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Pyridoxine (VB

Skin transparency is a cosmetic asset highly considered by Asian women. Resulting from complex light interactions within the skin, but still not fully understood, there is no simple method to measure it objectively. In this study, skin parameters from digital images were analysed to build a model predicting transparency.Initially, 71 Japanese women (between ages 50 and 60 years) were recruited. This group was then extended to 262 women (between ages 21 and 60 years). Pictures of their faces were taken with the ColorfaceIn the initial group of 71 subjects, 109 parameters correlated with transparency. Half of them are from the cheek and relate to colour or colour homogeneity. If the cheek presented the largest proportion of correlated parameters, best correlations were usually found in other facial regions. Multiple regressions from some cheek parameters can predict up to 80% of transparency. Stepwise regression on parameters from 262 subjects led to a six-parameter model, which is highly correlated (R = 84.1%) with transparency. It combines skin texture, colour, colour homogeneity and gloss parameters. If half of them are from the cheek, the others are from the tear trough, the full face and the cheekbone.Using parameters from digital pictures exclusively, we propose a model that accurately reflects transparency. Including parameters previously shown to relate to transparency, this model should be useful for future dermatology and cosmetic research.

The purpose of this study was to clarify the impact of protein carbonylation on the chemical characteristics of the hair surface focusing on hydrophobicity.First, we examined the validity of methods to evaluate hydrophobicity, one that utilizes the fluorescence of 1-anilinonaphtalene-8-sulfonic acid (1,8-ANS) compared with the contact angles against HThe fluorescence intensity of 1,8-ANS of the hair surface modified chemically correlated well with the contact angles against HThe results suggest that the carbonylation of proteins at the hair surface with aldehydes decreases hydrophobicity and promotes further damage as does bleaching.OBJECTIF: l’objectif de cette étude était de clarifier l’impact de la carbonylation des protéines sur les caractéristiques chimiques de la surface des cheveux en se concentrant sur l’hydrophobicité. MÉTHODES: nous avons d’abord examiné la validité de la méthode d’évaluation de l’hydrophobicité, une méthode qui utilise la fluorescence de l’acide 1-anilinonaphtalène-8-sulfonique (1,8-ANS) par rapport aux angles de contact avec l’H2O, de la surface des cheveux chimiquement modifiés par hydrolyse alcaline ou traités par chlorure d’ammonium stéarylique. Nous avons mesuré la fluorescence provenant du 1,8-ANS, l’angle de contact et les modifications des groupes fonctionnels, aldéhydes (le degré de carbonylation), NH2, COOH et SH des cheveux décolorés à l’H2O2 ou traités par acroléine, à l’aide de méthodes de marquage par fluorescence. RÉSULTATS: l’intensité de la fluorescence du 1,8-ANS de la surface des cheveux modifiés chimiquement était bien corrélée aux angles de contact avec l’H2O. Les résultats ont indiqué que le 1,8-ANS était adapté à l’évaluation de l’hydrophobicité de la surface des cheveux. L’hydrophobicité des cheveux décolorés à l’H2O2 ou carbonylés à l’acroléine a diminué. De plus, les modifications des groupes fonctionnels des cheveux carbonylés par l’acroléine ont augmenté, tout comme celles des cheveux décolorés à l’H2O2. CONCLUSION: les résultats suggèrent que la carbonylation des protéines à la surface des cheveux par des aldéhydes diminue l’hydrophobicité et favorise d’autres dommages, tout comme la décoloration.

Residues of olive fruit (ROF) after the extraction of oils are an increasing source of industrial waste, because olive oil is becoming more popular as a healthy food. It has been reported that olives have some polyphenols that have an antioxidation capability. On the other hand, excess oxidative stress disrupts epidermal barrier function. This study was conducted to determine whether ROF could be utilized as an antioxidant source to reduce industrial wastes and to identify possible active materials to maintain healthy skin. Olive fruits are categorized into two groups depending on the time of harvest, young fruit (YF) and mature fruit (MF). Thus, we examined the antioxidant potentials of extracts from YF and from MF to remove reactive oxygen species (ROS) from biological and chemical aspects. HaCaT keratinocytes cultured with extracts of YF or MF had reduced levels of intracellular ROS in spite of the relatively low chemical capability against ROS scavenging. The biological effects of the YF extract were superior to those of the MF extract. The YF extract showed effective reductions of intracellular ROS and carbonylated proteins that were elevated by the stress-related hormone cortisol. In addition, the YF extract reinforced the intracellular antioxidation capability through the activation of Nrf2 signaling. Taken together, the YF extract was an effective source to reinforce the intracellular antioxidation capability. We conclude from these results that utilizing ROF would lead to the reduction of industrial wastes and would supply active materials to maintain healthy skin.

