An Interleukin-4 and Interleukin-13 Induced Atopic Dermatitis Human Skin Equivalent Model by a Skin-On-A-Chip

Currently, the mechanism of progression of atopic dermatitis (AD) is not well understood because there is no physiologically appropriate disease model in terms of disease complexity and multifactoriality. Type 2 inflammation, mediated by interleukin (IL)-4 and IL-13, plays an important role in AD. In this study, full-thickness human skin equivalents consisting of human-derived cells were fabricated from pumpless microfluidic chips and stimulated with IL-4 and IL-13. The morphological properties, gene expression, cytokine secretion and protein expression of the stimulated human skin equivalent (HSE) epidermis were investigated. The results showed epidermal and spongy formations similar to those observed in lesions in AD, and decreased expression of barrier-related filaggrin, loricrin and involucrin genes and proteins induced by IL-4Rα signaling. In addition, we induced the expression of carbonic anhydrase II (CAII), a gene specifically expressed in the epidermis of patients with AD. Thus, AD human skin equivalents can be used to mimic the key pathological features of atopic dermatitis, overcoming the limitations of existing studies that rely solely on mouse models and have been unable to translate their effects to humans. Our results will be useful for future research on the development of therapeutic agents for atopic dermatitis.     

Coenzyme Q10 Efficacy Test for Human Skin Equivalents Using a Pumpless Skin-On-A-Chip System

A human skin equivalent (HSE) composed of the epidermis and dermis is cultured using a pumpless skin-on-a-chip system to supply cultures the desired flow rate using gravity flow without a pump or an external tube connection. Coenzyme Q10 efficacy is tested by adjusting its concentration, as it is known to have anti-aging and antioxidant effects in culture solutions. The relationship between the contraction rate of a full-thickness human skin equivalent and secreted transforming growth factor (TGF) β-1 is analyzed via enzyme-linked immunosorbent assay (ELISA). Following hematoxylin and eosin (H&E) staining, an image of the skin equivalent is analyzed to measure the epidermal layer’s thickness. The cell density and differentiation of the dermis layer are investigated. Gene and protein expression in the dermal and epidermal layers are quantitatively analyzed using quantitative real time polymerase chain reaction (qPCR) and immunohistochemical staining. As the coenzyme Q10 treatment concentration increased, the number of cells per unit area and the thickness of the epidermal layer increased, the expression level of filaggrin increased, and the contraction rate of full-thickness HSE was proportional to the amount of TGF β-1 secreted.     

Revisiting the Roles of Filaggrin in Atopic Dermatitis

The discovery in 2006 that loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and can predispose to atopic dermatitis (AD) galvanized the dermatology research community and shed new light on a skin protein that was first identified in 1981. However, although outstanding work has uncovered several key functions of filaggrin in epidermal homeostasis, a comprehensive understanding of how filaggrin deficiency contributes to AD is still incomplete, including details of the upstream factors that lead to the reduced amounts of filaggrin, regardless of genotype. In this review, we re-evaluate data focusing on the roles of filaggrin in the epidermis, as well as in AD. Filaggrin is important for alignment of keratin intermediate filaments, control of keratinocyte shape, and maintenance of epidermal texture via production of water-retaining molecules. Moreover, filaggrin deficiency leads to cellular abnormalities in keratinocytes and induces subtle epidermal barrier impairment that is sufficient enough to facilitate the ingress of certain exogenous molecules into the epidermis. However, although FLG null mutations regulate skin moisture in non-lesional AD skin, filaggrin deficiency per se does not lead to the neutralization of skin surface pH or to excessive transepidermal water loss in atopic skin. Separating facts from chaff regarding the functions of filaggrin in the epidermis is necessary for the design efficacious therapies to treat dry and atopic skin.     

皮肤细胞培养技术在化妆品功效研究中的应用

皮肤细胞的体外培养技术一直是化妆品相关皮肤领域研究的重要手段。从传统的各种皮肤细胞单层培养以皮肤细胞的三维培养技术等多种培养模型。重建表皮具有分化的特征，与真皮类似物组成简化的人的皮肤。皮肤的三维培养因其具有类似于人体皮肤的特点而成为化妆品研究中的理想模型。这些培养技术的发展对延缓皮肤衰老、美白化妆品配方研制及护发产品开发具有广泛应用价值，更是基础研究必不可少工具。     

Deacetylasperulosidic Acid Ameliorates Pruritus,Immune Imbalance,and Skin Barrier Dysfunction in 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis NC/Nga Mice

The prevalence of atopic dermatitis (AD), a disease characterized by severe pruritus, immune imbalance, and skin barrier dysfunction, is rapidly increasing worldwide. Deacetylasperulosidic acid (DAA) has anti-atopic activity in the three main cell types associated with AD: keratinocytes, mast cells, and eosinophils. Our study investigated the anti-atopic activity of DAA in 2,4-dinitrochlorobenzene-induced NC/Nga mice. DAA alleviated the symptoms of AD, including infiltration of inflammatory cells (mast cells and eosinophils), epidermal thickness, ear thickness, and scratching behavior. Furthermore, DAA reduced serum IgE, histamine, and IgG1/IgG2a ratio and modulated the levels of AD-related cytokines and chemokines, namely interleukin (IL)-1β, IL-4, IL-6, IL-9, IL-10, IL-12, tumor necrosis factor-α, interferon-γ, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, macrophage-derived chemokine, and regulated on activation the normal T cell expressed and secreted in the serum. DAA restored immune balance by regulating gene expression and secretion of Th1-, Th2-, Th9-, Th17-, and Th22-mediated inflammatory factors in the dorsal skin and splenocytes and restored skin barrier function by increasing the expression of the pro-filaggrin gene and barrier-related proteins filaggrin, involucrin, and loricrin. These results suggest DAA as a potential therapeutic agent that can alleviate the symptoms of AD by reducing pruritus, modulating immune imbalance, and restoring skin barrier function.     

高科技让消费者留住青春

综述了抗衰老方面的各种美容手段,介绍了全球各大知名化妆品公司先进的抗衰老美容产品的性能,对化妆品市场进行了分析。     

The Discovery and Function of Filaggrin

Keratohyalin granules were discovered in the mid-19th century in cells that terminally differentiate to form the outer, cornified layer of the epidermis. The first indications of the composition of these structures emerged in the 1960s from a histochemical stain for histidine, followed by radioautographic evidence of a high incidence of histidine incorporation into newly synthesized proteins in cells containing the granules. Research during the next three decades revealed the structure and function of a major protein in these granules, which was initially called the ‘histidine-rich protein’. Steinert and Dale named the protein ‘filaggrin’ in 1981 because of its ability to aggregate keratin intermediate filaments. The human gene for the precursor, ‘profilaggrin,’ was reported in 1991 to encode 10, 11 or 12 nearly identical repeats. Remarkably, the mouse and rat genes encode up to 20 repeats. The lifetime of filaggrin is the time required for keratinocytes in the granular layer to move into the inner cornified layer. During this transition, filaggrin facilitates the collapse of corneocytes into ‘building blocks’ that become an impermeable surface barrier. The subsequent degradation of filaggrin is as remarkable as its synthesis, and the end-products aid in maintaining moisture in the cornified layer. It was apparent that ichthyosis vulgaris and atopic dermatitis were associated with the absence of this protein. McLean’s team in 2006 identified the cause of these diseases by discovering loss-of-function mutations in the profilaggrin gene, which led to dysfunction of the surface barrier. This story illustrates the complexity in maintaining a healthy, functional epidermis.     

化妆品功效评价(Ⅷ)——3D皮肤模型在化妆品功效评价中的应用

概述了3D皮肤模型在化妆品功效评价中的应用,对3D皮肤模型的类型以及基于各种3D皮肤模型的主要体外功效检测方法进行了介绍。3D表皮模型由角质形成细胞构成,用于皮肤屏障、保湿、抗炎、UV防护、经皮吸收等功效性检测;3D黑素皮肤模型由角质形成细胞和黑素细胞构成,主要用于美白功效检测;3D全层皮肤模型由角质形成细胞和成纤维细胞构成,主要用于抗衰功效检测。基于3D皮肤模型,可获得多维度检测结果,实现对化妆品受试物功效的综合评价。     

Synthesis of Kisspeptin-Mimicking Fragments and Investigation of their Skin Anti-Aging Effects

In recent years, a number of active materials have been developed to provide anti-aging benefits for skin and, among them, peptides have been considered the most promising candidate due to their remarkable and long-lasting anti-wrinkle activity. Recent studies have begun to elucidate the relationship between the secretion of emotion-related hormones and skin aging. Kisspeptin, a neuropeptide encoded by the KISS1 gene, has gained attention in reproductive endocrinology since it stimulates the reproductive axis in the hypothalamus; however, the effects of Kisspeptin on skin have not been studied yet. In this study, we synthesized Kisspeptin-10 and Kisspeptin-E, which are biologically active fragments, to mimic the action of Kisspeptin. Next, we demonstrated the anti-aging effects of the Kisspeptin-mimicking fragments using UV-induced skin aging models, such as UV-induced human dermal fibroblasts (Hs68) and human skin explants. Kisspeptin-E suppressed UV-induced 11 beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) stimulation leading to a regulation of skin aging related genes, including type I procollagen, matrix metalloproteinases-1 (MMP-1), interleukin-6 (IL-6), and IL-8, and rescued the skin integrity. Taken together, these results suggest that Kisspeptin-E could be useful to improve UV-induced skin aging by modulating expression of stress related genes, such as 11β-HSD1.     

A Connecting Link between Hyaluronan Synthase 3-Mediated Hyaluronan Production and Epidermal Function

Hyaluronan (HA), an essential component of the extracellular matrix of the skin, is synthesized by HA synthases (HAS1-3). To date, epidermal HA has been considered a major player in regulating cell proliferation and differentiation. However, a previous study reported that depletion of epidermal HA by Streptomyces hyaluronidase (St-HAase) has no influence on epidermal structure and function. In the present study, to further explore roles of epidermal HA, we examined effects of siRNA-mediated knockdown of HAS3, as well as conventional HA-depletion methods using St-HAase and 4-methylumbelliferone (4MU), on epidermal turnover and architecture in reconstructed skin or epidermal equivalents. Consistent with previous findings, HA depletion by St-HAase did not have a substantial influence on the epidermal architecture and turnover in skin equivalents. 4MU treatment resulted in reduced keratinocyte proliferation and epidermal thinning but did not seem to substantially decrease the abundance of extracellular HA. In contrast, siRNA-mediated knockdown of HAS3 in epidermal equivalents resulted in a significant reduction in epidermal HA content and thickness, accompanied by decreased keratinocyte proliferation and differentiation. These results suggest that HAS3-mediated HA production, rather than extracellularly deposited HA, may play a role in keratinocyte proliferation and differentiation, at least in the developing epidermis in reconstructed epidermal equivalents.     

基因芯片技术在化妆品中的应用

基因芯片技术具有对大量基因进行平行和快速分析检验的优势,广泛应用于新基因的发现、基因诊断以及药物筛选等领域。基因芯片技术越来越受到化妆品研发人员的关注,已成功应用于揭示功效成分作用靶点、人皮肤基因表达水平变化等研究。总结了近年来基因芯片技术在化妆品新产品开发、功效评价和分析检测3个方面的研究现状和应用实例。     

CYLD Inhibits the Development of Skin Squamous Cell Tumors in Immunocompetent Mice

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.     

聚天冬氨酸钠在护肤品中的应用

利用皮肤水分测试仪和皮肤水分流失测试仪,考察了添加低、高分子量及不同质量分数聚天冬氨酸钠(PASP)的保湿化妆品作用于人体皮肤后的水分含量和经表皮水分散失情况。结果表明,高分子量PASP对皮肤的保湿效果较好、锁水效果显著,高分子量PASP在质量分数为0.8%时,使皮肤水分含量的增长率达到0.5%以上,对皮肤的保湿效果与透明质酸相当。使用皮肤弹性测试仪对新鲜猪皮进行了体外透皮实验,测试了使用PASP前后猪皮弹性状况的变化,表明PASP具有提高皮肤弹性的效果。PASP代替目前广泛使用的透明质酸作为化妆品中的保湿剂具有良好的发展前景。     

化妆品赋形剂对皮肤的临床效果

运用赋形剂配制和使用效果的实例，通过屏障保护和恢复上的一些关键问题，说明了化妆品赋形剂在皮肤保护和治疗中的不同功效。讨论了羊毛脂、矿脂和生理脂质在化妆品赋形剂里的重要作用。指出为治疗不同的皮肤病，选择适当的化妆品赋形剂的重要性。论述了有最佳脂质含量的化妆品赋形剂对特应性皮炎的明显疗效。     

FAK Shutdown: Consequences on Epithelial Morphogenesis and Biomarker Expression Involving an Innovative Biomaterial for Tissue Regeneration

By employing an innovative biohybrid membrane, the present study aimed at elucidating the mechanistic role of the focal adhesion kinase (FAK) in epithelial morphogenesis in vitro over 4, 7, and 10 days. The consequences of siRNA-mediated FAK knockdown on epithelial morphogenesis were monitored by quantifying cell layers and detecting the expression of biomarkers of epithelial differentiation and homeostasis. Histologic examination of FAK-depleted samples showed a significant increase in cell layers resembling epithelial hyperplasia. Semiquantitative fluorescence imaging (SQFI) revealed tissue homeostatic disturbances by significantly increased involucrin expression over time, persistence of yes-associated protein (YAP) and an increase of keratin (K) 1 at day 4. The dysbalanced involucrin pattern was underscored by ROCK-II^Ser1366 activity at day 7 and 10. SQFI data were confirmed by quantitative PCR and Western blot analysis, thereby corroborating the FAK shutdown-related expression changes. The artificial FAK shutdown was also associated with a significantly higher expression of filaggrin at day 10, sustained keratinocyte proliferation, and the dysregulated expression of K19 and vimentin. These siRNA-induced consequences indicate the mechanistic role of FAK in epithelial morphogenesis by simultaneously considering prospective biomaterial-based epithelial regenerative approaches.     

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii^+/−, wild type: WT, transgenic Flii overexpressing: Flii^Tg/Tg) using Western blot and immunohistochemistry. Flii^+/− embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. Flii^Tg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.     

Ex Vivo Live Full-Thickness Porcine Skin Model as a Versatile In Vitro Testing Method for Skin Barrier Research

Since the European Union (EU) announced their animal testing ban in 2013, all animal experiments related to cosmetics have been prohibited, creating a demand for alternatives to animal experiments for skin studies. Here, we investigated whether an ex vivo live porcine skin model can be employed to study the safety and skin barrier-improving effects of hydroxyacids widely used in cosmetics for keratolytic peels. Glycolic acid (1–10%), salicylic acid (0.2–2%), and lactobionic acid (1.2–12%) were used as representative substances for α-hydroxyacid (AHA), β-hydroxyacid (BHA), and polyhydroxyacid (PHA), respectively. When hydroxyacids were applied at high concentrations on the porcine skin every other day for 6 days, tissue viability was reduced to 50–80%, suggesting that the toxicity of cosmetic ingredients can be evaluated with this model. Based on tissue viability, the treatment scheme was changed to a single exposure for 20 min. The protective effects of a single exposure of hydroxyacids on skin barrier function were evaluated by examining rhodamine permeability and epidermal structural components of barrier function using immunohistochemistry (IHC) and immunofluorescence (IF) staining. Lactobionic acid (PHAs) improved skin barrier function most compared to other AHAs and BHAs. Most importantly, trans-epidermal water loss (TEWL), an important functional marker of skin barrier function, could be measured with this model, which confirmed the significant skin barrier-protective effects of PHAs. Collectively, we demonstrated that the ex vivo live full-thickness porcine skin model can be an excellent alternative to animal experiments for skin studies on the safety and efficacy of cosmetic ingredients.     

miR-155 Contributes to Normal Keratinocyte Differentiation and Is Upregulated in the Epidermis of Psoriatic Skin Lesions

The role of microRNAs (miRNAs) during keratinocyte (KC) differentiation and in skin diseases with epidermal phenotypes has attracted strong interest over the past few years. However, combined mRNA and miRNA expression analyses to elucidate the intricate mRNA–miRNA networks of KCs at different stages of differentiation have not been performed yet. In the present study, we investigated the dynamics of miRNA and mRNA expression during KC differentiation in vitro and in normal and psoriatic epidermis. While we identified comparable numbers of up- and downregulated mRNAs (49% and 51%, respectively), miRNAs were predominantly upregulated (76% vs 24%) during KC differentiation. Further bioinformatics analyses suggested an important inhibitory role for miR-155 in KC differentiation, as it was repressed during KC differentiation in normal skin but strongly upregulated in the epidermis of psoriatic skin lesions. Mimicking the inflammatory milieu of psoriatic skin in vitro, we could show that the pro-inflammatory cytokines IL17, IL1β and INFγ synergistically upregulated miR-155 expression in KCs. Forced over-expression of miR-155 in human in vitro skin models specifically reduced the expression of loricrin (LOR) in KCs, indicating that miR-155 interferes with the establishment of a normal epidermal barrier. Together, our data indicate that downregulation of miR-155 during KC differentiation is a crucial step for epidermal barrier formation. Furthermore, its strong upregulation in psoriatic lesions suggests a contributing role of miR-155 in the altered keratinocyte differentiation observed in psoriasis. Therefore, miR-155 represents as a potential target for treating psoriatic skin lesions.