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Skin yellowness is a hallmark of dull or unhealthy skin, particularly among Asians. Previous research has indicated a link between skin glycation and skin yellowness. However, the specific glycated chemicals contributing to yellowish skin appearance have not been identified yet. Using HPLC-PDA-HRMS coupled with native and artificially glycated human epidermal explant skin, we identified intensely yellow colored glycated chromophores "(1R, 8aR) and (1S, 8aR)-4-(2-furyl)-7-[(2-furyl)-methylidene]-2-hydroxy-2H,7H,8AH-pyrano-[2,3-B]-pyran-3-one" (abbreviated as AGEY) from human skin samples for the first time. The abundance of AGEY was strongly correlated with skin yellowness in the multiple skin explant tissues. We further confirmed the presence of AGEY in cultured human keratinocytes and 3D reconstructed human epidermal (RHE) models. Additionally, we demonstrated that a combination of four cosmetic compounds with anti-glycation properties can inhibit the formation of AGEY and reduce yellowness in the RHE models. In conclusion, we have identified specific advanced glycation end products with an intense yellow color, namely AGEY, in human skin tissues for the first time. The series of study results highlighted the significant contribution of AGEY to the yellow appearance of the skin. Furthermore, we have identified a potential cosmetic solution to mitigate AGEY formation, leading to a reduction in yellowness in the in vitro RHE models.
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Produtos Finais de Glicação Avançada , Queratinócitos , Pele , Humanos , Produtos Finais de Glicação Avançada/metabolismo , Pele/metabolismo , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Glicosilação , Epiderme/metabolismo , Cosméticos/química , Feminino , Adulto , Pigmentação da Pele/efeitos dos fármacosRESUMO
Chemical leukoderma is an acquired depigmentation of the skin caused by repeated exposure to specific agents damaging to epidermal melanocytes. Case reports of chemical leukoderma have been associated with some consumer products. To date, there are no well-accepted approaches for evaluating and minimizing this risk. To this end, a framework is presented that evaluates the physical and chemical characteristics of compounds associated with chemical leukoderma and employs structure-activity relationship (SAR) read-across and predictive metabolism tools to determine whether a compound is at increased risk of evoking chemical leukoderma. In addition to in silico approaches, the testing strategy includes in chemico quinone formation and in vitro melanocyte cytotoxicity assays to dimension the risk as part of an overall weight of evidence approach to risk assessment. Cosmetic ingredients raspberry ketone, undecylenoyl phenylalanine, tocopheryl succinate, p-coumaric acid, resveratrol, resveratrol dimethyl ether, sucrose dilaurate, tranexamic acid, niacinamide and caffeic acid are evaluated in this framework and compared to positive controls rhododendrol and hydroquinone. Overall, this framework is considered an important step toward mitigating the risk of chemical leukoderma for compounds used in consumer products.
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Hipopigmentação , Butanóis , Epiderme/metabolismo , Humanos , Hipopigmentação/induzido quimicamente , Hipopigmentação/metabolismo , Melanócitos/metabolismo , Resveratrol/metabolismo , Pele/metabolismoRESUMO
Sallow and/or dull skin appearance is greatly attributable to the yellow components of skin tone. Bilirubin is a yellow chromophore known to be made in the liver and/or spleen and is transported throughout the body via the blood stream. Recent publications suggest bilirubin may be synthesized in other cells/organs, including the skin. We found human keratinocytes express the transcripts involved in bilirubin biosynthesis. In parallel, we also found human keratinocytes could indeed synthesize bilirubin in monolayer keratinocytes and in a 3D human skin-equivalent model. The synthesized amount was substantial enough to contribute to skin yellowness. In addition, oxidative stress enhanced bilirubin production. Using UnaG, a protein that forms a fluorescent species upon binding to bilirubin, we also visualized the intracellular expression of bilirubin in keratinocytes. Finally, we screened a compound library and discovered that the sucrose laurate/dilaurate (SDL) combination significantly reduced bilirubin levels, as well as bilirubin-mediated yellowness. In conclusion, bilirubin is indeed synthesized in epidermal keratinocytes and can be upregulated by oxidative stress, which could contribute to chronic or transient yellow skin tone appearance. Application of SDL diminishes bilirubin generation and may be a potential solution to mitigate yellowish and/or dull skin appearance.
