Melanin Content Of Melanocytes Research Articles

Aktivitas pegagan (Centella asiatica) pada dermatologi

Pegagan (Centella asiatica L.) is a herbal plant used in dermatology that has activity in treating skin diseases and skin lesions such as excoriations, burns, hypertrophic scars, antioxidants, anti-aging, skin whitening, and as a cosmetic ingredient. This review aims to collect data regarding the activity of Centella asiatica herb extracts in dermatology in both preclinical and clinical trial identification is done by searching literature through media Science Direct and Google Scholar using the keywords ''Centella asiatica'', ''Gotu kola'', ''dermatology'', ''cosmetics'', and ''whitening''. The search was based on the inclusion and exclusion criteria that had been set. The literature results show that the chemical constituents of Centella asiatica, such as asiaticoside, madecassoside, asiatic acid, and madecassic acid, are phytochemicals that play an essential role in the pharmacological activity of Centella asiatica in dermatology as a treatment and skin care. In both preclinical and clinical tests, it was shown that administration of Centella asiatica was capable of proliferating fibroblasts, activating the Smads pathway, increasing collagen synthesis, reducing metalloproteinase activity by increasing collagen deposition, and reducing melanin content in melanocytes so that it can modulate melanogenesis by inhibiting the expression of tyrosinase mRNA.

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.

The Whitening Properties of the Mixture Composed of Pomegranate, Osmanthus and Olive and the Protective Effects Against Ultraviolet Deleterious Effects.

BackgroundUltraviolet (UV) rays are the major environmental factor that damage skin physiology causing deleterious effects such as oxidation, photoaging and pigmentation. There has been considerable interest in using botanicals to prevent skin damages caused by UV irradiation.AimIn this study, three plant extracts were tested either individually or combined together (mixture) as well as their corresponding main active compound: pomegranate/punicalagin, osmanthus/verbascoside and olive/hydroxytyrosol. We evaluated the whitening and anti-photoaging properties of the nutritional mixture using 2D human culture model and a 3D full-thickness pigmented skin model exposed to UVB and UVA.MethodsFor exploring skin pigmentation, oxidation and aging, we performed cell viability, tyrosinase activity and melanin content assays as well as histology analysis (Whartin–Starry staining), immunodetection (PMEL, MDA, collagen type I and elastin) and carbonylated proteins analysis by electrophoresis separation.ResultsResults showed that the pomegranate extract and the active molecule punicalagin could reduce the tyrosinase activity and melanin content in melanocytes (P < 0.05). The mixture, pomegranate extract and punicalagin inhibited the melanin production and pre-melanosomal protein (PMEL) expression in the 3D skin pigmented model (P < 0.001). Furthermore, the mixture treatment repaired the expressions of collagen I and elastin decrease by UV exposure (P < 0.01). The mixture also significantly decreased lipid peroxidation (P < 0.001) and carbonylated proteins (P < 0.05) in the skin model compared to the UV-exposed condition.ConclusionTo conclude, the mixture composed of pomegranate, osmanthus and olive extracts protects human skin from UV rays deleterious effects and exhibits antioxidative, anti-aging and skin whitening properties. Our data suggested pomegranate contributed to the whitening properties of the mixture notably through its main active compound, punicalagin. The mixture might be a good candidates for further development as natural antioxidant and skin care product.

The Antioxidant and Tyrosinase-Inhibiting Activities of 4-Hydroxycinnamoyl-Malate from a Citrus junos Callus Extract.

We evaluated the antioxidant and skin-lightening activities of 4-hydroxycinnamoyl-malate (4-HCM) in vitro; the material is a water-soluble component of Citrus junos callus. 4-HCM was waterextracted from callus grown on MS medium containing picloram to enhance growth. The antioxidant activity of 4-HCM was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzoline-6-sulfonate) (ABTS) free-radical-scavenging assays. 4-HCM-mediated inhibition of extracellular matrix protein glycation was assessed using the elastin and collagen glycation assays. Inhibition of skin pigmentation was evaluated by measuring anti-tyrosinase activity and melanogenesis in melanoma cells. 4-HCM was then incorporated into elastic nanoliposomes (ENLs) and delivered topically to enhance percutaneous absorption. At 5 mM, the free-radical-scavenging activity of 4-HCM was 54.2±0.631% in the ABTS assay, comparable to that of 1 mM ascorbic acid (46.5± 0.17%). The IC50 value for inhibition of advanced glycation end-product formation from elastin was 1.25±0.23 mM; collagen glycation was completely inhibited when the 4-HCM level was >5 mM. At 0.5 mM, the material afforded 49.2±3.09% inhibition of tyrosinase activity and, at 10 mM, reduced the melanocyte melanin content by 78% without significant cellular toxicity. Moreover, 4-HCM-loaded ENLs exhibited 569% enhanced permeation of 4-HCM across the human epidermis, which may afford skin-lightening if included in cosmetic formulations.

