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445 A set of plant essential oils have antioxidant activity in ultraviolet B-irradiated epidermal keratinocytes by upregulating antioxidant and detoxifying enzymes
Epidermal Structure and Barrier Function | ABSTRACTS 444
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Reactive oxygen species induce Th2 allergic inflammation in the skin by activating PI3K-HIF-a pathway J Choi1, M Piao1, E Park2, S Yun1, J Lee1 and S Lee1 1 Chonnam National University Medical School, Gwangju, Republic of Korea and 2 Hisol co., Ltd, Namwon-si, Republic of Korea Background: Reactive oxygen species (ROS) plays an important role to activate the Th2 allergic inflammation in asthma or rhinitis. Purpose: This study was aimed to evaluate whether ROS is implicated in the pathogenesis of Th2 inflammation in the skin. Compound 48/80-treated HaCaT cells were tested for a new cell model for atopic dermatitis (AD). Materials: In compound 48/80-treated HaCaT cells, ROS production, PI3K-HIF-1a activation, and expression levels of biomarkers for Th2 inflammation in the skin were evaluated. The role of ROS on Th2 inflammation in the skin were verified in the compound 48/80-treated HaCaT cells, which were treated with N-acetyl cysteine (NAC). Results: Compound 48/80 (0.1 mg/ ml) induced the ROS production, PI3K-HIF-1a, as it modulated biomarkers for Th2 inflammation in the skin: upregulation of KLK5 and PAR2 (pro-Th2 inflammatory markers), TSLP and IL-33 (Th2 inflammatory markers), and NGF and CGRP (neurogenic inflammatory markers); and downregulation of filaggrin (barrier function marker). The compound 48/80-induced modulation of those biomarkers could be suppressed by antioxidant treatment (NAC), indicating that ROS plays a crucial role during Th2 inflammation in the skin. The modulatory pattern of biomarkers for Th2 inflammation in the compound 48/80-treated HaCaT cells mimics to the AD skin, which was further confirmed in the house dust mite-treated human keratinocytes as control experiments. Conclusion: Compound 48/80-producing ROS induces Th2 inflammation in the skin through the activation of the PI3K-HIF-1a pathway.
A set of plant essential oils have antioxidant activity in ultraviolet B-irradiated epidermal keratinocytes by upregulating antioxidant and detoxifying enzymes J Choi1, S Yun2, J Lee1, M Piao1, E Park3 and S Lee1 1 Chonnam National University Medical School, Gwangju, Republic of Korea, 2 Chonnam National University, Gwangju, Republic of Korea and 3 Hisol co., Ltd, Namwon-si, Republic of Korea Background: Plant-derived extracts including essential oils have long been used as important ingredients of cosmetics. In recent years, there has been a demand for safe cosmeceuticals with excellent antiaging activity. Aim: This study aimed to develop candidate plant extracts with excellent antioxidant and detoxifying activities in keratinocytes with a view to protecting photoaging of the skin. Methods: The ROS-scavenging activities of candidate plant essential oils were determined in ultraviolet B (UVB)-irradiated normal human epidermal keratinocytes (NHEKs). Their antioxidant and detoxifying activities were determined by measuring enzymatic antioxidants, phase 2 enzymes, and total polyphenol content. Results: In DCF-DA and confocal microscopy studies, the plant essential oils including Lavender oil, Lemongrass oil, Rosemary oil, Chamomile oil, and Peppermint oil showed excellent ROS-scavenging activity in UVB-irradiated NHEKs. In RT-PCR studies, all these oils upregulated the expression of enzymatic antioxidants (CuSOD, GPx II, and Prx I) and phase 2 detoxifying enzymes (HO-1, NQO-1, GSTpi, GSTA4, and GCLM) via Nrf2 in NHEKs. Conclusions: All of the five essential oils can be regarded as good candidate natural products with potential for use as new cosmeceuticals.