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Bilirrubina , Queratinócitos , Bilirrubina/metabolismo , Bilirrubina/farmacologia , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Pele/metabolismo , Sacarose/análogos & derivadosRESUMO
BACKGROUND: Hyperpigmentation has varied aetio-pathologies. Hence, accurate and reproducible diagnosis of the type of hyperpigmentation is essential for effective management. It is typically made clinically by dermatologists but the rate of inter- and intra-observer agreement/variability is unknown. Hyperpigmented facial lesions are extremely common but access to dermatological services is difficult or costly in most countries. Thus, it is desired to evaluate dermatologists' inter- and intra-observer agreement in the diagnosis and to develop an algorithm for automated diagnosis. MATERIALS AND METHODS: Hyperpigmented lesions on 392 facial images were diagnosed by three experienced dermatologists either jointly or independently, and this process was subsequently repeated for 52 randomly selected images. When there was non-concordance amongst the dermatologists for the diagnosis, a majority decision was taken as correct diagnosis. Inter-observer and intra-observer agreement were analysed for the diagnosis of the hyperpigmented lesions. Thereafter, a multiclass classification method was developed to perform the task in an automatic manner. The developed algorithm was compared and validated against the ground truth derived from the dermatologists. RESULTS: Both inter- and intra-observer agreements are in the moderate range. The algorithm agreed well with the derived ground truth, with a Kappa value of 0.492, which is similar to the Kappa values of inter- and intra- observer agreements. CONCLUSION: The rates of inter- and intra-observer agreement in the diagnosis of hyperpigmented facial lesions amongst dermatologists were moderate in this study. Compared to visual assessment from the dermatologists, automated diagnosis using the developed algorithm achieved a high rate of concordance.
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Dermatologistas/estatística & dados numéricos , Face/diagnóstico por imagem , Hiperpigmentação/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Adulto , Algoritmos , Feminino , Humanos , Pessoa de Meia-Idade , Variações Dependentes do Observador , Fotografação , Reprodutibilidade dos TestesRESUMO
Geotrichum candidum is a dimorphic yeast used in cheese processing. To our knowledge, no major metabolites have been identified to date in G. candidum except for some amino acid and fatty acid metabolites. This has limited research on the commercial use of G. candidum. In this study, we aimed to analyze temporal changes in the intra- and extra-cellular metabolites of G. candidum and Saccharomyces cerevisiae cultured in YM medium as reference. As a result of metabolite analysis, it was observed that G. candidum tends to accumulateãpentose phosphate pathway compounds, which are involved in nucleic acid synthesis, after 48 h of cultivation when compared to S. cerevisiae. In addition, G. candidum accumulated higher amounts of the antioxidant glutathione in the medium than did S. cerevisiae. In addition, G. candidum accumulated large amounts of B vitamins such as pantothenic acid and nicotinic acid in the medium. Finally, we examined the potential of G. candidum as a host for the production of useful compounds such as pantothenic acid. When cultured in medium supplemented with the pantothenic acid precursor ß-alanine, G. candidum produced 12-fold higher amounts of pantothenic acid (30 µM) than that by S. cerevisiae. This study indicates that G. candidum accumulates various useful compounds that are dissimilar to those produced by S. cerevisiae. Furthermore, G. candidum has the potential to produce useful chemicals under appropriate culture conditions.
