Articles et publications

24th World Congress of Dermatology, Milan, 10-15 June, 2019. A. F. C. Pereira, M. H. Igarashi, R. Lage, S. G. Silva, M. Mercuri, A. F. Pereira, M. S. Silva, G. Facchini, A. L. T. A. Pinheiro, S. Eberlin. Environmental factors play a key role in the extrinsic hair aging. A classic example is ultraviolet (UV) radiation, which is responsible for the degradation of collagen XVII?1 (COL17A1) present in hair follicle (HF), resulting in an aging hair phenotype characterized by thinning, depigmentation, and hair loss. In addition, dermal papilla cells, also present in HF, play a crucial role in morphogenesis and hair cycle through mechanisms involving the Wnt signaling pathway. This signaling pathway is required for the development, regeneration of HF, and hair growth, mediated by the stabilization and translocation of the key signal transducer ?-catenin. In this study, we evaluated the ability of a hair treatment including a shampoo (ShPi) and a tonic (HtPi) to prevent hair aging using ex vivo human scalp submitted to UV radiation. COL17A1 semi-quantification and stimulation of hair shaft growth were performed. In parallel, we evaluated the effects of an active complex (AcPi) present in hair products to improve Wnt signaling by ?-catenin quantification in dermal papilla cells. Our results demonstrated that the associated treatment with ShPi and HtPi was able to stimulate hair shaft growth by 31.49% in relation to the basal group and to prevent the excessive degradation of COL17A1 in HF exposed to UV radiation by 2.6-fold compared to the UV radiation group. In addition, AcPi acted to prolong the anagen phase by increasing ?-catenin production by 47.79% in dermal papilla cells. Taken together, our results show that the associated treatment with ShPi and HtPi products promotes growth, strengthening, and vitality of the hair shaft, besides protecting HF from the deleterious effects of UV radiation, preventing the appearance of an aging hair phenotype and hair loss.

XXIV Congreso Latinoamericano e Ibérico de Químicos Cosméticos, São Paulo-SP, 21-23 Maio 2019. Araújo, Roberta B.; Silva, Michelle S.; Facchini, Gustavo; Silva, Gustavo H.; Pinheiro, Ana L. T. A.; Eberlin, Samara. A transição da camada basal para o estrato córneo é um processo essencial para a formação da barreira epidérmica e envolve a síntese de queratinas basais (K14), supra-basais (K10) e proteínas de envelope - involucrina, loricrina e profilagrina. Durante o processo de envelhecimento, a barreira cutânea e a matriz extracelular são precocemente afetadas pela ação de agentes extrínsecos, resultando em uma pele de aparência desidratada e rugosa. Neste estudo, avaliamos os efeitos de uma formulação hidratante sobre a produção de proteínas de envelope filagrina, involucrina e loricrina, queratinas basais 10 e 14 e matriz extracelular - pró-colágeno tipo I utilizando um modelo in vitro de cultura de células humanas. Queratinócitos e fibroblastos humanos foram incubados com a formulação hidratante para posterior quantificação dos marcadores propostos por meio de ensaio imunoenzimático (Elisa). Nossos resultados demonstraram que a incubação das culturas de queratinócitos com a formulação hidratante aumentou significativamente os níveis de involucrina, filagrina, loricrina e queratina 10, em relação ao grupo controle não tratado. Da mesma maneira, a formulação hidratante promoveu um aumento na produção proteica de pró-colágeno tipo I em cultura de fibroblastos humanos. O desenvolvimento de cosméticos multifuncionais é uma ferramenta que confere praticidade e aderência na rotina do cuidado da pele. Nesse trabalho, apresentamos uma formulação hidratante que é capaz de equilibrar a síntese de proteínas de barreira cutânea e proteger contra a regulação negativa da matriz extracelular. Os resultados indicam uma atividade biológica deste produto na promoção da hidratação, coesão celular, integridade da barreira da pele e restauração da matriz extracelular.