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Early atopic dermatitis biological analysis G Bellemere1, G Boyer1, A Moga2, M Fontanie´3, C de Belilovsky1 and C Baudouin1 1 Laboratoires Expanscience, Epernon, France, 2 SYNELVIA, Labege, France and 3 VIBIOSPHEN, Labege, France Background: Pathophysiology of atopic dermatitis (AD) is characterized by decreased levels of Natural Moisturizing Factors (NMFs) and ceramides, with microbiota dysbiosis (colonization by staphylococci species). Little is known about these parameters before the onset of AD and during first AD flare in children with and without a genetic risk of AD. Objectives: The main objective was to quantify NMFs, ceramides, S aureus (SA) and S epidermidis (SE) colonization from birth until first AD flare. The impact of daily applications of emollients is reported. Methods: Swabs were taken on forearms, face and AD lesions in 45 infants for staphylococci, 76 for NMF/ceramides out of 180 newborns involved in a 6-month AD prevention study with daily emollient applications. SA and SE were analyzed for by quantitative PCR and for NMF and ceramides by LC/UV and LC/MS. Results: A depletion of NMFs has been observed on forearms and face of children at genetic risk of developing AD compared to control. Daily application of emollients normalized NMFs. No significant difference in NMFs levels has been observed in AD children compared to non-AD. Ceramide levels were not different in children at risk of developing AD nor in AD children compared to control. nfants were poorly colonized with SA: only 6 children among 45 on forearms and 17 on the face. In contrast, infants were largely colonized with SE: 42 infants on the forearm and 40 on the face. Only 2 AD lesions out of 11 were found positive for SA and 9 for SE. Levels of SA and SE colonization were not modified by emollients. Conclusion: This clinical study showed limited colonization of SA before and during early AD. Specific AD emollients improved hydration and skin barrier function and did not alter skin microbiota.
Collagen XVIII, a key interfacial component of the skin architecture I Bonnet1, S Cadau1, N Berthelemy2, V Bardey3, V Andre-Frei1, M Chavan4, H Zahouani5 and P Rousselle6 1 BASF BCS, Lyon, France, 2 BASF BCS, Essey les Nancy, France, 3 BASF BCS, Pulnoy, France, 4 BASF CORP, Tarrytown, NY, 5 Tribology - System Dynamics Laboratory ECL, Ecully, France and 6 Laboratoire de Biologie Tissulaire et Inge´nierie The´rapeutique, CNRS/UCBL1, Lyon, France Collagen XVIII holds structural properties of both collagen and heparan sulfate proteoglycan. It is expressed ubiquitously in various basement membrane (BM) structures of the body. In the skin, it could be synthetized by keratinocytes, endothelial cells, epithelial cells of the sweat glands and by adipocytes. Collagen XVIII complete role is still unknown but its localization and ultrastructural organization reveal that it is an important component of all BM molecular networks. Understanding its expression modulation with age could provide new strategies to counteract the loss of tissue structure and cohesion observed during ageing. To study chrono and photoageing impact on collagen XVIII maintenance, its protein and gene expression were analyzed in keratinocytes and skin biopsies. To specifically evaluate the collagen XVIII protein, and not its endostatin domain, an affinity-purified rabbit antibody against two collagenic domains was developed for the immunoassay. At the dermo-epidermal junction level, collagen XVIII protein expression was strongly decreased as skin ages (-43.5%) and this decrease could be explained by a decrease of COL18A1 gene expression in keratinocytes exposed or not to UVB. We then observed the stimulating effect of a Khaya senegalensis bark extract from the skin surface to deeper layers on different cells (keratinocytes, endothelial cells, adipocytes). The mechanical properties of reconstructed skins were evaluated with a totally new designed miniaturized non-touch device using air bluster system. The results proved that increasing collagen XVIII synthesis impacts the skin structural properties consequently improving skin elasticity, surface homogeneity, and reducing wrinkles.
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Involvement of catecholamine in the differentiation of sebaceous glands in vitro K Mizuno1, K Okuyama2, N Akimoto1 and T Sato1 1 Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan and 2 Department of Dermatology, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan Sebaceous glands secret sebum onto the skin surface and as such a thin lipid layer is formed as a physiological barrier. Sebaceous gland cells (sebocytes) differentiate to accumulate abundant cytoplasmic lipids. Since sebocyte differentiation has been reported to be regulated by hormones, growth factors, and cytokines, whether the autonomic nervous system is associated with the regulation of sebum production in sebaceous glands remains unclear. In the present study, we examined the effect of norepinephrine and epinephrine on the differentiation of hamster sebocytes. First, immunohistochemical staining using a neuron-specific class III beta-tubulin antibody showed that nerve filaments were detectable adjacent to sebaceous glands in human skin, assuming an association of the peripheral nervous system with sebaceous gland functions. In addition, when hamster sebocytes were treated for 10 days with norepinephrine or epinephrine (0.1-1 mM), the intracellular lipid-accumulation was augmented in a dose-dependent manner. Furthermore, the production of triacylglycerols (TG), a major sebum component, was increased in norepinephrine- and epinephrine-treated hamster sebocytes. Moreover, the expression of perilipin, which is a sebaceous gland differentiation marker, was transcriptionally augmented by norepinephrine and epinephrine in hamster sebocytes. Therefore, these results provide novel evidence that sympathetic nerve neurotransmitters participate in sebaceous gland differentiation due to the upregulation of TG production and perilipin expression.
