Non-invasive Microneedle Application Increases Ceramide and Natural Moisturizing Factors in a Reconstructed Human Skin Model.

Recently, microneedling as a cosmetic product has attracted attention as one way to improve skin barrier function and moisturizing function to reduce wrinkle formation. However, some cases of erythema and edema have been reported as side effects. In order to develop safer microneedle cosmetics, we investigated whether microneedles can improve skin barrier function and moisturizing function even when applied in a non-invasive manner that does not penetrate the stratum corneum. We established the condition of non-penetrating microneedle application on reconstructed human full-thickness skin models and examined the effect on the skin models when microneedles were applied under this condition. Microneedle application increased the gene expression of serine palmitoyltransferase long chain base subunit (SPTLC) 3, filaggrin, and transglutaminase 1. The amount of ceramide produced by SPTLC was also increased by microneedle application. Gene expression of filaggrin-degrading enzymes and the amount of free amino acids, a product of filaggrin degradation, were also increased by microneedling. These results suggest that non-invasive microneedle application can improve skin barrier function and moisturizing function by increasing the amount of ceramide and natural moisturizing factors.

Enhancement of Cornified Envelope-Related Gene and Protein Expression by Carba Cyclic Phosphatidic Acid in Normal Human Epidermal Keratinocytes.

The aim of this study was to examine the effects of carba cyclic phosphatidic acid (ccPA) on cornified envelope (CE) formation and keratinocyte differentiation. ccPA-treated keratinocytes showed higher mRNA and protein levels of differentiation markers and CE components than untreated cells. These results suggest that ccPA could serve as therapeutic targets for treating skin barrier dysfunction because of their roles in upregulating genes and proteins associated with CE formation and keratinocyte differentiation.

Effect of Lactic Fermentation Products on Human Epidermal Cell Differentiation, Ceramide Content, and Amino Acid Production.

INTRODUCTION: Lactic fermentation products (LFPs) are thought to affect "good" bacteria in the gut. We previously reported that oral administration of LFPs has beneficial therapeutic effects in a mouse model of atopic dermatitis. However, it is unclear how LFPs affect human epidermal cell differentiation, ceramide (Cer), and amino acid production. OBJECTIVE: The aim of this study was to determine the effects of LFPs on epidermal cell differentiation, by assessing amino acid and Cer production. METHODS: A 3-dimensional cultured human epidermis model and normal human epidermal keratinocytes were used. Cytotoxicity tests were performed using alamar Blue. Transepidermal water loss (TEWL) was used as an index to assess barrier function. Keratin 1 (K1), keratin 5 (K5), keratin 10 (K10), involucrin (INV), calpain 1, and transglutaminase (TGase) (markers of differentiation) and profilaggrin (proFLG) and bleomycin hydrolase (amino acid synthesis-related genes) expression levels were quantified by RT-PCR. In addition, TGase protein levels were measured by Western blotting. The intercellular lipid content of the stratum corneum was measured by high-performance thin-layer chromatography. Amino acids were quantified using an amino acid analyzer. Finally, bound water content in the stratum corneum was measured by differential scanning calorimetry. RESULTS: Cell viability did not change, but TEWL was significantly decreased in the cells treated with LFPs compared with the control cells. Treatment with LFPs significantly increased expression of the late-differentiation markers INV and TGase at the RNA level. Furthermore, TGase protein expression was significantly increased by treatment with LFPs. Treating a 3-dimensional cultured epidermis model with LFPs significantly increased the intercellular lipid content of the stratum corneum and production of the amino acid arginine (Arg). The amount of bound water in the stratum corneum was increased significantly in the LFP application group. CONCLUSION: Treatment with LFPs promotes human epidermal cell differentiation and increases the intercellular content of the free fatty acid, Chol, Cer [NS], Cer [AS], and Cer [AP]. This may result in improved skin barrier function. The increased amount of Arg observed in keratinocytes may help improve water retention.

