Effect of Plantago asiatica L. extract on the anagen phase in human hair follicle dermal papilla cells

The hair growth cycle consists of the anagen, catagen, and telogen phases, and hair follicle dermal papilla (HDP) cells of human hair play a role in the initiation and maintenance of the anagen phase. Reduction in HDP cells contributes to hair loss; however, the limited treatment options are associated with negative side effects. Therefore, a naturally derived substance with hair loss‐preventing properties is needed.


| INTRODUC TI ON
Hair protects the scalp from UV rays and cold temperatures, and plays an important role in determining the appearance of an individual. Hair loss is caused by genetic issues, or acquired factors such as stress, environmental pollution, and abnormal hormone secretion.
However, treatment methods for hair loss remain limited. 1 Hair growth occurs in cycles, consisting of the anagen stage for 2-6 years, catagen phase for 1-2 weeks, and telogen stage for 2 months, followed by hair loss.
The hair follicle dermal papilla (HDP) cells of human hair signal stem cells of the follicular bulge to initiate the anagen phase. 2 When the number of HDP cells decrease, the initiation of the anagen phase is delayed and the telogen phase is maintained. 3 Hair thinning and loss are partly related to the number of HDP cells, 4 which is essential for anagen initiation, maintenance, and restoration of hair morphology and prevention of hair loss.
HDP cells regulate the hair growth cycle through the secretion of growth factors and cytokines. Among the several hair growth factors, vascular endothelial growth factor (VEGF) improves follicular vascular formation 5 ; keratinocyte growth factor (KGF; also known as FGF7) serves as an important endogenous medium for normal follicular growth development and differentiation 6 ; hepatocyte growth factor (HGF) stimulates keratinocyte differentiation and follicular growth 7 ; and MYC plays a critical role in proliferation and anagen development. 8 These growth factors are regulated by the Wnt/β-catenin signaling pathway in HDP cells. Furthermore, Wnt/β-catenin signaling enhances hair follicle morphogenesis and regeneration. 9 During the Wnt-off state, GSK-3β phosphorylates β-catenin, leading to proteasomal degradation. However, during the Wnt-on state, β-catenin is not phosphorylated by GSK-3β and becomes stabilized in the cytosol, and is then imported into the nucleus. β-Catenin, which can bind to the T-cell factor (Tcf)/LEF site in the nucleus, promotes transcriptional activity to upregulate target genes. 10,11 Seven TCF binding sites were found in the VEGF gene promoter. 12 In addition, hair growth factors are regulated by mitogenactivated protein kinase (MAPK)/cAMP response element-binding protein (CREB) signaling. 13 CREB phosphorylated by MAPK migrates to the nucleus and serves as a transcription factor for different growth factors. 14 Blood vessels play an important role in hair growth because they supply nutrients to the HDP cells. Additionally, angiogenesis is a feature of the anagen stage, and HDP cells produce angiogenic factors to aid vessels. Furthermore, Wnt/β-catenin signaling affects angiogenesis in various organs. 15 The US Food and Drug Administration has approved finasteride and minoxidil as hair growth-promoting drugs. Finasteride is a 5αreductase type II inhibitor, and minoxidil is a local vasodilator that promotes hair growth by activating hair cell division. The side effects of finasteride include decreased sexual function in men, infertility, and birth defects in women. Moreover, the side effects of minoxidil include itching and dry dermatitis. 16 Owing to these problems and the poor stability of chemical drugs, researchers are searching for naturally derived hair growth promoters.
Plantago asiatica L. shows antioxidative properties and a skin whitening effect by inhibiting tyrosinase activity. 17,18 Moreover, the constituents of P. asiatica L. have antibacterial properties. 19,20 Therefore, P. asiatica is widely used in feminine cleansers. However, the hair growth-promoting effect of P. asiatica L. and its underlying mechanism are poorly understood. Thus, in this study, the hair growth-promoting effects of P. asiatica L. extract (PAE) in HDP cells and the mechanisms underlying these effects were investigated.

| Cell culture
Hair follicle dermal papilla cells (Cell Engineering for Origin) were cultured in basal medium supplemented with 4% serum and 0.5% penicillin and streptomycin (CEFOgro™ HDP; Cell Engineering for Origin).