XXIV Congreso Latinoamericano e Ibérico de Químicos Cosméticos, São Paulo-SP, 21-23 Maio 2019. Facchini, Gustavo; Silva, Michelle S.; Silva, Gustavo H.; Pinheiro, Ana L. T. A.; Pinheiro, Adriano S.; Eberlin, Samir; Vieira, André S.; Eberlin, Samara. A radiação infravermelha-A (rIV-A) vem sendo confirmada como agravante do processo de envelhecimento cutâneo devido à indução de alterações fisiológicas que culminam desde desordens inestéticas até a formação de neoplasias malignas. A identificação de estruturas alvo dessa radiação na pele tornou-se uma ferramenta importante com o intuito de disponibilizar uma metodologia precisa para avaliação da eficácia de dermocosméticos na proteção contra os efeitos nocivos da rIV-A. O objetivo deste estudo foi investigar a modulação de genes envolvidos com as alterações funcionais promovidas pela rIV-A em explantes de pele humana, através da expressão gênica em larga escala utilizando o sequenciamento de mRNA (RNA-seq - next generation sequencing). Fragmentos de pele obtidos de cirurgia plástica eletiva foram expostos a rIV-A e submetidos a uma análise de expressão gênica em larga escala sequenciando todo o transcriptoma (RNA-Seq). A comparação entre o transcriptoma dos fragmentos irradiados e o controle indicou a existência de 214 genes diferencialmente expressos. A rIV-A levou ao enriquecimento de vias envolvidas com resposta imunológica, adesão celular, ciclo celular, metabolismo esfingolipídico, biossíntese de ácidos graxos, estresse oxidativo, vias de sinalização ERK e AKT, além de outras vias relacionadas à progressão de melanoma. Dentre as patologias apontadas na análise dos genes diferencialmente expressos (MetaCore®) estão hiperalgesia, dermatite de contato irritativa, psoríase, neoplasia de células escamosas e carcinoma de células de Merkel. A rIV-A promove alterações relevantes no metabolismo da pele que propiciam a resposta inflamatória e o estresse oxidativo, impactando principalmente na formação do estrato córneo e estrutura da epiderme. Baseado nesses achados e nos biomarcadores apontados abrem-se novas metodologias de avaliação mais abrangente para comprovar a eficácia de produtos que protejam a pele dos efeitos deletérios da rIV-A.

XXIV Congreso Latinoamericano e Ibérico de Químicos Cosméticos, São Paulo-SP, 21-23 Maio 2019. Igarashi, Mamy H.; Pereira, Andréia F. C.; Silva, Sheila G.; Mercuri, Maurizio; Silva, Michelle S.; Facchini, Gustavo; Silva, Gustavo H.; Pinheiro, Ana L. T. A.; Eberlin, Samara. O envelhecimento cutâneo representa um processo multifatorial que tem como mecanismo primordial a produção excessiva de radicais livres, caracterizando um ambiente de estresse oxidativo após exposição da pele às diversas agressões externas, particularmente radiação UV (rUV) e a fumaça de cigarro (FC). A ação aditiva dos diferentes agentes extrínsecos na pele culmina no aumento da susceptibilidade do tecido evidenciando as alterações inestéticas. Avaliar a eficácia de um produto cosmético na prevenção de peroxidação lipídica (síntese de malondialdeído - MDA), da translocação nuclear do receptor aril hidrocarboneto (AhR) e determinação do estado radicalar da pele/formação de proteínas radicaladas (RSF) em cultura de queratinócitos humanos expostos à rUV e FC. Adicionalmente, a marcação proteica de pró-colágeno tipo I foi avaliada em explantes de pele humana. Queratinócitos humanos foram incubados com o produto cosmético e expostos a rUV e a FC para avaliação da translocação de AhR, síntese de MDA e RSF. Os fragmentos de pele humana, obtidos de cirurgia plástica eletiva, foram incubados com o produto para posterior avaliação por imunofluorescência e semi-quantificação de pró-colágeno tipo I. Os resultados demonstraram que o produto cosmético apresentou proteção contra a translocação nuclear de AhR, reduziu a produção de MDA e de proteínas radicaladas (RSF) induzidos pela exposição do sistema teste à radiação UV e FC. Além disso, o produto demonstrou habilidade em aumentar a síntese de pró-colágeno tipo I em modelo de pele humana. Estes resultados em conjunto revelam que o produto avaliado exerce um efeito antipoluição, antienvelhecimento e antioxidante, protegendo a pele dos efeitos deletérios da exposição à radiação UV e fumaça de cigarro.