Decreases of collagen fibers and the disappearance of oxytalan fibers are typical symptoms of photoaged skin. Although a low quality of mitochondria (MT) in photoaged skin cells has been observed, it is unknown whether the decreased quality of MT is responsible for the insufficient formation of dermal fibers.To identify the role of mitochondrial quality in skin photoaging focusing on the formation of dermal fibers.Type I collagen and fibrillin-1 fibers in normal human dermal fibroblasts (NHDFs) were observed by immunostaining. Type I collagen and fibrillin-1 proteins in NHDFs were quantified by ELISA. Mitochondrial quality was evaluated by measuring levels of intracellular ATP and MITOL, which regulate mitochondrial quality.UVA-irradiated NHDFs formed insufficient type I collagen and fibrillin-1 fibers and had a decreased ratio of extracellular versus intracellular levels of those proteins. Although expression levels of motor proteins that transport those proteins intracellularly were not affected by UVA, intracellular ATP levels, which is the driving force of motor proteins, were decreased by UVA along with decreased MITOL protein. Knockdown of MITOL in NHDFs decreased the level of intracellular ATP and caused the insufficient formation of type I collagen and fibrillin-1 fibers due to interfering with the secretion of those proteins.These results indicate that a low quality of MT with ATP depletion in dermal fibroblasts caused by irradiation with UVA induces the insufficient formation of type I collagen and fibrillin-1 fibers due to the decreased extracellular secretion of those proteins.

Carbonylated proteins (CPs) are synthesized by reactions between amino groups in proteins and reactive aldehyde compounds (RAC) yielded from lipid peroxidation initiated by reactive oxygen species (ROS). In the skin, CPs are detected in a higher frequency at sun-exposed sites of the skin in elderly subjects. Since CPs in the stratum corneum (SC) have been reported to correlate with skin water content and transepidermal water loss, it is considered that the accumulation of CPs in the SC involves the loss of skin moisture functions. However, the roles of CPs in the dermis on skin physiology are still unclear. The purpose of this study was to investigate the roles of CPs in the dermis during the progression of photoaged skin and to propose a method to prevent or reduce the synthesis of CPs. The exposure of human normal dermal fibroblasts to CPs increased intracellular ROS levels and the synthesis of intracellular CPs. In addition, CPs caused morphological changes of fibroblasts. Furthermore, CPs caused alterations of mRNA expression levels of dermal matrix-related proteins, such as upregulating MMP-1 and IL-8. These results indicated that CPs disrupt construction of the dermal matrix. On the other hand, α-tocopherol and β-carotene suppressed the synthesis of RAC during lipid peroxidation which resulted in the reduction of UVA-induced CPs in the SC. From these results, we propose that extracellular CPs increase intracellular ROS levels and contribute to alterations of the dermal matrix. To prevent the synthesis of CPs, the application of α-tocopherol or β-carotene could be effective.

Background Pyridoxine (VB6 ), which acts as a coenzyme in the biosynthesis of niacin, is formulated in pharmaceuticals to treat skin roughness. However, the mechanism of action of VB6 is not known precisely. Objective This study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VB6 focusing on antioxidant behaviour. Methods Intracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2',7'-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VB6 . Intracellular glutathione was quantified using an assay based on the glutathione recycling system with 5,5'-dithiobis (2-nitrobenzoic acid) reagent and glutathione reductase. Carbonylated proteins (CPs) were semi-quantified by detecting aldehyde residues. Results Treatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VB6 restored SPT mRNA expression in BSO-treated NHEKs. VB6 decreased the level of intracellular CPs with or without BSO treatment in a dose-dependent manner. In addition, VB6 increased levels of intracellular NADH/NADPH and glutathione through the activation of nuclear factor E2-related factor 2 (Nrf2) signalling. Conclusion These results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB6 improved the down-regulation of SPT mRNA expression initiated by highly oxidative conditions by enhancing the intracellular antioxidant system.

Although extracellular carbonylated proteins (CPs) are found at higher levels in sun-exposed skin, their impact on the cellular functions of fibroblasts and their involvement in the progression of photoaging skin are not fully clarified. In our previous study, we reported that extracellular CPs increase levels of intracellular oxidative stress and result in the accumulation of newly synthesized CPs in normal human dermal fibroblasts (NHDF). Furthermore, fibroblasts exposed to CP-BSA, which is a model of extracellular CPs, had upregulated expression levels of mRNAs encoding matrix metalloproteinase-1 (MMP-1) and interleukin-8/CXCL8 (IL-8/CXCL8). These facts suggested the possibility that extracellular CPs induce a fragile structure in the dermis through the degradation of collagen and elastin. The purpose of this study was to characterize the efficacy of natural carotenoids, such as astaxanthin analogs, produced by Hematococus pluvialis (CHPs) to improve the impaired functions of fibroblasts exposed to CPs. CHPs suppressed the intracellular CP levels elevated by CP-BSA, restored mRNA expression levels of factors involved in the formation and assembly of collagen and elastin fibers and improved the formation of those fibers impaired by CP-BSA. We conclude that CHPs function as antiaging substances due to their restoration of the impaired formation of collagen and elastin fibers caused by extracellular soluble CPs.

Background Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. Objective To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. Methods The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. Results The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. Conclusion Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.

