2 Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing 211198, China;
3 School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou 325035, China;
4 State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou 325035, China
Vitiligo is a progressive pigmentation disorder that occurs with the death of melanocytes in the epidermis. The prevalence of the disorder is 1%-2% worldwide, with no racial or sex differences . Several theories for the etiology of vitiligo have been proposed, suggesting that melanocyte self-destruction occurs due to biochemical, neural, auto- immune or genetic factors. However, the pathogenesis of vitiligo is not fully understood, as these factors cannot clearly explain melanocyte loss in different forms of the disorder . Moreover, a recent study has reported that 55% of patients with vitiligo have other autoimmune disorders, suggesting a common etiological basis .
Many compounds with different chemical structures have been found to induce re-pigmentation in subjects with vitiligo. To distinguish the most effective among these compounds in terms of re-pigmentation potential, we first conducted an exhaustive literature review to identify compounds with reported melanogenic effects. Ten compounds belonging to the following five chemical families were identified in our search: purines, piperidines, flavonoids, coumarins, and steroids. Upon considering the melanogenic properties and toxic effects of these compounds, we selected six for further screening in order to establish their mechanism of action: 6-benzylaminopurine (6-bap), piperine (pip), chrysin (chr), kaempferol (kaem), scopoletin (sco), and vitamin D3 (VitD3).
The selected compounds are briefly introduced as follows. 6-bap is a first-generation synthetic cytokinin that elicits plant growth and development responses by stimulating cell division. Moreover, 6-bap can lead to the activation of protein kinase A (PKA) via a cyclic adenosine monophosphate (cAMP) independent pathway, subsequently stimulating melanogenesis by up-regulating microphthalmia associated transcription factor (MITF) and tyrosinase expression . The next two compounds, pip and chr, have recently been used as screened ligands that allow docking against vitiligo associated protein-1 (VIT1) in a docking study . Pip is an alkaloid with a piperidine skeleton, which is responsible for the pungency of both black and long peppers, while chr is a naturally occurring flavone type of flavonoid. The fourth compound, kaem, is a natural flavonol, found in a variety of plants and plant- derived foods, and has been identified as a melanogenesis promoter . Sco is a coumarin that helps to stimulate tyrosinase (the rate-limiting enzyme of melanogenesis) activity . Finally, VitD3 is a steroid that plays a key role in endocrine, paracrine and autocrine functions, and regulates several functions involving the epidermis, hair follicles, skin, and immune system .
The application of B16F10 murine melanoma cells (in vitro) and the development of a zebrafish model (in vivo) have improved our understanding of the physiopathology of vitiligo. More importantly, the transgenic approach has become popular for developmental analysis in the fruit fly, mouse, and zebrafish . The green fluorescent transgenic zebrafish is typically preferred for screening angiogenic drugs, anti-arterial sclerosis drugs, and detecting environmental toxicity, etc [10-11]. Zebrafish gene tyrosinase-related protein 1 (tyrp1) is specifically expressed in the retinal pigment epithelium and melanocytes, and can be divided into two types, namely tyrp1a and tyrp1b. In medaka fish, tyrp1a is simultaneously expressed in the retinal pigment epithelium and melanocytes, while tyrp1b is only expressed in melanocytes . Furthermore, Lister et al.  have identified a second MITF; one of the homologous gene sequences found in zebrafish, mitfb, which is expressed in the eye and other sites in the mouse. MITF expression, can rescue melanophore development in the neural crest. Zebrafish mitfa and mitfb arose by gene duplication and current roles for these genes represent subfunctionalization of an ancestral locus .
