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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.

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.

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.

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.

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.

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.

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。

Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavonoid, was found to prevent human dermal fibroblast cell damage induced by reactive oxygen species such as hydrogen peroxide (H2O2), tert-butyl hydroperoxide (BuOOH) and superoxide anions (.O2-) in a concentration-dependent manner, and was more effective than the iron chelator, deferoxamine, hydroxyl radical (.OH) scavengers such as dimethyl sulfoxide (DMSO) and ethanol (EtOH), the lipid peroxidation chain blocker, alpha-tocopherol (Vit. E) and the xanthine oxidase inhibitor, allopurinol. To probe the mechanism of cell defense, the reaction of baicalein with oxygen free radicals was investigated using electron spin resonance (ESR) spectrometry. Baicalein decreased the signal intensities due to the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adducts of .OH, .O2- and tert-butyl peroxyl (BuOO.) radicals in a concentration-dependent manner. The IC50 values, which are the 50% inhibition concentrations of baicalein for the free radicals, were 10, 45 and 310 microM, respectively. These results suggested that baicalein possesses free radical scavenging ability which prevents the fibroblast damage induced by these free radical species.

長期間にわたるUVA暴露は, 皮膚の「たるみ」や「深いしわ」に特徴付けられる光老化を引き起こす。このような臨床的症状は細胞外マトリックスの構造変化に由来すると考えられる。正常な真皮構造を維持するために, コラーゲン, エラスチンおよびグリコサミノグリカンなどの細胞外マトリックス成分は産生・分解を繰り返している。この産生・分解のアンバランスが真皮構造の変化の原因となると考えられる。光老化による真皮マトリックスの構造変化を阻止あるいは改善する方法として, われわれは紫外線暴露により活性が上昇する細胞外マトリックス成分分解酵素 (コラゲナーゼおよびエラスターゼ) の活性をコントロールすることに注目した。そこで, それぞれの酵素活性に対し阻害作用を有する有効成分を探索した。その結果, ユーカリ抽出物がI型コラゲナーゼおよびヒト線維芽細胞由来コラゲナーゼに対して, またセイヨウニワトコ抽出物が炎症に伴い浸潤してくる好中球由来エラスターゼ (セリンプロテアーゼ) の活性に対して高い阻害作用を示すことがわかった。これらのことはユーカリ抽出物がUVA暴露後に線維芽細胞が分泌するI型コラゲナーゼ (MMP-1) を阻害することによりI型コラーゲンの分解を阻害することを示唆する。さらに、セイヨウニワトコ抽出物は好中球由来エラスターゼ活性を阻害する成分として有効である。

It has been reported by Von Scott that α-hydroxy acids have potent effects on wrinkle-repairing. MTT (3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium Bromide) method has been used for estimation of cytotoxicity, mitogenic activity and cell viability. We attempted the development of the cell activity estimating method by using MTT method and the investigation of α-hydroxy acids-wrinkle repairing mechanism on human dermis fibroblast culture system. It has been found that glycolic acid increased the activity which was estimated by the MTT method. Subsequently the proliferation effect of glycolic acid was examined. Glycolic acid showed high proliferating activity.Furthermore, the effect on collagen synthesis was examined.However, the enhancement on collagen synthesis by glycolic acid was not found.From these results, it was speculated that the wrinkle-repairing of glycolic acid was realized by the enhancement of proliferation in dermal fibroblasts.

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.

Oxidative stress caused by ultraviolet (UV) radiation generates reactive oxygen species (ROS) in the skin, induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyper-pigmentation. Thus, increasing the anti-oxidative ability of skin cells is expected to be a good strategy for skin-lightening cosmetics. Metallothionein (MT) is one of the stress-induced proteins and is known to exhibit a strong anti-oxidative property. We previously reported that a zinc(II) complex with glycine (Zn(II)(Gly)(2)) effectively induces MT expression in cultured human keratinocytes. To determine its potential as a new skin lightening active, we examined whether Zn(II)(Gly)(2) regulates the release of melanocyte-activating factors from UVB-irradiated keratinocytes and affects melanin production in a reconstructed human epidermal equivalent. Conditioned medium from UVB-irradiated keratinocytes accelerated melanocyte proliferation to 110%, and that increase could be prevented by pre-treatment with Zn(II)(Gly)(2). In addition, Zn(II)(Gly)(2) significantly reduced both the production of prostaglandin E(2) and proopiomelanocortin expression in UVB-irradiated keratinocytes. Zn(II)(Gly)(2) also decreased melanin production in a reconstructed human epidermal equivalent. These results indicate that MT-induction in the epidermis effectively up-regulates tolerance against oxidative stress and inhibits the secretion of melanocyte growth and activating factors from keratinocytes. Thus, Zn(II)(Gly)(2) is a good candidate as a new skin-lightening active.