Tyrosinase activity and melanogenic effects of Lespedeza bicolor extract in vitro and in vivo

Lespedeza bicolor (L. bicolor) is used for medicinal purposes because of its various biological and pharmacological activities. In this study, the effects of L. bicolor ethanol extract on the treatment of vitiligo were investigated. The determination of melanin content in melanocytes was measured using B16 melanoma cells and C57BL/6J Ler-vit/vit mice. Finally, the quercetin content in L. bicolor were qualitatively analyzed using HPLC. The results obviously indicated that the L. bicolor extract enhanced melanogenesis and increased tyrosinase activity in cultured melanoma cells and C57BL/6J Ler-vit/vit mice. Treatment with L. bicolor extract led to a higher content of melanin and eumelanin in C57BL/6J Ler-vit/vit mice hair than in control (untreated) mice, which demonstrated the therapeutic effect of hair-graying associated with vitiligo. There was a notable increase in melanocytes in the skin of C57BL/6J Ler-vit/vit mice treated with L. bicolor extract compared with the control. L. bicolor extract was a potent tyrosinase and melanin synthesis activator in B16 melanoma cells. C57BL/6J Ler-vit/vit mice treated with L. bicolor extract had significantly higher melanin content in hair than the untreated control. The results suggest that L. bicolor extract is a potential alternative treatment for improvement of vitiligo.

Dysfunction of ATG7-dependent autophagy dysregulates the antioxidant response and contributes to oxidative stress-induced biological impairments in human epidermal melanocytes

Autophagy is a process involving the self-digestion of components that participates in anti-oxidative stress responses and protects cells against oxidative damage. However, the role of autophagy in the anti-oxidative stress responses of melanocytes remains unclear. To investigate the role of autophagy in human epidermal melanocytes, we knocked down and overexpressed ATG7, the critical gene of autophagy, in normal human epidermal melanocytes. We demonstrated that ATG7-dependent autophagy could affect melanin content of melanocytes by regulating melanogenesis. Moreover, suppression of ATG7-dependent autophagy inhibits proliferation and promotes oxidative stress-induced apoptosis of melanocytes, whereas enhancement of ATG7-dependent autophagy protects melanocytes from oxidative stress-induced apoptosis. Meanwhile, deficiency of ATG7-dependent autophagy results in premature senescence of melanocytes under oxidative stress. Notably, we verified that ATG7-dependent autophagy could alter oxidative stress homeostasis by regulating reactive oxygen species (ROS) production, nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway, and the activity of several antioxidant enzymes in melanocytes. In conclusion, our study suggested that ATG7-dependent autophagy is indispensable for redox homeostasis and the biological functions of melanocytes, such as melanogenesis, proliferation, apoptosis, and senescence, especially under oxidative stress.

Tyrosinase Activity and Melanogenic Effects of Rhododendron schlippenbachii Extract In vivo and In vitro

Rhododendron schlippenbachii have been used as a medicine because of their various biological activities. In this study, R. schlippenbachii ethanol extract was evaluated for the treatment of vitiligo. The R. schlippenbachii ethanol extract did not show any cell cytotoxicity. The effect on mushroom tyrosinase and cellular tyrosinase activities were further assessed. In addition, the determination of melanin content in melanocytes was measured using both the B16 melanoma cells and C57BL/6J Ler-vit/vit mice. Finally, the existence of quercetin in R. schlippenbachii was confirmed by qualitative analysis using HPLC. The results clearly demonstrated the R. schlippenbachii extract enhanced melanogenesis and also increased tyrosinase activity in cultured melanoma cells and C57BL/6J Ler-vit/vit mice. In addition, treatment with R. schlippenbachii extract led to a higher content of melanin and eumelanin in C57BL/6J Ler-vit/vit mice hair than in control (untreated) mice, which demonstrated the therapeutic effect of hair-graying associated with vitiligo. Finally, we confirmed a notable increase in melanocytes in the skin of C57BL/6J Ler-vit/vit mice treated with R. schlippenbachii extract compared with the control. Extracts of R. schlippenbachii was shown to be potent tyrosinase and melanin synthesis activator in B16 melanoma cells. The R. schlippenbachii extract have significantly higher melanin content than the untreated control in C57BL/6J Ler-vit/vit mice hair. The results suggest that R. schlippenbachii extract might be considered as an alternative treatment for improvement of vitiligo.

