The effects of centipedegrass extract on hair growth via promotion of anagen inductive activity

Abstract

To investigate the CGE on hair growth and to explore the mechanism that is involved in the acceleration of anagen induction, we investigated the effects of CGE studied on cell proliferation and molecular mechanism in human hair dermal papilla cells (hDPCs) and keratinocytes (HaCaT cells). Additionally, hair growth evaluation was carried out following topical treatment of the dorsal skin of telogen C57BL/6 mice with CGE for 14 days. As result, CGE increased cell viability and ALP activity in hDPCs. Moreover, CGE increased the expression of catenin beta 1 (CTNNB1), ALP, sex-determining region Y-box 2 (SOX2), insulin-like growth factor 1 (IGF1), and vascular endothelial growth factor A (VEGFA) genes in hDPCs. CGE increased the expression of proteins such as ALP, β-catenin, and phosphorylation of glycogen synthase kinase 3β (pGSK3β), and protein kinase B (pAKT) in hDPCs. Furthermore, CGE induced the proliferation of HaCaT cells and up-regulated AKT-ERK-GSKβ-β-catenin signaling in HaCaT cells. Additionally, the anagen induction effects of CGE were confirmed on the telogen-anagen transition mice model. these findings demonstrated that CGE promoted the entering the growth phase of hair follicle via activation of β-catenin signaling pathways in vivo. Thus, this study suggests that CGE might be a potential therapeutic reagent for hair growth.

Fig 1. CGE increased anagen properties in hDPCs via up-regulating β-catenin signaling.

(A) Morphological changes in hDPCs after CGE treatment. MNX used as a positive control. Scale bar: 200 μm (B) Cell viability. CGE treatment increased hDPCs viability at 24 h after treatment. CGE treatment increased ALP expression in hDPCs. ALP expression was measured by (C) ALP activity assay KIT and (D) western blot using anti-ALP. Anti-GAPDH used as a loading control. (E) mRNA transcription of CTNNB1, ALP, SOX2, IGF-1 and VEGF were measured by RT-qPCR after 12 h with each treatment. (F) Representative bands of Western blot analysis for the protein expression of β-catenin, pGSK3β, GSK3β, pAKT, AKT, or GAPDH antibodies. (G) Densitometry analysis of western blot. Data are presented as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01 vs. Veh. Veh, vehicle control; MNX, minoxidil 10 μM; SD, standard deviation.

Fig 2. CGE increased HaCaT proliferation via up-regulating β-catenin signaling.

(A) Cell viability. CGE treatment increased HaCaT viability after 24 h. (B) Representative images of Ki-67 expression. Scale bar: 50 μm (C) Representative images of Western blot analysis for the protein expression of β-catenin, pGSK3β, GSK3β, pAKT, AKT, pERK, ERK, PCNA, or GAPDH antibodies. (D) Densitometry analysis of western blot. Data are presented as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01 vs. Veh. Veh, vehicle control; MNX, minoxidil 10 μM; SD, standard deviation.

Fig 3. CGE promoted hair re-growth in C57BL/6 dorsal skin.

Each candidate was applied to the back skin for 14 days. 5% minoxidil was used as a positive control. After depilation, the observation was preceded for 14 day, at the end of experiment, the sacrifice was conducted. (A) Gross images, at 7^th, 11^th, and 14^th day after CGE treatment. (B) Gross images were measured for hair growth score. (C) Schematic diagram of the experimental schedule. Data are presented as the mean ± SD. **p ≤ 0.01 vs. Veh. Veh, vehicle control; MNX, minoxidil 10 μM; SD, standard deviation.

Fig 4. CGE treatment accelerated entry into the anagen phase via up-regulating β-catenin signaling in vivo.

(A) Representative images of H&E staining. IHC and IF were conducted for observing (B) β-catenin, (E) SHH, (F) ALP, and (G) Ki-67 expression in mouse skin tissues. Scale bar: 100 μm. Histological quantitative score, (C) hair follicle length and (D) skin thickness were measured for evaluating anagen properties. Data are presented as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01 vs. Veh. Veh, vehicle control; MNX, 5% minoxidil; SD, standard deviation.