Tyrosinase Activity In Melanocytes Research Articles

Emerging Roles of Dermal Fibroblasts in Hyperpigmentation and Hypopigmentation: A Review.

Skin pigmentation disorders may increase patients' psychological burdens. Consequently, they are increasingly attracting attention. Dermal fibroblasts have been shown to regulate pigmentation by secreting soluble factors. This study aimed to summarize recent findings on the effects of dermal fibroblasts on hyperpigmentation and hypopigmentation, enabling the discovery of new therapeutic targets. PubMed was searched for literature on fibroblast factors, hyperpigmentation, and hypopigmentation, and a comprehensive summary and analysis were performed. Fibroblasts secrete both cytokines that promote pigmentation, including stem cell factor (SCF) and keratinocyte growth factor (KGF), and small amounts of those that inhibit pigmentation, such as Dickkopf1 (DKK1) and transforming growth factor (TGF)-β. Fibroblast-derived extracellular matrix (ECM) can also affect melanocyte tyrosinase activity and the transfer of melanosomes. In hyperpigmentation disorders, such as melasma and solar lentigines, the secretion of pigmentation-promoting factors increases, and the activity of key enzymes in melanin production is elevated. In hypopigmentation disorders, including vitiligo, the secretion of melanogenic factors decreases while the factors that inhibit pigmentation increase. Fibroblasts may serve as a new therapeutic target, providing new insights to precisely treat pigmentary disorders. Fibroblasts synthesize and secrete various cytokines and proteins that modify melanin synthesis and transfer through different signaling pathways, playing prominent roles in pigmentary skin disorders, such as photoaging, melasma, solar lentigo, and vitiligo.

Combine with RNA-seq Reveals the Effect of Melatonin in the Synthesis of Melanin in Primary Melanocytes of Silky Fowls Black-Bone Chicken.

(1) Background: It was found that the melanin of black-bone chicken has various effects such as scavenging DPPH free radicals and anti-oxidation, and the synthesis of melanin is affected by various factors including hormones. In addition, several studies have found that melatonin affects the melanoma cell synthesis of melanin, which has not been reported in chicken primary melanocytes; so, relevant studies were conducted. (2) Methods: In this study, chicken primary melanocytes were isolated and characterized, and then melanocytes were treated with different concentrations of melatonin to investigate the effects of melatonin on melanin synthesis in chicken melanocytes in terms of melanin synthesis-related genes, melanin content, and tyrosinase activity, and combined with RNA seq to detect the change in gene expression level of chicken melanocytes after melatonin treatment. (3) Results: We isolated and characterized primary melanocytes, and indirect immunofluorescence assay results showed positive melanocyte marker genes. RT-qPCR results showed that melatonin decreased the expression of melanin synthesis-related genes. In addition, melatonin reduced the melanin content and decreased the tyrosinase activity of melanocytes in the treated group. A total of 1703 differentially expressed genes were screened by RNA-seq, and in addition, in the KEGG results, the signaling pathway associated with melanin synthesis, and the mTOR signaling pathway were enriched. (4) Conclusions: Melatonin could decrease the synthesis of melanin in chicken primary melanocytes.

A Randomized Trial of Oral Tranexamic Acid With Fluocinolone-Based Triple Cream Versus Fluocinolone Based Triple Cream Alone for the Treatment of Melasma.

Oral tranexamic acid (TXA) is a relatively new treatment option for melasma. It is thought to reduce hyperpigmentation through inhibition of the plasminogen/plasmin pathway with resulting decreases in epidermal melanocyte tyrosinase activity, inflammatory mediators, dermal neovascularization, and mast cell numbers.

The Biochemical and Molecular Analysis of Changes in Melanogenesis Induced by UVA-Activated Fluoroquinolones-In Vitro Study on Human Normal Melanocytes.