The Long Noncoding RNA UCA1 Negatively Regulates Melanogenesis in Melanocytes

The long noncoding RNA UCA1 was first discovered in bladder cancer and is known to regulate the proliferation and migration of melanoma. However, its role in melanogenesis is unclear. In this study, we aimed to explore the role and mechanism of UCA1 in melanogenesis. Our findings showed that the expression of UCA1 was negatively correlated with melanin content in melanocytes and pigmented nevus. Overexpression of UCA1 in melanocytes decreased melanin content and the expression of melanogenesis-related genes, whereas knockdown of UCA1 in melanocytes had the opposite effect. High-throughput sequencing revealed thatmicrophthalmia-associated transcription factor (MITF), an important transcription factor affecting melanogenesis, was also negatively correlated with the expression of UCA1. Furthermore, the transcription factor CRE-binding protein (CREB), which promotes MITF expression, was negatively regulated by UCA1. The cAMP/protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) signaling pathways, which are upstream of the CREB/MITF/melanogenesis axis, were activated or inhibited in response to silencing or enhancing UCA1 expression, respectively. In addition, enhanced UCA1 expression downregulates the expression of melanogenesis-related genes induced by UVB in melanocytes. In conclusion, UCA1 may negatively regulate the CREB/MITF/melanogenesis axis through inhibiting the cAMP/PKA, ERK, and JNK signaling pathways in melanocytes. UCA1 may be a potential therapeutic target for the treatment of pigmented skin diseases.

Sulfated hyaluronan-containing artificial extracellular matrices promote proliferation of keratinocytes and melanotic phenotype of melanocytes from the outer root sheath of hair follicles.

The aim of this work was to establish improved cultivation conditions for human keratinocytes (HUKORS) and melanocytes (HUMORS) from the outer root sheath (ORS) of human hair follicles for purposes of generating an epidermal graft. To this end, the cells were cultivated on artificial extracellular matrix coatings composed of collagen (COLL) and hyaluronan (HA) with varying sulfation degrees. HUKORS and HUMORS were characterized based on their morphology and proliferation, marker gene expression, protein expression and melanin content in melanocytes. Depending on the sulfation degree, the matrices provided a favorable proliferation environment for HUKORS and improved the balance between proliferation and the exertion of melanotic phenotype (gene expression and melanin content) in HUMORS. Based on the increased gene expression of microphthalmia-associated transcription factor, as well as the downstream-affected melanotic genes premelanosome protein and tyrosinase in HUMORS cultivated on collagen matrices with high-sulfated HA, we assume that sulfated HA enhanced melanotic phenotype either by directly binding the CD44 receptor or by concentrating signaling mediators on site. Being a promising cultivation environment for both HUKORS and HUMORS, collagen matrices with sulfated HA have a potential of significantly improving the development of ORS-based epidermal grafts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1640-1653, 2019.

Exosomal miRNA derived from keratinocytes regulates pigmentation in melanocytes

BackgroundPigmentation is controlled by complex mechanisms. Evidence suggests that miRNAs can regulate pigmentation. However, the mechanism has not been fully elucidated. Objective In this study, we revealed a novel mechanism that regulates pigmentation involving exosomes, miRNAs and the crosstalk between keratinocytes and melanocytes. MethodsThe expression and localization of exosome specific marker TSG101 in keratinocytes and melanocytes; Changes of melanin content in melanocytes after co-culture of exosome and melanocytes; Expression changes of target gene TYR and its related genes and inhibitory effect of miR-330-5p on pigmentation were studied by using various molecular biological techniques. ResultsIn this experiment, we used miR-330-5p in keratinocytes to verify the effect of keratinocyte derived exosome on melanocyte pigmentation. First, we found that keratinocytes secrete exosomes carrying miR-330-5p; moreover, greater miR-330-5p expression was found in exosomes derived from keratinocytes that overexpressed miR-330-5p. Second, we found that exosomes derived from keratinocytes with overexpression of miR-330-5p caused a significant increase in miR-330-5p in melanocytes. Finally, exosomes derived from keratinocytes that overexpressed miR-330-5p induced a significant decrease in the production of melanin and expression of TYR in melanocytes. Meanwhile, we overexpressed miR-330-5p in melanocytes, which also proved the inhibitory effect of miR-330-5p on pigmentation. ConclusionThese findings suggest that keratinocytes crosstalk with melanocytes in the epidermal melanin unit via exosomal miRNAs. These studies reveal an important role of exosomes in melanocyte pigmentation, which opens a new pathway of melanogenesis.

