Bax expression and not Bcl-2 in longer ex vivo culture experiments. These results suggested that PG extract antagonizes DKK-1-induced catagen-like changes, in part, through the regulation of apoptosis-related factor expression in HFs.