Comprehensive Analysis of Exosomal MicroRNAs Derived from UVB-Irradiated Keratinocytes as Potential Melanogenesis Regulators.

The exosomes derived from keratinocytes can have a substantial impact on melanogenesis by influencing melanocytes. MicroRNAs (miRNAs) encapsulated within exosomes are implicated in the control of melanogenesis, particularly when under the influence of UVB irradiation. This investigation explores UVB-induced exosomal miRNAs from keratinocytes as potential regulators of melanogenesis. UVB-irradiated, keratinocyte-derived exosomes were observed to augment melanogenesis in melanocytes, resulting in an upregulation of MITF, TRP1, TRP2, and TYR expression compared to non-UVB-irradiated exosomes. Additionally, a subset of exosomal miRNAs was differentially selected and confirmed to exert both enhancing and inhibitory effects on melanogenesis through functional assays. Notably, hsa-miR-644a, hsa-miR-365b-5p, and hsa-miR-29c-3p were found to upregulate melanogenesis, while hsa-miR-18a-5p, hsa-miR-197-5p, and hsa-miR-4281 downregulated melanogenesis. These findings suggest the involvement of keratinocyte-derived exosomal miRNAs in melanogenesis regulation within melanocytes. The expression levels of exosomal miRNAs from keratinocytes exhibited a UVB-dependent increase, indicating a potential role for these miRNAs as regulators of melanogenesis in response to UVB irradiation. Furthermore, melanogenesis was found to be dependent on exosomes derived from keratinocytes. This underscores the potential of UVB-induced exosomal miRNAs derived from keratinocytes as regulators of melanogenesis. Moreover, this study unveils a significant role for exosomes in melanocyte pigmentation, presenting a novel pathway in the intricate process of melanogenesis.

Phosphorylation of glucocorticoid receptor induced by 16-kauren-2-beta-18, 19-triol decreases expression of Melanophilin through JNK signalling.

16-kauren-2-beta-18, 19-triol (16-kauren) is a natural diterpenoid substance derived from Asteraceae psiadia punctulata, a small tropical shrub in Africa and Asia, and it can reduce Mlph expression without affecting the expression of Rab27a and MyoVa in melanocytes. Melanophilin (Mlph) is an important linker protein in the melanosome transport process. However, the signal transduction pathway for the regulation of Mlph expression has not been fully established. We examined the mechanism of 16-kauren on Mlph expression. Murine melan-a melanocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, and luciferase assay were performed. The inhibition of Mlph expression by 16-kauren-2ß-18,19-triol (16-kauren) occurs through the JNK signal and is reversed following glucocorticoid receptor (GR) activation by dexamethasone (Dex). Especially, 16-kauren activates JNK and c-jun signalling, part of the MAPK pathway, with subsequent Mlph repression. When the JNK signal is weakened by siRNA, the inhibition of Mlph expression by 16-kauren was not seen. JNK activation by 16-kauren induces GR phosphorylation, which leads to Mlph repression. These results demonstrate that 16-kauren regulates Mlph expression through the phosphorylation of GR via the JNK signalling pathway.

Evaluation of facial skin age based on biophysical properties in vivo.

OBJECTIVE: The evaluation of skin age, reflecting overall facial characteristics, has not been established. Previous studies focused on visual assessment or individual-specific feature such as wrinkles or skin color. We studied the evaluation model of skin age index (SAI) including the overall aging features including wrinkles, skin color, pigmentation, elasticity, and hydration. METHODS: Total 300 healthy women aged between 20 and 69 years included in this study. Pearson correlation analysis performed to identify the key factors among the biophysical properties with aging and developed the prediction model of SAI. Statistical regression analysis and machine learning technique applied to build the prediction model using the coefficient of determination (R2 ) and root mean square error (RMSE). Validation study of the SAI model performed on 24 women for 6 weeks application with anti-aging product. RESULTS: Prediction model of SAI consisted of skin elasticity, wrinkles, skin color (brightness, Pigmented spot, and Uv spot), and hydration, which are major features for aging. The cforest model to assess a SAI using machine learning identified the highest R2 and lowest RMSE compared to other models, such as svmRadial, gaussprRadial, blackboost, rpart, and statistical regression formula. The cforest prediction model confirmed a significant decrease of predicted SAI after 6 weeks of application of anti-aging product. CONCLUSION: We developed a prediction model to evaluate a SAI using machine learning, and led to accurate predicted age for overall clinical aging. This model can a good standard index for evaluating facial skin aging and anti-aging products.

