Eight-week-old male C57BL/6 mice were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China), and then allowed to adapt for a week with food and water ad libitum. Mice were cared under 26 ± 2°C room temperature and 12 hours of a light/dark cycle. This study had been reviewed and approved by the Foshan University, Institutional Animal Care and Use Committee (FSUeae2018014).

An androgenetic alopecia model was used with modifications (Truong et al., 2017; Zhang et al., 2018). Fifty mice at 9 weeks of age were used as the animal model to determine the effect of Jagged1 and/or EGF on androgenetic alopecia. After adapting to the environment, mice were anesthetized with 200 µl of 4% chloralhydrate intraperitoneally, and the dorsal area (2 × 2 cm) was shaved with an animal clipper and applied with hair removal cream externally to allow hair follicle synchronization. The dorsal depilatory mice were randomly assigned to five treatment groups. As testosterone was dissolved in 30% (v/v) ethanol, the control group received 30% ethanol on the depilation area alone; the testosterone group received 0.5% testosterone (Patel et al., 2014); the Jagged1 group received 0.5% testosterone followed by 1 mg/ml of Jagged1 one hour later; the EGF group received 0.5% testosterone followed by 30 µg/ml of EGF 1 hour later; the Jagged1 and EGF group received 0.5% testosterone followed by 1 mg/ml of Jagged1 and 30 µg/ml of EGF 1 hour later. Synthesized Jagged1 (CDDYYYGFGCNKFCRPR; Shanghai Top-peptide Biotechnology Co., Ltd.) and EGF (Levesque et al., 2018) were dissolved in sterile water to a final concentration of 1 mg/ml and 30 µg/ml, respectively. Testosterone (Dalian Meilun Biotechnology Co., Ltd; No: MB1636) was dissolved in 30% ethanol to a final concentration of 5 μg/ml. From day 1 after hair removal, each compound (100 µl) was topically applied to the shaved dorsal area once a day for 7 or 14 days. Mice in the control group were firstly applied with 100 µl of 30% alcohol. After 1 h, 100 µl of sterile water was applied. The testosterone group and the treatment group were applied with 100 µl of 0.5% testosterone, followed by the sterile water and peptide(s) as indicated above 1 h later, respectively.

To examine the hair growth promoting effect, the darkening of mice skin color was observed every day. At day 0, 7, and 14 after topical application, the mice were photographed. Hair re-growth efficacy score was measured as 0, 1, 2, 3, 4, and 5 in correspondence to 0%, 20%, 20–40%, 40–60%, 60–80%, 80–100% of hair growth, respectively.

Five mice of each group were euthanized on day 7 and 14 with diethyl ether, respectively, and skin tissue was extracted from the shaved dorsal area for tissue and following gene expression analysis. The dorsal skin samples were fixed in 4% formaldehyde for 24 hours and then processed by paraffin block embedding using standard techniques. General histology was visualized by H&E staining, and the dermis thickness was subsequently observed. Then, the number of hair follicles was determined under a microscope (Nikon, ECLIPSE E200).

In order to describe Ki67-positive hair matrix cells in mouse hair follicles, immunohistochemistry was performed on 5-µm-thick, paraffin-embedded skin tissue sections. The sections were deparaffinized in xylene, and rehydrated by a series of graded ethanol rinses. After antigen retrieval in sodium citrate buffer, endogenous peroxidase activity was blocked with 10% bovine serum for 2 hours, followed by incubation overnight at 4°C with rabbit anti-Ki67 (Abcam, Cambridge, UK; No.: ab15580, 1:500) primary antibody. Subsequently, horseradish peroxidase conjugated secondary antibody (Service Bio, GB23303, 1:200) was pipetted on the sections and incubated for 30 minutes at 37℃. The negative control was subjected to the same steps as described above, but the primary antibody was replaced by 10% bovine serum. With diaminobenzidine (DAB), the reaction product was visualized as brown staining. Then, the examined sections were counterstained by hematoxylin. The expression level of Ki67 in the tissue sections were assessed and quantified using an Open Source Plugin based on the staining intensity, and then assigned a score as high positive (3+), positive (2+), low positive (1+), and negative (0) (Varghese et al., 2014).