Photoaged skin is characterized by the appearance of pigmented spots such as solar lentigos, deep wrinkles and sags, and progresses due to chronic sun exposure. Among the wavelengths of sunlight, UVA is responsible for the appearance of wrinkles and sags that originate from structural alterations in the dermis of photoaged skin such as the depletion of collagen fibers. Thus, improving and restoring collagen fibers is an effective approach to reduce skin photoaging and maintain a youthful appearance. This study was conducted to evaluate the potential of an extract of Ocimum basilicum (OC), which contains rosmarinic acid (RA), as an anti-photoaging material focusing on the capacity to restore collagen fibers that are disrupted due to intracellular oxidative stress. In spite of their relatively low capacities for chemical scavenging of reactive oxygen species (ROS), both OC and RA showed efficient removal of biological oxidative stress by reducing levels of intracellular ROS and carbonylated proteins (CPs) in fibroblasts following exposure to single or repetitive UVA irradiations. Fibroblasts irradiated with repetitive UVA as a model for chronic sun-exposed cells showed significant increases in matrix metalloproteinase-1 and decreases in type I collagen synthesis and formed reduced numbers of collagen fibers. Since both OC and RA restored the adverse phenomena caused by repetitive UVA irradiation, we conclude that OC containing RA is an effective anti-photoaging material.

Sacran, a polysaccharide isolated from the alga Aphanothece sacrum (Suizenji-nori), has unique physical and physiological characteristics. In a previous study, we reported that sacran improves skin conditions in individuals who suffer from atopic dermatitis (AD), focusing on its trapping function against extrinsic stimuli compared with hyaluronic acid (HA). First, we examined the penetration of sacran through stratum corneum (SC) with an impaired barrier function using immature reconstructed human epidermal equivalents. Sacran penetrates the SC to living cell layers of the epidermis, which suggested that sacran would attenuate adverse influences in keratinocytes caused by extracellular factors such as irritants or proinflammatory cytokines such as interleukin 1α (IL-1α). Sacran markedly reduced the cell damage induced by a nonionic detergent, sodium lauryl sulfate (SLS). Moreover, sacran restored the elevation of intracellular reactive oxygen species (ROS) levels stimulated by SLS and by IL-1α. These effects of sacran were superior to those of HA. In order to investigate the restoration effects of sacran, the influence of sacran on the physical properties of lipid bilayers was evaluated by measuring the order parameter using the ESR spin-labeling method. Because sacran failed to cause changes in the order parameters of liposomes and HaCaT keratinocytes, these results indicate that sacran does not interact with lipid bilayers although it restored changes in the order parameter caused by SLS. The sum of these results demonstrates that sacran reduces the influence of extracellular stimuli by its trapping effects. We conclude that the improving action of sacran is based on its trapping effect.

The formation of skin pigmentation requires multiple steps, namely the activation of melanocytes, the synthesis of melanin, the transport of melanosomes to the tips of melanocyte dendrites and the transfer of melanosomes from melanocytes to surrounding keratinocytes. Recently, we reported that melanosomes accumulate in melanocytes when melanosome transport is disrupted and that they are then degraded by the autophagy system. In this study, we examined whether 3-O-glyceryl-2-O-hexyl ascorbate (VC-HG) suppresses melanogenesis through the activation of autophagy since VC-HG interferes with melanosome transport through the down-regulated expression of MyosinVa and Kinesin. The results demonstrate that VC-HG-treated B16 cells show an activation of autophagy through an increased expression level of Microtubule-associated protein 1 light chain 3 (LC3)-II and a decreased expression level of p62. Furthermore, the decrease of melanin content elicited by VC-HG was partially abolished by hydroxychloroquine or pepstatin A which are inhibitors of autophagy. Taken together, we conclude that VC-HG suppresses melanogenesis by activating the autophagy system.

Photoaging of the skin is marked by obvious wrinkles and mainly depends on degradation of the extracellular matrix (ECM) in the dermis. Matrix metalloproteinase (MMP)-1 is one of the most important factors involved in degradation of the ECM; however, its mechanism of activation is not fully understood. It has been thought that MMP-1 is expressed by dermal fibroblasts as an inactive precursor protein that is activated by proteinases produced by keratinocytes in the epidermis. In this study, we constructed a 3D model of the dermis using collagen-embedded fibroblasts with or without ultraviolet (UV)-A exposure to mimic photoaging in the dermis. Collagen lattices embedded with UV-A-irradiated fibroblasts miniaturized and collagen was degraded to a greater extent than collagen lattices embedded with non-irradiated fibroblasts. The results demonstrate that fibroblasts in this 3D model express activated MMP-1 in the absence of keratinocytes. Moreover, the results confirm that activation of MMP-1 depends on increased plasmin activity in this model and lattice miniaturization was inhibited by the plasmin inhibitor tranexamic acid. Our results suggest that plasmin acts as an activator of MMP-1 and the inhibition of plasmin prevents collagen degradation.