Kanamycin induces free radicals formation in melanocytes: An important factor for aminoglycosides ototoxicity.

Ototoxicity is well-documented but not fully understood undesirable side effect of aminoglycoside antibiotic, kanamycin. Kanamycin is capable of binding to melanin biopolymers-natural pigments of the skin, hair,and eyes. Melanin-producing cells, melanocytes, are also present in the inner ear and are known to be necessary for normal hearing. It was considered that melanin content in the inner ear may influence aminoglycoside-induced ototoxic effect. The impact of kanamycin on melanocytes homeostasis may thus play role in the antibiotic-induced ototoxic effect. Previously, we demonstrated that kanamycin disturbs homeostasis in light-pigmented melanocytes. To investigate if/how melanization contributes to this phenomenon, the study using in vitro model of dark-pigmented melanocytes is required. Spectrophotometric measurements and electron paramagnetic resonance (EPR) spectroscopy analysis were performed. Kanamycin induced aconcentration-dependent loss in HEMn-DP melanocytes viability. The value of IC 50 was estimated to be 5.0 mM. Modulation of the activity of analyzed antioxidant enzymes and increased production of free radicals as well as the decrease of the melanin content were observed. Our results confirmed that kanamycin generates oxidative stress in melanocytes. The increased level of free radicals caused by kanamycin may be responsible for the imbalance of antioxidant defense and the reduction of melanin content in melanocytes. The role of melanin in the mechanism of kanamycin-induced hearing impairment was discussed and the obtained results were compared with the previously demonstrated data concerning light-pigmented melanocytes.

High correlation between skin color based on CIELAB color space, epidermal melanocyte ratio, and melanocyte melanin content.

BackgroundTo treat skin color disorders, such as vitiligo or burns, melanocytes are transplanted for tissue regeneration. However, melanocyte distribution in the human body varies with age and location, making it difficult to select the optimal donor skin to achieve a desired color match. Determining the correlations with the desired skin color measurement based on CIELAB color, epidermal melanocyte numbers, and melanin content of individual melanocytes is critical for clinical application.MethodFifteen foreskin samples from Asian young adults were analyzed for skin color, melanocyte ratio (melanocyte proportion in the epidermis), and melanin concentration. Furthermore, an equation was developed based on CIELAB color with melanocyte ratio, melanin concentration, and the product of melanocyte ratio and melanin concentration. The equation was validated by seeding different ratios of keratinocytes and melanocytes in tissue-engineered skin substitutes, and the degree of fitness in expected skin color was confirmed.ResultsLinear regression analysis revealed a significant strong negative correlation (r = − 0.847, R2 = 0.717) between CIELAB L* value and the product of the epidermal melanocyte ratio and cell-based melanin concentration. Furthermore, the results showed that an optimal skin color match was achieved by the formula.DiscussionWe found that L* value was correlated with the value obtained from multiplying the epidermal melanocyte ratio (R) and melanin content (M) and that this correlation was more significant than either L* vs M or L* vs R. This suggests that more accurate prediction of skin color can be achieved by considering both R and M. Therefore, precise skin color match in treating vitiligo or burn patients would be potentially achievable based on extensive collection of skin data from people of Asian descent.

Preparation, Characterization, and Biological Activities of Topical Anti-Aging Ingredients in a Citrus junos Callus Extract

In this study, we prepared and characterized a callus extract from Citrus junos and assessed its utility as a source of topical anti-aging ingredients. Callus extract was produced by aqueous extraction from Citrus junos grown on Murashige and Skoog medium with picloram as a growth regulator. After measuring the total phenolic and flavonoid contents, the major phenolic compound in calli was identified as p-hydroxycinnamoylmalic acid (1) by spectroscopic analysis. The total phenol content in the extract was determined to be 24.50 ± 0.43 mg/g of gallic acid equivalents; however, the total flavonoid content of the extract was not determined. The biological activities of the callus extract, in terms of skin anti-aging, were assessed by measuring the anti-tyrosinase activity in, and melanogenesis by, melanoma cells; and proliferation of, and procollagen synthesis by, human fibroblasts. The callus extract was incorporated into nanoliposomes (NLs) to improve its percutaneous absorption. Addition of the callus extract resulted in a 1.85-fold decrease in the melanin content of melanocytes compared with that with arbutin. The extract (500 μg/mL) significantly promoted the proliferation of, and procollagen synthesis by, fibroblasts (by 154% and 176%, respectively). In addition, the flux through the human epidermis of Citrus junos callus extract incorporated into NLs was 17.67-fold higher than that of the callus extract alone. These findings suggest that Citrus junos callus extract-loaded NLs have promise as an anti-aging cosmetic, as well as having a skin-lightening effect.