The primary aim of the present study was to determine the effectiveness, toxicity, and mechanism of action for the six compounds with previously reported melanogenic effects. This was done by evaluating the effect of each of these compounds on melanogenesis in zebrafish, followed by an investigation of melanogenic effects in vitro. Moreover, we screened the effects of the most potent compound on tyrp1a and mitfa in transgenic zebrafish, and finally, investigated the antioxidant activity of this compound in vivo. Notably, this was the first study to investigate melanogenesis in transgenic zebrafish.Materials and Methods Reagents
Chr, sco, kaem, VitD3, and pip were purchased from Aladdin industrial corporation (Shanghai, China). 6-bap was purchased from Sigma-Aldrich, life science & technology Co., Ltd., (Wuxi, China). Methylcellulose, alpha-melanocyte stimulating hormone (α-MSH), Phenylthiourea (PTU), tricaine methanesulfonate and hydrogen peroxide were purchased from Sigma- Aldrich (Burlington, USA). Dimethylsulfoxide (DMSO), L-3, 4-dihydroxyphenylalanine (L-DOPA), 3-(4, 5-dimethyl-thiazol- 2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), were purchased from Sigma-Aldrich (burlington, USA). DiChloro-dihydro- Fluorescein Diacetate (Beyotime Biotechnology-China).In vivo zebrafish model Maintenance of zebrafish
Zebrafish (Danio rerio) farming was carried out in accordance with methods detailed in "The Zebrafish Book"  with a 14-h light (6: 00 a.m. to 8: 00 p.m.) and 10-h dark (8: 00 p.m. to 6: 00 a.m.) cycle at a temperature of 28.5 ℃; the animals were fed twice a day (7: 00 a.m. and 5: 00 p.m.). The experiments were started at 8: 00 p.m. by placing one female and one male in the spawning tank, divided by a separator plate. The following day, the lights were turned on at 6: 00 a.m. and the separator plate was removed for 20 min. The zebrafish were then returned to their respective tanks, and the embryos in the spawning tank were collected and cleaned before they were cultured in zebrafish embryo medium and placed in an illuminator at 28.5 ℃.Toxicity assays in zebrafish
Zebrafish embryo medium (3000 μL) was added to each well of a six-well plate, followed by adding 10 embryos. After 1 h, a 0.1% DMSO solution of each test compound was added at concentrations ranging from 0-1000 μmol·L-1. The survival rate of embryos was observed for the next 48 h i.e., 9 to 57 h post-fertilization (hpf) at 28 ℃. The dead embryos were removed from the well after 24 and 48 h and their exact number was recorded in a data table of living and dead embryos in each well for the given period.Melanogenesis activity
To characterize the melanogenic effects of these com- pounds, total melanin content of whole zebrafish extracts was measured. The effect of these compounds on the pigmentation of zebrafish embryos was determined according to a previous report . Briefly, zebrafish embryos were collected, synchronized, and arrayed by pipette into a 6-well plate. Each well was filled with ten embryos along with 3000 μL of zebrafish embryo medium. The embryos were pre-treated with 0.2 mmol·L-1 PTU from 9 to 38 hpf (29 h) in each well. PTU was then removed, embryos were immediately washed and treated with different concentrations of the experimental compounds (0.1% DMSO); untreated embryos were used as a control and kept undisturbed for the next 48 h i.e., up to 86 hpf. Phenotype-based evaluation of body pigmentation was performed at 86 hpf. Embryos were anesthetized in tricaine methanesulfonate solution (Sigma-Aldrich, Wuxi, China) for 5-10 sec and transferred to 3% methyl cellulose, which was mounted on a depression slide and photographed under a SZX16 stereo microscope (Olympus, Japan).Effect of 6-bap on tyrp1a and mitfa in transgenic zebrafish
We established a tyrp1a green fluorescent transgenic zebrafish strain by the microinjection of zebrafish embryos at one-cell stage (30 min postfertilization) with Tol2 (tyrp1a: eGFP) vector as described by Kawakami . Embryos were then pre-treated with PTU to remove melanin via inhibition of tyrosinase-dependent signaling  and analyzed with 6-bap to check the specificity of the fluorescent protein, which was primarily driven by TYRP1 and MITF promoters. At 83 hpf, the expressions of fluorescence were observed under a fluorescence stereomicroscope SZX16 (Japan, Olympus).Cell culture
The murine melanoma cell line B16-F10 was purchased from the Chinese Cell Bank (Shanghai, China) and maintained as a monolayer culture in Dulbecco's Modified Eagle's Medium (Gibico, USA) supplemented with 10% (V/V) heat-inactivated fetal bovine serum (Gibico), 100 U·mL-1 penicillin, 100 μg·mL-1 streptomycin (Gibico) at 37 ℃ in a humidified 5% CO2 incubator.Cell viability assay
Cell viability was determined using the MTT assay. After B16F10 cells were plated in 96-well plates at a density of 2 × 104 cells/well, the cells were incubated with the compounds for 48 h, the culture medium was removed and replaced with 20 mL of MTT solution (5 mg·mL-1) dissolved in phosphate- buffered saline (PBS) and incubated for 2 h at 37 ℃. The medium was removed, and 150 μL of DMSO was added to each well and gently shaken for 5 min. Optical absorbance was set at 570 nm with a microplate spectrophotometer (BD Bioscience, USA). Absorbance of cells without treatment was considered as 100% of cell survival. Each treatment was performed in quintuplicate and each experiment was replicated thrice.TYR activity and melanin content assay in B16F10 cells
TYR activity and melanin content assay was carried out according to a previous report  with slight modifications. TYR activity in B16F10 cells was examined by measuring the rate of oxidation of L-DOPA. Briefly, the cells were treated with a test compound; after 48 h they were washed with ice-cold PBS and lysis was performed by incubation in a cell lysis buffer (1 nmol·L-1 PMSF) at 4 ℃ for 20 min. The lysates were then centrifuged at 14 000 r·min-1 for 15 min and the protein content in the supernatant was determined by Bradford assay, with bovine serum albumin BSA as the protein standard. Tyrosinase activity was then determined as follows: 100 mL of supernatant containing total 10 mg of centrifuged proteins was added to each well in a 96-well plate, and then mixed with 100 mL 0.1% L-DOPA in 0.1 mol·L-1 PBS (pH 6.8) (M/V). After incubation at 37 ℃ for 0.5 h, dopachrome formation was monitored by measuring absorbance at 475 nm. Specific tyrosinase activity was normalized with protein content in the reaction.