[Pathogenic and Compensatory Mechanisms in Epidermis of Sphingomyelin Synthase 2-Deficient Mice].

Sphingomyelin (SM) is a constituent of cellular membranes, while ceramides (Cer) produced from SM on plasma membranes serve as a lipid mediator that regulates cell proliferation, differentiation, and apoptosis. In the skin, SM also is a precursor of Cer, an important constituent of epidermal permeability barrier. We investigated the role of epidermal SM synthase (SMS)2, an isoform of SMS, which modulates SM and Cer levels on plasma membranes. Although SMS2-knockout (SMS2-KO) mice were not neonatal lethal, an ichthyotic phenotype with epidermal hyperplasia and hyperkeratosis was evident at birth, which persisted until 2 weeks of age. These mice showed abnormal lamellar body morphology and secretion, and abnormal extracellular lamellar membranes in the stratum corneum. These abnormalities were no longer evident by 4 weeks of age in SMS2-KO mice. Our study suggests that (1) exposure to a dry terrestrial environment initiates compensatory responses, thereby normalizing epidermal ichthyotic abnormalities and (2) that a nonlethal gene abnormality can cause an ichthyotic skin phenotype.

Epidermal permeability barrier function and sphingolipid content in the skin of sphingomyelin synthase 2 deficient mice.

Sphingomyelin synthase (SMS) is an enzyme that generates sphingomyelin (SM) from ceramide (CER) and phosphatidylcholine. SM in the epidermis is a precursor of CER, an important lipid for epidermal permeability barrier function. However, the physiological role of SMS in skin is unclear. To uncover the function of SMS in skin, we investigated sphingolipid metabolism enzyme activity in skin, SM content in the epidermis, CER content in the stratum corneum (SC) and transepidermal water loss (TEWL) as an indicator of barrier function in SMS2-knockout (KO) mice. The activities of sphingolipid metabolism enzymes in skin homogenates were measured using a fluorescently labelled substrate. Enzymatic reaction products were detected by high-performance liquid chromatography (HPLC). Lipids in the epidermis or SC were extracted and quantified by high-performance thin layer chromatography (HPTLC). TEWL was measured using a Tewameter TM300. In SMS2-KO mice, SMS activity in skin homogenates, epidermal SM content and SC CER content were significantly decreased relative to wild-type (WT) mice. The TEWL of SMS2-KO mice was significantly increased compared to WT mice. Our data indicate that SMS2 generates SM in the epidermis and contributes to epidermal permeability barrier function and will support understanding of SM-related metabolic disorders.

Upregulation of gene expression levels of ceramide metabolic enzymes after application of sphingomyelin-based liposomes to a three-dimensional cultured human epidermis model.

BACKGROUND/AIMS: We have previously reported that the application of sphingomyelin-based liposomes (SM-L) to a three-dimensional cultured skin model increase the content of ceramides NS, NP, AS and AP. However, the mechanism responsible for these increased ceramide levels was not identified. METHODS: SM-L and sphingomyelinase (SMase) were combined and incubated at 37 °C for 24 h. SM-L were also applied to three-dimensional cultured skin for 24 h and quantification of SMase and ß-glucocerebrosidase (ß-GCase) mRNA expression levels performed using real-time PCR. Additionally, three dimensional cultured skin was incubated with SM-L and the ß-GCase inhibitor conduritol B epoxide (CBE) and the ceramide content determined by high performance thin layer chromatography. RESULTS: We observed generation of ceramide NS after reaction of SM-L and SMase. However, the other ceramide classes were not detected. Notably, SMase and ß-GCase mRNA expression levels were significantly increased in cells of the skin model following application of SM-L. The levels of ceramides NS, NP, AS and AP were decreased by treatment with CBE. However, only ceramide NS was significantly increased by treatment with CBE and SM-L in combination. CONCLUSION: These findings indicate that application of SM-L to cultured skin upregulates the expression of SMase and ß-GCase and increases ceramide content.

The Effect of Glycation on Epidermal Lipid Content, Its Metabolism and Change in Barrier Function.