Fluoroquinolones cause phototoxic reactions, manifested as different types of skin lesions, including hyperpigmentation. The disturbances of melanogenesis indicate that fluoroquinolones may affect cellular processes in melanocytes. It has been reported that these antibiotics may bind with melanin and accumulate in pigmented cells. The study aimed to examine the changes in melanogenesis in human normal melanocytes exposed to UVA radiation and treated with lomefloxacin and moxifloxacin, the most and the least fluoroquinolone, respectively. The obtained results demonstrated that both tested fluoroquinolones inhibited melanogenesis through a decrease in tyrosinase activity and down-regulation of tyrosinase and microphthalmia-associated transcription factor production. Only lomefloxacin potentiated UVA-induced melanogenesis. Under UVA irradiation lomefloxacin significantly enhanced melanin content and tyrosinase activity in melanocytes, although the drug did not cause an increased expression of tyrosinase or microphthalmia-associated transcription factor. The current studies revealed that phototoxic activity of fluoroquinolones is associated with alterations in the melanogenesis process. The difference in phototoxic potential of fluoroquinolones derivatives may be connected with various effects on UVA-induced events at a cellular level.

Imiquimod induced vitiligo-like lesions-A consequence of modified melanocyte function.

IntroductionImiquimod plays an important role in the management of condyloma and premalignant lesions. Successively, an increase of hypopigmented lesions following imiquimod application has been reported. However, the mechanisms of imiquimod on melanocytes remain unclear. This study was designed to assess the effect of Imiquimod on the functions of melanocytes in vitro.MethodsPrimary cultured melanocytes were isolated from normal control skin tissue. After incubation with imiquimod for 48 h in vitro, cell viability was analyzed by cell counting kit‐8 assay. Apoptosis was detected using the Annexin V‐fluorescein‐5‐isothiocyanate flow cytometry assay. Melanin content and tyrosinase activity in melanocytes were measured by colorimetric method and the modified dopachrome method. The production of inflammatory cytokine interleukin 8 (IL‐8), IL‐6, and soluble ICAM‐1 (soluble Intercellular Adhesion Molecule‐1[sICAM‐1]) in melanocytes were measured by enzyme‐linked immunosorbent assay (ELISA). Toll‐like receptor 7 (TLR7), toll‐like receptor 9 (TLR9) protein, and autophagy‐related proteins microtubule‐associated protein 1A/1B‐light chain 3 (LC3‐II), p62, mechanistic target of rapamycin (mTOR), and Atg5 were assessed using western blot analysis.ResultsImiquimod significantly inhibited the activity of tyrosinase activity and decreased melanin content in melanocytes and significantly increased apoptosis and IL‐6, IL‐8, and sICAM‐1 production in melanocytes. Moreover, the expression of TLR7 and TLR9 proteins were significantly increased, and the expression of mTOR, p62 protein were markedly decreased, but the expression of LC3II/I and Atg5 protein were significantly increased in melanocytes after incubating with imiquimod.ConclusionsThis study shows that imiquimod directly inhibits melanogenesis and increases melanocyte apoptosis rates. These effects combined with the upregulation of TLR7 and TLR9 together with increased autophagy activity and inflammatory cytokines production, might be the main reasons leading to hypopigmented lesions after imiquimod application.

Glucose Exerts an Anti-Melanogenic Effect by Indirect Inactivation of Tyrosinase in Melanocytes and a Human Skin Equivalent.

Sugars are ubiquitous in organisms and well-known cosmetic ingredients for moisturizing skin with minimal side-effects. Glucose, a simple sugar used as an energy source by living cells, is often used in skin care products. Several reports have demonstrated that sugar and sugar-related compounds have anti-melanogenic effects on melanocytes. However, the underlying molecular mechanism by which glucose inhibits melanin synthesis is unknown, even though glucose is used as a whitening as well as moisturizing ingredient in cosmetics. Herein, we found that glucose significantly reduced the melanin content of α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells and darkly pigmented normal human melanocytes with no signs of cytotoxicity. Furthermore, topical treatment of glucose clearly demonstrated its whitening efficacy through photography, Fontana-Masson (F&M) staining, and multi-photon microscopy in a pigmented 3D human skin model, MelanoDerm. However, glucose did not alter the gene expression or protein levels of major melanogenic proteins in melanocytes. While glucose potently decreased intracellular tyrosinase activity in melanocytes, it did not reduce mushroom tyrosinase activity in a cell-free experimental system. However, glucose was metabolized into lactic acid, which can powerfully suppress tyrosinase activity. Thus, we concluded that glucose indirectly inhibits tyrosinase activity through conversion into lactic acid, explaining its anti-melanogenic effects in melanocytes.

