Neobavaisoflavone Inhibits Melanogenesis through the Regulation of Akt/GSK-3ß and MEK/ERK Pathways in B16F10 Cells and a Reconstructed Human 3D Skin Model.

Previous studies have confirmed the anti-melanogenic effect of the aerial part of Pueraria lobata, however, due to its inherent color, P. lobata has limited commercial use. In this study, an extract (GALM-DC) of the aerial part of P. lobata having improved color by the use of activated carbon was obtained. Furthermore, the active compound neobavaisoflavone (NBI) was identified from GALM-DC. The effect of NBI on melanogenesis, tyrosinase activity, α-glucosidase activity, and mechanism of action in melanocytes was investigated. Tyrosinase activity, melanin contents and the expression of melanin-related genes and proteins were determined in B16F10 cells. NBI reduced melanin synthesis and tyrosinase activity. Furthermore, NBI treatment reduced the mRNA and protein expression levels of MITF, TRP-1, and tyrosinase. NBI also works by phosphorylating and activating proteins that inhibit melanogenesis, such as GSK3ß and ERK. Specific inhibitors of Akt/GSK-3ß (LY294002) and MEK/ERK (PD98059) signaling prevented the inhibition of melanogenesis by NBI. NBI inhibited melanin production through the regulation of MEK/ERK and Akt/GSK-3ß signaling pathways in α-MSH-stimulated B16F10 cells. NBI suppresses tyrosinase activity and melanogenesis through inhibition of α-glucosidase activity. Besides, NBI significantly reduced melanogenesis in a reconstructed human 3D skin model. In conclusion, these results suggest that NBI has potential as a skin-whitening agent for hyperpigmentation.

Anti-Aging Potential of Substance P-Based Hydrogel for Human Skin Longevity.

Skin aging is generally caused by a decline in the components of the extracellular matrix (e.g., collagen and elastin) and due to inflammatory phenomena. Many growth factors and peptides with cell-growth and collagen-synthesis activities have shown promise in their application in anti-aging materials. However, the effect of collagen production, without anti-inflammatory effect, and skin penetration may not be enough for their use in anti-aging agents. Previously, we reported a substance P (SP)-based hydrogel (SP gel) that had potential wound-healing activities via induction of skin cell regeneration and collagen synthesis. Here, we analyzed the anti-aging activities and skin absorption effects of SP gel to extend its characterization. Toxicity tests, performed on human dermal fibroblasts (HDFs) and on a reconstructed 3D human skin model, indicated SP gel to be safe for long-term use, without causing irritation, even at high concentrations. In-vitro analysis revealed that SP gel elicited stronger collagen production activities than SP alone, and promoted anti-inflammatory effects with increased skin absorption properties. Moreover, SP gel did not induce melanin synthesis in a keratinocyte-melanocyte co-culture system. Together, the results suggest that SP gel has potential cosmetic effects and applicability as a novel ingredient in anti-aging products.

Histological and functional characterization of 3D human skin models mimicking the inflammatory skin diseases psoriasis and atopic dermatitis.

Three-dimensional (3D) human skin equivalents have emerged as valuable tools in skin research, replacing animal experimentation and precluding the need for patient biopsies. In this study, we advanced 3D skin equivalents to model the inflammatory skin diseases atopic dermatitis and psoriasis by cytokine stimulation, and were successful in integrating TH1 T cells into skin models to develop an immunocompetent 3D psoriasis model. We performed in-depth histological and functional characterization of 3D skin equivalents and validated them in terms of tissue architecture, pathological changes, expression of antimicrobial peptides and Staphylococcus aureus colonization using 3D reconstruction by multiphoton microscopy and phenotyping by highly multiplexed 'co-detection by indexing' (CODEX) microscopy. We show that our skin equivalents have a structural architecture with a well-developed dermis and epidermis, thus resembling human skin. In addition, the skin models of atopic dermatitis and psoriasis show several phenotypic features of inflammatory skin disease, including disturbed epidermal differentiation and alterations in the expression of epidermal barrier genes and antimicrobial peptides, and can be reliably used to test novel treatment strategies. Therefore, these 3D equivalents will be a valuable tool in experimental dermatological research.

Successful proof of concept of a micronucleus genotoxicity assay on reconstructed epidermis exhibiting intrinsic metabolic activity.

We investigated the commercially available Episkin LM™ reconstructed epidermis test system as a potential 3D model for human genotoxicity assessment by cytokinesis-block micronucleus assay to mitigate limitations of the currently accepted micronucleus test. We established appropriate culture conditions for cytokinesis-block micronucleus assay in maximizing the frequency of binucleated cells by choice of culture medium and calibration of the system exposure to the cytokinesis inhibitor Cytochalasin B, without affecting the basal frequency of micronuclei in the model. We confirmed that the application of the classic solvents had no significant effect on this basal level of micronuclei. We determined the performance of cytokinesis-block micronucleus assay in Episkin LM™ reconstructed epidermis to predict in vivo genotoxins by testing the genotoxicity potential of 17 well known in vivo genotoxic, progenotoxic and non-genotoxic reference chemicals over a 48 h and 72 h exposure period. We found that cytokinesis-block micronucleus assays in Episkin™ reconstructed epidermis following the 48 h-topical regimen had a specificity of 60-75% and a sensitivity of 83-85%, resulting in an overall accuracy of 76-82% for genotoxicity assessment in tissues depending on the assessment of the reference chemicals with equivocal genotoxic profiles in the literature. The positive micronucleus test results obtained without addition of any exogenous metabolic activation system confirmed the ability of Episkin LM™ reconstructed epidermis to intrinsically bioactivate progenotoxic chemicals. The evidence showed that the 72-h exposure protocol significantly improved the detection of progenotoxins. Taken together, our data demonstrated that the Episkin LM™ reconstructed epidermis system is a relevant in vitro tool in the study of genetic toxicology.

Three-dimensional experiments and individual based simulations show that cell proliferation drives melanoma nest formation in human skin tissue.

BACKGROUND: Melanoma can be diagnosed by identifying nests of cells on the skin surface. Understanding the processes that drive nest formation is important as these processes could be potential targets for new cancer drugs. Cell proliferation and cell migration are two potential mechanisms that could conceivably drive melanoma nest formation. However, it is unclear which one of these two putative mechanisms plays a dominant role in driving nest formation. RESULTS: We use a suite of three-dimensional (3D) experiments in human skin tissue and a parallel series of 3D individual-based simulations to explore whether cell migration or cell proliferation plays a dominant role in nest formation. In the experiments we measure nest formation in populations of irradiated (non-proliferative) and non-irradiated (proliferative) melanoma cells, cultured together with primary keratinocyte and fibroblast cells on a 3D experimental human skin model. Results show that nest size depends on initial cell number and is driven primarily by cell proliferation rather than cell migration. CONCLUSIONS: Nest size depends on cell number, and is driven primarily by cell proliferation rather than cell migration. All experimental results are consistent with simulation data from a 3D individual based model (IBM) of cell migration and cell proliferation.