BACKGROUND/AIMS: Advanced glycation end products, which are linked to both aging and hyperglycemia, cause marked functional and structural alterations in human skin. Though it is well known that the metabolism of glucose is closely associated with that of fatty acid (FA), sharing the same energy-yielding reaction pathways as glucose, its effect on the epidermis has been unclear so far. METHODS: Content of ceramides, cholesterol and FA in a reconstructed epidermal model glycated by glyoxal was analyzed by high-performance thin-layer chromatography. FA species extracted from HaCaT keratinocytes was determined by gas chromatography/mass spectrometry. Regulation of FA synthesis was analyzed by real-time PCR. For physiological analysis, excised mouse skin was glycated using a vertical diffusion cell and used for the evaluation of barrier function by transepidermal water loss measurement and observation of penetration of sodium fluorescein. RESULTS: Saturated FA content was significantly increased in glycated epidermis, and glycation upregulated mRNA expression of FA elongases 2 and 3 and FA synthase in HaCaT cells. Further, both inside-out and outside-in barriers were disrupted in glycated excised skin. CONCLUSION: Biological and physical change in the epidermis, especially upregulation of FA synthesis by glycation, contributed to barrier disruption, and inhibiting glycation may offer an effective treatment option for aged or glycated skin.

Permeation of Hydrophilic Molecules across Glycated Skin Is Differentially Regulated by the Stratum Corneum and Epidermis-Dermis.

The effects of glycation on skin permeation and accumulation of compounds were evaluated using an in vitro glycated skin model. Glycation of the skin of hairless mice was induced using vertical diffusion cells and incubation with phosphate-buffered saline containing 50 mM glyoxal for 24 h. Flux and accumulation in the skin were determined by applying hydrophilic and lipophilic molecules (Sodium fluorescein; FL-Na and Nile red, respectively) to this in vitro glycated skin model. Furthermore, to investigate the effect of glycation on epidermal-dermal barrier properties, we conducted diffusion experiments with FL-Na and fluorescein isothiocyanate-dextran using stratum corneum (SC)-stripped glycated skin. The in vitro glycated skin model demonstrated characteristic glycation alterations like a yellowish change in skin color and surface roughness. For low-molecular weight (MW) hydrophilic molecules, flux across glycated full-thickness skin was higher than that across normal skin, although there was no difference with lipophilic molecules. However, glycated epidermis-dermis showed lower flux, and the difference increased with the MW of the compound. Furthermore, the amount of high-MW hydrophilic molecules accumulated in glycated epidermis-dermis was decreased. These results suggest that glycated SC and epidermis-dermis differentially regulate the permeability of hydrophilic molecules and highlight the importance of controlling drug delivery by modifying the formulation or method of application depending on skin condition.

Enhancement of Skin Penetration of Hydrophilic and Lipophilic Compounds by pH-sensitive Liposomes.

PURPOSE: Enhance skin penetration of hydrophilic and lipophilic compounds using liposomes that are responsible to the pH of the skin surface. METHODS: pH-sensitive liposomes were prepared by a thin layer and freeze-thaw method with dioleoyl phosphatidyl ethanolamine and cholesteryl hemisuccinate. Liposomal fusion with stratum corneum lipid liposomes was measured using fluorescence resonance energy transfer. Particle diameter and zeta potential of the liposomes after fusion were measured by dynamic light scattering and electrophoresis. RESULTS: Under neutral pH conditions, the diameter of the pH-sensitive liposomes was 130 nm and their zeta potential was -70 mV. In weakly acidic conditions, the diameter was larger than 3,000 nm and the zeta potential was -50 mV. In contrast, the particle diameter and the zeta potential of the non-pH-sensitive liposomes remained constant under various pH conditions. A skin penetration study was performed on hairless mice skin using vertical diffusion cells, showing that the fusion ability of pH-sensitive liposomes was higher than that of non-pH-sensitive liposomes. In the skin penetration study was carried out using hydrophilic (calcein) and lipophilic (N-(7-nitrobenz- 2-oxa-1,3-diazol-4yl)-PE) (NBD-PE) model compounds which were applied to the skin with pH-sensitive liposomes as carrier. The fluorescent compounds contained within the pH-sensitive liposomes permeated the skin more effectively than those within non-pH-sensitive liposomes, and this ability was further enhanced with the lipophilic compound. CONCLUSION: These studies suggest that pH-sensitive liposomes have potential as an important tool for delivery of compounds into the skin.

