Table 1.
Primer sequences used for RT-PCR.
Fig 1.
Effect of oleuropein on DPC proliferation.
DPCs (3 × 104 cells/well) were treated with various concentrations of oleuropein (OP) or minoxidil (MXD), as indicated. (A) Viable cells were determined by MTT assay. (B) Cells were counted by trypan blue exclusion. The values are the mean ± SD of triplicate determinations from three independent wells. * = P < 0.05 and ** = P < 0.01 vs. control.
Fig 2.
Effect of oleuropein on the activation status of the Wnt/β-catenin pathway in DPCs.
DPCs were treated with 20 μM OP or 100 μM MXD for 24 h. (A) The protein level of β-catenin in the DPCs was determined by western blotting. A representative image of triplicate experiments is shown in the upper panel. The lower panel shows the intensity of the bands that were densitometrically measured and normalized against the protein level of β-actin. (B) DPCs were immunocytochemically stained with β-catenin antibody (left column panel), and the corresponding DAPI nuclear staining is also shown (middle column panel); merged images are shown in the right column panel. Original magnification: 20×. (C) LEF1 and Cyc-D1 mRNA levels were measured by RT-PCR. A representative image of triplicate experiments is shown in the left panel. The right panel shows the intensity of the bands that were densitometrically measured and normalized against the mRNA expression level of GAPDH. The western blotting and RT-PCR results are the mean ± SD from duplicates of three independent experiments. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 (one-way ANOVA, Tukey's test).
Fig 3.
Hair growth-promoting effect of oleuropein in C57BL/6N mice.
The back skin of male C57BL/6N mice (eight weeks old) was shaved and treated daily with CON, 0.4 mg OP, or 3 mg MXD for 28 days. (A) The back skin was photographed at 0, 7, 14, 21, and 28 days. (B) The length of randomly plucked hairs was measured at different time intervals (0, 7, 14, 21, and 28 days) after topical application of CON, OP, or MXD. Values are the mean ± SD of eight mice. ns = p > 0.05, *** = p < 0.001 (one-way ANOVA, Tukey's test).
Fig 4.
Effect of oleuropein on hair follicle growth in C57BL/6N mice.
The CON, 0.4 mg/mL OP, or 3 mg/mL MXD was topically applied to the shaved back skin of C57BL/6N mice for 28 days. (A) The effect of oleuropein on the hair follicles of the mice was analyzed by using hematoxylin and eosin staining. Longitudinal sections of the back skin were stained, and representative photomicrographs of skin sections are shown. Bars, 200 μm. Number (B) and diameter (C) of hair follicles in deep subcutis. Values are the mean ± SD of eight mice. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 (one-way ANOVA, Tukey's test).
Fig 5.
Effect of oleuropein on mRNA and protein expression of growth factors in C57BL/6N mouse skin tissue.
(A) The IGF-1, HGF, VEGF, and KGF mRNA expression levels were measured by RT-PCR. A representative image of triplicate experiments is shown in the left panel. The right panel shows the intensity of the bands that were densitometrically measured and normalized against the mRNA expression level of GAPDH. (B) Dermal levels of IGF-1 were assessed by ELISA. (C) Immunohistochemical analysis of IGF-1 expression in hair follicle. Original magnification: 40×. The RT-PCR results are the mean (n = 8) ± SD of three independent experiments (n = 2 or 3 per experiment) for each group. ns = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001 (one-way ANOVA, Tukey's test).
Fig 6.
Effect of oleuropein on expression of genes related to hair growth in C57BL/6N mice skin tissue.
(A) The Wnt10b, DKK1, LRP5, FZDR1, LEF1, and Cyc-D1 mRNA expression levels were measured by RT-PCR. A representative image of triplicate experiments is shown in the left panel. The right panel shows the intensity of the bands that were densitometrically measured and normalized against the mRNA expression level of GAPDH. (B) The protein level of β-catenin in C57BL/6N mouse skin tissue was determined by western blotting. The lower panel shows the intensity of the bands that were densitometrically measured and normalized against the protein level of β-actin. (C) Immunohistochemical analysis of β-catenin in hair follicles. Original magnification: 40×. Western blotting and RT-PCR results are the mean (n = 8) ± SD of three independent experiments (n = 2 or 3 per experiment) for each group. ns = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001 (one-way ANOVA, Tukey's test).
Fig 7.
Effects of cycloheximide on β-catenin accumulation in the nucleus of DPCs induced by oleuropein treatment.
DPCs were allowed to attach overnight and then treated (60 min) with cycloheximide (10 μg/ml) and thereafter switched to medium with or without oleuroepin (20 μM) in the presence of cycloheximide (10 μg/ml) for 24 h. Then cells were stained for β-catenin immunofluorescence (red) and counterstained with DAPI (blue). Merged image of β-catenin-rhodamine and DAPI staining is also shown. Original magnification: 20×.
Fig 8.
Effects of cycloheximide on gene expression changes in DPCs induced by oleuropein treatment.
DPCs were allowed to attach overnight and then treated (60 min) with cycloheximide (10 μg/ml) and thereafter switched to medium with or without oleuroepin (20 μM) in the presence of cycloheximide (10 μg/ml) for 24 h. mRNA expression levels of LEF1, Cyc-D1, IGF-1, HGF, VEGF, and KGF were measured by RT-PCR. A representative image of triplicate experiments is shown in the left panel. The right panel shows the intensity of the bands that were densitometrically measured and normalized against the mRNA expression level of GAPDH. The RT-PCR results are the mean ± SD from duplicates of three independent experiments. ns = P > 0.05, * = p < 0.05, ** = p < 0.01 (student’s t-test).