SummaryBackground Ascorbic acid (AsA) has multifunctional benefits on skin beauty, such as the reduction in oxidative stress and the induction of collagen production. Among them, the prevention and improvement of skin pigmentation by AsA is a most important benefit for people. However, it is well known that AsA not only is quite unstable in formulations but it also has a low capability of skin penetration due to its hydrophilic property. In addition, existing water-soluble AsA derivatives that were developed to improve its stability also have low skin penetration. Aim To investigate the potential of a newly synthesized amphiphilic derivative of AsA, 3-O-Glyceryl-2-O-hexyl ascorbate (VC-HG), which has an added glyceryl group and a hexyl group, on skin beauty focusing on its skin lightening/whitening effects. Methods DNA microarray analysis and real-time PCR were used to clarify the effects of VC-HG on melanogenesis using B16 mouse melanoma cells. The effects of VC-HG on melanin synthesis, tyrosinase protein levels, and the inhibition of tyrosinase activity were evaluated. Results DNA microarray analysis revealed that treatment with VC-HG downregulated the expression of genes encoding tyrosinase and MyosinVa. Further, real-time PCR analysis showed the downregulation of tyrosinase, MyosinVa, Rab27a, and Kinesin mRNAs following VC-HG treatment. In addition, VC-HG caused decreases in tyrosinase protein levels and melanin synthesis. Conclusion We conclude that VC-HG has an impact on skin lightening/whitening by inhibiting tyrosinase protein synthesis and interfering with intracellular melanosome transport.

Melanosomes containing melanin are transported from the perinuclear area to the tips of dendrites in epidermal melanocytes, and are then transferred to keratinocytes. Thus, skin color is determined by the amount of melanin synthesized in melanocytes and the subsequent dispersion of melanosomes in the epidermis. Therefore, disrupting intracellular melanosome transport in melanocytes is considered an effective approach to regulate skin color. However, the fate of melanosomes that accumulate in melanocytes due to disrupted intracellular transport is unclear. In this study, we disrupted melanosome transport by knockdown of the motor protein MyosinVa. Knock-down of MyosinVa (M-KD) in cells treated with theophylline significantly down-regulated the mRNA and protein expression levels of tyrosinase. Interestingly, intracellular melanin contents in M-KD cells were decreased. Furthermore, M-KD cells showed activation of autophagy through increased expression of Microtubule-associated protein 1 light chain 3 (LC3) -II and decreased expression of p62. The sum of these results indicate that disruption of melanosome transport causes their degradation by autophagy.

Background Autophagy is known as an intracellular cleanup system necessary to maintain homeostasis of the skin. Many studies have pointed out the relationship between aging and the inactivation of autophagy function, which suggests that the inactivation of autophagy occurs in aged skin. However, the aging process of the skin is complicated compared with other organs, because the skin is localized at the border between the inside of the body and the environment. Thus, skin aging is strongly affected by environmental factors, and it is well recognized that ultraviolet (UV) radiation is an important environmental factor that promotes skin aging. Therefore, characterizing the autophagic phenotypes induced by environmental factors is important to understand the process of skin aging. Methods In order to demonstrate the status of autophagy during environment-induced aging of the skin, we investigated the autophagy profiles of normal human dermal fibroblasts (NHDFs) treated with repetitive UVA irradiation as model fibroblasts in photoaged skin. Results Repetitively UVA-irradiated NHDFs showed increased numbers of autophagosomes, which coincided with the accumulation of p62 and increased levels of LAMP-1 and lysosomes. The behavior of repetitively UVA-irradiated NHDFs on autophagy was similar to that of NHDFs treated with hydroxychloroquine (HCQ), which is an inhibitor of lysosomal proteinase. Conclusion In summary, these results demonstrate that repetitively UVA-irradiated fibroblasts have reduced autophagy function due to the dysfunction of lysosomes.

Recent increases in air pollution have raised concerns about its adverse effects on human health. Sacran is a natural polysaccharide isolated from a cyanobacterium. We previously reported that sacran improves skin conditions because of its effects as an artificial barrier against external stimuli, which suggested that sacran might protect the skin against air pollutants. The goal of this study was to characterize the potential of sacran to protect human skin against damage from air pollutants and to compare sacran with hyaluronic acid (HA). Sacran that was topically applied on the skin stayed on the surface or in the stratum corneum. Sacran-treated filters had a shielding effect against benzo[a]pyrene (BaP) and aldehyde compounds contained in tobacco smoke. Sacran suppressed the upregulation of cytochrome P4501A1 messenger ribonucleic acid (mRNA), which is a xenobiotic-metabolizing enzyme induced by BaP, and other responses against tobacco smoke in HaCaT keratinocytes. Furthermore, topical application of a serum containing 0.04% sacran on the skin reduced levels of carbonylated proteins in corneocytes of tobacco smokers. Sacran showed superior effects in every characteristic measured, compared with HA. We conclude that sacran ameliorates the oxidative stress initiated by tobacco smoke by shielding the skin surface and protects human skin.