A role for nuclear-localized keratin 17 in the response of skin tumor keratinocytes to DNA damaging agents RP Hobbs, J Hsu, JT Jacob and PA Coulombe Johns Hopkins University, Baltimore, MD The intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and tumors originating in stratified and pseudostratified epithelia. We have previously reported that the genetic loss of K17 is sufficient to delay tumor onset in two distinct mouse models for spontaneous skin tumorigenesis (Gli2tg, BCC-like; HPV16tg, SCClike). In part, this delay is related to the ability of K17 to localize to the nucleus of tumor-prone keratinocytes, where it associates with chromatin and promotes the expression of several proinflammatory cytokines. Multiple lines of evidence suggest that K17’s pro-tumorigenic role is likely to be significant at an early time of transformation, e.g., the initiation stage. To explore this possibility, we subject human tumor cell lines, parental and CRISPR-generated KRT17 null, to three distinct modalities of DNA damage [1 mg DMBA for 12 hr, 100 mM H2O2 for 30 min, and 2 gy ionizing radiation (IR)] and assessed both the extent of damage and the ensuing repair response. All treatments induced DNA Damage and repair Response (DDR), as assessed by the comet assay along with immunoblotting or immunostaining for gH2AX and phosphorylation of DDR-associated proteins (e.g. p53, ATM, ATR, BRCA1, CHK1, CHK2) in skin tumor keratinocytes. In striking contrast to parental cells, we observed a significant attenuation of DDR in KRT17 null keratinocytes, irrespective of the damaging agent used. Reexpression of K17 into KRT17 null cells completely restored the ability of DNA damaging agents to induce DDR. In contrast, re-expression of a K17 variant lacking a functional nuclear localization signal, or of wildtype K14 or K16, proved incapable of restoring the ability of DNA damage agents to induce DDR. These findings uncover a novel and specific role for nuclear-localized K17 as a coordinator of the DNA damage response in tumor keratinocytes.
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Reactive oxygen species induce Th2 allergic inflammation in the skin by activating PI3K-HIF-a pathway J Choi1, M Piao1, E Park2, S Yun1, J Lee1 and S Lee1 1 Chonnam National University Medical School, Gwangju, Republic of Korea and 2 Hisol co., Ltd, Namwon-si, Republic of Korea Background: Reactive oxygen species (ROS) plays an important role to activate the Th2 allergic inflammation in asthma or rhinitis. Purpose: This study was aimed to evaluate whether ROS is implicated in the pathogenesis of Th2 inflammation in the skin. Compound 48/80-treated HaCaT cells were tested for a new cell model for atopic dermatitis (AD). Materials: In compound 48/80-treated HaCaT cells, ROS production, PI3K-HIF-1a activation, and expression levels of biomarkers for Th2 inflammation in the skin were evaluated. The role of ROS on Th2 inflammation in the skin were verified in the compound 48/80-treated HaCaT cells, which were treated with N-acetyl cysteine (NAC). Results: Compound 48/80 (0.1 mg/ ml) induced the ROS production, PI3K-HIF-1a, as it modulated biomarkers for Th2 inflammation in the skin: upregulation of KLK5 and PAR2 (pro-Th2 inflammatory markers), TSLP and IL-33 (Th2 inflammatory markers), and NGF and CGRP (neurogenic inflammatory markers); and downregulation of filaggrin (barrier function marker). The compound 48/80-induced modulation of those biomarkers could be suppressed by antioxidant treatment (NAC), indicating that ROS plays a crucial role during Th2 inflammation in the skin. The modulatory pattern of biomarkers for Th2 inflammation in the compound 48/80-treated HaCaT cells mimics to the AD skin, which was further confirmed in the house dust mite-treated human keratinocytes as control experiments. Conclusion: Compound 48/80-producing ROS induces Th2 inflammation in the skin through the activation of the PI3K-HIF-1a pathway.