Human umbilical vein endothelial cells (HUVEC; Cell Engineering
for Origin) were cultured in basal medium supplemented with 7.2% serum and 0.5% penicillin and streptomycin (CEFOgro™ HUVEC; Cell Engineering for Origin).
The cells were seeded on 100 mm culture dishes until they reached approximately 80% confluence. Cells were incubated in 5% CO 2 in a humidified atmosphere at 37°C.
Before PAE treatment, the medium was replaced with fresh Dulbecco's modified Eagle medium (Welgene) supplemented with 2% fetal bovine serum and 1% penicillin-streptomycin.
After discarding the supernatant, formazan crystals were dissolved in 100 μL DMSO. Absorbance at 590 nm was measured using a microplate reader (SpectraMax ABS Plus, Molecular Devices).

| Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Hair follicle dermal papilla cells were seeded at a density of 1.2 × 10 5 cells/well in a 6-well plate and treated with PAE at concentrations of 10, 25, and 50 μg/mL for 48 h, with untreated cells serving as controls. Total RNA was extracted using NucleoZOL (MACHEREY-NAGEL) according to the manufacturer's protocols. Briefly, 500 μL NucleoZOL was added to the 6-well plate that the RNA was removed from, and lysed cells were transferred into a 1.5 mL tube. RNase-free water (200 μL) was added and shaken for 15 s and then incubated for 15 min at room temperature. Samples were centrifuged for 15 min at 12 000 g. We prepared 500 μL isopropanol per sample, and 500 μL supernatant of each sample was then added to the prepared isopropanol. Samples were incubated for 10 min at room temperature and then centrifuged for 10 min at 12 000 g. The supernatant of samples was discarded and the pellets were washed with 700 μL 75% ethanol and centrifuged for 3 min at 8000 g. Ethanol was removed from the pellet and the RNA pellet was dissolved using RNase-free water (Thermo Fisher Scientific). cDNA was synthesized using the  Table 1.

| Western blotting
Hair follicle dermal papilla cells were seeded in 60-mm dishes and treated with 50 μg/mL PAE. The cells were then lysed and total protein was extracted using radioimmunoprecipitation assay buffer (iN-tRON Biotechnology). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% skim milk (BD Difco™) in Tris-buffered saline containing 0.08% Tween 20 and then washed three times. The membranes were incubated with primary antibody at 4°C overnight.
After washing, secondary antibody was reacted with each membrane for 2 h at room temperature. The blot was visualized with an enhanced chemiluminescence reagent (GE Healthcare). Immunoblots were ana- (1:2500) (Thermo Fisher Scientific). GAPDH was used as an internal control to verify the uniform loading of the proteins.

| Tube formation assay
We coated 96-well plates with Matrigel® matrix and dried them for

| Statistical analysis
The data are presented as the mean ± standard deviation. p < 0.05 was considered statistically significant compared with the samples from the non-treated groups (control).

| PAE increased the mRNA expression levels of cytokines related to hair growth
Hair growth is regulated by various cytokines. 6,22,23 To assess the effect of PAE treatment on the levels of cytokines, we estimated TA B L E 1 Specific primer sequences used for RT-qPCR.

| PAE activated the GSK-3β/β -catenin signaling pathway in HDP cells
The Wnt/β-catenin signaling pathway regulates hair growth factors, and β-catenin plays an important role in stimulating the stem cells of hair follicles and hair regeneration. 11,24 To determine the effect of PAE on this signaling cascade, we performed western blotting for these factors after treating HDP cells with 50 μg/mL PAE for 0, 1, 2, 5, 10, and 20 min. GSK-3β was phosphorylated after 1 min of PAE treatment; total GSK-3β levels did not change ( Figure 3A,B). As GSK-3β was phosphorylated, the amount of total β-catenin accumulated and peaked at 10 min ( Figure 3A-C). In addition, it was confirmed that GSK-3β was suppressed and intracellular β-catenin was increased in a PAE dose-dependent manner ( Figure 3D,E).