To measure apoptotic cells in mouse hair follicles, terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labeling (TUNEL) staining was performed following the recommendations of the manufacturer (In Situ Cell Death Detection, POD Kit (Roche,11684817910)). Deparaffinized skin tissue sections were randomly taken from each group and treated with 20 μg/ml proteinase K for 25 min at 37℃ to strip proteins from nuclei, and then rinsed three times with PBS (pH = 7.4). Inactivation of endogenous peroxidase was performed by incubating with 3% H₂O₂ for 15 min. After hematoxylin counterstaining for 3 min, the normal cell nuclei were stained blue, and the TUNEL positive cell revealed by DAB were brownish yellow. All sections were examined immediately after the reaction and photographed with microscope (Nikon, ECLIPSE E200). For quantitative analyses, the number of TUNEL positive or Ki-67 positive cells was counted by using ImageJ software.

The extracted skin tissues in the shaved dorsal area were collected and stored at −80°C for RNA analysis. Total RNA was isolated from the skin samples with RNAzol^® RT (Molecular Research Center, Inc. Cincinnati, OH; No.: RN 190) in accordance with the protocol. RNA was quantitated using a NanoPhotometer-NP80 (Implen, Germany). Eight hundred nanograms of total RNA of each sample was reversely transcribed into complementary DNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TakaRa, Beijing, China; NO.: RR047A) and conducted in Biometra T professional gradient Thermocycler (Biometra, Germany). Quantitative PCR was performed using SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Beijing, China; No.: RR820A). Thermal cycling was performed for 30 seconds at 95°C for enzyme activation, denaturation for 5 seconds at 95°C, and annealing for 30 seconds at 60°C. The real-time PCR was performed for at least 40 cycles. A dissociation curve was generated to assure the absence of non-specific products or primer dimers. All primer sets used in this study were obtained from Sangon Biotech Co., Ltd. Primers (Shanghai) and expected product size are shown in Table 1 . Product sizes were verified by agarose gel electrophoresis. Relative quantification was conducted with the 2^−∆∆Ct method (Livak and Schmittgen, 2001). Expression data of the genes of interest were normalized with the housekeeping gene β-actin. All real-time PCRs were performed in triplicate, and the changes in gene expression were reported as fold-increases relative to testosterone group.

Each experiment was repeated at least three times. The data were statistically analyzed using one-way ANOVA and the least squares difference test for the comparison among groups using SPSS 20. Significant differences were defined at p values of <0.05.

The effect of Jagged1 and/or EGF on testosterone-suppressed hair growth in vitro was investigated. As shown in Figure 1 , hair growth was significantly suppressed by testosterone. Addition of Jagged1 partially revered the inhibition effect testosterone, although not to the level of control. EGF, and EGF plus Jagged1 both revered testosterone inhibition completely ( Figure 1 ).

We then further examined the in vivo effect of the two factors in vivo. To achieve synchronized anagen development over the back skin area, 50 mice were depilated at the dorsal back area, and randomly divided into five groups with the following topical treatments in the same volume: control (30% ethanol); testosterone to induce androgenetic alopecia (negative control; 0.5% testosterone (Patel et al., 2014); Jagged1 (0.5% testosterone + 1 mg/ml Jagged1); EGF (0.5% testosterone + 30 μg/ml EGF); and Jagged1 + EGF (0.5% testosterone + 1 mg/ml Jagged1 + 30 μg/ml EGF).

These treatments were applied twice a day for 14 days, and hair regrowth was assessed. As showed in Figure 2 , testosterone (testo) significantly suppressed hair growth compared to control. The suppression was partially reversed by topical application of Jagged1 and EGF, respectively. In the presence of both Jagged1 and EGF, the hair growth stimulating effect was stronger than that of Jagged1 or EGF alone. Hair growth was further evaluated using the hair grow scoring system (Varghese et al., 2014). It was found that Jagged1 and EGF tended to partially reverse testosterone-suppressed hair growth, however, the revision was not significant statistically. In the presence of both the bioactive factors (Jagged1 + EGF), the hair growth score was increased to the level that was significantly higher than that of the testo group (p < 0.05), and not different from the non-testosterone positive control ( Figure 2B ).