A set of plant essential oils have antioxidant activity in ultraviolet B-irradiated epidermal keratinocytes by upregulating antioxidant and detoxifying enzymes J Choi1, S Yun2, J Lee1, M Piao1, E Park3 and S Lee1 1 Chonnam National University Medical School, Gwangju, Republic of Korea, 2 Chonnam National University, Gwangju, Republic of Korea and 3 Hisol co., Ltd, Namwon-si, Republic of Korea Background: Plant-derived extracts including essential oils have long been used as important ingredients of cosmetics. In recent years, there has been a demand for safe cosmeceuticals with excellent antiaging activity. Aim: This study aimed to develop candidate plant extracts with excellent antioxidant and detoxifying activities in keratinocytes with a view to protecting photoaging of the skin. Methods: The ROS-scavenging activities of candidate plant essential oils were determined in ultraviolet B (UVB)-irradiated normal human epidermal keratinocytes (NHEKs). Their antioxidant and detoxifying activities were determined by measuring enzymatic antioxidants, phase 2 enzymes, and total polyphenol content. Results: In DCF-DA and confocal microscopy studies, the plant essential oils including Lavender oil, Lemongrass oil, Rosemary oil, Chamomile oil, and Peppermint oil showed excellent ROS-scavenging activity in UVB-irradiated NHEKs. In RT-PCR studies, all these oils upregulated the expression of enzymatic antioxidants (CuSOD, GPx II, and Prx I) and phase 2 detoxifying enzymes (HO-1, NQO-1, GSTpi, GSTA4, and GCLM) via Nrf2 in NHEKs. Conclusions: All of the five essential oils can be regarded as good candidate natural products with potential for use as new cosmeceuticals.
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Early atopic dermatitis biological analysis G Bellemere1, G Boyer1, A Moga2, M Fontanie´3, C de Belilovsky1 and C Baudouin1 1 Laboratoires Expanscience, Epernon, France, 2 SYNELVIA, Labege, France and 3 VIBIOSPHEN, Labege, France Background: Pathophysiology of atopic dermatitis (AD) is characterized by decreased levels of Natural Moisturizing Factors (NMFs) and ceramides, with microbiota dysbiosis (colonization by staphylococci species). Little is known about these parameters before the onset of AD and during first AD flare in children with and without a genetic risk of AD. Objectives: The main objective was to quantify NMFs, ceramides, S aureus (SA) and S epidermidis (SE) colonization from birth until first AD flare. The impact of daily applications of emollients is reported. Methods: Swabs were taken on forearms, face and AD lesions in 45 infants for staphylococci, 76 for NMF/ceramides out of 180 newborns involved in a 6-month AD prevention study with daily emollient applications. SA and SE were analyzed for by quantitative PCR and for NMF and ceramides by LC/UV and LC/MS. Results: A depletion of NMFs has been observed on forearms and face of children at genetic risk of developing AD compared to control. Daily application of emollients normalized NMFs. No significant difference in NMFs levels has been observed in AD children compared to non-AD. Ceramide levels were not different in children at risk of developing AD nor in AD children compared to control. nfants were poorly colonized with SA: only 6 children among 45 on forearms and 17 on the face. In contrast, infants were largely colonized with SE: 42 infants on the forearm and 40 on the face. Only 2 AD lesions out of 11 were found positive for SA and 9 for SE. Levels of SA and SE colonization were not modified by emollients. Conclusion: This clinical study showed limited colonization of SA before and during early AD. Specific AD emollients improved hydration and skin barrier function and did not alter skin microbiota.
Collagen XVIII, a key interfacial component of the skin architecture I Bonnet1, S Cadau1, N Berthelemy2, V Bardey3, V Andre-Frei1, M Chavan4, H Zahouani5 and P Rousselle6 1 BASF BCS, Lyon, France, 2 BASF BCS, Essey les Nancy, France, 3 BASF BCS, Pulnoy, France, 4 BASF CORP, Tarrytown, NY, 5 Tribology - System Dynamics Laboratory ECL, Ecully, France and 6 Laboratoire de Biologie Tissulaire et Inge´nierie The´rapeutique, CNRS/UCBL1, Lyon, France Collagen XVIII holds structural properties of both collagen and heparan sulfate proteoglycan. It is expressed ubiquitously in various basement membrane (BM) structures of the body. In the skin, it could be synthetized by keratinocytes, endothelial cells, epithelial cells of the sweat glands and by adipocytes. Collagen XVIII complete role is still unknown but its localization and ultrastructural organization reveal that it is an important component of all BM molecular networks. Understanding its expression modulation with age could provide new strategies to counteract the loss of tissue structure and cohesion observed during ageing. To study chrono and photoageing impact on collagen XVIII maintenance, its protein and gene expression were analyzed in keratinocytes and skin biopsies. To specifically evaluate the collagen XVIII protein, and not its endostatin domain, an affinity-purified rabbit antibody against two collagenic domains was developed for the immunoassay. At the dermo-epidermal junction level, collagen XVIII protein expression was strongly decreased as skin ages (-43.5%) and this decrease could be explained by a decrease of COL18A1 gene expression in keratinocytes exposed or not to UVB. We then observed the stimulating effect of a Khaya senegalensis bark extract from the skin surface to deeper layers on different cells (keratinocytes, endothelial cells, adipocytes). The mechanical properties of reconstructed skins were evaluated with a totally new designed miniaturized non-touch device using air bluster system. The results proved that increasing collagen XVIII synthesis impacts the skin structural properties consequently improving skin elasticity, surface homogeneity, and reducing wrinkles.