30 th IFSCC Congress (International Federation of Societies of Cosmetic Chemists), Munich, 18-21 setembro, 2018. Renan Lage, Andreia Feital da Costa Pereira, Mamy Honda Igarashi, Sheila Gomes da Silva, Maurizio Mercuri, Elis Angela de Alcantara Viana, Giulia Okamoto Maciente, Amanda Francielli Pereira, Suelma Natalie de Melo Oliveira, Michelle Sabrina Silva, Gustavo Facchini, Ana Lúcia Alves Tabarini Pinheiro, Samara Eberlin. The skin is a complex system consisting of epidermis, dermis, and appendages intertwined between the two layers. The most superficial layer of the skin, the epidermis, is avascular and receives nutrients from the capillaries of the dermis. The corneal layer, considered the outermost of the epidermis, is about 15-20 microns thick and consists of corneocytes (differentiated, anucleated, and keratinized keratinocytes) and lipid layers. This morphofunctional structure confers the barrier property of the skin, and thus it is the stratum corneum that limits permeation of substances. In this sense, cosmetic or pharmaceutical excipients and actives have been researched so that skin barriers are preserved when the scope of an investigation is safety, or diminished when the goal is permeation. In this study, we evaluated the cutaneous permeation of a shampoo and a hair tonic, formulated with a nanoencapsulated active, through fluorescence microscopy using human scalp fragments from elective plastic ritidoplasty. Skin fragments were treated with shampoo and a hair tonic, formulated with a nanoencapsulated active and labeled with Nile Red, for a period of 5 consecutive days. Histological procedures were carried out for posterior evaluation of skin permeation through visualization, recording, and semi-quantification of the fluorescence produced by the nanoencapsulated active labeled with the Nile Red dye. The evaluation of the images obtained revealed a greater intensity of the gray scale emitted by the red channel throughout the histo-anatomical region of hair follicles in the groups treated with both cosmetic formulations (shampoo and hair tonic) when compared to control and respective placebo formulations. These results were corroborated by the semi-quantification of the fluorescence intensity obtained in the fresh histological sections in the control group and treated with the investigational products. According to the obtained results, we can conclude that the products applied, isolated or associated, on the human scalp demonstrate permeation after 5 days of application throughout the hair follicle area. The association of the products showed a higher intensity of fluorescence when compared to the isolated treatments.

Cosmetics & Toiletries Revista de Cosméticos & Tecnologia 2018, 30(1): 46-49. ISSN 0103-4030. Michelle Sabrina da Silva; Gustavo Facchini; Ana Lúcia Tabarini Alves Pinheiro; Adriano da Silva Pinheiro; Samir Eberlin; Samara Eberlin. Neste estudo analisamos o efeito da radiação UV no envelhecimento do folículo capilar, em fragmentos de escalpo por meio da semi-quantificação de COL17A1. Nossos resultados demonstraram a aceleração da proteólise de COL17A1 devido à radiação, resultando na miniaturização deste mini-órgão a qual compromete o desenvolvimento e crescimento capilar.

23ª Radesp - 11º Simpósio de Cosmiatria & Laser da SBD, 11-13 outubro, 2018. Mamy Honda Igarashi, Andréia Feital da Costa Pereira, Sheila Gomes da Silva, Maurizio Mercuri, Ana Lúcia Tabarini Pinheiro, Gustavo Facchini. Substâncias que atuam na homeostase mitocondrial e equilíbrio energético são de grande importância para a manutenção das funções biológicas desempenhadas pelas células cutâneas. Nessa investigação avaliamos a eficácia pré-clínica de um complexo de ativos antiqueda (CAAq) no equilíbrio energético mitocondrial por meio da quantificação de ATP e semi-quantificação da biogênese mitocondrial e espécies reativas de oxigênio mitocondrial (mtROS) em cultura de células da papila dérmica humana (HHDPCs) expostas à radiação UV. HHDPCs foram tratadas com 3 concentrações de CAAq e posteriormente submetidas a radiação UV. Após 24 horas as células foram coletadas para quantificação de ATP, marcação e semi-quantificação de biogêneses e ROS mitocondrial. CAAq promoveu um aumento de até 107,70% na síntese de ATP em HHDPCs submetidas à radiação UV. Além disso, reduziu a produção de ROS em 48,34% e proporcionou um aumento na biogênese mitocondrial de até 100%, ambos em relação às células irradiadas. Estes resultados nos permitem inferir que o produto avaliado CAAq apresenta um resultado positivo no equilíbrio energético mitocondrial, restaurando aos níveis basais a produção de ATP, ROS e biogênese mitocondrial.