Assuntos
Queijo , Saccharomyces cerevisiae , Ácido Pantotênico , AminoácidosRESUMO
Sleep shortage is a major concern in modern life and induces various psycho-physical disorders, including skin problems. In cosmeceutics, females are aware that sleep deprivation worsens their facial skin tone. Here, we measured the effects of sleep deprivation on facial skin yellowness and examined yellow chromophores, such as bilirubin and carotenoids, in blood serum as potential causes of yellowness. Total sleep deprivation (0 h sleep overnight, N = 28) and repeated partial sleep deprivation (4 h sleep for 5 consecutive days, N = 10) induced significant increases in facial skin yellowness. The higher yellowness was sustained even after both sleep deprivation types stopped. However, circulating levels of yellow chromophores were unchanged in the total sleep deprivation study. Neither circulating interleukin-6 nor urinary biopyrrin levels were affected by total sleep deprivation, suggesting that apparent oxidative stress in the body was not detected in the present total deprivation protocol. Facial redness was affected by neither total nor repeated partial sleep deprivation. Therefore, blood circulation may play a limited role in elevated yellowness. In conclusion, facial skin yellowness was indeed increased by sleep deprivation in our clinical studies. Local in situ skin-derived factors, rather than systemic chromophore change, may contribute to the sleep deprivation-induced elevation of facial skin yellowness.
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Galactomyces ferment filtrate (GFF, Pitera™) is a cosmetic ingredient known to have multiple skin care benefits, such as reducing redness and pore size via the topical application of its moisturizer form. Although GFF is known to act partly as an antioxidative agonist for the aryl hydrocarbon receptor (AHR), its significance in keratinocyte biology is not fully understood. In this study, we conducted a transcriptomic analysis of GFF-treated human keratinocytes. Three different lots of GFF consistently modulated 99 (22 upregulated and 77 downregulated) genes, including upregulating cytochrome P450 1A1 (CYP1A1), a specific downstream gene for AHR activation. GFF also enhanced the expression of epidermal differentiation/barrier-related genes, such as small proline-rich proteins 1A and 1B (SPRR1A and SPRR1B), as well as wound healing-related genes such as serpin B2 (SERPINB2). Genes encoding components of tight junctions claudin-1 (CLDN1) and claudin-4 (CLDN4) were also target genes upregulated in the GFF-treated keratinocytes. In contrast, the three lots of GFF consistently downregulated the expression of inflammation-related genes such as chemokine (C-X-C motif) ligand 14 (CXCL14) and interleukin-6 receptor (IL6R). These results highlight the beneficial properties of GFF in maintaining keratinocyte homeostasis.
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INTRODUCTION: Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70 years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. METHODS: Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. RESULTS: In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. CONCLUSION: Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.
RESUMO
Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, beta-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2 .- in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.
Assuntos
Espécies Reativas de Oxigênio , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Antioxidantes/farmacologia , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biossíntese , Humanos , Medições Luminescentes , Pele/metabolismoRESUMO
BACKGROUND: Yeast extracts have been shown to perform anti-inflammatory and cytoprotective activities. However, the effects of yeast extracts on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and epidermal damages are still unclear. OBJECTIVE: To investigate the effect of Saccharomycopsis Ferment Filtrate (SFF) on LPS-induced NO production in RAW264.7 macrophages and epidermal damages. METHOD: RAW264.7 cells are incubated with LPS (25 ng/mL) and different concentrations of SFF. The amount of NO production is detected by Griess reaction. Additionally, the expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) are detected by Western blotting. Artificial epidermis is also used to mimic the in vivo condition to investigate the protective effects of SFF on LPS- or ultraviolet radiation (UVR)-induced damages by histology and electron microscopy. RESULTS: The results show that SFF addition inhibits LPS-induced NO production and iNOS protein expression in a concentration-dependent manner without notable cytotoxicity in RAW264.7 cells, and induction of HO-1 protein expression by SFF was observed. Interestingly, both HO-1 inducers, hemin and CoCl2, significantly attenuated LPS-induced NO production and iNOS protein expression. The addition of CoCl2 potentiated the inhibitory effect of SFF on LPS-induced NO production. It seems that HO-1 protein participates in SFF inhibition of LPS-induced NO production. Furthermore, SFF exhibits significant protective effect on LPS- or UVR-induced damages in the artificial epidermis via histological and electron microscopic observations. CONCLUSION: This study provided the first evidence to indicate the beneficial effects of SFF in preventing NO production in macrophages and damages in epidermis, respectively. It suggests that SFF possesses potential to be further developed.