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Involvement of catecholamine in the differentiation of sebaceous glands in vitro K Mizuno1, K Okuyama2, N Akimoto1 and T Sato1 1 Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan and 2 Department of Dermatology, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan Sebaceous glands secret sebum onto the skin surface and as such a thin lipid layer is formed as a physiological barrier. Sebaceous gland cells (sebocytes) differentiate to accumulate abundant cytoplasmic lipids. Since sebocyte differentiation has been reported to be regulated by hormones, growth factors, and cytokines, whether the autonomic nervous system is associated with the regulation of sebum production in sebaceous glands remains unclear. In the present study, we examined the effect of norepinephrine and epinephrine on the differentiation of hamster sebocytes. First, immunohistochemical staining using a neuron-specific class III beta-tubulin antibody showed that nerve filaments were detectable adjacent to sebaceous glands in human skin, assuming an association of the peripheral nervous system with sebaceous gland functions. In addition, when hamster sebocytes were treated for 10 days with norepinephrine or epinephrine (0.1-1 mM), the intracellular lipid-accumulation was augmented in a dose-dependent manner. Furthermore, the production of triacylglycerols (TG), a major sebum component, was increased in norepinephrine- and epinephrine-treated hamster sebocytes. Moreover, the expression of perilipin, which is a sebaceous gland differentiation marker, was transcriptionally augmented by norepinephrine and epinephrine in hamster sebocytes. Therefore, these results provide novel evidence that sympathetic nerve neurotransmitters participate in sebaceous gland differentiation due to the upregulation of TG production and perilipin expression.
A role for nuclear-localized keratin 17 in the response of skin tumor keratinocytes to DNA damaging agents RP Hobbs, J Hsu, JT Jacob and PA Coulombe Johns Hopkins University, Baltimore, MD The intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and tumors originating in stratified and pseudostratified epithelia. We have previously reported that the genetic loss of K17 is sufficient to delay tumor onset in two distinct mouse models for spontaneous skin tumorigenesis (Gli2tg, BCC-like; HPV16tg, SCClike). In part, this delay is related to the ability of K17 to localize to the nucleus of tumor-prone keratinocytes, where it associates with chromatin and promotes the expression of several proinflammatory cytokines. Multiple lines of evidence suggest that K17’s pro-tumorigenic role is likely to be significant at an early time of transformation, e.g., the initiation stage. To explore this possibility, we subject human tumor cell lines, parental and CRISPR-generated KRT17 null, to three distinct modalities of DNA damage [1 mg DMBA for 12 hr, 100 mM H2O2 for 30 min, and 2 gy ionizing radiation (IR)] and assessed both the extent of damage and the ensuing repair response. All treatments induced DNA Damage and repair Response (DDR), as assessed by the comet assay along with immunoblotting or immunostaining for gH2AX and phosphorylation of DDR-associated proteins (e.g. p53, ATM, ATR, BRCA1, CHK1, CHK2) in skin tumor keratinocytes. In striking contrast to parental cells, we observed a significant attenuation of DDR in KRT17 null keratinocytes, irrespective of the damaging agent used. Reexpression of K17 into KRT17 null cells completely restored the ability of DNA damaging agents to induce DDR. In contrast, re-expression of a K17 variant lacking a functional nuclear localization signal, or of wildtype K14 or K16, proved incapable of restoring the ability of DNA damage agents to induce DDR. These findings uncover a novel and specific role for nuclear-localized K17 as a coordinator of the DNA damage response in tumor keratinocytes.
www.jidonline.org S77