Summary Background Vitamin C, which is a strong anti-oxidant, plays an important role in maintaining physiological states. In dermatology, Vitamin C is used for treatment of various skin problems such as de-pigmentation of hyperpigmented spots. However, Vitamin C has limited stability and permeability, and development of a Vitamin C derivative with improved properties is needed. Objective We evaluated the effect of a lipophilic Vitamin C derivative, tetra-isopalmitoyl ascorbic acid (VC-IP), on ultraviolet (UV)-induced skin pigmentation, to determine its potential as a more effective form of Vitamin C. Methods The release of Vitamin C from VC-IP was examined using a reconstructed skin model following topical application of VC-IP. Anti-oxidative and anti-inflammatory activities of VC-IP were tested in cultured human keratinocytes. Subsequently, clinical test was done to clarify the effect of VC-IP on UVB-induced skin pigmentation. Results VC-IP released Vitamin C in physiological conditions and worked as pro-Vitamin C. In subsequent experiments, we found that VC-IP suppressed the elevation of intracellular peroxide after UVB irradiation, and enhanced cellular tolerance against UVB and reactive oxygen species such as hydrogen peroxide and tert-butyl hydroperoxide. Furthermore, VC-IP reduced the production of interleukin-1α and prostaglandin E2 in UVB-irradiated keratinocytes and suppressed melanocyte proliferation in conditioned culture medium prepared from UVB-irradiated keratinocytes. Finally, in a clinical study, topical application of a 3% VC-IP cream for 3 weeks suppressed pigmentation after UVB irradiation. Conclusions These results demonstrate that VC-IP is a precursor of Vitamin C, and effectively suppresses UVB-induced skin pigmentation, possibly through its anti-oxidative activity.

Summary Background It is well established that the alterations of dermal matrix contributes to skin aging characterized by wrinkles. On the other hand, physiological NO is useful to maintain skin homeostasis such as a vasodilatation. However, a role of NO on production of dermal matrix has been clarified. Objective In this study, we have attempted to analyze the role of NO on type I collagen synthesis of normal human dermal fibroblasts including expression of procollagen αI S(1) mRNA/protein and heat shock protein 47 (HSP47). Methods The effects of NO which was generated by two types of NO donors, sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP), on type I collagen and HSP47 and their related mRNA expression were examined with ELISA and RT-PCR. Results NO was significantly accelerated the production of type I collagen by fibroblasts corresponding with up-regulation of procollagen αI (1) mRNA. Furthermore, NO increased both levels of HSP47 protein and mRNA in fibroblasts in a dose-dependent manner. Conclusions These results suggest that NO has dual effects on collagen synthesis by fibroblasts as follows; one is the direct stimulation of collagen synthesis due to the up-regulation of procollagen αI(1) mRNA, and the other is an indirect effect through the increase of HSP47 mRNA expression. This is the first report that exogenous NO stimulates HSP47 production by dermal fibroblasts.

Advanced glycation end-products (AGEs) have been reported to accumulate in the dermal skin. However, it remains unknown whether the AGEs interact with the dermal fibroblasts and influence their function. Previously, we demonstrated that AGEs hastened photoaging of the skin by means of active oxygen species such as *O(2)(-), H(2)O(2), and *OH, generated during UVA irradiation. The purpose of the present study was to clarify the influence of AGEs on the functions of dermal fibroblasts under physiological conditions. It was found that AGEs decreased both hyaluronic acid (HA) synthesis and activity of elastase-type matrix metalloproteinase (ET-MMP). Because the reactions of both HA synthesis and ET-MMP were found to take place at the cell membrane region, it appeared that AGEs modulated cellular dysfunction through an interaction with the cell membrane. To clarify the mechanisms of these dysfunction in relation to AGEs, we examined the interaction between AGEs and cell membranes, and obtained the following results: (1) AGEs associated with the cell membranes and liposomal membrane prepared with phosphatidyl choline; (2) AGEs hydrophobically modified the circumstances of the cell membrane and liposome membrane as evaluated by experiments using a fluorescence probe; (3) AGEs increased the fluidity of the cell membrane and liposomal membrane as estimated by ESR spin-labeling using 5-doxylstearic acid; and (4) AGEs accelerated lactate dehydrogenase (LDH) leakage from the cells. On the basis of these experimental results, we proposed that AGEs modulated cell function through a nonspecific interaction with the membranes of dermal fibroblasts.