JAAD Game Changers: Randomized, placebo-controlled, double-blind study of oral tranexamic acid in the treatment of moderate-to-severe melasma

Capsule Summary•Melasma is often recalcitrant to therapy.•Oral tranexamic acid was found to be effective and superior to placebo in patients with moderate-to-severe melasma.•Tranexamic acid should be considered in the treatment of patients with moderate to severe melasma who do not respond to standard therapy.How did this article change the practice of dermatology?Tranexamic acid is a fibrinolytic agent that causes decreased tyrosinase activity in melanocytes. When used in combination with sunscreen, tranexamic acid, 250 mg twice daily for 3 months, improved moderate to severe melasma. Before initiating treatment, it is important to screen patients for thromboembolism risk.1

Intramelanocytic Acidification Plays a Role in the Antimelanogenic and Antioxidative Properties of Vitamin C and Its Derivatives.

Although vitamin C (VC, L-ascorbic acid) has been widely used as a skin lightening agent for a long time, the mechanism by which it inhibits melanogenesis remains poorly understood. It is well-documented that the intramelanocytic pH is an important factor in regulating tyrosinase function and melanosome maturation. The activity of tyrosinase, the rate-limiting enzyme required for melanin synthesis, is generally minimal in an acidic environment. Given that VC is an acidic compound, we might speculate that the intracellular acidification of melanocytes induced by VC likely reduces melanin content through the suppression of tyrosinase activity. The results of this study reveal that treatment of melanocytes with VC or its derivatives, magnesium ascorbyl phosphate (MAP) and 3-O-ethyl-L-ascorbic acid (AAE), resulted in significant decreases in the tyrosinase activity and melanin content and in the levels of intracellular reactive oxygen species (ROS), indicating that VC and its derivatives possess antimelanogenic and antioxidative activities. Western blotting analysis indicated that VC, MAP, and AAE exert their antimelanogenic activity by inhibiting the tyrosinase activity rather than by downregulating the expression of melanogenic proteins such as tyrosinase, premelanosome protein 17 (Pmel17) and microphthalmia-associated transcription factor (MITF). Further, we found that the reduced tyrosinase activity of melanocytes treated with VC or its derivatives could be reactivated following intracellular neutralization induced by ammonium chloride (NH4Cl) or concanamycin A (Con A). Finally, we examined the expression of sodium-dependent VC transporter-2 (SVCT-2) using western blotting and qPCR, which revealed that there was a significant increase in the expression of SVCT-2 in melanocytes following treatment with VC. VC-mediated intracellular acidification was neutralized by phloretin (a putative SVCT-2 inhibitor) in a dose-dependent manner. Taken together, these data show that VC and its derivatives suppress tyrosinase activity through cytoplasmic acidification that potentially results from enhanced VC transmembrane transport via the VC transporter SVCT-2.

Transdermal BQ-788/EA@ZnO quantum dots as targeting and smart tyrosinase inhibitors in melanocytes

Tyrosinase inhibitors could effectively limit the activity of tyrosinase in melanocytes to reduce the excessive synthesis and deposition of melanin. However, low skin permeability and lacking in targeting greatly restricted their application. Herein, ZnO quantum dots were synthesized by gel-sol method and grafted with BQ-788, which have been employed as transdermal and targeting carrier to delivery ellagic acid to melanocytes. Ellagic acid loaded ZnO quantum dots with the size distribution of around 9 nm could targetedly bind to melanocytes and enter the melanocytes by endocytosis within 1 h. The ellagic acid release behavior was controlled by the decreasing of pH via the rapid dissolution of ZnO. When the concentration of BQ-788/EA@ZnO was 12.5 μg/mL, the inhibition rate on tyrosinase activity and melanin deposition were up to 44.23 ± 4.97% and 37.50 ± 5.23%, respectively. In view of their good biocompatibility, they were of great potential in clinically external application for tyrosinase inhibition.

An In-Vitro Assay Estimating Changes in Melanin Content of Melanoma Cells due to Ultra-Dilute, Potentized Preparations.