30th IFSCC Congress (International Federation of Societies of Cosmetic Chemists), Munich, 18-21 September, 2018. Silva, Michelle Sabrina1; Facchini, Gustavo1; Buzalaf, Marilia Afonso Rabelo2; Leite, Aline Lima2; Eberlin, Samir3; Pinheiro, Ana Lucia Tabarini Alves1; Pinheiro, Adriano da Silva; Eberlin, Samara1. Hair is an important and evolutionarily conserved structure. It originates from hair follicles deep within the dermis and is mainly composed of hair keratins and KAPs (Keratin-Associated Proteins), which form a complex network that contributes to rigidity and mechanical properties. Hair damage is a multifactorial process influenced by genetic and environmental factors. A classic example of an environmental factor is ultraviolet (UV) radiation, which results in the induction of oxidative stress and the production of free radicals capable of causing damage to structures present in the hair follicle (HF) and hair fiber. However, the mechanisms associated with hair photodamage are still not completely clear. In this study, we investigate the effects of UVA/B radiation in human scalp with the purpose of developing novel biomarkers for healthy and damaged hair using proteomic analysis. Scalp fragments from healthy female subjects, aged 40-60 years, submitted to rhytidoplasty, were fractionated using a punch nº 5 and cultured overnight. After this period, they were exposed to daily doses of 10 J/cm² UVA/B radiation for five consecutive days, using UVA Cube 400, SOL 500 H2 filter, and UV Meter. After 24 hours of the last dose of UVA/B, scalp fragments were collected. Total proteins were extracted and analyzed by a high-resolution label-free mass spectrometry (MSE) approach to identify differential protein expression. The results were analyzed by ProteinLynx Global Server (PLGS), and Cytoscape combined with Cluster Marker and ClueGO plugins were used to perform bioinformatic analysis. Proteomic analysis is a tool capable of identifying and quantifying the proteome of a biological system. The results obtained for proteomic analysis between control and UVA/B groups revealed alterations in several proteins correlated with important biological processes associated with hair growth, development, loss, and pigmentation. We observed that UVA/B radiation, at the experimental dose proposed in this study, significantly altered proteins associated with the cell cycle, cornified envelope formation, telomere maintenance, WNT signaling, cell response to stress, oxidative stress-induced senescence, and cellular response to external stimuli. In this study, we used a proteomics platform to analyze the effects of UVA/B radiation on the hair, allowing an overview of the damage in fiber and hair follicle. Oxidative stress induced by UV radiation impacts directly the dermal papilla cells from HF, inducing a senescent state and suppressing follicular differentiation of keratinocytes, which accelerates hair follicle loss. Furthermore, oxidative stress compromises the niche of melanocytes in HF, leading to changes in melanogenic pathways and cell death, culminating in hair depigmentation. Additionally, we observed an important alteration in histones and intermediate keratin filaments that compromise the structure of the hair fiber, impairing strength and mechanical resistance. Taken together, our results of proteomic analysis demonstrated a compromise in proteins associated with basic mechanisms of hair development, suggesting a direct impact of UVA/B radiation on hair growth, loss, pigmentation, thinning, and morphology. These results provide sustained new markers for evaluating the effectiveness of dermocosmetics for hair treatment.