Sodium lauryl sulfate (SLS), a representative anionic surfactant, is well-known to induce rough skin following single or multiple topical applications. The mechanism by which SLS induces rough skin is thought to result from the disruption of skin moisture function consisting of NMF and epidermal lipids. However, a recent study demonstrated that topically applied SLS easily penetrates into the living cell layers of the epidermis, which suggests that physiological alterations of keratinocytes might cause the SLS-induced rough skin. This study was conducted to clarify the effects of SLS on keratinocytes to demonstrate the contribution of SLS to the induction of rough skin. In addition, the potentials of other widely used anionic surfactants to induce rough skin were evaluated. HaCaT keratinocytes treated with SLS had increased levels of intracellular ROS and IL-1α secretion. Application of SLS on the surface of a reconstructed epidermal equivalent also showed the increased generation of ROS. Further, SLS-treated cells showed an increase of intracellular calpain activity associated with the increase of intracellular Ca2+ concentration. The increase of intracellular ROS was abolished by the addition of BAPTA-AM, a specific chelator of Ca2+. In addition, IL-1α also stimulated ROS generation by HaCaT keratinocytes. An ESR spin-labeling study demonstrated that SLS increased the fluidity of membranes of liposomes and cells. Together, those results indicate that SLS initially interacts with cell membranes, which results in the elevation of intracellular Ca2+ influx. Ca2+ stimulates the secretion of IL-1α due to the activation of calpain, and also increases ROS generation. IL-1α also stimulates ROS generation by HaCaT keratinocytes. We conclude from these results that the elevation of intracellular ROS levels is one of the causes of SLS-induced rough skin. Finally, among the other anionic surfactants tested, sodium lauryl phosphate has less potential to induce rough skin because of its lower generation of ROS.

Skin sensitivity is a serious problem for many people, and it can be induced by various factors such as UV irradiation, physical and mental stresses, air pollution, dry air and so on. Skin dryness triggered by UV and dry air is one of the most important causes inducing the development of sensitive skin, and it has been reported that oxidative stress contributes to skin dryness. In this study, we investigated whether treatment with 3-O-laurylglyceryl ascorbate (VC-3LG), which is an amphipathic ascorbic acid derivative, can suppress the development of sensitive skin. The results demonstrate that VC-3LG restores the expression levels of interleukin-1α, nerve growth factor and matrix metalloprotease-9 in the dry skin models of reconstructed human epidermal equivalents (RHEEs) and in H2 O2 -treated keratinocytes. In addition, VC-3LG suppresses the dendrite elongation of nerve cells induced in RHEEs by dry skin conditions and by H2 O2 treatment of keratinocytes. Therefore, we consider that treatment of the skin with VC-3LG is an effective approach to improve the development of sensitive skin.

Pyridoxine (VBThis study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VBIntracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2',7'-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VBTreatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VBThese results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB

The formation of skin pigmentation requires multiple steps, namely the activation of melanocytes, the synthesis of melanin, the transport of melanosomes to the tips of melanocyte dendrites and the transfer of melanosomes from melanocytes to surrounding keratinocytes. Recently, we reported that melanosomes accumulate in melanocytes when melanosome transport is disrupted and that they are then degraded by the autophagy system. In this study, we examined whether 3-O-glyceryl-2-O-hexyl ascorbate (VC-HG) suppresses melanogenesis through the activation of autophagy since VC-HG interferes with melanosome transport through the down-regulated expression of MyosinVa and Kinesin. The results demonstrate that VC-HG-treated B16 cells show an activation of autophagy through an increased expression level of Microtubule-associated protein 1 light chain 3 (LC3)-II and a decreased expression level of p62. Furthermore, the decrease of melanin content elicited by VC-HG was partially abolished by hydroxychloroquine or pepstatin A which are inhibitors of autophagy. Taken together, we conclude that VC-HG suppresses melanogenesis by activating the autophagy system.

Background The induction of skin pigmentation by ultraviolet (UV) radiation has been shown to result from factors secreted from UV-exposed keratinocytes that enhance melanogenesis in melanosomes (MSs) and stimulates their transfer to keratinocytes. Among those factors, it has been reported that α-melanocyte stimulating hormone, which is converted from the precursor proopiomelanocortin (POMC) following UV exposure, stimulates the transfer of MSs from melanocytes to surrounding keratinocytes. Objective The purpose of this study was to evaluate the effects of a red pumpkin seed (RPS) extract on the transfer of MSs to keratinocytes and to clarify the involvement of reactive oxygen species (ROS) on the UVB-induced transfer of MSs. Methods The transfer of MSs into keratinocytes was examined by measuring the incorporation of fluorescent beads, which were used as pseudo-MSs. mRNA expression levels of POMC, Nrf2, and Nrf2-related genes were determined using real-time PCR. Intracellular ROS levels were estimated with H2 DCFDA. Results The incorporation of fluorescent beads into keratinocytes was enhanced by treatment with the conditioned medium (CM) from keratinocytes exposed to UVB or H2 O2 . UVB or H2 O2 exposed keratinocytes had an up-regulated mRNA expression level of POMC. Treatment of keratinocytes with the RPS extract enhanced their intracellular antioxidant system via the activation of Nrf2 signaling and suppressed their incorporation of fluorescent beads that had been stimulated by the CM from UVB or H2 O2 exposed keratinocytes. Conclusion These results indicate that the RPS extract suppresses MS transfer stimulated by ROS generated following UVB exposure through the activation of Nrf2 signaling.

