Image2_Epimedium brevicornum Maxim. Extract exhibits pigmentation by melanin biosynthesis and melanosome biogenesis/transfer.TIF

Epimedium brevicornum Maxim. (Epimedii Folium) is a traditional medicine widely utilized in China for sexual dysfunction and osteoporosis treatment. Recently, studies have reported that Epimedium flavonoid icariin displayed hair growth and melanogenic ability by targeting tyrosinase activity. Nevertheless, icariin hydrolysate icariside II and icaritin cause depigmentation due to their tyrosinase inhibition. These pigment functional discrepancies from Epimedium constituents arouse our great interest. Then, this study focused on the pigmentation effects of Epimedii Folium extract (EFE) on melanin synthesis and melanosome biogenesis/transfer, and further identified the bioactive constituents. First, in in vitro systemic studies, we discovered that the potent melanogenic and repigmented effects of EFE were dependent on concentration and amount of time in multi-melanocytes, normal human skin tissue, and vitiligo perilesional areas. In vivo, EFE exhibited repigmented effect on two kinds of depigmented models of N-phenylthiourea-induced zebrafish and hydroquinone-induced mice. Mechanistically, EFE strongly promoted tyrosinase activity and upregulated the protein expression of tyrosinase families which finally contribute to melanin biosynthesis by activating the MAPK/ERK1/2 signal pathway. In addition, EFE effectively increased melanosome number, accelerated melanosome maturity and cytoplasmic transport through the growth/extension of melanocyte dendrites, and induced melanosome transfer from melanocyte to keratinocyte for pigmentation. The six main flavonoid ingredients were identified among EFE. Compared to others, epimedin B (EB) was confirmed as a high-content, low-toxicity, and effective melanogenic compound in EFE. Taking all these together, this study systematically demonstrates the potential pigmentation effect of Epimedium brevicornum Maxim., and clarifies its related molecular mechanisms and melanogenesis basis. These results give additional insight into Epimedium herb pharmacology and may provide a novel therapy basis for hypopigmentation disorders.</p

Time table of the experiments.

<p>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.</p

Effects of chronic stress on mice body weight gain and serum corticosterone levels.

<p><b>A</b>: 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>B</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. ** <i>P<0.01</i>, compared with control.</p

Primer sequences.

<p>Primer sequences.</p

Chronic stress causing reduction of melanogenesis in mice dorsal skin.

<p><b>A</b>: 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>B</b>: The mRNA expression levels of microphthalmia-associated transcription factor (MITF) in mouse skin. <b>C</b>: 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. * <i>P<0.05</i>, ** <i>P<0.01</i>, *** <i>P<0.001</i>, compared with control.</p

Chronic stress causing disrupted expressions of cutaneous HPA axis elements.

<p><b>A</b>: 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>B–K</b>: 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. * <i>P<0.05</i>, ** <i>P<0.01</i>, compared with control.</p

Effects of HPA axis-related hormones on melanin synthesis in vitro.

<p><b>A</b>: Measurement of melanin contents in normal human epidermal melanocytes (NHEMs) after treatment with 50 nM α-MSH or 1 µM DEX for 72 h. <b>D</b>: Measurement of melanin contents in B16F10 cells after treatment with 50 nM α-MSH or 1 µM DEX for 72 h. <b>B–C</b>: The mRNA expression levels of MITF and TYR in NHEMs after treatment with 50 nM α-MSH or 1 µM DEX for 24 h. <b>E–F</b>: 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. * <i>P<0.05</i>, ** <i>P<0.01</i>, *** <i>P<0.001</i>, compared with control; ^&&<i>P<0.01</i>, compared with α-MSH.</p

Effects of RU486 on the dorsal skin of stressed mice.

<p><b>A</b>: 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>B</b>: MITF expression was analyzed by immunoblotting. <b>C</b>: TYR expression was analyzed by immunoblotting. <b>D</b>: 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>E</b>: 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. * <i>P<0.05</i>, *** <i>P<0.001</i>, compared with control; ^&<i>P<0.05</i>, ^&&<i>P<0.01</i>, ^&&&<i>P<0.001</i>, compared with CUMS.</p

Developmental Neurotoxic Effects of Percutaneous Drug Delivery: Behavior and Neurochemical Studies in C57BL/6 Mice

<div><p>Dermatosis often as a chronic disease requires effective long-term treatment; a comprehensive evaluation of mental health of dermatology drug does not receive enough attention. An interaction between dermatology and psychiatry has been increasingly described. Substantial evidence has accumulated that psychological stress can be associated with pigmentation, endocrine and immune systems in skin to create the optimal responses against pathogens and other physicochemical stressors to maintain or restore internal homeostasis. Additionally, given the common ectodermal origin shared by the brain and skin, we are interested in assessing how disruption of skin systems (pigmentary, endocrine and immune systems) may play a key role in brain functions. Thus, we selected three drugs (hydroquinone, isotretinoin, tacrolimus) with percutaneous excessive delivery to respectively intervene in these systems and then evaluate the potential neurotoxic effects. Firstly, C57BL/6 mice were administrated a dermal dose of hydroquinone cream, isotretinoin gel or tacrolimus ointment (2%, 0.05%, 0.1%, respectively, 5 times of the clinical dose). Behavioral testing was performed and levels of proteins were measured in the hippocampus. It was found that mice treated with isotretinoin or tacrolimus, presented a lower activity in open-field test and obvious depressive-like behavior in tail suspension test. Besides, they damaged cytoarchitecture, reduced the level of 5-HT-5-HT1A/1B system and increased the expression of apoptosis-related proteins in the hippocampus. To enable sensitive monitoring the dose-response characteristics of the consecutive neurobehavioral disorders, mice received gradient concentrations of hydroquinone (2%, 4%, 6%). Subsequently, hydroquinone induced behavioral disorders and hippocampal dysfunction in a dose-dependent response. When doses were high as 6% which was 3 times higher than 2% dose, then 100% of mice exhibited depressive-like behavior. Certainly, 6% hydroquinone exposure elicited the most serious impairment of hippocampal structure and survival. The fact that higher doses of hydroquinone are associated with a greater risk of depression is further indication that hydroquinone is responsible for the development of depression. These above data demonstrated that chronic administration of different dermatology drugs contributed into common mental distress. This surprising discovery of chemical stressors stimulating the hippocampal dysfunction, paves the way for exciting areas of study on the cross-talk between the skin and the brain, as well as is suggesting how to develop effective and safe usage of dermatological drugs in daily practice.</p></div

Effect of 2% HQ, 0.05% INN and 0.1% TAC on neuronal damage and apoptosis of the hippocampus in C57BL/6 mice.

<p>(A) HE staining showed a higher percentage of cell loss in CA1 region of INN and TAC treated mice. Scale bar = 100μm. (B) Representative photomicrographs showed a striking increase in TUNEL-positive apoptotic cells (indicated by arrow) of hippocampus (CA1, CA3 and DG). Scale bar = 100μm. (C) Hippocampal cell was lysed and total cell extracts were analyzed by western blot. Relative changes in protein expression of pro- or anti-apoptotic proteins (caspase 8, 9, cleaved caspase 3, Bax and Bcl-2) in the hippocampus. (D) Quantitative results for protein expression and bax/bcl-2 ratio following HQ, INN and TAC treatment. Densitometric scanning of band intensities obtained from three separate experiments was used to quantify protein expression. Data were analyzed by One-Way Analysis of Variance (ANOVA). *<i>P</i> < 0.05, **<i>P</i> < 0.01 <i>vs</i>. control group.</p