Figure 1.
Time table of the experiments.
CRS or CUMS was administered beginning on day 1 and continued for 21 days. Drugs were also administered from day 1 for 21 days. All mice received epilation to induce anagen of the hair cycle at day 9. Mice were photographed on days 10 and 22, which were 2 and 13 days post-depilation. Tissue samples were collected on day 22.
Table 1.
Primer sequences.
Figure 2.
Chronic stress causing reduction of melanogenesis in mice dorsal skin.
A: Photographs of mice back skin on day 2 and day 13 after epilation showing the reduction of melanin in the skin of CRS and CUMS group mice on day 13. B: The mRNA expression levels of microphthalmia-associated transcription factor (MITF) in mouse skin. C: The mRNA expression levels of tyrosinase (TYR) in mouse skin. The expression levels of each gene were normalized against β-Actin then calculated as fold change using the comparative 2-ΔΔCT method. Data are showed in mean ± SEM, n = 8, and the data were analyzed by one-way ANOVA with Tukey's post hoc test. * P<0.05, ** P<0.01, *** P<0.001, compared with control.
Figure 3.
Chronic stress causing disrupted expressions of cutaneous HPA axis elements.
A: POMC expression was analyzed by immunoblotting. β-Actin expression was indicated as a loading control. Western blot assay are representative of three experiments. Densitometric scanning of band intensities obtained from three separate experiments was used to quantify change of proteins expression. Three animals were used for each data point. Data are showed in mean ± SEM. B–K: The mRNA expression levels of POMC, UCN1, MC1R, MC2R, CRHR1, CRHR2, CYP11A1, Hsd11b1, Hsd11b2, and Nr3c1 in mouse skin. The expression levels of each gene were normalized against β-Actin then calculated as fold change using the comparative 2-ΔΔCT method. Data are showed in mean ± SEM, n = 8. Data were analyzed by one-way ANOVA with Tukey's post hoc test. * P<0.05, ** P<0.01, compared with control.
Figure 4.
Effects of chronic stress on mice body weight gain and serum corticosterone levels.
A: On days 3, 6, 9, 12, 15, 18, and 21, CRS and CUMS did not inhibit mice body weight gain significantly compared with control. B: The serum corticosterone levels in mice of different group. Serum for corticosterone measurement was collected on day 22, one day after the final stressor. Data are showed in mean ± SEM, n = 6, and the data were analyzed by one-way ANOVA with Tukey's post hoc test. ** P<0.01, compared with control.
Figure 5.
Corticosterone causing reduction of melanogenesis and disrupted expressions of cutaneous HPA axis elements.
A: Photographs of mice back skin on day 2 and day 13 after epilation showing the reduction of melanin in corticosterone treated mice on day 13. B: MITF expression was analyzed by immunoblotting. C: POMC expression was analyzed by immunoblotting. β-Actin expression was indicated as a loading control. Western blot assay are representative of three experiments. Densitometric scanning of band intensities obtained from three separate experiments was used to quantify change of proteins expression. Three animals were used for each data point. Data are showed in mean ± SEM. D: The mRNA expression levels of UCN1, POMC, CYP11A1, CRHR1, CRHR2, MC1R, MC2R, Hsd11b1, Hsd11b2, and Nr3c1 in mouse skin. The expression levels of each gene were normalized against β-Actin then calculated as fold change using the comparative 2-ΔΔCT method. Data are showed in mean ± SEM, n = 8. Data were analyzed by Student's t test. * P<0.05, ** P<0.01, *** P<0.001, compared with control.
Figure 6.
Effects of RU486 on the dorsal skin of stressed mice.
A: Photographs of mice back skin on day 2 and day 13 after epilation showing that the mice received RU486 injection had a significantly darker observable skin color than CUMS mice. B: MITF expression was analyzed by immunoblotting. C: TYR expression was analyzed by immunoblotting. D: POMC expression was analyzed by immunoblotting. β-Actin expression was indicated as a loading control. Western blot assay are representative of three experiments. Densitometric scanning of band intensities obtained from three separate experiments was used to quantify change of proteins expression. Three animals were used for each data point. Data are showed in mean ± SEM. E: The mRNA expression levels of UCN1, POMC, and CYP11A1 in mouse skin. The expression levels of each gene were normalized against β-Actin then calculated as fold change using the comparative 2-ΔΔCT method. Data are showed in mean ± SEM, n = 8. The data were analyzed by one-way ANOVA with Tukey's post hoc test. * P<0.05, *** P<0.001, compared with control; &P<0.05, &&P<0.01, &&&P<0.001, compared with CUMS.
Figure 7.
Effects of HPA axis-related hormones on melanin synthesis in vitro.
A: Measurement of melanin contents in normal human epidermal melanocytes (NHEMs) after treatment with 50 nM α-MSH or 1 µM DEX for 72 h. D: Measurement of melanin contents in B16F10 cells after treatment with 50 nM α-MSH or 1 µM DEX for 72 h. B–C: The mRNA expression levels of MITF and TYR in NHEMs after treatment with 50 nM α-MSH or 1 µM DEX for 24 h. E–F: The mRNA expression levels of MITF and TYR in B16F10 cells after treatment with 50 nM α-MSH or 1 µM DEX for 24 h. The expression levels of each gene were normalized against β-Actin or GAPDH then calculated as fold change using the comparative 2-ΔΔCT method. Data are combined from three separate experiments and showed in mean ± SEM, and the data were analyzed by one-way ANOVA with Tukey's post hoc test. * P<0.05, ** P<0.01, *** P<0.001, compared with control; &&P<0.01, compared with α-MSH.