Background Skin hydration is generally assessed using the parameters of skin surface water content (SWC) and trans-epidermal water loss (TEWL). To date, few studies have characterized skin conditions using correlations between skin hydration parameters and corneocyte parameters. Aims The parameters SWC and TEWL allow the classification of skin conditions into four distinct Groups. The purpose of this study was to assess the characteristics of skin conditions classified by SWC and TEWL for correlations with parameters from corneocytes. Methods A human volunteer test was conducted that measured SWC and TEWL. As corneocyte-derived parameters, the size and thick abrasion ratios, the ratio of sulfhydryl groups and disulfide bonds (SH/SS) and CP levels were analyzed. Results Volunteers were classified by their median SWC and TEWL values into 4 Groups: Group I (high SWC/low TEWL), Group II (high SWC/high TEWL), Group III (low SWC/low TEWL), and Group IV (low SWC/high TEWL). Group IV showed a significantly smaller size of corneocytes. Groups III and IV had significantly higher thick abrasion ratios and CP levels. Group I had a significantly lower SH/SS value. The SWC/TEWL value showed a decline in order from Group I to Group IV. Conclusion Groups classified by their SWC and TEWL values showed characteristic skin conditions. We propose that the SWC and TEWL ratio is a comprehensive parameter to assess skin conditions.

A Kampo prescriptions, hochuekkito (HET) has been utilized for treating functional conditions such as general fatigue, compromised state and gastrointestinal motility disorder. Recently, HET has attracted the attention of dermatologists because of its clinically positive effects in atopic dermatitis (AD) treatment. To explain this positive effect of HET, we examined its protective ability against oxidative skin stress using a murine model. The dorsal region of 8-week-old male HR-1 hairless mice, which were raised on a HET (0%, 2% and 10%) mixed diet, was irradiated once with 70 mJ/cm(2) of ultraviolet (UV)-B light. After 4 days, transepidermal water loss (TEWL) and stratum corneum water content (SCWC), were determined as a measure of degree of skin dysfunction. To estimate the amount of active oxygen generated, the stratum corneum catalase activity (SCCA) and stratum corneum carbonylated protein (SCCP) content in the tape-stripped stratum corneum samples were measured. We also measured the H(2) O(2) scavenging ability of HET, and analyzed the changes in the expression levels of several inflammation and oxidative stress-related genes in the skin of HET-fed mice. In control mice, exposure to UV-B led to significant increases in TEWL and SCCP and significant decreases in SCWC and SCCA. These UV-B-induced changes were reduced in mice administrated HET, and the reduction was HET dose-dependent. Our results suggested that HET offered a protective effect against UV-B-induced skin damage. We also found that HET had relatively low ability to scavenge H(2) O(2) , and expression level of cyclooxygenase-2 mRNA decreased in HET-fed mouse.

Background Carbonylated proteins (CPs) are generated by the reaction of basic amino acid residues in proteins with aldehyde compounds produced during lipid peroxidation. CPs in the stratum corneum (SC) impact skin conditions such as skin moisture functions including water content and transepidermal water loss (TEWL). In addition, CPs can be frequently seen in the SC from sun-exposed sites compared with sun-protected sites. Objective The aim of this study was to reveal whether CPs could be a generation source of reactive oxygen species (ROS) in the SC following exposure to ultraviolet (UV) radiation and to identify the type of ROS and its generation mechanism. Methods ROS generation was detected using a methyl cypridina luciferin analog (MCLA) chemiluminescence system and an ESR spin-trapping method. CPs in porcine SC, in a keratin film and in bovine serum albumin (BSA) were prepared by reaction with acrolein. Levels of protein carbonylation were quantified by detecting aldehyde residues. Results CP levels in the SC were increased in a UVA energy-dependent manner. That result suggested that a source of ROS generation existed in the SC initiated and produced the carbonylation of SC proteins. Carbonylated BSA and carbonylated porcine SC sheets exhibited fluorescence spectra at an excitation wavelength of 430 nm and an emission wavelength of 520 nm. Irradiation of the SC with UVA increased protein carbonylation and the amount of autofluorescence in the SC. ROS generation in the SC caused by UVA and by short-wavelength visible light (blue light, 400–470 nm) was detected by the MCLA chemiluminescence system. Artificially carbonylated porcine SCs and keratin films had increases of chemiluminescence intensity after exposure to both light sources as well. The addition of superoxide dismutase to the MCLA system completely abolished the incremental chemiluminescence intensity after both UVA and blue light exposure of the SC. In addition, acrolein-treated BSA gave ESR signals like hydroxyl radicals ( OH) converted from superoxide anion radicals ( O 2 − ) during irradiation with a xenon arc lamp containing UVA and visible light. From the sum of these results, we consider that CPs are produced from O 2 − initially generated from exposure to UVA and blue light. Conclusion CPs are excited by absorbing sunlight, particularly UVA and blue light, and result in the generation of O 2 − through a CPs progress new protein carbonylation in stratum corneum through ROS generation. photosensitizing reaction. Further, the results suggest that the O 2 − produces CPs in the SC through lipid peroxidation in the sebum, and finally affects skin conditions including color and moisture functions.