In vivo method to evaluate antioxidative activity using UVA-induced carbonylated protein on human skin.

BACKGROUND: Skin is continuously exposed to oxidative stress caused by reactive oxygen species (ROS) produced by the ultraviolet (UV) light, and it is important to evaluate the antioxidant activity. Carbonylated proteins (CPs) are candidate markers of oxidative modification as a result from the ROS. We aimed to develop the CP-based method to assess the efficacy of antioxidants in human skin. METHODS: Ten healthy females were enrolled in the study to determine the UVA dosage for CP production, and another 10 females were included to evaluate the antioxidative activity. The stratum corneum was collected from test skin using D-Squame tape, and CPs from the SC were stained by fluorescence labeling and observed using a fluorescence microscope. RESULTS: CP level significantly increased with UVA irradiation from 15J/cm2 to 50J/cm2 compared to the control (non-UVA) area. CP production significantly increased by 34.38% and 35.22% in UVA irradiation and squalene (vehicle) areas. 5% α-tocopherol and ß-carotene significantly increased the CP production by 20.77% and 19.34% after 2 hours of 30J of UVA irradiation compared to control area. Inhibition rate of CPs in 5% α-tocopherol and 5% ß-carotene showed 41.45% and 45.37% after 2 hours of UVA irradiation. CONCLUSION: This study developed the simple, visual, and direct in vivo method to evaluate the antioxidative activity for products in human skin by measuring the CP level as an oxidative modification caused by UVA-induced ROS generation.

Age-related biophysical changes of the epidermal and dermal skin in Korean women.

INTRODUCTION: The clinical characteristics of skin were investigated to study the inter-relationship and changes in the biophysical properties of the epidermal and dermal layers associated with aging using noninvasive methods. METHODS: Our study included 100 healthy women aged between the early 20s and late 60s. Biophysical characteristics of skin such as color (brightness and spots), transparency, wrinkle on crow's feet, elasticity, hydration, sebum content, glossiness, and transepidermal water loss measured under controlled conditions. RESULTS: This study performed in a Korean population demonstrated that aging significantly affects human skin in terms of parameters such as wrinkles, skin color, elasticity, and epidermal hydration. Age-related changes in skin hydration showed varying patterns between the epidermis and dermis. Skin color showed heterogeneous characteristics between the upper and lower epidermal layers associated with aging. Skin elasticity and wrinkles were observed to show and inversely proportional relationship in the early 40s. CONCLUSIONS: We confirmed the significant influence of aging on the biophysical properties of skin and determined the distinct age-related biophysical changes in the epidermal and dermal layers of skin using noninvasive method. This study indicates the need for further research to investigate the distinctive age-related changes in characteristics of the epidermal and dermal layers of human skin.

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Substance P restores normal skin architecture and reduces epidermal infiltration of sensory nerve fiber in TNCB-induced atopic dermatitis-like lesions in NC/Nga mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. Substance P (SP) is an 11-amino-acid endogenous neuropeptide that belongs to the tachykinin family and several reports recently have supported the anti-inflammatory and tissue repairing roles of SP. OBJECTIVE: In this study, we investigated whether SP can improve AD symptoms, especially the impaired skin barrier function, in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced chronic dermatitis of NC/Nga mice or not. METHOD: AD-like dermatitis was induced in NC/Nga mice by repeated sensitization with TNCB for 5 weeks. The experimental group designations and topical treatments were as follows: vehicle group (AD-VE); SP group (AD-SP); and SP with NK1R antagonist CP99994 (AD-SP-A) group. Histological analysis was performed to evaluate epidermal differentiation, dermal integrity, and epidermal nerve innervation in AD-like lesions. The skin barrier functions and pruritus of NC/Nga mice were evaluated by measuring transepidermal water loss (TEWL) and scratching behavior, respectively. RESULT: Topical SP treatment resulted in significant down-regulation of Ki67 and the abnormal-type keratins (K) K6, K16, and K17, restoration of filaggrin and claudin-1, marked reduction of TEWL, and restoration of basement membrane and dermal collagen deposition, even under continuous sensitization of low dose TNCB. In addition, SP significantly reduced innervation of itch-evoking nerve fibers, gelatinase activity and nerve growth factor (NGF) expression in the epidermis but upregulated semaphorin-3A (Sema3A) expression in the epidermis, along with reduced scratching behavior in TNCB-treated NC/Nga mice. All of these effects were completely reversed by co-treatment with the NK1R antagonist CP99994. In cultured human keratinocytes, SP treatment reduced expression of TGF-α, but upregulated TGF-ß and Sema3A. CONCLUSION: Topically administered SP can restore normal skin barrier function, reduce epidermal infiltration of itch-evoking nerve fibers in the AD-like skin lesions, and alleviate scratching behavior. Thus, SP may be proposed as a potential medication for chronic dermatitis and AD.