Effect of ß-cyclodextrin derivatives on the diosgenin absorption in Caco-2 cell monolayer and rats.

Orally administrated diosgenin, a steroidal saponin found in the roots of Dioscorea villosa, improves reduced skin thickness in ovariectomized mice, and plays an important role in the treatment of hyperlipidemia. Diosgenin has been noticed as an active element in cosmeceutical and dietary supplements. We have already elucidated that the absolute oral bioavailability of diosgenin is very low; however, a high skin distribution of diosgenin was also observed. The aim of the present study was to examine and compare the effects of ß-cyclodextrin (ß-CD) and 3 kinds of its derivatives such as hydroxypropyl ß-CD on the diosgenin permeability using a Caco-2 model and rat jejunal perfusion. These derivatives of ß-CD greatly improved the low solubility of diosgenin. No significant increase was observed in the lactate dehydrogenase leakage from Caco-2 cell, while a slight decrease was found on the transepithelial electrical resistance by diosgenin and ß-CD derivatives. However, ß-CD derivatives, especially hydroxyethyl ß-CD and hydroxypropyl ß-CD, markedly enhanced diosgenin permeability across the Caco-2 monolayer and rat jejunum. The bioavailability of diosgenin in the presence of ß-CD derivatives were about 4 to 11 fold higher than diosgenin suspension. The mechanisms of these enhancement effects may be due to improvements in solubility and tight junction opening.

Effects of low molecular weight soybean peptide on mRNA and protein expression levels of differentiation markers in normal human epidermal keratinocytes.

Low molecular weight soybean peptide (LSP) was applied to normal human epidermal keratinocytes, and the results showed a significant increase in the gene expression levels of involucrin, transglutaminase, and profilaggrin. Filaggrin protein levels were also significantly higher. It is possible that LSP has an epidermal cell differentiation-promoting effect and may be able to regulate metabolism of the epidermis.

Ionic composition of Shotokuseki extract alters cell differentiation and lipid metabolism in three-dimensional cultured human epidermis.

Corneocytes and intercellular lipids form the stratum corneum. The content and composition of intercellular lipids in the stratum corneum significantly affect skin barrier function. The purpose of this study was to demonstrate the effect of Shotokuseki extract (SE) on intercellular lipid production and metabolism in human three-dimensional cultured human epidermis. SE or ion mixtures containing five common ions were applied to three-dimensional cultured human epidermis for 2-8 days for each assay. The mRNA expression levels of epidermal differentiation markers and lipid metabolism genes were quantified by real-time PCR. After extraction of lipids from the epidermis, ceramide, sphingosine, free fatty acids, and cholesterol were quantified by LC-MS/MS, GC-MS, or HPLC. The results showed that the application of SE increased the gene expression levels of epidermal differentiation markers keratin10 and transglutaminase. Elongation of very long-chain fatty acids protein 3, serine palmitoyl transferase, ceramide synthase 3, and acid ceramidase mRNA expression levels increased and fatty acid synthase mRNA expression decreased. The content of each lipid, [EOS] ceramide decreased and total sphingosine content increased on day 4. On day 8 of application, ceramide [NDS], [NP], and [EODS] increased and total free fatty acid content decreased. These results show that SE alters the lipid composition of the epidermis, increasing ceramides and decreasing free fatty acids in the epidermis. The composition of the ions in the SE may be responsible for the changes in lipid composition. These behaviors were different from those observed when the ion mixture was applied. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-024-00616-3.

