Identification of potential hub genes associated with atopic dermatitis-like recombinant human epidermal model using integrated transcriptomic and proteomic analysis.

Atopic dermatitis (AD) is a severe inflammatory skin disorder, characterized by elevated levels of proinflammatory cytokines that fuel a vicious cycle of inflammation. While inflammatory recombinant human epidermal (RHE) models relevant to AD have been established, comprehensive understanding remains limited. To illuminate changes and identify potential hub genes involved in AD-related inflammation, RHE models, stimulated by an inflammatory cocktail including polyinosinic-polycytidylic acid, tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4) and interleukin 13 (IL-13), were constructed and examined using tandem mass tags-proteomic coupled with RNA-seq transcriptomic analyses. Principal component analysis (PCA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment were employed for the analysis of related genes and proteins. Protein-protein interaction networks helped identify hub genes, which were further confirmed by qPCR and western blot. We observed high expression of thymic stromal lymphopoietin in the inflammatory RHE. Our study identified 2369 differentially expressed genes and 880 differentially expressed proteins in the cocktail-induced group versus the normal control group. A total of 248 overlapping symbols were enriched in various biological processes and signaling pathways, including cornification envelope, cell-cell junction, calcium ion binding, extracellular matrix receptor, terpenoid backbone biosynthesis, and peroxisome proliferator-activated receptors signaling pathway, among others. Among the 248 overlapping symbols, CytoHubba identified 10 hub molecules, namely signal transducer and activator of transcription 3 (STAT3), integrin subunit beta 1 (ITGB1), filaggrin (FLG), involucrin (IVL), DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58), small proline rich protein 1B (SPRR1B), interferon induced with helicase C domain 1 (IFIH1), desmoglein 1 (DSG1), collagen type XVII alpha 1 chain (COL17A1), and integrin subunit alpha 6 (ITGA6), based on the degree. These integrated results offer valuable insights into the molecular mechanisms of AD and present potential tools for screening cosmetic formulations intended for the treatment of AD.

Unsaturated fatty acid-enriched extract from Hippophae rhamnoides seed reduces skin dryness through up-regulating aquaporins 3 and hyaluronan synthetases 2 expressions.

BACKGROUND: Seed oil of sea buckthorn (SBT) is well known to contain high amount of polyunsaturated fatty acid (PUFA), and PUFA is generally acknowledged to promote skin hydration by reducing trans-epidermal water loss (TEWL). AIMS: The present study is aimed to investigate that skin hydration offered by SBT seed oil is whether through up-regulating AQP3 or HAS2 expression. METHODS: MTT assay was performed to detect cytotoxicity of SBT seed oil, and then, PCR was carried out to explore whether SBT seed oil can increase AQP3 mRNA expression in normal human epidermis keratinocytes (NHEK) cells or not. Immunofluorescence (IF) and Western blot analysis were used to test the protein level expression of AQP3 and HAS2 influenced by SBT seed oil in NHEK cells or in reconstructed epidermis skin model. RESULTS: According to the result of MTT assay, all test concentration of SBT seed oil showed no cytotoxicity to cells. 10 µg/mL SBT seed oil treatment evidently increased AQP3 mRNA level compared to negative control (NC). IF and Western blot analysis results demonstrated that AQP3 and HAS2 protein levels in NHEK cells treated with 10 µg/mL SBT seed oil were much higher than that of NC. Finally, treatment with 10 µg/mL SBT seed oil substantially up-regulated expression of AQP3 and HAS2 protein in reconstructed epidermis skin model in comparison to NC. CONCLUSIONS: In summary, our study first proved that SBT seed oil can improve skin hydration through increasing AQP3 and HAS2 expressions.

Development of a new eczema-like reconstructed skin equivalent for testing child atopic dermatitis-relieving cosmetics.