30th IFSCC Congress (International Federation of Societies of Cosmetic Chemists), Munich, 18-21 September, 2018 - Apresentação Oral Eberlin S1, Facchini G1, Silva MS1, Pinheiro ALTA1, Vieira AS2, Eberlin S3, Pinheiro AS1 Skin photoaging has been attributed to other spectra of solar radiation such as infrared A (IRA). This fact has aroused the interest of the scientific community to evaluate the biological changes promoted by IRA in the skin. Considering the advent of the 3R policy, the need for development of alternative methods that reflect more accurately the effects of a real condition becomes imminent. In this sense, an alternative to study the mechanisms of skin biology is the use of skin explants obtained from plastic surgery. The aim of this study was to investigate the modulation of genes involved with the functional alterations promoted by IRA radiation in human skin explants, through large-scale gene expression using mRNA sequencing (RNA-seq - next generation sequencing). Skin fragments from ten (10) healthy female subjects, aged 30-60 years, phototype II - IV, submitted to abdominoplasty, were fractionated using a punch nº 5 and cultured overnight. After this time, they were exposed to daily doses of 360 J/cm² IRA radiation for four consecutive days, using Hydrosun 750 and HBM1 devices (Hydrosun Medizintechnik GmbH, Müllheim, Germany). Tissue viability was measured using the MTT assay at all experimental times (24, 48, 72, 96, and 120 hours after the surgical procedure). After 24 hours of the last dose of IRA, skin fragments were subjected to a large-scale gene expression analysis by sequencing the whole transcriptome (RNA-Seq). RNA was extracted, followed by fractionation of mRNA and cDNA library construction. Differentially expressed genes were submitted to enrichment pathways analysis using the MetaCore® database (Thomas-Reuters). Regarding tissue viability, there was no difference between irradiated skin fragments and control groups throughout the experiment. Comparison between the transcriptome of irradiated fragments and controls indicated the existence of 214 differentially expressed genes (9 upregulated and 205 downregulated). Regarding upregulated genes, IRA radiation led to the enrichment of pathways involved with immune response, development, and cell signaling related to neoplasms, specifically with the progression of melanoma and insulin-like growth factor in cancer. For the downregulated genes, 72 pathways were found to be significantly enriched, such as those involved with cell adhesion, immune response, cell cycle, sphingolipid metabolism, fatty acid biosynthesis, oxidative stress, and signaling pathways involving ERK and AKT. These results showed that IRA radiation is able to modulate genes that correlate with neoplasms, such as squamous cell neoplasia and Merkel cell carcinoma. Additionally, hyperalgesia, irritant contact dermatitis, and psoriasis presented significant enrichment. Assessments of gene expression on a large scale revealed relevant changes in skin metabolism, development of aesthetic changes, and appearance of cutaneous pathologies on the IRA-exposed skin. Important changes are associated with an increase in inflammatory profile, immune response, formation of inflammasome, and exacerbation of oxidative stress. In addition, genes that directly impact the formation of the stratum corneum and structure of the cutaneous epidermis were also compromised. The results obtained reveal different ways to better understand the impact of IRA radiation on human skin, allowing the development of more accurate evaluation methods to check the effectiveness of actives and products in protecting the skin from the deleterious effects of IRA radiation.

30th IFSCC Congress (International Federation of Societies of Cosmetic Chemists), Munich, 18-21 September, 2018. Alberto De Lago, Eduardo Hernandez, Tania Z Reyes, Pinto JR, Albarici VC, Facchini G, Silva MS, Pinheiro ALTA, Pinheiro AS, Eberlin S. Studies have shown that many factors, such as skin inflammatory processes and prolonged exposure to sunlight, could affect the skin barrier and extracellular matrix, inducing a decrease in the synthesis of major dermal proteins like collagen, elastin, and hyaluronic acid, which are clinically characterized by wrinkles, rough skin, loss of water, and skin tone. In this study, we evaluated the in vivo effects of a dermocosmetic formulation in increasing firmness, elasticity, and hydration of the skin using instrumental techniques and perceived efficacy. Preclinical studies consisted of assessing the production of collagen and hyaluronic acid using an in vitro model of human fibroblasts. Clinical evaluation was performed after 14, 30, and 60 days of treatment with the dermocosmetic product and included a sensorial analysis of perceived efficacy through a questionnaire answered by the participants. In addition, the following instrumental analyses were performed: cutometry (to evaluate skin firmness and elasticity), corneometry (for hydration), and image analysis (to evaluate wrinkles and expression lines). Human dermal fibroblasts were incubated with non-cytotoxic concentrations of the dermocosmetic formulation. Cell culture medium and treatments were replaced every 2 days. Culture supernatants were collected after 1, 14, and 30 days of incubation. The levels of total collagen and hyaluronic acid were measured using commercially available kits. ANOVA test was used, followed by Bonferroni post-test with a 5% significance level. The instrumental results obtained for firmness and elasticity parameters revealed progressive increases after 14, 30, and 60 days of cosmetic treatment on the face and neck. The analysis of cutaneous hydration, evaluated after 24 hours of application, revealed a 12% increase using the corneometry technique. Imaging of the depth and size of wrinkles also revealed significant reductions. In the evaluation of perceived efficacy, over 80% of the volunteers reported improvements in attributes such as nutrition, softness, luminosity, and the appearance of wrinkles and expression lines after 14, 30, and 60 days of cosmetic treatment. In vitro results corroborated the clinical findings, demonstrating an increase in the production of total collagen and hyaluronic acid in cultures of fibroblasts treated with the dermocosmetic formulation. According to the results obtained, we conclude that the dermocosmetic formulation has the ability to stimulate the synthesis of total collagen and hyaluronic acid when compared to the untreated group. This effect is directly related to improved skin support, favoring tissue repair and regeneration. In addition, the stimulation of extracellular matrix components contributes to the reduction in the formation of expression lines and wrinkles, one of the most important changes in skin aging, conferring anti-aging activity to the evaluated product.