Assuntos
Produtos Biológicos/uso terapêutico , Dermatite/terapia , Inflamação/terapia , Óxido Nítrico/metabolismo , Saccharomycopsis , Animais , Células Cultivadas , Cobalto , Dermatite/metabolismo , Dermatite/prevenção & controle , Expressão Gênica , Heme Oxigenase-1/metabolismo , Hemina , Humanos , Lipopolissacarídeos , Camundongos , Modelos Biológicos , Óxido Nítrico Sintase Tipo II/metabolismo , Pele/efeitos da radiaçãoRESUMO
Degradation of melanosomes in light skin (LS, i.e. phototype I/II) appears to occur more rapidly than dark skin (DS, i.e. phototype IV/V). Hydrolytic enzymes known to reside and be expressed in a differential pattern within the interfollicular epidermis are implicated in playing a role in epidermal differentiation and potentially melanosome degradation. The aim of this present study was to evaluate the differential expression of hydrolytic enzymes that may correlate with physiological and phenotypic differences seen between DS and LS. Expression of six hydrolytic enzymes was confirmed by microarray analysis of the suprabasal epidermis from LS and DS. Specific lysosomal hydrolases identified by microarray analysis were analyzed by indirect immunofluorescence (IIF) and immunoblot analysis. Immunogold electron microscopy (IEM) was completed to visualize cellular expression of the hydrolytic enzyme cathepsin L2 (Cath L2) and biochemical assay was performed to ascertain Cath L2 activity. Immunoblotting of light and dark epidermal lysates demonstrated that of the six enzymes initially analyzed, both prostatic acid phosphatase (ACPP) and Cath L2 were reproducibly upregulated in DS and LS, respectively. IIF and IEM analyses of Cath L2 in tissue confirmed this differential expression. Biochemical analysis of Cath L2 in light and dark epidermal lysates displays increased activity of Cath L2 in LS. The results of this study confirm differential expression of ACPP and Cath L2 in DS and LS at gene and protein level. Additionally, Cath L2 displays increased activity in LS-derived epidermal lysates. This study indentified two acid hydrolases that may play a role in melanosome degradation and pigment processing.
Assuntos
Epiderme/enzimologia , Melanossomas/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Pigmentação da Pele/genética , Fosfatase Ácida , Catepsinas/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Prepúcio do Pênis , Humanos , Hidrólise , Immunoblotting , Imuno-Histoquímica , Recém-Nascido , Masculino , Melanossomas/genética , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , FenótipoRESUMO
Modification of skin complexion coloration has traditionally been accomplished by interruption or attenuation of melanogenesis and/or melanosome transfer. Post-transfer modification of pigmented melanosomes provides an attractive and distinct avenue of modulating skin pigmentation. The processing of melanosomes during keratinocyte (KC) terminal differentiation and the degradative variability observed between light and dark skin (LS and DS) remains enigmatic. To evaluate this, we developed a model system to investigate the loss of fluorescently labeled and isolated melanosomes by cultured human KCs. The extent of melanosome loss has been qualitatively assessed using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Results show that melanosomes are incorporated into the cytoplasm of both light and dark keratinocytes (LKCs and DKCs) and trafficked to a perinuclear region. Within 48 hours, confocal microscopy images suggest that LKCs display accelerated melanosome loss. This time-dependent decrease in carboxyfluorescein diacetate (CFDA) fluorescence was then quantitatively analyzed using flow cytometry. Consistent with the results of the confocal analysis, over a 48-hour time frame, LKCs appear to lose melanosomes more efficiently than DKCs. These experiments show that melanosomes are more rapidly lost in KCs derived from LS as opposed to DS.