Recent studies have demonstrated that lysophospholipids (LPL) play critical roles in several biological signal transduction pathways to maintain the homoeostasis of cells, tissues and organs. Among them, lysophosphatidic acid (LPA) has been identified as a lipid mediator that induces morphological improvement in the epidermis in mice. In this study, we examined the effects of LPL (soybean-derived phospholipids modified with phospholipase A2 and C) compared with LPA. We initially examined the effects of LPA on normal human epidermal keratinocytes (NHEK) focusing on the expression of profilaggrin and serine palmitoyltransferase (SPT) mRNAs. LPA enhanced the expression of profilaggrin and SPT mRNAs via the modulation of Ca(2+) influx. Based on those results, the influence of LPL on NHEK was examined and was expanded to analyse the expression of two tight junction-related proteins, occludin and claudin-1. LPL had similar effects to increase profilaggrin and SPT mRNA expression and also stimulated the expression of occludin and claudin-1 at the mRNA and protein levels. In accordance with these results, LPL elicited significant improvements in surface water content in human skin. These findings indicate that LPL has the potential to strengthen the skin moisturizing capability by up-regulating the expression of mRNAs encoding components important to skin barrier function and skin hydration.

Summary Background All-trans-retinoic acid (RA) is well known as a potent anti-aging drug. However, RA is generally difficult to use topically due to its irritancy and mutagenicity. Therefore, we synthesized a new RA derivative, d -δ-tocopheryl retinoate (TR). Objective To evaluate the potency of TR on anti-aging effects, investigating responses of human skin cells, keratinocytes and fibroblasts. Method To examine the potential for TR to remove reactive oxygen species (ROS), we used an electron spin resonance (ESR) spin-trapping method and a chemical assay. To investigate the effects of TR on skin cells, we used HaCaT cells, human keratinocytes, and human fibroblasts. Further, clinical test was carried out. Results In ESR study, TR showed the quenching abilities against singlet oxygen. In a biological system, TR significantly suppressed the elongation of DNA comet tails in HaCaT keratinocytes after UVB irradiation. The sum of those results suggests that TR has a capability to remove oxidative stress. TR had no effect on matrix metalloprotainase-1 (MMP-1) expression by UVA-irradiated fibroblasts. TR increased collagen production by fibroblasts and hyaluronic acid (HA) production by keratinocytes. We conducted a clinical study on wrinkle improvement using eight human volunteers. Treatment with TR exhibited a significant reduction in the depth of linear wrinkles, showing the potency of TR to improve wrinkles. Conclusion Anti-wrinkle efficacy of TR is suggested to involve removing oxidative stress and modulating the metabolism of the extracellular matrix.

Summary Background Heat shock protein 47 (HSP47) is a specific chaperone of procollagen. It is important to elucidate the effects of heat on collagen synthesis both in vitro and in vivo. Objectives We examined the effects of heat on collagen synthesis and the role of HSP47 using an in vitro system, and we also characterized the efficacy of wrinkle removal by heat treatment of human skin. Methods Normal human fibroblasts were used to evaluate the relationship between heat-induced collagen synthesis and HSP47 using an enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and antisense. Heat stimulation of 6-week-old hairless mice (HOS:HR-1) was performed at varying temperatures (38, 40 and 42 °C) 3 days a week for 4 weeks, then the amount of collagen was determined by hydroxyproline content. For clinical evaluation, the left side of the face of 31 women (aged 36–55 years), was treated with heat 10 min a day for 2 months using hot steam which kept the skin surface temperature at 40–42 °C. Evaluations were performed using a visual analog scale, by replica taking, and with a Cutometer, prior to and 4 and 8 weeks after the heat treatment. Results The in vitro study showed that heat treatment enhanced collagen biosynthesis by up-regulating HSP47 mRNA and protein expression but not procollagenα1(I). Antisense inhibition of HSP47 prevented the increase of collagen synthesis induced by heat. Heat treatment at 40–42 °C enhanced hydroxyproline content and improved wrinkles/sags of the facial skin. Conclusions These findings indicate that heat treatment at 40–42 °C has a beneficial therapeutic potential to repair wrinkles and sags in the skin through the up-regulation of collagen synthesis.

Glycolic acid (GA), one of the alpha-hydroxy acids, is widely used as an agent for chemical peeling. Although there are several reports about the clinical effects of GA in the literature, its biological mechanism remains mostly unclear, and there are only a few reports about its effects on skin rejuvenation mediated by keratinocytes. The aim of this study was to investigate the effect of GA on the dermal matrix metabolism of keratinocytes and fibroblasts using in vitro and ex vivo systems. Our study shows that GA not only directly accelerates collagen synthesis by fibroblasts, but it also modulates matrix degradation and collagen synthesis through keratinocyte-released cytokines. We confirm that IL-1alpha is one of the primary mediators for matrix degradation released from keratinocytes after GA treatment. These results suggest that GA contributes to the recovery of photodamaged skin through various actions, depending on the skin cell type.