Enhancement of diosgenin distribution in the skin by cyclodextrin complexation following oral administration.

Orally administrated diosgenin, a steroidal saponin found in several plants including Dioscorea villosa, recovers skin thickness reduced in ovariectomized mice, and plays an important role in the treatment of hyperlipidemia. Thus, diosgenin is an active element of cosmeceutical and dietary supplements. However, we have already elucidated that the skin distribution and absolute oral bioavailability of diosgenin is very low. The aim of this study is to evaluate the efficacy of diosgenin-cyclodextrin (CD) complexes in improving the skin concentration of diosgenin. The formation of the CD complex was indicated by powder X-ray diffraction (XRD), differential scanning calorimetry (DSC), and scanning electron microscope (SEM) studies. Oral administration of the diosgenin/ß-CD complex resulted in a significant enhancement in terms of the skin distribution of diosgenin, maximum plasma level (C(max)), area under the plasma concentration-time curve (AUC), and absolute oral bioavailability over those of the drug alone. These results suggest that the inclusion complex of diosgenin/ß-CD can be used to improve low skin content of diosgenin.

Sphingomyelin reduces melanogenesis in murine B16 melanoma cells through indirect suppression of tyrosinase.

Growing consumer interest in skin whitening has led to intensive investigations of whitening methods. In this study, we evaluated the effect of sphingomyelin, a component of cell membranes, on melanin production. B16 mouse melanoma cells were treated with lauroyl-sphingomyelin (SM) or its metabolite lauroyl-ceramide (CER) and measured for cell viability, melanin content, and direct and indirect tyrosinase activity. Expression of melanin synthesis-related genes encoding tyrosinase (Tyr), tyrosinase-related proteins (Trp1 and Trp2), and microphthalmia-associated transcription factor (Mitf) were quantified by real-time PCR, and SM content in cells was measured by fluorescence high-performance liquid chromatography. SM treatment decreased melanin content in a concentration-dependent manner, without significantly altering the number of viable cells. By contrast, treatment with CER at the same concentrations did not decrease melanin content. SM inhibited the activity of intracellular tyrosinase, but not mushroom-derived tyrosinase. Gene expression levels of Tyr and Mitf were significantly reduced by treatment with SM, while those of Trp2 and Mitf were significantly reduced by CER. Fluorescence-labeled SM was converted to fluorescence-labeled CER in cells over time. In conclusion, CER was found to inhibit melanogenesis without inhibiting tyrosinase activity, suggesting that SM is more water soluble than CER, and is more effectively taken up into cells.

2-kDa hyaluronan ameliorates human facial wrinkles through increased dermal collagen density related to promotion of collagen remodeling.

BACKGROUND/AIMS: Hyaluronan (HA) oligosaccharides are involved in several biological processes, primarily collagen remodeling and wound healing. Collagen remodeling is retarded in aging skin and causes wrinkles. The aim of this study was to evaluate the effect of 2-kDa HA oligosaccharides (HA2k) on wrinkles by permeation through the stratum corneum and promotion of collagen remodeling. METHODS: A 3D skin model and excised human skin were used to evaluate the permeation of fluorescein-labeled HA2k. The effect of HA2k on collagen metabolism was evaluated by measuring the protein level of type 1 pro-collagen (COL1A1) and matrix metalloproteinase-1 (MMP-1) in the 3D skin model. 0.1% HA2k solution and vehicle control was applied to the human forearm for 8 weeks to evaluate dermal collagen density. To evaluate the effect of HA2k on depth of facial wrinkles, a randomized controlled trial was conducted with 0.1% HA2k lotion and vehicle lotion for 8 weeks. RESULTS: HA2k was confirmed to permeate through the stratum corneum by fluorescent microscopy. Both COL1A1 and MMP-1 were upregulated by HA2k application in a 3D skin model culture. The collagen density was higher for the HA2k-treated forearm than for the vehicle control-treated forearm after 4 weeks. The maximum wrinkle depths in the nasolabial fold and crow's feet area were significantly shallower in the HA2k lotion group than in the control group. CONCLUSION: HA2k permeated the stratum corneum, activated collagen synthesis and degradation simultaneously, and ameliorated wrinkles.