The authors had previously conducted an in-vitro study to observe the effect of homeopathic medicines on melanogenesis, demonstrating anti-vitiligo potential by increasing the melanin content in murine B16F10 melanoma cells. A similar experiment was performed using further homeopathic preparations sourced from kojic acid (KA), hydrogen peroxide (H2O2; HP), 6-biopterin (BP), and [Nle4, D-Phe7]-α-melanocyte-stimulating hormone (NLE), some of which are known to induce vitiligo or melano-destruction at physiological dose. The homeopathic preparations of BP, KA, NLE, and HP were used in 30c potency. Alcohol and potentized alcohol were used as vehicle controls. Prior to starting the main experiment, the viability of B16F10 melanoma cells after treatment with study preparations was assayed. Melanin content (at 48 h and 96 h) and tyrosinase activity in melanocytes were determined. At the end of 48 hours, NLE and HP in 30c potency had a significantly greater melanin content (p = 0.015 and p = 0.039, respectively) compared with controls; BP and KA in 30c potency had no significant effects. No significant changes were seen at the end of 96 hours. KA, NLE, HP, and vehicle controls showed an inhibition of tyrosinase activity. The study demonstrated melanogenic effects of two homeopathic preparations. Further research to evaluate the therapeutic efficacy of these medicines is warranted.

Baicalin and berberine ultradeformable vesicles as potential adjuvant in vitiligo therapy.

0.5-1% of the world's population is affected by vitiligo, a disease characterized by a gradual depigmentation of the skin. Baicalin and berberine are natural compounds with beneficial activities, such as antioxidant, anti-inflammatory and proliferative effects. These polyphenols could be useful for the treatment of vitiligo symptoms, and their efficacy can be improved by loading in suitable carriers. The aim of this work was to formulate and characterize baicalin or berberine loaded ultradeformable vesicles, and demonstrate their potential as adjuvants in the treatment of vitiligo. The vesicles were produced using a previously reported simple, scalable method. Their morphology, size distribution, surface charge and entrapment efficiency were assessed. The ability of the vesicles to promote the permeation of the polyphenols was evaluated. The antioxidant and photoprotective effects were investigated in vitro using keratinocytes and fibroblasts. Further, the stimulation of melanin production and tyrosinase activity in melanocytes after treatment with the vesicles were assessed. Ultradeformable vesicles were small in size, homogeneously dispersed, and negatively charged. They were able to incorporate high amounts of baicalin and berberine, and promote their skin permeation. In fact, the polyphenols concentration in the epidermis was higher than 10%, which could be indicative of the formation of a depot in the epidermis. The vesicles showed remarkable antioxidant and photoprotective capabilities, presumably correlated with the stimulation of melanin production and tyrosinase activity. In conclusion, baicalin or berberine ultradeformable vesicles, and particularly their combination, may represent promising nanosystem-based adjuvants for the treatment of vitiligo symptoms.

Anti-aging effects of black raspberry extract on cataract, alopecia, skin whitening, and weight loss

Background: To maintain good health, it is important to eliminate extra reactive oxygen generated in the body. Furthermore, ingesting foods containing antioxidants is beneficial. The oxygen radical absorbance capacity (ORAC) values for black raspberry extract (BRE), blueberry extract (BBE), and raspberry extract (RBE) are 62, 66, and 49 µM Trolox equivalents (TE)/g respectively. These values are higher than those for typical antioxidant foods that have been discovered so far (3–30 µM TE/g). Our aim was to find new functionality from the food with the high ORAC value. Therefore, we have prepared these four berry extracts and examined whether they have anti-aging effects and if those effects correlate with their antioxidant activities.Methods: We studied the following effects of 4 berry extracts: 1) lens cell protective effects; 2) effects against alopecia; 3) induction of uncoupling protein-1 (UCP1), a regulator of fat and energy consumption in adipocytes, and stimulation of irisin secretion from skeletal muscle cells; and 4) inhibitory effects on melanocyte tyrosinase activity. The evaluation method was based on below; 1) a-crystallin, type 17 collagen, heat shock protein 47 (HSP47), UCP1 and Irisin - mRNA by qRT-PCR, 2) the amount of the UCP1 and Irisin protein by ELISA. 3) Inhibition of tyrosinase activity was measured by dopachrome production using L-tyrosine.Results: In lens cells, a-crystallin mRNA expression was induced 1 hour after treatment of the cell with Blabina (a powdered formulation containing BRE) and BRE. The extracts of all four berry species promoted the growth of follicle dermal papilla cells by 3-20% in a concentration-dependent manner. These berry extracts were also discovered to markedly induce the expression of mRNAs of type 17 collagen and HSP47 in the hair follicle stem cell and elevate the expression levels of UCP1 mRNA and its protein in adipocytes in a concentration-dependent manner. BRE and Blabina inhibited 5a-reductase in follicle dermal papilla cells and tyrosinase activity in melanocytes at the concentrations which inhibited dopachrome production by at least 50%. Finally, Blabina was discovered to stimulate the irisin secretion from skeletal muscles.Conclusion: These results suggest that berry extracts, particularly BRE, have anti-aging effects through their higher antioxidant activities.Keywords: Anti-aging; antioxidant; alopecia; black raspberry; weight loss; oxygen radical, absorbance capacity; skin whitening