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Células Epidérmicas , Queratinócitos/ultraestrutura , Melanossomas/ultraestrutura , Pigmentação da Pele , Células Cultivadas , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia Confocal , Microscopia Eletrônica de TransmissãoRESUMO
OBJECTIVES: N-undecyl-10-enoyl-L-phenylalanine (Sepiwhite), N-undecylenoyl phenylalanine), a reported alpha-melanocyte-stimulating hormone (MSH) receptor antagonist, has been observed to reduce melanin production in cultured melanocytes. In other testing, niacinamide has been found to inhibit melanosome transfer in cultured cells and to reduce the appearance of hyperpigmented spots in clinical studies. Since these two agents function by different mechanisms, we conducted two studies to determine if their combination is more effective than niacinamide alone in reducing facial hyperpigmentation. METHODS: Two double-blind, 10-week (2-week washout + 8-week treatment), left-right randomized, split-face clinical studies were conducted. In one, two groups of Japanese women applied one of two pairs of test emulsion formulations: a vehicle control and a 5% niacinamide formulation (n= 40), or a 5% niacinamide and a 5% niacinamide plus 1%N-undecylenoyl phenylalanine formulation (n = 40). Each formulation was applied to the randomly assigned side of the face. In the second study, Caucasian women applied one of three emulsions: vehicle control, 5% niacinamide formulation, or combination 5% niacinamide plus 1%N-undecylenoyl-phenylalanine formulation to the randomly assigned side of the face (n = approximately 60 treatment sites per formulation). In both studies, hyperpigmented spots were evaluated at weeks 4 and 8 by quantitative image analysis. RESULTS: In both studies, the combination formulation was significantly more effective than the vehicle and the 5% niacinamide formulation in reducing the appearance of hyperpigmentation after 8 weeks. CONCLUSIONS: The combination of 5% niacinamide and 1%N-undecylenoyl phenylalanine is an effective anti-aging technology for use on facial skin.
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Dermatoses Faciais/tratamento farmacológico , Hiperpigmentação/tratamento farmacológico , Niacinamida/administração & dosagem , Fenilalanina/análogos & derivados , Administração Tópica , Adulto , Idoso , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Humanos , Pessoa de Meia-Idade , Fenilalanina/administração & dosagemRESUMO
Glucosamine has been reported to inhibit melanin production in melanocyte culture. It thus has a potential to reduce hyperpigmentation via topical use. Due to stability limitations of glucosamine, we chose to clinically evaluate the stable derivative N-acetyl glucosamine (NAG). Based on in vitro Franz cell testing, NAG is a good skin penetrant. In an 8-week, double-blind, placebo-controlled, left-right randomized, split-face clinical test, topical 2% NAG reduced the appearance of facial hyperpigmentation. In a second clinical study involving the topical combination of 2% NAG with 4% niacinamide, an agent previously shown to be clinically active, the effect on hyperpigmentation was greater. Both of these agents are well tolerated by the skin. This high tolerance coupled with relative ease of formulation and stability in solution make NAG, especially in combination with niacinamide, a suitable cosmetic ingredient for use in skin care products dealing with issues of skin hyperpigmentation.
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Acetilglucosamina/uso terapêutico , Dermatoses Faciais/tratamento farmacológico , Hiperpigmentação/tratamento farmacológico , Niacinamida/uso terapêutico , Administração Tópica , Adulto , Idoso , Povo Asiático , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Quimioterapia Combinada , Dermatoses Faciais/diagnóstico , Feminino , Seguimentos , Humanos , Hiperpigmentação/diagnóstico , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Valores de Referência , Medição de Risco , Índice de Gravidade de Doença , Resultado do Tratamento , População BrancaRESUMO
BACKGROUND/PURPOSE: Cutaneous hyperpigmentation occurs in multiple conditions. There is a strong need for the improvement of hyperpigmentation especially among Asian women. However, the effect of existing skin-lightening agents is not sufficient. One reason attributes to the limited capability of active agents to be delivered transepidermally. Ultrasound is one promising approach to enhance transepidermal transport. In this work, we investigate the effect of the use of high-frequency ultrasound together with coupling gel containing skin-lightening agents (ascorbyl glucoside and niacinamide) on facial hyperpigmentation in vivo in Japanese women. METHODS: The effect of ultrasound on the absorption of skin-lightening agents into the stratum corneum was evaluated in a tape-stripping method on human forearms in vivo. The skin efficacy was assessed in a facial clinical trial involving 60 subjects with hyperpigmentation in a paired design. Subjects were assigned to two groups, each group using two treatments (one on each facial cheek): (1) skin-lightening gel with ultrasound vs. no treatment or (2) skin-lightening gel with ultrasound vs. skin-lightening gel treatment. Changes in facial hyperpigmentation were objectively quantified by computer analysis and visual grading of high-resolution digital images of the face in addition to the subjective assessment via questionnaire. RESULTS: Ultrasound radiation enhanced the absorption of skin-lightening agents in the stratum corneum in a radiation-time-dependent manner. In the facial clinical trial, use of ultrasound radiation together with the skin-lightening gel significantly reduced facial hyperpigmented spots compared with both no treatment and skin-lightening gel alone after 4 weeks. CONCLUSIONS: The data suggest that use of high-frequency ultrasound radiation together with skin-lightening gel is effective to reduce hyperpigmentation via enhancing transepidermal transport of skin-lightening agents.