Possibility of alveolar bone promoting enhancement by using lipophilic and/or hydrophilic zinc related compounds in zinc-deficient osteoporosis rats.

The purpose of this research is improvement of therapeutic treatment for periodontitis by using lipophilic and/or hydrophilic zinc materials. The sample suspension injections were prepared from zinc octanate (C8Zn), zinc stearate (C18Zn), zinc sulfate hepta-hydrate (ZnSO4) and tricalcium phosphate (ZnTCP) containing 6.17 w/w% zinc. After administrating of all injections to around alveolar bone of zinc-deficient osteoporosis rats, plasma Zn concentration, bone mineral content (BMC) of jawbone, BMC and bone mechanical strength (BMS) of femur and permeability tests for hairless rat stripped skin were measured as therapeutic scores. BMC and BMS were measured by using an X-ray computing tomography and the three-point bending method, respectively. The body weight, plasma Zn concentrations and the area under curve (AUC) for Zn of C8Zn, C18Zn and ZnTCP group rats were higher than those of control group, but those of ZnSO4 group were not changed. BMC of alveolar bone and femur and BMS of femur for C8Zn and C18Zn groups for 12 weeks were significantly higher than those of the control group, but those of ZnSO4 group were not changed. Stripped rat skin permeability treated by the hydrophilic creams containing C8Zn was 5-times higher than that of ZnTCP.

Effects of soybean peptide and collagen peptide on collagen synthesis in normal human dermal fibroblasts.

The collagen present in the dermis of the skin is a fibrous protein that fills the gaps between cells and helps maintain tissue flexibility. Effectively increasing the collagen present in the skin is an important goal for cosmetic research. Recent research has shown that soybean peptide (SP) has anti-fatigue activity, antioxidant activity, and the ability to increase type I collagen, while collagen peptide (CP) has the ability to enhance corneal moisture content and viscoelasticity, as well as to increase levels of hyaluronic acid synthesizing enzymes in human skin. Little documented research, however, has been conducted on collagen formation in relation to these peptides. Therefore, this research applied SP and CP with molecular weights primarily around 500 and preparations containing both SP and CP to normal human dermal fibroblasts together with magnesium ascorbyl phosphate (VC-PMg), and used real-time PCR to determine the gene expression of type I collagen (COL1A1), which contributes to collagen synthesis, and Smad7, which contribute to collagen breakdown. In addition, enzyme linked immuno sorbent assay (ELISA) was used to measure collagen content in the media. COL1A1 gene expression at 24 h after sample addition showed higher tendency in all samples and increased with time at 4, 8 and 24 h after addition. Smad7 gene expression was not substantially different at 4 h after addition. matrix metalloproteinase-1 gene expression was higher following SP addition, but was lower after the addition of CP and SP+CP. Medium collagen content was higher in all samples and increased with time at 8 h after addition. Collagen levels were higher when SP and CP were added together.

Modulation of function and structure of stratum corneum in sphingomyelin synthase 2-deficient mice.