Histologically, consistent with what was observed in the hair growth in general, at day 14 of treatment the overall hair follicle appeared to be better developed in the following order: control > Jagged1 + EGF > Jagged1 > EGF > Testo group ( Figure 3A ). To better quantitate the histological response to treatment, follicle number was counted. As to be expected, significantly less hair follicle number was found in the testosterone treated group compared to the control (p < 0.05). Although there is a trend of follicle number increase in the EGF group, it is however, still not statistically significantly different than that of the testo group alone ( Figure 3B ). The follicle number decreased by testosterone was reversed to the level that it was not significantly different to that of non-testosterone control in the Jagged1, and Jagged1 + EGF groups ( Figure 3B ). When the height of the subcutis area was also measured, it was found that the thickness of subcutis decreased significantly by testosterone. While this decrease was partially, but not significantly reversed by Jagged1 and EGF, respectively, a significant reversion was observed in the presence of both Jagged1, and EGF ( Figure 3C ), suggesting more follicles are in the anagen stage in the presence of both of the bioactive factors.

Ki67 is expressed in the G1, S, G2, and M phase of the cell cycle, but not in resting cells in G0. Ki67 is thus regarded as proliferation marker (Lopez et al., 1991). To study if the improved hair growth may be associated with increased proliferation of hair follicle cell by Jagged1 and EGF, immunohistochemistry was performed using antibody against Ki67. At day 7 of treatment, while no increase of Ki67 positive score was observed in the Jagged1 and EGF group, respectively, the Ki67 staining score is the highest in the Jagged1 + EGF group, compared to that of the other four groups, suggesting that follicle cell proliferation was significantly enhanced when both of the bioactive factor are presence ( Figure 4 ). By day 14, the Ki67 score in the Jagged1 + EGF is the same as that of the control group. Interesting, the score is lower in the EGF group compared to that of the control and Jagged1 + EGF groups at this time point ( Figure 4 ).

To study if Jagged1 and/or EGF may have an effect on apoptosis of the hair follicle cell, TUNEL analysis was perform. Figure 5A showed the representative images of TUNEL staining. Quantitation of apoptosis rate in hair follicle using Image-pro plus 6.0 software revealed that while EGF by itself suppressed apoptosis in hair follicle cells (p < 0.05), there was no difference in apoptosis rate among control, Testo, Jagged1, and Jagged1 + EGF groups ( Figure 5B ).

As Jagged1 and EGF promoted hair follicle growth, we next sought to study if they regulate the expression of genes such as Notch1, Notch2, Sonic hedgehog (Shh), keratinocyte growth factor (FGF7), vascular endothelial growth factor (VEGF), and β-catenin, which are associated with follicle cell proliferation and differentiation. RNAs were isolated from back skin of mice from days 7 and 14 treatment, and RT-PCR was performed. As shown in Figure 5 , while Notch2 expression was increased by Jagged1 at day 7, its expression in other groups was unchanged. At day 14, testosterone increased Notch1 and Notch2 expression, and this induction was suppressed by Jagged1 and EGF, respectively and in combination to the level of control ( Figures 6A, B ). At day 7, no change was observed on the expression of Shh and FGF7 across various groups. At day 14, testosterone-induced Shh and FGF7 expression, and this induction was suppressed by Jagged1, EGF, and the combination of the two factors ( Figures 6C, D ). The expression of VEGF was increased in the Jagged1 + EGF group at day 7, although its mRNA level was down regulated in the Jagged1 + EGF group at day 14 ( Figure 6E ). Similar expression pattern was also observed for the expression of β-catenin ( Figure 6F ).

It is known that inflammation plays an important role in androgenetic alopecia (Trueb, 2002). To study if the hair growth promoting effect of Jagged1 and EGF may be associated with their involvement in inflammation modulation, we examined the expression of tumor necrosis factor alpha (TNF-α) and inducible T-cell costimulatory (Icos), which are both are known to be immunomodulators. TNF-α expression was increase in the Jagged1 + EGF group at day 7. By day 14, testosterone-induced TNF-α expression, and this induction was suppressed in the Jagged1 and Jagged1 + EGF groups to the level that is the same as the non-testosterone control ( Figure 7A ). Jagged1 increase Icos expression at day 7. However, by day 14, testosterone-induced Icos expression, and this induction was suppressed by Jagged1, EGF, and their combination to the level that is the same as the non-testosterone control ( Figure 7B ).