Table 1.
Forward and reverse primers, and the expected sizes of the 5α-R enzymes, the AR, and β-actin.
Table 2.
Forward and reverse primers and the expected sizes of growth factors and β-actin.
Fig 1.
The culture characteristics of DPCs isolated from human hair follicles obtained by AGA patients.
(A) (a) Intact hair follicle bulb (red arrows indicate the dermal papilla in the hair bulb region; 10×). (b) The pear-like shape of the typical membrane-bound DPCs (40×). (c) The initial outgrowth of isolated DPCs after 2 weeks of culturing (40×). (d) The isolated DPCs before full cell confluency after 1 month of culturing (40×). (e) The isolated DPCs at full cell confluency (40×). (f and g) The isolated DPCs at low cell density after culturing for 1 and 3 days, exhibiting a flatted and spindle-shaped morphology (100×). (h) The isolated DPCs forming multi-layered aggregations before reaching full confluency (40×). (B) RT-PCR showing the expression of target genes by DPCs. (a) The expression of 5α-R1 (1,025 bp) and 5α-R2 (801 bp) at passage 7. (b) The expression of AR (801 bp) at passage 5.
Fig 2.
Cytotoxicity assay of AM and AC on DPCs.
DPCs were treated with various concentrations of (a) AM (0–40 μg/mL) and (b) AC (0–40 μM) for 48 h. Cell viability was evaluated by PrestoBlue® assay. The data is presented as mean + SD (n = 3). * p < 0.05 versus non-treated cells.
Fig 3.
Cell cycle analysis of DPCs after a treatment with the AM extract or with AC.
The cells were treated with either the AM extract (10 μg/mL) or AC (10 μM). After 48 h, the cells were analyzed for DNA content by a FACSort flow cytometer, and their sub G0/G1 phase population was evaluated for its DNA distribution in the apoptotic process. The data are presented as mean ± standard deviation (SD) (n = 3). *p<0.05 and **p<0.01 versus non-treated cells.
Fig 4.
5α-R inhibitory effects of AM and AC by performing on TLC plate (a) A TLC plate showing the 5α-R inhibitory activity of the AM extract (10 μg/mL) (lane 3) and of AC (10 μM) (lane 4), with dustasteride (1 μM) being used as a positive control (lane 2). The internal control (cells without the inhibitor) is shown in lane 5, while the controls with standard DHT and cells with T are shown in lanes 1 and 6, respectively. (b) The bar graphs represent the percentage of DHT produced in the presence of the AM extract and the AC relatively to the internal control, with the obtained values presented in the Table. All data were obtained from experiments performed in triplicate, and are presented as mean ± SD (n = 3). ***p<0.005 versus non-treated cells.
Fig 5.
The immunofluorescent staining of the AR and the nucleus.
The first column shows phase contrast pictures of DPCs, the second column shows the pictures of the AR green fluorescence staining by using an antibody against AR, the third column shows the blue fluorescence nuclear staining, and the fourth column shows the merged images of the second and third columns. Rows: (a) DPCs under normal conditions (without a DHT treatment); (b) DPCs treated with 30 μM of DHT for 12 h; (c) DPCs treated with 30 μM of DHT and 10 μM of flutamide (positive control); (d) DPCs treated with 30 μM of DHT and 10 μg/mL of the AM extract; (e) DPCs treated with 30 μM of DHT and 10 μM of AC. The nucleus was stained by Hoechst dye blue fluorescence and observed under a fluorescence microscope (40×).
Fig 6.
The effect of the AM extract (10 μg/mL) and of the AC (10 μM) on the gene expression of VEGF and HGF.
DPCs were incubated with 30 μM of T, and were treated with the AM extract or with AC at various times before determining the gene expression of (a) VEGF and (b) HGF. The expression levels of each of these growth factors were analyzed by using the ImageJ 1.53e software. All data were performed in triplicate, and are herein presented as mean ± SD (n = 3). *p<0.05, **p<0.01, and ***p<0.005 versus non-treated cells.
Fig 7.
The effect of the AM extract on the protein expression of the hair growth factors.
(a) VEGF, (b) HGF and (c) KGF. DPCs were incubated with T (30 μM), and were treated with the AM extract (10 μg/mL) for various periods (6, 12, and 24 h). DPCs incubated with only T (30 μM) acted as a control. The protein expression is represented relatively to the control group levels at 0 h. The bar graph data analysis of the undertaken Western blotting shows the expression of VEGF (a), HGF (b), and KGF (c). All data are presented as mean ± SD (n = 3). *p<0.05, **p<0.01, and ***p<0.005 versus non-treated cells.
Fig 8.
The effect of the AM extract on the protein expression of anti-apoptotic mediators.
DPCs were incubated with T (30 μM) as a control, and were treated with the AM extract (10 μg/mL) for various periods (6, 12, and 24 h). The bar graph data analysis of the undertaken Western blotting is presented relatively to the control group at 0 h. The expression levels of Bcl-2 (a), Bax (b), and cleaved caspase-3 (c) are presented. All data are presented as mean ± SD (n = 3). *p<0.05, and ***p<0.005 versus non-treated cells.
Fig 9.
Effect of AM on the protein expression of BMP-4.
DPCs were incubated with T (30 μM) and were treated with the AM extract (10 μg/mL) for 6, 12, and 24 h. DPCs were incubated only with T (30 μM) as a control. The histograms represent the relative levels of the protein expression (relative to the control group). Quantitative analysis of the protein expression was determined by using the ImageJ software. Western blot showing the protein expression of BMP-4 in each condition, and quantitative analysis of the protein expression. The data are presented as mean ± SD (n = 3). *p<0.05, and ***p<0.005 versus non-treated cells.
Fig 10.
A proposed mechanism of the AM extract on the modulation of the hair cycle.