OBJECTIVE: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, and it has serious effects on children's and families' quality of life. We aimed to screen and evaluate the efficacy of different formulas on relieving of atopic dermatitis clinical symptoms by developing an eczema-like reconstructed human skin equivalent in vitro. METHOD: Some research has reported that thymic stromal lymphopoietin (TSLP) may be a potential therapeutic target for the treatment of AD. We developed an eczema-like in vitro skin equivalent by coculturing the cocktails polyinosinic-polycytidylic acid sodium salt (poly(I:C)) and lipopolysaccharides (LPS). The eczema-like skin equivalent was characterized by overexpression of TSLP and impaired skin barrier function. Three cosmetic formulas with the potential of anti-inflammation and skin barrier promotion were topically applied onto the eczema-like skin equivalent, mimicking in vivo application. The inhibitory effect on TSLP was examined by ELISA. Effects on tissue viability and skin barrier function were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. CONCLUSION: The results show that eczema-like skin equivalent induced by cocktails of poly(I:C) and LPS can mimic the skin characters of the atopic dermatitis. The cocktails can induce high TSLP expression, impaired cell viability, and skin barrier function. The cosmetic formulas with the potential of anti-inflammation and skin barrier promotion were evaluated to be helpful to decrease and relieve the impact of AD with the decreased TSLP and the higher tissue viability than the eczema-like skin equivalent without any cosmetic application. The eczema-like skin equivalent can be used to screen and evaluate formulas on AD relieving.

Treatment with Docosahexaenoic Acid Improves Epidermal Keratinocyte Differentiation and Ameliorates Inflammation in Human Keratinocytes and Reconstructed Human Epidermis Models.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that can cause skin barrier function damage. Although co-incubation with docosahexaenoic acid (DHA) exerts a positive effect on deficient skin models, no studies have investigated the effects of topical treatment with DHA in an inflammatory reconstructed human epidermis (RHE) model. The effects of DHA on monolayer normal human epidermal keratinocyte (NHEK) cells were evaluated using cell counting kit-8 (CCK-8), real-time quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). The skin-related barrier function was assessed using hematoxylin-eosin (HE) staining, Western blot (WB), immunohistofluorescence (IF), and ELISA in normal and inflammatory RHE models. Docosahexaenoic acid upregulated filaggrin and loricrin expression at mRNA levels in addition to suppressing overexpression of tumor necrosis factor-α (TNF-α), interleukin-α (IL-1α), and interleukin-6 (IL-6) stimulated by polyinosinic-polycytidylic acid (poly I:C) plus lipopolysaccharide (LPS) (stimulation cocktail) in cultured NHEK cells. After topical treatment with DHA, cocktail-induced inflammatory characteristics of skin diseases, including barrier morphology, differentiation proteins, and thymic stromal lymphopoietin (TSLP) secretion, were alleviated in RHE models. Supplementation with DHA can improve related barrier function and have anti-inflammation effects in monolayer keratinocytes and RHE models, which indicates that DHA may have potential value for the treatment of inflammation-associated skin diseases.

A Novel Effect of Lipids Extracted from Vernix Caseosa on Regulation of Filaggrin Expression in Human Epidermal Keratinocytes.

BACKGROUND: Vernix caseosa (VC), which is known as a unique human substance, is a biofilm that covers the skin of most human newborns. VC has many biological functions including anti-infective, skin cleansing and skin barrier repair. OBJECTIVE: In the study, we purpose to investigate the novel effect of lipids extracted from VC on the regulation of filaggrin (FLG) expression and anti-inflammation in normal human epidermal keratinocyte (NHEK) cells. METHODS: The lipids were extracted by chloroform/methanol (Folch method) and the major properties of fatty acid methyl esters were determined with gas chromatography-mass spectrometer. The relative viability of NHEK cells was evaluated by Cell Counting Kit 8 assay. The related expression of skin barrier protein was accessed with real-time quantitative polymerase chain reaction, Western blot and Immunofluorescence in NHEK cells with or without poly (I:C). Meanwhile, the changes of thymic stromal lymphopoietin (TSLP) and tumor necrosis factor alpha (TNF-α) are analyzed by enzyme-linked immunosorbent assay. RESULTS: VC lipids mostly contained saturated and branched chains fatty acids. The expression of mRNA and protein of FLG were significantly increased after the supplement with lipid in NHEK cells. Meanwhile, lipids reversed the inhibition of poly (I:C) on FLG. Moreover, lipids suppressed the over secretion of TSLP and TNF-α induced by poly (I:C). CONCLUSION: These results indicate that lipids extracted from VC has positive effects on the expression of FLG and anti-inflammation, suggesting that lipids of VC may be used for a reference for novel therapeutic method in reducing and remedying skin disease like atopic disease.