Our previous study reported that advanced glycation end-products (AGE)-modified BSA produced active oxygen species, *O2-, H2O2, and *OH under UVA irradiation and enhanced the cytotoxicity of UVA light. We examined whether pentosidine in AGE-modified BSA was involved in one of the mechanisms generating the active oxygen species. In biological investigations, fibroblasts exposed to UVA (20 J/cm2) in the presence of pentosidine-rich compounds (PRCs), which were prepared with L-arginine, L-lysine and glucose, showed a time-dependent leakage of the cytosolic enzyme LDH. In addition, release of LDH was suppressed by addition of DMSO and deferoxamine under UVA irradiation. From these results, it was determined that PRCs exposed to UVA damaged the plasma membrane of human dermal fibroblasts due to the conversion of *OH from H2O2 via a Fenton-like reaction. These features of PRCs exposed to UVA were consistent with those of AGE-modified BSA. In an ESR study, PRCs under UVA irradiation yielded DMPO-OH (DMPO-OH adduct) using DMPO as a spin-trapping reagent. *O2- generation from UVA-irradiated PRCs was also indicated by the combination of NBT reduction and SOD. When PRCs were exposed to UVA light controlled with a long-pass filter, WG-360, it was found that their production of *O2- was prohibited less than 50% in the NBT reduction assay. The *O2- production profile of PRCs depending on the wavelength of UVA light was similar to that of AGE-modified BSA. Furthermore, it was found that the H2O2 level was increased by PRCs exposed to UVA. These results indicated that pentosidine is an important factor of AGE-modified BSA in active oxygen generation under UVA irradiation.

Advanced glycation end-products (AGEs) have been reported to be accumulated in dermal skin. However, the role of AGEs in the photoaging of human skin remains unknown, and for this reason, we have examined the interaction between AGEs and ultraviolet A light (UVA) from both the chemical and biological aspects. Previously, we reported that exposing human dermal fibroblasts to UVA in the presence of AGEs that were prepared with bovine serum albumin (BSA) decreased the cell viability due to superoxide anion radical s (.O2(-)) and hydroxyl radicals (.OH) generated by AGEs under UVA irradiation, and active oxygen species are detected with ESR spin-trapping. To identify the active oxygen species in detail and to clarify the cell damaging mechanism, we performed several experiments and the following results were obtained. (1) In ESR spin-trapping, by addition of dimethyl sulfoxide and superoxide dismutase, ESR signals due to .O2(-) -derived DMPO-OOH and .OH-derived DMPO-OH adducts, respectively, were detectable. (2) UVA-irradiated AGEs elevated the lipid peroxide levels in both fibroblasts and liposomes. But the peroxidation in liposomes was inhibited by addition of deferoxamine. (3) Survival of fibroblasts exposed to UVA in the presence of AGEs was elevated by addition of deferoxamine. And finally, (4) survival of fibroblasts was found to be regulated by the level of H2O2. On the basis of these results, we propose a possible mechanism in which AGEs under UVA irradiation generate active oxygen species involving .O2(-), H2O2, and .OH, and the .OH species plays a harmful role in promoting cell damage.

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S,R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 microM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 microM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.

Ergothioneine (EGT) is a sulfur-containing amino acid, and is presumed to function as a natural antioxidant. The purpose of this study was to identify the nature of the antioxidant activity and investigate the effects of EGT on UV-induced cellular response. In chemical studies, EGT scavenged the superoxide anion radical (*O(2)(-)) and singlet oxygen ((1)O(2)). In cultured fibroblasts, EGT suppressed TNF-alpha up-regulation by UVB irradiation. In addition, in fibroblasts exposed to UV-A, EGT suppressed the expression of matrix metalloproteinase 1 (MMP-1) protein by nearly 50% and reduced MMP-1 mRNA expression. From these results, we conclude that EGT scavenges reactive oxygen species generated by both Type I and Type II photosensitization and suppresses both TNF-alpha expression and MMP-1 at their transcriptional level. EGT may reduce skin anti-aging effects after UV irradiation by the scavenging of *O(2)(-) and (1)O(2), and reducing signals for protease and inflammatory activity.

The sensory irritation such as biting appears after application of cosmetics. This irritation is the passing stimulation without inflammation. We investigated many cosmetic materials, and made researches on the substance and mechanism of this stimulation.In the results, the sensitivity among the population showed the individual differences. The intensity of irritation is directly related to the number of molecules, is effected by temperature and viscosity of vehicles.We considered that the causative substances must have the high affinity to the skin. This high affinity is dependent on suitable molecular size, polarity, structure and electronic property of the molecule.The detail of this stimulation was reported.