The levels of intracellular and secreted melanin were measured as described previously . Total melanin in the cell pellet was dissolved in 100 mL of 1 mol·L-1 NaOH/10% DMSO for 1 h at 80 ℃. The absorbance at 405 nm was measured. Melanin content was calculated as a percent of the control. Specific melanin content was adjusted by the amount of protein in the same reaction.Antioxidative activities of 6-bap compared to chr
At 30 days post fertilization, synchronized zebrafish were divided into four groups and treated in four different tanks, namely, control, model, 6-bap, and chr. Control zebrafish was simply grown in the medium. Model, 6-bap and chr group zebrafish were treated with 200 μmol·L-1 H2O2 to expose oxidation processes. After 24 h, the medium in model, 6-bap and chr were replaced with medium. 30 μmol·L-1 of 6-bap and 30 μmol·L-1 of chr were added to 6-bap and chr groups, respectively. After 24 h of treatment, the zebrafish were grinded by an Ultrasonic Cell Disruptor, then transferred into 96-well plates and treated with DCFH-DA solution (20 μmol·mL-1). After 30 min of incubation in the dark at 28.5 ℃ DCF fluorescence was measured at 485-528 nm.Statistical analysis
All data were expressed as means ± SE. Statistical analysis was performed with a one-way ANOVA followed by Tukey's post hoc test for correction of multiple comparisons. Values with P < 0.05 were considered statistically significant.Results Effect of compounds on melanogenesis in zebrafish
Prior to the investigation of melanogenesis, a toxicity assay was performed to determine the toxicity of the selected compounds to zebrafish. As shown in Fig. 1A, a significantly lower toxicity rate was observed in embryos at the following concentrations: sco 0-30 μmol·L-1, chr 0-30 μmol·L-1, pip 0-1 μmol·L-1, VitD3 0.01-0.1 μmol·L-1, kaem 0-30 μmol·L-1, and 6-bap 0-30 μmol·L-1, for a 48-h exposure, indicating their optimal effective concentration toward zebrafish embryos.
Pigmentation was analyzed by photography as shown in Fig. 1. These compounds exhibited significantly potent stimulating effects on melanogenesis at the following concentrations: sco 20 μmol·L-1, chr 20 μmol·L-1, pip 0.5 μmol·L-1, VitD3 0.05 μmol·L-1, kaem 20 μmol·L-1, and 6-bap 20 μmol·L-1, compared to the control group. In addition, 6-bap showed the strongest re-pigmentation action, which increased markedly after treatment with PTU over 29 h.Cytotoxicity toward B1610 cells
To examine whether sco, chr, pip, vit-d3, kaem and 6-bap have cytotoxic effects, we treated B16 cells with these compounds at various concentrations; cell viability was determined using the MTT assay. As shown in Fig. 2, the compounds were non-toxic at concentrations ranging from 1 to 150 μmol·L-1 i.e., sco 0-50 μmol·L-1, chr 0-25 μmol·L-1, pip 0-25 μmol·L-1, VitD3 0-1 μmol·L-1, kaem 0-150 μmol·L-1 and 6-bap 0-100 μmol·L-1.
The compounds were examined against B16 cell lysate to identify their influence on stimulation of melanin pigmentation. As shown in Fig. 3, the cellular melanin contents were significantly increased at concentrations of sco 10 μmol·L-1, chr 25 μmol·L-1, pip 10 μmol·L-1, vit-d3 1 μmol·L-1, kaem 100 μmol·L-1, and 6-bap 100 μmol·L-1. Furthermore, 6-bap at 100 μmol·L-1 was identified as the most potent compound both in vitro and in vivo, resulting in an approximately two- fold increase in melanin content.