30th IFSCC Congress (International Federation of Societies of Cosmetic Chemists), Munich, 18-21 September, 2018. Mamy Honda Igarashi, Andreia Feital da Costa Pereira, Renan Lage, Sheila Gomes da Silva, Maurizio Mercuri, Elis Angela de Alcantara Viana, Giulia Okamoto Maciente, Amanda Francielli Pereira, Suelma Natalie de Melo Oliveira, Michelle Sabrina Silva, Gustavo Facchini, Ana Lúcia Alves Tabarini Pinheiro, Samara Eberlin. Extrinsic aging is attributed to changes in the skin, mainly influenced by ultraviolet radiation (UV). Proinflammatory cytokines, such as interleukin 1 (IL-1), produced as a result of exposure to UV radiation, induce the production of MMPs in fibroblasts, resulting in an exacerbation of the degradation of extracellular matrix (ECM) components, contributing to photoaging. In this study, we evaluated the effects of a cosmetic formulation containing nanoencapsulated glycolic acid on the production of collagen type I and III, MMP-1 and 9, and IL-1? using an in vitro model of human keratinocytes, fibroblasts, and skin explant. Human fibroblasts and keratinocytes were incubated for 48 hours with three concentrations of the cosmetic formulation previously determined by the MTT method. Cell cultures were exposed to a dose of 10 J/cm² UVA/B radiation and incubated with fresh culture medium for 24 hours for the measurement of collagen type I and III, MMP-1 and 9 (fibroblast), and IL-1? (keratinocytes). At the same time, human skin fragments originating from elective plastic surgery were treated with the cosmetic formulation for semi-quantification of pro-collagen type I through immunofluorescence. As expected, our results demonstrate that UV radiation reduced the production of collagen type I and III and elevated the synthesis of MMP-1, MMP-9, and IL-1?. However, in comparison to the group exposed only to UV radiation, the cosmetic formulation significantly stimulated the production of collagen type I and III in all evaluated concentrations. In relation to MMP-1 and -9 synthesis, the cosmetic formulation was able to prevent the rises induced by UV radiation exposure. The increase in IL-1? production was also reduced when keratinocyte cultures were pretreated with the cosmetic product. Immunofluorescence for pro-collagen type I was shown to be strongly increased in the treated group when compared to the control group. The results obtained allow us to infer that the cosmetic formulation containing nanoencapsulated glycolic acid exerts a positive effect on skin protection against photoaging, increasing the production of collagen and reducing the production of IL-1?, MMP-1, and MMP-9 induced by exposure to UV radiation, thus protecting and restoring the extracellular matrix.

30th IFSCC Congress (International Federation of Societies of Cosmetic Chemists), Munich, 18-21 September, 2018. Andreia Feital da Costa Pereira, Renan Lage, Mamy Honda Igarashi, Sheila Gomes da Silva, Maurizio Mercuri, Elis Angela de Alcantara Viana, Giulia Okamoto Maciente, Amanda Francielli Pereira, Michelle Sabrina Silva, Gustavo Facchini, Ana Lúcia Alves Tabarini Pinheiro, Samara Eberlin. Skin aging represents a multifactorial, progressive, and degenerative process of inevitable course, and practically irreversible. The primordial mechanism involved in this process is the excessive production of free radicals, characterizing an oxidative stress environment, after intense exposure of the skin to various external aggressions and other intrinsic elements. Lipid peroxidation is a consequence of free radicals attack that leads to cell membrane damage. In addition, oxidative stress results in an increase in the glycation process - formation of advanced glycation end products (AGEs), which not only influences the properties of collagen and the extracellular matrix, but also affects the interactions of cells with the matrix, resulting in cellular alterations, such as migration, growth, proliferation, differentiation, and gene expression. In this study, we evaluated the antiaging effectiveness of a cosmetic formulation in preventing photodamage caused by UV radiation in human keratinocyte culture. Cells were incubated for 48 hours with three concentrations of the cosmetic formulation previously determined by the MTT method and further exposed to a dose of 10 J/cm² UVA/B radiation. After irradiation, test systems were incubated with fresh culture medium and maintained for 24 hours for the measurement of the receptor for AGE (rAGE) and lipid peroxidation by quantification of malondialdehyde (MDA). As expected, our results demonstrate that UV radiation increases the production of MDA and decreases the amount of free receptor for AGE (rAGEs). However, in comparison to the group only exposed to UV radiation, the cosmetic formulation significantly reduced the production of MDA in all evaluated concentrations. Regarding the synthesis of rAGEs, the cosmetic formulation was able to prevent depletion induced by exposure to UV radiation. The results obtained allow us to infer that the cosmetic formulation exerts a protective effect against UV radiation. The mechanisms are not fully elucidated, but it is possible to suggest that an antioxidant action is involved considering the reduction of lipid peroxidation by decreasing the formation of the lipid hydroperoxide malondialdehyde. In addition, the cosmetic formulation demonstrated an anti-glycating action since it increased the amount of free receptor for AGE (rAGEs).