Development and in vitro assessment of psoralen and resveratrol co-loaded ultradeformable liposomes for the treatment of vitiligo

Vitiligo is a de-pigmenting skin disorder characterized by white patches on skin due to partial or complete loss of melanocytes. Psoralen in combination with ultraviolet-A (PUVA) acts by stimulation of melanin content and tyrosinase activity in melanocytes. Resveratrol, a sirtuin activator and a potential anti-oxidant reduce oxidative stress which is one of the triggering factors for initiation of vitiligo. Despite their therapeutic activity, weak percutaneous permeability of psoralen and poor solubility of resveratrol hinders their effective topical administration. The aim of present study is to formulate ultradeformable liposomes (UDL) co-loaded with psoralen and resveratrol for evaluation of PUVA and anti-oxidant combination in vitiligo treatment. For this purpose, UDL composed of DC-Chol, cholesterol and sodium deoxy cholate were prepared for their co-delivery. Liposomal carriers were characterized and evaluated for their efficacy using B16F10 cell line. Free radical scavenging potential was also determined for these carriers by in vitro anti-oxidant assays. Optimal co-loaded UDL with particle size ranging from 120 to 130nm, zeta potential of +46.2mV, entrapment efficiency of 74.09% (psoralen) and 76.91% (resveratrol) were obtained. Compared to control, co-loaded UDL showed significant stimulation of melanin and tyrosinase activity with major contribution of psoralen. Further, co-loaded UDL also exhibited potential free radical scavenging activity where resveratrol played a key role. Hence, psoralen and resveratrol co-loaded UDL acts in vitiligo through dual mechanisms of action viz., stimulation of melanin and tyrosinase activity as well as by anti-oxidant activity. These findings indicate that psoralen and resveratrol co-loaded UDL has the promising therapeutic potential for the treatment of vitiligo.

Cosmeceutical Effects of Galactomannan Fraction from Arenga pinnata Fruits In vitro.

Background:Cosmeceuticals refer to natural cosmetics with medical-like benefits due to their bioactive contents. Sugar palm fruit (Arenga pinnata) extract has been claimed for its anti-aging effect in vitro. However, its active compounds for cosmeceuticals is still unclear.Objective:This study was aimed to extract galactomannan from A. pinnata fruits and test its efficacy for tyrosinase inhibition, antioxidant, and anti-photoaging activities in vitro.Materials and Methods:Galactomannan from A. pinnata fruits was extracted by freeze drying and identified for its chemical compounds by using pyrolysis gas chromatography-mass spectrometry (py-GC/MS). Galactomannan was tested for its tyrosinase inhibition in both cell-based (melanocytes) and enzymatic assays, antioxidant activity using ferrous ion chelating assay (FCA) assay, and anti-photoaging activity for inhibiting the gene expression of matrix metalloproteinase-1 (MMP-1) and MMP-13 in macrophages using quantitative real-time polymerase chain reaction (qRT-PCR) analysis.Results:Identification of galactomannan fraction from A. pinnata fruits by py-GC/MS mainly consisted of oxonium ion and glucosides. For cellular assay, galactomannan at 5 μg/mL inhibited >50% of tyrosinase activity in melanocytes induced by phorbol myristate acetate. At the enzymatic level, galactomannan at similar concentration showed less tyrosinase activity inhibition (~20%). FCA results showed that galactomannan at 10 μg/mL exerted >50% of antioxidant activity. The qRT-PCR data indicated that galactomannan at 5 μg/mL inhibited >50% of MMP-1 and MMP-13 gene expressions in ultraviolet B-treated macrophages.Conclusion:Galactomannan fraction from A. pinnata fruits has efficacy for enlightening effect, antioxidant, and anti-photoaging activity in the dose-independent pattern, indicating its cosmeceutical effects for skin healthcare.SUMMARY A. pinnata fruit containing galactomannan has cosmeceutical potentials through enlightening effect, antioxidant, and anti-photoaging activity in vitro.Galactomannan fraction has inhibitory effect on tyrosinase activity in both cellular melanocytes and enzymatic systems.Galactomannan fraction has strong protection against UVB-irradiation effect by inhibiting collagenase genes (MMP-1 and MMP-13) in macrophages. Abbreviations Used: Py-GC/MS: Pyrolysis-Gas Chromatography-Mass Spectrometry; FCA: Ferrous chelating activity; MMP: Matrix metalloproteinase; qRT-PCR: Quantitative Real-Time Polymerase Chain Reaction; PMA: Phorbol myristate acetate; UV: Ultraviolet; RPMI: Roswell Park Memorial Institute; DMEM: Dulbecco's modified eagle media; FBS: Fetal bovine serum; PBS: Phosphate buffered saline; MTT: 3-(4,5-diethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide; L-DOPA: L-3,4-dihydroxyphenylalanine; EDTA: Ethylenediaminetetraacetic acid; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; DPPH: 1,1-diphenyl-2-picryl-hydrazyl; SPF: Sun protection factor