一酸化窒素 (NO) は皮膚血流量の調節や血管の弛緩などに重要な役割を果たしていることが知られている。NOは生体内でNO合成酵素 (NOS) の作用によりL-arginine (L-Arg) から合成される。われわれはヒト血管内皮細胞において, (1) L-ピロリドンカルボン酸 (L-PCA) は濃度依存的に, NO産生量を促進する, (2) L-PCAは血管内皮細胞へのL-Argの取り込みを促進する, そして (3) この促進効果はカチオン性アミノ酸トランスポーター (CAT) 阻害剤であるL-NMMAによって抑制されることを見出した。これらの結果にもとついて, L-PCAはCATを介したL-Argの細胞への取り込みを促進することにより, NO生成量を適度に増加させ血流を調節していることがわかった。しかし, これらのNO合成促進およびL-Arg取り込み促進効果はL-PCAにのみ見られ, D-体には見られなかった。さらに, ヒト前腕内側部での閉塞パッチテストにおいても, L-PCAは皮膚の赤み・血流量を一時的に増加させることが認められた。これまでL-PCAは天然保湿因子の主要な成分の一つであることから保湿を目的としたスキンケアに広く用いられてきたが, 血流促進を目的とした用途においても有用であることが明らかとなった。

alpha-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of alpha-tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with alpha-tocopherol for 24 h, the intracellular GSH was increased at every concentration of alpha-tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with alpha-tocopherol at 50 microM for 24 h exhibited resistance against H2O2. However, a short exposure of HaCaT keratinocytes to alpha-tocopherol for 1 h did not influence either the GSH level or the resistance to H2O2. These findings suggest that GSH, which is inductively synthesized by alpha-tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of alpha-tocopherol on the GSH level, BSO, which is a typical inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of alpha-tocopherol on the GSH level was observed. On the other hand, alpha-tocopherol resulted in the up-regulation of gamma-GCS-HS (heavy subunit) mRNA. In addition, water soluble alpha-tocopherol derivatives (alpha-tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that alpha-tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of gamma-GCS-HS mRNA.

We previously reported that epidermal glycation causes an increase in saturated fatty acid (FA) content in a differentiated reconstructed skin model and HaCaT cells. However, the relationship between ceramides (CERs) and glycation and their effects on stratum corneum (SC) barrier function was not elucidated. In this study, we investigated the effect of glycation on lipid content in 6-day-old cultured reconstructed skin. We used the EPISKIN RHE 6D model and induced glycation using glyoxal. In addition to transepidermal water loss, content of CERs, cholesterol and FA in the reconstructed epidermal model were analyzed by high performance thin layer chromatography. Expression of genes related to ceramide metabolism was determined by real time RT-PCR. Membrane fluidity of stratum corneum lipid liposomes (SCLL) that mimic glycated epidermis was analyzed using an electron spin resonance technique. It was found that FA was significantly increased by glycation. CER[NS], [AP], and cholesterol were decreased in glycated epidermis. Expression of ceramide synthase 3 (CERS3) was significantly decreased while fatty acid elongase 3 was increased by glyoxal in a dose dependent manner. Membrane fluidity of SCLL mimicking the lipid composition of glycated epidermis was increased compared with controls. Therefore, disruption of CER and FA content in glycated epidermis may be regulated via CERS3 expression and contribute to abnormal membrane fluidity.

皮膚の老化をより印象づける, 「しわ」や「たるみ」といった現象は, 種々の真皮マトリックスの構造変化に由来すると考えられている。真皮マトリックスの中でもグリコサミノグリカンは, 膠原線維 (collagen) や弾性線維 (elastin) などの構造基盤を成す成分の間隙を埋めるものであり, 皮膚の水分保持や柔軟性に関わりが深い。今回われわれはこれらグリコサミノグリカンの一種であるヒアルロン酸 (HA) に注目した。線維芽細胞を用いたin vitroの系でHA合成を促進させる植物抽出物の探索を行ったところ, 数種の植物抽出物に有効なHA合成促進効果を認め, その中でもキダチアロエに高い活性を認めた。成分分画を行い活性を追ったところ, 比較的高極性画分に活性が移行することを確認した。また, 分画に伴いその活性は増大した。さらに線維芽細胞にその存在が認められているHA合成酵素 (HAS) のmRNA発現について検討を行い, 分画後のフラクションにHAS 2 mRNA発現量の増加を確認した。

シワは加齢に伴って現れる皮膚の老化の特徴であり, シワを改善することは化粧品の大きな課題である。シワの予防効果や改善効果を評価するためには, シワ深さを定量化することが必要となる。しかし, これまではシワ深さ定量法は試験者各様の手法で行われており, 個々の手法の測定条件などの違いにより安定した測定値を得ることが非常に困難であった。そこで今回, われわれは標準スケールを用いたシワ深さの定量法について検討した。その結果, 光量や照射角度などの画像撮影条件や, 撮影した画像からシワを抽出する画像処理条件にかかわらず, 再現性の高いシワ深さ定量値を得ることができたので報告する。