27th EADV (European Academy of Dermatology and Venereology) Congress, Paris, France, 12-16 September, 2018. Andreia Feital da Costa Pereira, Renan Lage, Mamy Honda Igarashi, Sheila Gomes da Silva, Maurizio Mercuri, Elis Angela de Alcantara Viana, Lucia de Fatia Holanda Paiva de Araújo, Amanda Francielli Pereira, Suelma Natalie de Melo Oliveira, Michelle Sabrina Silva, Gustavo Facchini, Ana Lúcia Alves Tabarini Pinheiro, Samara Eberlin. The most important function of the skin is to maintain homeostasis, protecting the body from external aggressions such as solar radiation, pollution, and xenobiotics. Excessive exposure to environmental factors culminates in changes in normal biological functions resulting in aesthetic changes, such as the appearance of wrinkles, skin dryness, excessive oiliness, and acne formation. The stratum corneum is the main layer responsible for protecting the skin against these external agents, and its formation is the result of epidermal proliferation and differentiation. Additionally, to prevent the aged appearance of the skin, it is essential to stimulate dermal layer cells to produce collagen, the main component of the extracellular matrix responsible for maintaining tissue structure and firmness. Finally, an important aspect to be considered is the effects of environmental factors on the natural cycle of the sebaceous gland, which is essential to control excessive sebum production. There is a trend to optimize cosmetic treatments by reducing the number of products to be applied daily and centralizing a single multi-benefit product for skin health. The purpose of this study was to evaluate the effects of a cosmetic product (GlyAHA) on epidermal renewal, collagen production, and sebum production. Human sebocytes were stimulated with insulin (10 nM) to induce sebaceous production and treated with GlyAHA at 0.100, 0.0316, and 0.0100 mg/ml for 6 days. Cell lysate was collected for quantification of triglycerides (TG) using a colorimetric assay. Ex vivo skin fragments were treated with GlyAHA for 5 consecutive days, with a 24-hour interval between each application. Fragments were collected, fixed, and cryopreserved for evaluation by immunofluorescence of Ki67, a cell proliferation marker of epidermal cells, and procollagen type I. Our results demonstrated that the treatment of ex vivo skin cultures with GlyAHA promotes a significant increase in the synthesis of Ki67 (2.65-fold) and pro-collagen type I (58%) when compared to the respective controls (p<0.01). Additionally, we observed that treatment of insulin-stimulated sebocyte cultures reduced TG production by 51.07%, 39.27%, and 41.76% at concentrations of 0.100, 0.0316, and 0.0100 mg/ml, respectively, when compared to the only insulin-stimulated group. The results obtained allow us to infer that GlyAHA protects the skin from the deleterious effects of exposure to environmental factors, preventing the appearance of unaesthetic characteristics associated with skin aging through the stimulation of collagen production and epidermal renewal. In addition, GlyAHA has a beneficial effect on oily skin and acne, as it promotes a reduction in triglycerides synthesis, which is the main component of sebum.