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Ácido Ascórbico/administração & dosagem , Hiperpigmentação/terapia , Niacinamida/administração & dosagem , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/efeitos da radiação , Terapia por Ultrassom/métodos , Adulto , Terapia Combinada , Fármacos Dermatológicos/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
The objective of this study is to image and detect reactive oxygen species (ROS) generated in the UVB-exposed three-dimensional human skin equivalent model (HSEM), EpiDermtrade mark 200, because the alternative system needs to be urgently established as a replacement for the skin of experimental animals. Evidence that the ROS generation is enhanced in the skin of live animals after the UVB exposure was already obtained, by using the real-time chemiluminescent (RT-CL) method consisting of a sensitive CL probe (CLA) and an ultra-low light imaging apparatus. In this study, CL emission due to the reaction of CLA with endogenously generated ROS increased significantly in the UVB-treated HSEM compared with that in the intact HSEM, the maximum level being observed at a dose of 27mJ/cm(2). ROS under UVB exposure was identified to be ()O2- and (1)O(2) as observed by suppressive effects of SOD and beta-carotene topically applied on sample surface before the UVB exposure. The results for UVB-induced ROS generation in HSEM were consistent with those observed in the skin of live animals. HSEM combined with the RT-CL method was shown to be useful system not only to predict UVB-induced ROS-related skin responses in human but also to find protective agents against UVB-stimulated oxidative stress in place of animals and ex vivo human skin.
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Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Medições Luminescentes/métodos , Espécies Reativas de Oxigênio/análise , Pele/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta , Células Cultivadas , Sistemas Computacionais , Relação Dose-Resposta à Radiação , Exposição Ambiental , Humanos , Doses de Radiação , Pele/citologia , Espectrofotometria Ultravioleta/métodosRESUMO
Skin pigmentation results in part from the transfer of melanized melanosomes synthesized by melanocytes to neighboring keratinocytes. Plasma membrane lectins and their glycoconjugates expressed by these epidermal cells are critical molecules involved in this transfer process. In addition, the derivative of vitamin B(3), niacinamide, can inhibit melanosome transfer and induce skin lightening. We investigated the effects of these molecules on the viability of melanocytes and keratinocytes and on the reversibility of melanosome-transfer inhibition induced by these agents using an in vitro melanocyte-keratinocyte coculture model system. While lectins and neoglycoproteins could induce apoptosis in a dose-dependent manner to melanocytes or keratinocytes in monoculture, similar dosages of the lectins, as opposed to neoglycoproteins, did not induce apoptosis to either cell type when treated in coculture. The dosages of lectins and niacinamide not affecting cell viability produced an inhibitory effect on melanosome transfer, when used either alone or together in cocultures of melanocytes-keratinocytes. Cocultures treated with lectins or niacinamide resumed normal melanosome transfer in 3 days after removal of the inhibitor, while cocultures treated with a combination of lectins and niacinamide demonstrated a lag in this recovery. Subsequently, we assessed the effect of niacinamide on facial hyperpigmented spots using a vehicle-controlled, split-faced design human clinical trial. Topical application of niacinamide resulted in a dose-dependent and reversible reduction in hyperpigmented lesions. These results suggest that lectins and niacinamide at concentrations that do not affect cell viability are reversible inhibitors of melanosome transfer.