Tranexamic acid: An emerging depigmenting agent

Tranexamic acid (TXA), an antifibrinolytic drug, is now gaining popularity as a depigmenting agent. It is a synthetic lysine amino acid derivative which mainly blocks the conversion of plasminogen to plasmin by inhibiting plasminogen activator. This results in less free arachidonic acid production, and hence a reduction in the prostaglandin (PG) levels as well. Thus, by reducing PG production, TXA reduces the melanocyte tyrosinase activity and plays an important role in the treatment of melasma, ultraviolet-induced hyperpigmentation, and other postinflammatory hyperpigmentation. It has been tried topically, orally, and intradermally in the management of melasma with minimal adverse effects. However, more randomized trials are needed to fully elucidate the exact mechanism of action, ideal route, frequency, and duration of administration of the drug, along with its potential to treat other pigmentary disorders.

Effects of diethylstilbestrol on the proliferation and tyrosinase activity of cultured human melanocytes.

The aim of the present study was to observe the effects of different exogenous estrogen diethylstilbestrol (DES) concentrations on the human melanocyte proliferation and tyrosinase activity. Skin specimens were obtained following blepharoplasty, and the melanocytes were primary cultured and passaged to the third generation. The melanocytes were seeded in 96-well plates, each well had 5×103 cells. The medium was changed after 24 h, and contained 10-4-10-8 M DES. After the melanocytes were incubated, the proliferation and tyrosinase activity were detected by the MTT assay and L-DOPA reaction. DES (10-8-10-6 M) enhanced the proliferation of cultured melanocytes. The intensity was positively correlated with the concentration of drug. DES, >10-5 M, inhibited the melanocytes proliferation or even produced the toxicity effect. Following the addition of 10-6 M DES to the medium, the tyrosinase activity of melanocytes was significantly increased, with P<0.05. In conclusion, a certain concentration of DES promoted the proliferation of melanocytes, enhanced the activity of tyrosinase and promoted pigment synthesis of melanocytes, with the optimal concentration of 10-6 M.

Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling

BackgroundParacrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect. ObjectiveTo resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action. Methods and ResultsRT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes. ConclusionsThese results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A).

Effects of Camellia reticulata, Chloranthus japonicas, Dodonaea viscosa and Paris polyphylla on the proliferation of and tyrosinase activity in a melanocyte cell line