27th EADV (European Academy of Dermatology and Venereology) Congress, Paris, France, 12-16 September 2018. Andreia Feital da Costa Pereira, Renan Lage, Mamy Honda Igarashi, Sheila Gomes da Silva, Maurizio Mercuri, Elis Angela de Alcantara Viana, Lucia de Fatia Holanda Paiva de Araújo, Amanda Francielli Pereira, Suelma Natalie de Melo Oliveira, Michelle Sabrina Silva, Gustavo Facchini, Ana Lúcia Alves Tabarini Pinheiro, Samara Eberlin. Atopic dermatitis (AD) is a chronic, recurrent, and inflammatory skin disease, characterized by the presence of eczematous lesions and pruritus. Despite the large number of clinical and experimental studies, and subsequent advances in this field, the pathophysiology of AD remains unclear. The most recent hypotheses about the pathogenesis of AD involve numerous factors: genetic predisposition, environmental factors, altered immune function, and skin barrier dysfunction. Innate and adaptive immunity has a definitive role in the development, maintenance, and crisis of AD. This process involves a complex interaction between T cells, antimicrobial peptides, cytokines, and keratinocytes. The alteration in the balance of the immune system directly influences the rupture of the skin barrier, characteristic of AD lesions, making individuals predisposed to skin infections. In the present study, we evaluated the preclinical efficacy of an active complex (AC) in anti-inflammatory response in cultures of human monocytes/macrophages (THP-1) stimulated with lipoteichoic acid (LTA) to induce the inflammatory microenvironment. Additionally, we evaluated the preclinical efficacy of a moisturizing formulation containing the AC (Hydra-AI) in the synthesis of filaggrin, involucrin, and loricrin in ex vivo skin fragments submitted to epidermal barrier rupture. Cultures of THP-1 were differentiated into phagocytes with 12-myristate 13-acetate phorbol. Afterward, the cultures were stimulated with LTA (10 μM) to induce the inflammatory microenvironment and treated with AC at 0.100, 0.0316, and 0.0100 mg/ml. Cell lysates and culture supernatants were collected for quantification of interleukins 4, 10, 13, 17, 31, and NF-κB using the ELISA technique. In parallel, ex vivo skin fragments were treated with Hydra-AI for two consecutive days, with a 24-hour interval between each application. The epidermis was ruptured using 4% sodium lauryl sulfate (SDS), and after 24 hours, the fragments were treated again with Hydra-AI. Fragments were collected, fixed, and cryopreserved for evaluation by immunofluorescence of filaggrin, involucrin, and loricrin. The results obtained demonstrated that the treatment with AC performed on the LTA-stimulated THP-1 cell cultures was able to significantly reduce the inflammatory mediators IL-4, 10, 13, 17, 31, and the signaling pathway NF-κB when compared to the stimulated group. In addition, immunofluorescence evaluations demonstrated that HYDRA-AI stimulates the synthesis of filaggrin, involucrin, and loricrin in fragments of ex vivo skin submitted to rupture of the epidermal barrier with SDS. The results obtained allow us to infer that AC presents anti-inflammatory activity, reducing the exacerbated production of mediators associated with the development of AD, which contribute to the impairment of the cutaneous barrier. In addition, HYDRA-AI upregulates the synthesis of filaggrin, loricrin, and involucrin, suggesting a positive effect on skin barrier protection, hydration, proliferation, differentiation, epidermal cohesion, and synthesis of NMF. Taken together, these results demonstrate that AC and the moisturizing formulation containing AC (HYDRA-AI) have a protective and restorative effect on sensitive skin, associated with atopic dermatitis.

2nd Pan-American Conference for Alternative Methods, Rio de Janeiro, 23-24 August 2018 - Apresentação Oral Silva, MS; Facchini, G; Pinheiro, ALTA; Eberlin, S; Pinheiro, AS; Eberlin, S. Fragments of ex vivo human skin constitute the experimental model that most closely approximates the real conditions to evaluate the efficacy and safety of products applied topically. In this model, all cell populations native to this tissue are present, including keratinocytes, melanocytes, Langerhans cells, and all dermal matrix components, containing fibroblasts, collagen, elastin, glycosaminoglycans, among others. The development of a test system using ex vivo skin fragments from elective plastic surgery, with standardized size and thickness cultured in an air-liquid interface, provides us with an important tool for understanding the diverse mechanisms involved in cutaneous disorders. The use of human skin has been reported in the literature, both by our research group and by other groups, as an important experimental model alternative to animal use, more reliable than the use of 2D culture of skin cells and less invasive than human skin biopsies. Our efforts to establish a model with high reproducibility and adapted to different experimental conditions revealed positive results in the use of ex vivo skin as a tool to evaluate different skin disorders such as photoaging, skin dryness, atopic dermatitis, hair follicle (HF) aging, skin and HF permeation, among others. Most fragments used in our experiments came from abdominoplasty (human skin abdominal explant cultured - hSaec) and rhytidoplasty (human skin scalp explant cultured - hSsec). All studies were conducted only after the approval of an ethics committee. In this work, we present a collection of results obtained by our group in recent years involving preclinical trials of topical product efficacy. These studies comprise specific methodologies in which mediators are evaluated by different techniques such as immunofluorescence and immunoassay, as well as more complex studies using omic platforms to understand the complex interaction of cutaneous tissue with the extrinsic environment.