Ocular Albinism Type 1 Regulates Melanogenesis in Mouse Melanocytes.

To investigate whether ocular albinism type 1 (OA1) is differentially expressed in the skin of mice with different coat colors and to determine its correlation with coat color establishment in mouse, the expression patterns and tissue distribution characterization of OA1 in the skin of mice with different coat colors were qualitatively and quantitatively analyzed by real-time quantitative PCR (qRT-PCR), immunofluorescence staining and Western blot. The qRT-PCR analysis revealed that OA1 mRNA was expressed in all mice skin samples tested, with the highest expression level in brown skin, a moderate expression level in black skin and the lowest expression level in gray skin. Positive OA1 protein bands were also detected in all skin samples by Western blot analysis. The relative expression levels of OA1 protein in both black and brown skin were significantly higher than that in gray skin, but there was no significant difference between black and brown mice. Immunofluorescence assays revealed that OA1 was mainly expressed in the hair follicle matrix, the inner and outer root sheath in the skin tissues with different coat colors. To get further insight into the important role of OA1 in the melanocytes’ pigmentation, we transfected the OA1 into mouse melanocytes and then detected the relative expression levels of pigmentation-related gene. Simultaneously, we tested the melanin content of melanocytes. As a result, the overexpression of OA1 significantly increased the expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TRP1) and premelanosome protein (PMEL). However, the tyrosinase-related protein 2 (TRP2) level was attenuated. By contrast, the level of glycoprotein non-metastatic melanoma protein b (GPNMB) was unaffected by OA1 overexpression. Furthermore, we observed a significant increase in melanin content in mouse melanocyte transfected OA1. Therefore, we propose that OA1 may participate in the formation of coat color by regulating the level of MITF and the number, size, motility and maturation of melanosome.

Modulation of Melanogenesis and Antioxidant Status of Melanocytes in Response to Phototoxic Action of Doxycycline.

Doxycycline is a commonly used tetracycline antibiotic showing the broad spectrum of antibacterial action. However, the use of this antibiotic is often connected with the risk of phototoxic reactions that lead to various skin disorders. One of the factors influencing the photosensitivity reactions is the melanin content in melanocytes. In this study, the impact of doxycycline and UVA irradiation on cell viability, melanogenesis and antioxidant defense system in cultured normal human epidermal melanocytes (HEMn-DP) was examined. The exposure of cells to doxycycline and UVA radiation resulted in concentration-dependent loss in melanocytes viability and induced melanin biosynthesis. Significant changes were stated in cellular antioxidant enzymes activity: SOD, CAT and GPx, which indicates alterations of antioxidant defense system. The results obtained in vitro may explain the mechanisms of phototoxic reactions that occur in normal human epidermal melanocytes in vivo after exposure of skin to doxycycline and UVA radiation.

MicroRNA-27a-3p Inhibits Melanogenesis in Mouse Skin Melanocytes by Targeting Wnt3a.

MicroRNAs (miRNAs) play an essential role in the regulation of almost all the biological processes, including melanogenesis. MiR-27a-3p is nearly six times higher in white alpaca skin compared to brown skin, which indicates that miR-27a-3p may be a candidate regulator for melanogenesis. Wnt3a plays an important role in promoting melanoblasts to differentiate into melanocytes and melanogenesis. To confirm the function of miR-27a-3p to melanogenesis in mammals, miR-27a-3p mimic, inhibitor and their negative control were transfected into mouse melanocytes. As a result, miR-27a-3p inhibits melanogenesis by repressing Wnt3a at post-transcriptional level. A significant decrease in Wnt3a luciferase activity was observed in 293T cells co-transfected with the matched luciferase reporter vector and pre-miR-27a. Furthermore, the presence of exogenous miR-27a-3p significantly decreased Wnt3a protein expression rather than mRNA and reduced β-catenin mRNA levels in melanocytes. The over-expression of miR-27a-3p significantly increased the melanin content of melanocytes. However, miR-27a-3p inhibitor performs an opposite effect on melanogenesis. Wnt3a is one target of miR-27a-3p. MiR-27a-3p could inhibit Wnt3a protein amount by post-transcriptional regulation and melanogenesis in mouse melanocytes. Previous studies reported that Wnt3a promoted melanogenensis in mouse melanocytes. Thus, miR-27-3p inhibits melanogenesis by repressing Wnt3a protein expression.