| PAE activated the MAPK/CREB signaling pathway in HDP cells
Hair growth factors are also regulated by MAPK/CREB signaling. 25,26 To determine the effect of PAE on growth factor-related signaling, we performed western blotting on components of the MAPK/CREB pathway after treating HDP cells with 50 μg/mL PAE for 0, 1, 2, 5, 10, and 20 min. ERK was phosphorylated at the Thr202/Tyr204 after 2 min of PAE treatment ( Figure 4A,B).
As ERK became activated, the Ser133 of CREB became phosphorylated after 5 min of PAE exposure ( Figure 4A-C).
Additionally, it was confirmed that ERK and CREB were phosphorylated in a dose-dependent manner during PAE treatment for 10 min (Figure 4D,E).

| PAE activated angiogenesis in HUVECs
Angiogenesis is significantly induced during hair growth and is decreased during the catagen and resting phases, 5

| DISCUSS ION
Until now, the effect of PAE on hair growth has rarely been investigated. Therefore, this study demonstrates the hair growthstimulating activities of PAE and the underlying molecular mechanism in HDP cells.
Many people suffer from hair loss, yet there is still no effective treatment or preventive method. Hair loss occurs when the anagen phase is not maintained or the hair cycle fails to restart from the telogen to the anagen phase. Therefore, maintaining the growth phase is important to prevent hair loss. HDP cells play an essential role in the formation and regeneration of hair and in maintaining the growth phase. 29 Plantago asiatica L. extract contains plantamajoside and verbascosid ( Figure S1). Verbascoside has anti-inflammatory activity, increases cell viability of dermal papilla cells, 30 and reduces testosterone. 31 In the current study, we discovered the proliferation of HDP cells was enhanced in a dose-dependent manner by PAE.
In hair follicles, cytokines related to hair growth induce and regulate hair formation and contribute to maintenance of the growth phase. KGF is necessary for hair growth and can induce proliferation and differentiation of normal hair follicles. 6
KGF peaks in the anagen phase. 33 Additionally, FGF topically applied to mice showed remarkable hair growth compared to the control. 34 VEGF is an index of angiogenesis, which stimulates cell proliferation and migration, provides nutrients to hair follicles, and promotes hair growth. 21 44 Therefore, we assume that the upregulation of growth factors caused by PAE is due to the activation of β-catenin and CREB.
In the current study, we confirmed that PAE is effective for increasing angiogenesis through HUVECs on Matrigel-coated plates.
VEGF is widely known as a factor that enhances angiogenesis, which is essential for the anagen phase. 45 As shown in Figure 2, PAE effectively increased VEGF. Therefore, we suggest that PAE may have induced angiogenesis by upregulating VEGF.
In conclusion, the effects of PAE were investigated to evaluate its potential application in hair care and hair loss prevention ( Figure 6). PAE promoted the proliferation of HDP cells by increasing the expression of KGF, VEGF, FGF2, and MYC, which was mediated by upregulation of the GSK-3β/β-catenin and MAPK/CREB signaling pathways. Moreover, PAE improved tube formation, thereby promoting the necessary angiogenesis required for the anagen phase.
Overall, these results indicate that PAE can be considered as a potential therapeutic agent for improving hair loss by inducing the anagen phase.

ACK N O WLE D G E M ENTS
None.

FU N D I N G I N FO R M ATI O N
No funding was obtained for this study.

CO N FLI C T O F I NTER E S T S TATEM ENT
None to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
This article does not contain any studies with human or animal subjects performed by any of the authors. The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to.