As a rate-limiting enzyme, tyrosinase plays a key role in melanin biosynthesis; the conversion of L-DOPA to dopa- quinone by mushroom tyrosinase was observed at 450 nm as shown in Fig. 4, and the tyrosinase expression levels of cells treated with sco 50 μmol·L-1, chr 25 μmol·L-1, pip 25 μmol·L-1, vit-d3 0.5 μmol·L-1, kaem 100 μmol·L-1, and 6-bap 100 μmol·L-1 was highly up-regulated compared with α-MSH 60 nmol·L-1 as a positive standard. This suggests that the augmentation of melanogenesis by sco, chr, pip, VitD3, kaem, and 6-bap in B16 cells occurs via stimulation of tyrosinase activity. The proliferation of cellular tyrosinase activity by these compounds may be caused either by direct stimulation of enzyme activity or by an augmentation in the amount of tyrosinase protein in the cells.
We initially investigated the effects of 6-bap on tyrp1a: eGFP and mitfa: eGFP transgenic zebrafish pre-treated with PTU 0.2 mmol·L-1. After 48 h of treatment with 6-bap we observed a strong stimulation of fluorescence as compared to a control group with zebrafish media without 6-bap (Figs. 5 A and B).
Based on previous reports, we expected chr to exhibit potent antioxidative properties. Accordingly, we found that chr exhibited reasonable antioxidative properties. The relative antioxidative property of 6-bap, however, was found to be very low (Fig. 6). This unexpected finding was indicated that the melanogenic property of 6-bap may be due to a direct mechanism.
Several reports have indicated that different classes of compounds, including flavonoid , alkaloid , coumarin  and cytokinin , may enhance melanogenesis. To identify the most potent among these and determine its mechanism, we investigated the effects of flavone (chrysin), flavonol (kaempferol), alkaloid (piperine), coumarin (scopoletin), cytokinin (6-benzylaminopurine), on melanogenesis in zebrafish and B16F10 cells.
Based on the results of toxicity tests, we conducted a melanogenesis assay on zebrafish within the tolerance limit. Zebrafish were treated with the compounds of interest at a range of concentrations extending to maximum tolerance levels of each compound. It is known that tyrosinase is responsible for the production and augmentation of melanin during melanogenesis and is the only essential enzyme required for melanin production . Augmented pigmentation was detected in zebrafish embryos exposed to the compounds for 2 days, and their melanogenic activity was visualized by stereomicroscope (Fig. 1C). All six compounds were found to exhibit potent melanogenic activity, with 6-bap emerging as the most potent one.
Similarly, in vitro results indicated that these chemical compounds were not cytotoxic to B16 cells at their optimal concentrations, while both total melanin and tyrosinase activity increased significantly at 100 μmol·L-1 of the cytokinin 6-bap (Fig. 2). These results suggest that by 6-bap may lead to melanin augmentation in cellular tyrosinase activity either by direct stimulation of enzyme activity or by stimulation of tyrosinase enzyme. A recent study showed that 6-bap activates PKA via a cAMP-independent pathway and subsequently stimulates melanogenesis by up-regulating MITF and tyrosinase expression . Activation of MITF itself was found to be a key transcriptional regulator of vertebrate melanin synthesis by members of the tyrosinase gene family: tyrosinase, TYRP1, and Dct .
Furthermore, we identified a possible mechanism underlying the melanogenesic effects of cytokinin 6-bap by activating fluorescence expression of tyrp1a in tyrp1a: eGFP zebrafish and mitfa in Mitfa: eGFP zebrafish. Our results indicated that increased GFP fluorescence in transgenic zebrafish occurs 48 h after treatment with 20 μmol·L-1 of 6-bap. This suggested that 6-bap increased melanogenesis in zebrafish through the up-regulation of mitfa, TYR and tyrp1a, which are required in melanocytes at several stages of melanogenesis [24-25].
We further showed that unlike chr flavones, 6-bap did not have significant antioxidative activity, indicating that this cytokinin N6-substituted adenine derivative has a direct effect on melanogenesis stimulation. Based on the structure-activity relationship of 6-bap, it is possible that the absence of hydroxyl groups linked with the adenine group of 6-bap may restrict its antioxidative properties and enhance its consequent melanogenic effect. These findings indicated that 6-bap may be a useful drug in studying the regulation of melanogenesis, and can be used for the treatment of vitiligo. Future studies should focus on the development of a new compound with both antioxidative and melanogenic properties.
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