Sphingomyelin synthase (SMS) synthesizes sphingomyelin (SM) from ceramide (Cer), a precursor of Cer. The effects of SMS deficiency on stratum corneum (SC) barrier function and SC lamellar structure are unknown. In this report, permeation of hydrophilic and lipophilic compounds through full-thickness skin or stripped skin of SMS2-knockout (KO) and wild-type (WT) mice was examined. Furthermore, small-angle and wide-angle X-ray scattering (SAXS and WAXS) measurements of the SC were performed as a function of temperature to analyze the lamellar structure and hydrocarbon chain packing, where a SC sample was changed from 10 °C to 120 °C at 2 °C/min and the X-ray diffraction profile in the small-angle region and the wide-angle region was observed. Skin permeability of the hydrophilic compound increased significantly for SMS2-KO mice when compared with that of WT mice. In contrast, no difference was observed in the penetration of lipophilic compounds in the skin of both SMS2-KO and WT mice. In SC of SMS2-KO mice, two sharp SAXS peaks were observed due to the lamellar structure with a repetition period of 4.8 nm. The WAXS revealed that the intensity ratio R0.42/0.37 of the 0.42 nm peak at 2.4 nm-1 to the 0.37 nm peak at 2.7 nm-1 was smaller in the SMS2-KO mouse than in the WT mouse. Due to the temperature dependence of the WAXS, the peaks of 2.4 and 2.7 nm-1 remained until the higher temperatures in SMS2-KO mouse SC than those in WT mouse SC. The results of X-ray diffraction suggest that deficiency of SMS2 may cause the appearance of highly ordered structures of SC, which in turn may reduce the barrier function of SC.

The ion balance of Shotokuseki extract promotes filaggrin fragmentation and increases amino acid production and pyrrolidone carboxylic acid content in three-dimensional cultured human epidermis.

Natural moisturizing factor (NMF) in the stratum corneum contributes to the retention of moisture there. The purpose of this study was to determine the penetration of ions in Shotokuseki extract (SE) into the three-dimensional cultured epidermis and the effect of NMF on the biosynthesis of amino acids and pyrrolidone carboxylic acid formation. Various ions, amino acids and pyrrolidone carboxylic acid were quantified by inductively coupled plasma mass spectrometry, fully automatic amino acid analyzer or high-performance liquid chromatography (HPLC) in three-dimensional cultured epidermis after application of SE. Gene expression levels of profilaggrin, calpain1, caspase14, and bleomycin hydrolase, which are involved in NMF production, were determined by reverse-transcription qPCR and bleomycin hydrolase activity was determined by aminopeptidase assay. The application of SE increased Na, K, Mg, Ca, Al, and Fe levels in three-dimensional cultured epidermis. The mRNA levels of the starting material of amino acid synthesis profilaggrin, and calpain1 and bleomycin hydrolase, which are involved in its fragmentation, increased. The activity of bleomycin hydrolase also increased. Furthermore, the levels of amino acids and pyrrolidone carboxylic acid increased in the three-dimensional cultured epidermis. This suggests that the ionic composition of SE may be involved in its moisturizing effect on the stratum corneum.

Effect of estrogen/progesterone ratio on the differentiation and the barrier function of epidermal keratinocyte and three-dimensional cultured human epidermis.

AIMS: Estrogen (E) and progesterone (P) are the major female hormones and are secreted with changing concentration ratios throughout the menstrual cycle. These hormones have been studied individually regarding their physiological function in the skin, but their concentration ratio (E/P) and its effect on the skin has not yet been investigated. The purpose of this study was to clarify the effect of the E/P ratio on skin barrier function. The menstrual cycle was divided into the menstrual, follicular, ovulation, and luteal phases. MATERIALS AND METHODS: The E/P concentration ratios corresponding with each phase were added to a three-dimensional epidermal model or normal human epidermal keratinocytes for 5 days. Gene and protein expression levels of several markers of cell differentiation, including loricrin (LOR) and transglutaminase (TGase), were quantified by real-time PCR and western blotting, respectively. Transepidermal water loss (TEWL) of the three-dimensional epidermal model was measured, and ceramide content was quantified by thin-layer chromatography. KEY FINDINGS: Gene expression of the epidermal differentiation markers, LOR and TGase, increased when applying the concentration ratio of E/P associated with the menstrual and luteal phases. The LOR protein level decreased from menstrual to luteal phases, and the TGase protein level increased from menstrual to luteal phases. During the same phases, ceramide NS increased and TEWL decreased. SIGNIFICANCE: Skin barrier function was improved by culturing cells at specific E/P concentration ratios, which would, therefore, be considered beneficial for female skin. This suggests that dysregulated E/P concentration ratios may be the cause of certain skin problems.