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.

Asiaticoside, a component of Centella asiatica, inhibits melanogenesis in B16F10 mouse melanoma.

Melanogenesis is the process of generating pigmentation via melanin synthesis and delivery. Three key enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1) and TRP2, metabolize melanin from L-tyrosine. Melanin synthesizing enzymes are regulated by microphthalmia-associated transcription factor (MITF). The titrated extract of Centella asiatica (TECA) contains the major components asiatic acid, asiaticoside and madecassic acid. The present study revealed that TECA reduces the melanin content in melanocytes. Moreover, the asiaticoside contained in TECA modulated melanogenesis by inhibiting tyrosinase mRNA expression. The decrease in tyrosinase mRNA levels was mediated through MITF. Uniquely, asiaticoside inhibited MITF by decreasing its DNA binding affinity. In conclusion, the results of the present study indicate that asiaticoside treatment may have beneficial effects in hyperpigmentation diseases or for skin whitening.

Effects of ginsenoside Rb1 on melanogenesis in human epidermal melanocytes.

Objective To estimate the effects of ginsenoside Rbl on melanogenesis in human melanocytes and underlying mechanisms. Methods Epidermal melanocytes were obtained from circumcision specimens of children, and subjected to primary culture. After 2 to 5 passages, the melanocytes were treated with different concentrations of ginsenoside Rbl, dimethyl sulfoxide （DMSO, vehicle control）, forskolin at 10μmol/L （positive control） or remained untreated （blank control）. After additional culture for 72 hours, methyl thiazolyl tetrazolium （MTT） assay and NaOH lysis method were used to evaluate cell viability and melanin content in melanocytes respectively, spectrophotometer to determine dopa oxidase activity of tyrosinase, Western blot to quantify the protein level of tyrosinase, microphthalmia-associated transcription factor （MITF）, phosphorylated and total cAMP response element binding protein （p-CREB and t-CREB） in melanocytes. Results After treatment with ginsenoside Rbl of 25, 50 and 100 μmol/L for 72 hours, the melanocytes experienced no significant changes in viability （P 〉 0.05）, but a significant dose-dependent increase in melanin content （112.4%±5.7%, 155.7%±6.3%, 217.2% ± 11.7% vs. 100%, P〈 0.05 or 0.01） and tyrosinase aetivity（117.9% ± 5.7%, 158.2%± 9.6%, 182.6%±10,0% vs. 100%, P 〈 0.05 or 0.01） compared with the vehicle control melanocytes. The protein expressions of tyrosinase, MITF and p-CREB were statistically higher in melanocytes treated with ginsenoside Rhl of 100 μmol/L for 72 hours than in the vehicle control melanocytes （225.4% ±12.8% vs. 100%±7.9%, 313.5%±16.7% vs. 100% ± 9.8%, 322.5% ±21.1% vs. 100%±9.1%, all P 〈 0.01）. The increase in MITF protein expression was inapparent in melanocytes at 8 hours after the treatment with ginsenoside Rbl of 100 μmol/L, but statistically significant at 24 hours compared with the melanocytes at baseline （P 〈 0.01 ）.The pretreatment with H-89 （a selective inhibitor of PKA） at 10μmol/L, significantly suppressed the ginsenoside Rbl （ 100μmol/L for 72 hours） -induced phosphorylation of CREB, increase in MITF, tyrosinase expression, as well as tyrosinase activity and melanin content in melanocytes （all P 〈 0.01）. Conclusions Ginsenoside Rbl could enhance the melanogenesis and tyrosinase activity in normal human melanocytes. The PKA/CREB/MITF/ tyrosinase signaling pathway may contribute to the pro-melanogenic effect of ginsenoside Rbl. Key words: Melanocytes; GINSENOSIDE; Monophenol monooxygenase; Microphthalmia-associated transcription factor, Melanogenesis

Science Signaling Podcast: 19 May 2009

TRPM1 is a cation channel found on vesicular membranes and associated with melanin content in melanocytes.