Objective To estimate the whitening effect of extracts of Camellia reticulata, Chloranthus japonicas, Dodonaea viscosa and Paris polyphylla. Methods A human epidermal melanocyte cell line HEM-m was cultured in vitro, and classified into several groups to be treated with 7 concentrations（10, 25, 50, 100, 200, 400, 800 mg/L）of different plant extracts, including Camellia reticulata leaf extract, 70% ethanol-eluted fraction of Camellia reticulata leaf extract, extract of Camellia reticulata alabastrum, 70% ethanol-eluted fraction of extract of Camellia reticulata alabastrum, Chloranthus japonicas extract, Dodonaea viscosa extract, and Paris polyphylla extract. At the same time, the negative control group, blank control group and positive control group（treated with 100 mg/L arbutin）were set up. After 72 hours of treatment, MTT assay was performed to detect cell proliferation, and dopa-oxidation assay to estimate tyrosinase activity in melanocytes. Results As far as the inhibitory effect on the proliferation of HEM-m cells was concerned, 400 mg/L Camellia reticulata leaf extract, 70% ethanol-eluted fraction of Camellia reticulata leaf extract at 10-100 mg/L, extract of Camellia reticulata alabastrum at 10-25 mg/L, 70% ethanol-eluted fraction of extract of Camellia reticulata alabastrum at 10 mg/L and Dodonaea viscosa extract at 10-25 mg/L were equivalent to（all P > 0.05）, 800 mg/L Camellia reticulata leaf extract was significantly weaker than（P 0.05）, stronger than 70% ethanol-eluted fraction of extract of Camellia reticulata alabastrum at 10-25 mg/L（P < 0.05）, but weaker than the other concentrations of these plant extracts（P < 0.05 or 0.01）. Conclusion Special concentrations of Camellia reticulate and Dodonaea viscose can inhibit tyrosinase activity. Key words: Plant extracts; Melanocytes; Cell proliferation; Tyrosinase

Effect of norfloxacin and moxifloxacin on melanin synthesis and antioxidant enzymes activity in normal human melanocytes.

Fluoroquinolone antibiotics provide broad-spectrum coverage for a number of infectious diseases, including respiratory as well as urinary tract infections. One of the important adverse effects of these drugs is phototoxicity which introduces a serious limitation to their use. To gain insight the molecular mechanisms underlying the fluoroquinolones-induced phototoxic side effects, the impact of two fluoroquinolone derivatives with different phototoxic potential, norfloxacin and moxifloxacin, on melanogenesis and antioxidant enzymes activity in normal human melanocytes HEMa-LP was determined. Both drugs induced concentration-dependent loss in melanocytes viability. The value of EC50 for these drugs was found to be 0.5 mM. Norfloxacin and moxifloxacin suppressed melanin biosynthesis; antibiotics were shown to inhibit cellular tyrosinase activity and to reduce melanin content in melanocytes. When comparing the both analyzed fluoroquinolones, it was observed that norfloxacin possesses greater inhibitory effect on tyrosinase activity in melanocytes than moxifloxacin. The extent of oxidative stress in cells was assessed by measuring the activity of antioxidant enzymes: SOD, CAT, and GPx. It was observed that norfloxacin caused higher depletion of antioxidant status in melanocytes when compared with moxifloxacin. The obtained results give a new insight into the mechanisms of fluoroquinolones toxicity directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical processes in melanocytes may be an explanation for various phototoxic activities of the analyzed fluoroquinolone derivatives in vivo.

Sequential release of salidroside and paeonol from a nanosphere-hydrogel system inhibits ultraviolet B-induced melanogenesis in guinea pig skin.

Melanin is the one of most important pigments for skin color in mammals. Excessive biosynthesis of melanin induces various pigment disorders. Much effort has been made to develop regulators to minimize skin pigmentation abnormalities. However, only a few of them are used, primarily because of safety concerns and low efficiency. In this study, we aimed to construct a novel nanosphere-gel for sequential delivery of salidroside and paeonol, to investigate the synergistic effects of these drugs in anti-melanogenesis, and to decrease their potential for toxicity in high dosage. Nanospheres were prepared and characterized for their particle size, polydispersity index, zeta potential, and morphological properties. The optimized nanospheres were incorporated in carbomer hydrogel with both paeonol and salidroside entrapped to form a dual drug-releasing nanosphere-gel. With this nanosphere-gel, rapid release of salidroside from the hydrogel followed by sustained release of paeonol from the nanosphere was achieved. Using a classical model of the melanogenesis response to ultraviolet exposure, it was shown that the anti-melanogenesis effects of the dual drug-releasing system, in which the doses of the individual drugs were decreased by half, was obviously enhanced when compared with the effects of the single drug preparations. Mechanistically, the burst release of salidroside from the hydrogel may enable prompt suppression of melanocyte proliferation on exposure to ultraviolet B radiation, while the paeonol released in a sustained manner can provide continuous inhibition of tyrosinase activity in melanocytes. Combined delivery of salidroside and paeonol was demonstrated to be a promising strategy for enhancing the therapeutic efficacy of these agents in anti-melanogenesis and reducing their toxicity, so may have great potential in nanomedicine.