Depigmenting Effect of Resveratrol Is Dependent on FOXO3a Activation without SIRT1 Activation.

Resveratrol exhibits not only anti-melanogenic property by inhibiting microphthalmia-associated transcription factor (MITF), but also anti-aging property by activating sirtuin-1 (SIRT1). In this study, the relationship between depigmenting effect of resveratrol and SIRT1/forkhead box O (FOXO) 3a activation and was investigated. Resveratrol suppressed melanogenesis by the downregulation of MITF and tyrosinase via ERK pathway. Results showed that the expression of both SIRT1 and FOXO3a were increased. It is reported that SIRT1 is critical regulator of FOXO-mediated transcription in response to oxidative stress. However in our study, FOXO3a activation appeared earlier than that of SIRT1. Furthermore, the effect of resveratrol on the levels of MITF and tyrosinase was suppressed when melanocytes were pre-treated with SP600125 (JNK inhibitor). However, pre-treatment with SIRT1 inhibitor (EX527, or sirtinol) did not affect the levels of MITF and tyrosinase. Therefore, resveratrol inhibits melanogenesis through the activation of FOXO3a but not by the activation of SIRT1. Although SIRT1 activation by resveratrol is a well-known mechanism of resveratrol-induced antiaging effects, our study showed that not SIRT1 but FOXO3a activation is involved in depigmenting effects of resveratrol.

Stem cell recovering effect of copper-free GHK in skin.

The peptide Gly-His-Lys (GHK) is a naturally occurring copper(II)-chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper-GHK promotes the survival of basal stem cells in the skin. However, the effects of copper-free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and ß1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK-treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper-free GHK showed similar effects with copper-GHK. Thus, it can be said that copper-free GHK can be used in industry to obtain the effects of copper-GHK in vivo. Further study is necessary to explore the relationship between copper-free GHK and copper-GHK.

Oligosaccharides of hyaluronic acid increased epidermal cell stemness by modulation of integrin expression.

BACKGROUNDS: Hyaluronic acid (HA) is an abundant matrix component and is degraded into polymers of various sizes. These generated fragments appear to have properties that affect wound healing of the skin. In industry, small-sized HA is used as a moisturizing agent but can have biologic effects when it is absorbed through the skin with barrier disruption. AIMS: In this study, the regenerative effects of these molecules were investigated using skin equivalent (SE) models. METHODS: Normal human keratinocytes and fibroblasts were isolated, and the effects of oligosaccharides of HA were tested in cultured keratinocytes and in the SE model. RESULTS: In the monolayer of cultured keratinocytes, oligosaccharides of HA did not affect the proliferation of keratinocytes. However, the epidermis became thicker when oligosaccharides of HA were added during the culture of SE models. The data also showed that oligosaccharides of HA promote the differentiation of the epidermis. Furthermore, the expression of p63, integrin-α6 and integrin-ß1 was increased. Western blot analysis also showed increased expression of both integrins. CONCLUSIONS: These findings suggest that oligosaccharides of HA increase the differentiation of the epidermis. In addition, increased number of p63, a putative stem cell marker of the skin, showed that oligosaccharides of HA promote the survival of basal stem cells by modulating the expression of integrin-α6 and integrin-ß1. Finally, it can be said that inflammation-induced small-sized oligosaccharides can have beneficial effects on epidermal regeneration and topically applied oligosaccharide of HA can have healing effects in skin problems.

Redox status is critical for stemness in skin equivalents.

The skin is constantly exposed to environmental oxidative stress. Skin equivalent (SE) models are three-dimensional systems in which cell-cell or cell-matrix interactions can be investigated. In this study, the effects of vitamin C or plant extracts with high antioxidant activities were tested. There was no significant difference in the epidermal thickness, but the basal cells became cuboidal when vitamin C or plant extracts were supplemented. Furthermore, immunohistochemical staining showed linear and intense staining of α6 and ß1 integrin along the basement membrane in vitamin C or plant extract treated models. The p63 and PCNA were also stained. Results showed that the number of p63 and PCNA positive cells was higher in the vitamin C or plant extract treated models than in the control SEs. Although the relationship between oxidative stress and stem cells is not known, our results suggest that redox status affects the stemness and the proliferative potential of epidermal basal cells by modulating microenvironment to epidermal basal stem cells.

Novel anti-inflammatory peptides as cosmeceutical peptides.

Ultraviolet (UV) radiation induced inflammation plays an important role in the aging of human skin. Prostaglandin (PG) E(2) is the primary mediator of UVB induced photoinflammation. We screened an internal library for dipeptides that inhibited UVB induced PGE(2) synthesis but showed no cytotoxicity toward human keratinocytes. We identified three highly active inhibitory sequences, LE (Leu+Glu), MW (Met+Trp) and MY (Met+Tyr). To evaluate their efficacy in human skin, 24 sites of abdomen skin were irradiated with a 308 nm excimer laser (300 mJ/cm(2)), after which 2% LE, MW, MY or a control were applied to the irradiated sites for 24h. The erythema index (EI) was measured before and 24h after treatment. The results showed that LE and MW significantly decreased UVB induced erythema (p=0.041 and p=0.036, respectively), but ME did not. Overall, LE and MW are candidate cosmeceutical peptides that can protect skin from UVB induced photoinflammation.

Copper-GHK increases integrin expression and p63 positivity by keratinocytes.

Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin.