Docosahexaenoic Acid Modulates Paracellular Absorption of Testosterone and Claudin-1 Expression in a Tissue-Engineered Skin Model

Healthy skin moLEdels produced by tissue-engineering often present a suboptimal skin barrier function as compared with normal human skin. Moreover, skin substitutes reconstructed according to the self-assembly method were found to be deficient in polyunsaturated fatty acids (PUFAs). Therefore, in this study, we investigated the effects of a supplementation of the culture media with docosahexaenoic acid (DHA) on the barrier function of skin substitutes. To this end, 10 μM DHA-supplemented skin substitutes were produced (n = 3), analyzed, and compared with controls (substitutes without supplementation). A Franz cell diffusion system, followed by ultra-performance liquid chromatography, was used to perform a skin permeability to testosterone assay. We then used gas chromatography to quantify the PUFAs found in the epidermal phospholipid fraction of the skin substitutes, which showed successful DHA incorporation. The permeability to testosterone was decreased following DHA supplementation and the lipid profile was improved. Differences in the expression of the tight junction (TJ) proteins claudin-1, claudin-4, occludin, and TJ protein-1 were observed, principally a significant increase in claudin-1 expression, which was furthermore confirmed by Western blot analyses. In conclusion, these results confirm that the DHA supplementation of cell culture media modulates different aspects of skin barrier function in vitro and reflects the importance of n-3 PUFAs regarding the lipid metabolism in keratinocytes.     

N-Palmitoyl Serinol Stimulates Ceramide Production through a CB1-Dependent Mechanism in In Vitro Model of Skin Inflammation

Ceramides, a class of sphingolipids containing a backbone of sphingoid base, are the most important and effective structural component for the formation of the epidermal permeability barrier. While ceramides comprise approximately 50% of the epidermal lipid content by mass, the content is substantially decreased in certain inflammatory skin diseases, such as atopic dermatitis (AD), causing improper barrier function. It is widely accepted that the endocannabinoid system (ECS) can modulate a number of biological responses in the central nerve system, prior studies revealed that activation of endocannabinoid receptor CB1, a key component of ECS, triggers the generation of ceramides that mediate neuronal cell fate. However, as the impact of ECS on the production of epidermal ceramide has not been studied, we here investigated whether the ECS stimulates the generation of epidermal ceramides in an IL-4-treated in vitro model of skin inflammation using N-palmitoyl serinol (PS), an analog of the endocannabinoid N-palmitoyl ethanolamine. Accordingly, an IL-4-mediated decrease in cellular ceramide levels was significantly stimulated in human epidermal keratinocytes (KC) following PS treatment through both de novo ceramide synthesis- and sphingomyelin hydrolysis-pathways. Importantly, PS selectively increases ceramides with long-chain fatty acids (FAs) (C22–C24), which mainly account for the formation of the epidermal barrier, through activation of ceramide synthase (CerS) 2 and Cer3 in IL-4-mediated inflamed KC. Furthermore, blockade of cannabinoid receptor CB1 activation by AM-251 failed to stimulate the production of total ceramide as well as long-chain ceramides in response to PS. These studies demonstrate that an analog of endocannabinoid, PS, stimulates the generation of specific ceramide species as well as the total amount of ceramides via the endocannabinoid receptor CB1-dependent mechanism, thereby resulting in the enhancement of epidermal permeability barrier function.     

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.     

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。     

Cigarette Smoke Affects ABCAl Expression via Liver X Receptor Nuclear Translocation in Human Keratinocytes

Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, the stratum corneum (SC), is formed by corneocytes embedded in a lipid matrix (cholesterol, ceramide and fatty acids). Therefore, the regulation of lipids and, in particular, of cholesterol homeostasis in the skin is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a key role in maintaining cellular cholesterol levels. Among the many factors that have been associated with skin diseases, the environmental stressor cigarette smoke has been recently studied. In the present study, we demonstrate that ABCA1 expression in human cells (HaCaT) was increased (both mRNA and protein levels) after CS exposure. This effect was mediated by the inhibition of NFkB (aldehydes adducts formation) that allows the translocation of liver X receptor (LXR). These findings suggest that passive smoking may play a role in skin cholesterol levels and thus affect cutaneous tissues functions.     

Rosmarinic Acid,as an NHE1 Activator,Decreases Skin Surface pH and Improves the Skin Barrier Function

Stratum corneum (SC) pH regulates skin barrier functions and elevated SC pH is an important factor in various inflammatory skin diseases. Acidic topical formulas have emerged as treatments for impaired skin barriers. Sodium proton exchanger 1 (NHE1) is an important factor in SC acidification. We investigated whether topical applications containing an NHE1 activator could improve skin barrier functions. We screened plant extracts to identify NHE1 activators in vitro and found Melissa officinalis leaf extract. Rosmarinic acid, a component of Melissa officinalis leaf extract, significantly increased NHE1 mRNA expression levels and NHE1 production. Immunofluorescence staining of NHE1 in 3D-cultured skin revealed greater upregulation of NHE1 expression by NHE1 activator cream, compared to vehicle cream. Epidermal lipid analysis revealed that the ceramide level was significantly higher upon application of the NHE1 activator cream on 3D-cultured skin, compared to application of a vehicle cream. In a clinical study of 50–60-year-old adult females (n = 21), application of the NHE1 activator-containing cream significantly improved skin barrier functions by reducing skin surface pH and transepidermal water loss and increasing skin hydration, compared to patients who applied vehicle cream and those receiving no treatment. Thus, creams containing NHE1 activators, such as rosmarinic acid, could help maintain or recover skin barrier functions.     

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.     

Increased Expression of 11β-Hydroxysteroid Dehydrogenase Type 1 Contributes to Epidermal Permeability Barrier Dysfunction in Aged Skin

Inactive cortisone is converted into active cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Excessive levels of active glucocorticoids could deteriorate skin barrier function; barrier impairment is also observed in aged skin. In this study, we aimed to determine whether permeability barrier impairment in the aged skin could be related to increased 11β-HSD1 expression. Aged humans (n = 10) showed increased cortisol in the stratum corneum (SC) and oral epithelium, compared to young subjects (n = 10). 11β-HSD1 expression (as assessed via immunohistochemical staining) was higher in the aged murine skin. Aged hairless mice (56-week-old, n = 5) manifested greater transepidermal water loss, lower SC hydration, and higher levels of serum inflammatory cytokines than the young mice (8-week-old, n = 5). Aged 11β-HSD1 knockout mice (n = 11), 11β-HSD1 inhibitor (INHI)-treated aged wild type (WT) mice (n = 5) and young WT mice (n = 10) exhibited reduced SC corticosterone level. Corneodesmosome density was low in WT aged mice (n = 5), but high in aged 11β-HSD1 knockout and aged INHI-treated WT mice. Aged mice exhibited lower SC lipid levels; this effect was reversed by INHI treatment. Therefore, upregulation of 11β-HSD1 in the aged skin increases the active-glucocorticoid levels; this suppresses SC lipid biosynthesis, leading to impaired epidermal permeability barrier.     

Atopic Dermatitis: The Fate of the Fat

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease in which dry and itchy skin may develop into skin lesions. AD has a strong genetic component, as children from parents with AD have a two-fold increased chance of developing the disease. Genetic risk loci and epigenetic modifications reported in AD mainly locate to genes involved in the immune response and epidermal barrier function. However, AD pathogenesis cannot be fully explained by (epi)genetic factors since environmental triggers such as stress, pollution, microbiota, climate, and allergens also play a crucial role. Alterations of the epidermal barrier in AD, observed at all stages of the disease and which precede the development of overt skin inflammation, manifest as: dry skin; epidermal ultrastructural abnormalities, notably anomalies of the lamellar body cargo system; and abnormal epidermal lipid composition, including shorter fatty acid moieties in several lipid classes, such as ceramides and free fatty acids. Thus, a compelling question is whether AD is primarily a lipid disorder evolving into a chronic inflammatory disease due to genetic susceptibility loci in immunogenic genes. In this review, we focus on lipid abnormalities observed in the epidermis and blood of AD patients and evaluate their primary role in eliciting an inflammatory response.     

Physicochemical analysis of liposome membranes consisting of model lipids in the stratum corneum

Lamellar lipid layers in the stratum corneum (SC), the outermost layer of the skin, act as a primary permeability barrier to protect the body. The roles of SC lipid composition and membrane structure in skin barrier function have been extensively investigated using ex-vivo SC samples and reconstructed SC lipids in the form of multi-lamellar lipids or liposomes. The primary lipids, especially ceramide, have been found to be highly important. Atopic dermatitis (AD) is a well-known chronic inflammatory skin disease with immunologic and epidermal abnormalities of the permeability barrier; therefore, a comparison of SC lipids in AD skin with those in normal skin is a promising method to explore the mechanisms of skin barrier function. Here, we focused on the effect of sphingoids (ceramide metabolites and a minor component of the SC lipids) and their content/species on skin barrier function. A significant difference in the leakage ratio was observed between model SC lipid liposomes with a different sphingolipid ratio (sphingosine/sphinganine), with a value of 5.43 for normal skin vs. 14.3 for AD skin. This result shows a good concordance with AD mouse experiments. Therefore, an alteration in the composition of minor SC lipids resulting from a ceramide metabolic abnormality can affect the membrane integrity (i.e., skin barrier function). Small angle X-ray scattering (SAXS) measurements revealed no distinct differences in the SAXS pattern between the 3 models, with all models forming a rigid membrane (i.e., a nearly hydrated solid). According to increasing the temperature, the peaks indicated that the lamellar structures decreased in all models and that the lateral packing of lipids decreased, which suggested annealing or melting of the gel to a liquid crystal, although no distinct phase transition was observed through fluorescence anisotropy measurements. Hence, we assume that the altered sphingoid composition triggers local membrane structural changes (i.e., formation of domains or clusters).     

hiPSC-Derived Epidermal Keratinocytes from Ichthyosis Patients Show Altered Expression of Cornification Markers

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.     

An improved flexible tolerance method for solving nonlinear constrained optimization problems:Application in mass integration

This paper proposes the use of the flexible tolerance method(FTM) modified with adaptive Nelder–Mead parameters and barrier to solve constrained optimization problems. The problems used to analyze the performance of the methods were taken from G-Suite functions, and the methods with the best performance were applied in mass integration problems. Four methods were proposed:(1) flexible tolerance method(FTM) using adaptive parameters(FTMA),(2) flexible tolerance method with scaling(FTMS) and with adaptive parameters(FTMAS),(3) FTMS including the barrier modification(MFTMS) and(4) MFTMS hybridized with PSO(MFTMS-PSO). The success rates of these methods were 100%(MFTMS), 85%(MFTMS-PSO), 40%(FTMAS) and 30%(FTMA).Numerical experiments indicated that the MFTMS could efficiently and reliably improve the accuracy of global optima. In mass integration, the method was able, from current process situation, to reach the optimum process configuration that includes integration issues, which was not possible using FTM in its standard formulation. The hybridization of FTMS with PSO(without barrier), FTMS-PSO, was also able to solve mass integration problems efficiently.     

Proteoglycan Combined with Hyaluronic Acid and Hydrolyzed Collagen Restores the Skin Barrier in Mild Atopic Dermatitis and Dry,Eczema-Prone Skin: A Pilot Study

Dry and eczema-prone skin conditions such as atopic dermatitis and xerotic eczema primarily indicate an impaired skin barrier function, which leads to chronic pruritus. Here, we investigated the effects of a novel emollient containing H.ECM^TM liposome, which contains a soluble proteoglycan in combination with hydrolyzed collagen and hyaluronic acid. A prospective, single-arm study was conducted on 25 participants with mild atopic dermatitis or dry skin to assess the hydration and anti-inflammatory effect of the novel emollient applied daily over four weeks. All efficacy parameters, including itching severity, transepidermal water loss, and skin hydration, improved significantly after four weeks. The in vitro and ex vivo studies confirmed the restoration of the skin’s barrier function. The study revealed the clinical and laboratory efficacy of H.ECM^TM liposome in reducing itching and improving the skin’s barrier integrity. Thus, the use of H.ECM^TM liposome can be considered a therapeutic option for dry and eczema-prone skin.     

Effect of α-Lipoic Acid on the Development of Human Skin Equivalents Using a Pumpless Skin-on-a-Chip Model

Owing to the prohibition of cosmetic animal testing, various attempts have recently been made using skin-on-a-chip (SOC) technology as a replacement for animal testing. Previously, we reported the development of a pumpless SOC capable of drug testing with a simple drive using the principle that the medium flows along the channel by gravity when the chip is tilted using a microfluidic channel. In this study, using pumpless SOC, instead of drug testing at the single-cell level, we evaluated the efficacy of α-lipoic acid (ALA), which is known as an anti-aging substance in skin equivalents, for skin tissue and epidermal structure formation. The expression of proteins and changes in genotyping were compared and evaluated. Hematoxylin and eosin staining for histological analysis showed a difference in the activity of fibroblasts in the dermis layer with respect to the presence or absence of ALA. We observed that the epidermis layer became increasingly prominent as the culture period was extended by treatment with 10 μM ALA. The expression of epidermal structural proteins of filaggrin, involucrin, keratin 10, and collagen IV increased because of the effect of ALA. Changes in the epidermis layer were noticeable after the ALA treatment. As a result of aging, damage to the skin-barrier function and structural integrity is reduced, indicating that ALA has an anti-aging effect. We performed a gene analysis of filaggrin, involucrin, keratin 10, integrin, and collagen I genes in ALA-treated human skin equivalents, which indicated an increase in filaggrin gene expression after ALA treatment. These results indicate that pumpless SOC can be used as an in vitro skin model similar to human skin, protein and gene expression can be analyzed, and it can be used for functional drug tests of cosmetic materials in the future. This technology is expected to contribute to the development of skin disease models.     

Elastic and Ultradeformable Liposomes for Transdermal Delivery of Active Pharmaceutical Ingredients (APIs)

Administration of active pharmaceutical ingredients (APIs) through the skin, by means of topical drug delivery systems, is an advanced therapeutic approach. As the skin is the largest organ of the human body, primarily acting as a natural protective barrier against permeation of xenobiotics, specific strategies to overcome this barrier are needed. Liposomes are nanometric-sized delivery systems composed of phospholipids, which are key components of cell membranes, making liposomes well tolerated and devoid of toxicity. As their lipid compositions are similar to those of the skin, liposomes are used as topical, dermal, and transdermal delivery systems. However, permeation of the first generation of liposomes through the skin posed some limitations; thus, a second generation of liposomes has emerged, overcoming permeability problems. Various mechanisms of permeation/penetration of elastic/ultra-deformable liposomes into the skin have been proposed; however, debate continues on their extent/mechanisms of permeation/penetration. In vivo bioavailability of an API administered in the form of ultra-deformable liposomes is similar to the bioavailability achieved when the same API is administered in the form of a solution by subcutaneous or epi-cutaneous injection, which demonstrates their applicability in transdermal drug delivery.     

Transcriptional Differences in Lipid-Metabolizing Enzymes in Murine Sebocytes Derived from Sebaceous Glands of the Skin and Preputial Glands

Sebaceous glands are adnexal structures, which critically contribute to skin homeostasis and the establishment of a functional epidermal barrier. Sebocytes, the main cell population found within the sebaceous glands, are highly specialized lipid-producing cells. Sebaceous gland-resembling tissue structures are also found in male rodents in the form of preputial glands. Similar to sebaceous glands, they are composed of lipid-specialized sebocytes. Due to a lack of adequate organ culture models for skin sebaceous glands and the fact that preputial glands are much larger and easier to handle, previous studies used preputial glands as a model for skin sebaceous glands. Here, we compared both types of sebocytes, using a single-cell RNA sequencing approach, to unravel potential similarities and differences between the two sebocyte populations. In spite of common gene expression patterns due to general lipid-producing properties, we found significant differences in the expression levels of genes encoding enzymes involved in the biogenesis of specialized lipid classes. Specifically, genes critically involved in the mevalonate pathway, including squalene synthase, as well as the sphingolipid salvage pathway, such as ceramide synthase, (acid) sphingomyelinase or acid and alkaline ceramidases, were significantly less expressed by preputial gland sebocytes. Together, our data revealed tissue-specific sebocyte populations, indicating major developmental, functional as well as biosynthetic differences between both glands. The use of preputial glands as a surrogate model to study skin sebaceous glands is therefore limited, and major differences between both glands need to be carefully considered before planning an experiment.     

保湿四部曲——从皮肤生理学到解决方案

综述了与保湿相关的皮肤生理学理论,包括皮肤各个层面的含水量及皮肤各层对保湿起关键作用的结构和组成。同时提出了针对不同皮肤保湿途径的解决方案,包括通过外源性补充角质层屏障的成分、结构或功能的快速保湿及针对角质屏障形成过程的靶点保湿机理。     

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。