Manifestation of atopic dermatitis-like skin in TNCB-induced NC/Nga mice is ameliorated by topical treatment of substance P, possibly through blockade of allergic inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. In this study, topically applied substance P (SP) significantly alleviated AD-like clinical symptoms in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced dermatitis in NC/Nga mice. This effect was nullified by pretreatment of the neurokinin-1 receptor (NK-1R) antagonist CP99994. SP treatment significantly reduced the infiltration of mast cells and CD3-positive T cells as well as inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and thymic stromal lymphopoietin (TSLP), in AD-like skin lesions and decreased the levels of IgE and thymus and activation-regulated chemokine in serum. This SP-induced alleviation of allergic inflammatory responses was also confirmed as reduced activation in the axillary lymph nodes (aLN) and spleen, suggesting the systemic effect of SP on immune responses in TNCB-induced NC/Nga mice. Furthermore, SP-mediated TSLP reduction was confirmed in human keratinocyte culture under pro-inflammatory TNF-α stimulation. Taken together, these results suggest that topically administered SP may have potential as a medication for atopic dermatitis.

Meta-markers for the differential diagnosis of lung cancer and lung disease.

UNLABELLED: Misdiagnosis of lung cancer remains a serious problem due to the difficulty of distinguishing lung cancer from other respiratory lung diseases. As a result, the development of serum-based differential diagnostic biomarkers is in high demand. In this study, 198 clinical serum samples from non-cancer lung disease and lung cancer patients were analyzed using nLC-MRM-MS for the levels of seven lung cancer biomarker candidates. When the candidates were assessed individually, only SERPINEA4 showed statistically significant changes in the serum levels. The MRM results and clinical information were analyzed using a logistic regression analysis to select model for the best 'meta-marker', or combination of biomarkers for differential diagnosis. Also, under consideration of statistical interaction, variables having low significance as a single factor but statistically influencing on meta-marker model were selected. Using this probabilistic classification, the best meta-marker was determined to be made up of two proteins SERPINA4 and PON1 with age factor. This meta-marker showed an enhanced differential diagnostic capability (AUC=0.915) for distinguishing the two patient groups. Our results suggest that a statistical model can determine optimal meta-markers, which may have better specificity and sensitivity than a single biomarker and thus improve the differential diagnosis of lung cancer and lung disease patients. BIOLOGICAL SIGNIFICANCE: Diagnosing lung cancer commonly involves the use of radiographic methods. However, an imaging-based diagnosis may fail to differentiate lung cancer from non-cancerous lung disease. In this study, we examined several serum proteins in the sera of 198 lung cancer and non-cancerous lung disease patients by multiple-reaction monitoring. We then used a combination of variables to generate a meta-marker model that is useful as a differential diagnostic biomarker.

A new moisturizer containing physiologic lipid granules alleviates atopic dermatitis.

BACKGROUND: Patients with atopic dermatitis show a defective barrier function. In atopic skin, ceramide is significantly decreased and the secretion of lamellar bodies is also impaired. To mimic lamellar bodies, we prepared lipid granules composed of ceramide, fatty acids and cholesterol. Because these lipid granules contain multiple lamellar structures, it is expected that they will have superior affinity to skin; hence, they should have a good moisturizing effect. This study was performed to evaluate the effects of moisturizer containing lipid granules on atopic dermatitis. METHODS: Patients with mild atopic dermatitis (n = 30, aged 5-19 years) were recruited and instructed to apply a moisturizer containing physiologic lipid granules for 4 weeks. The SCORing Atopic Dermatitis (SCORAD) score and general symptoms were evaluated. In addition, transepidermal water loss (TEWL) and stratum corneum (SC) hydration were also measured. RESULTS: Twenty-nine patients completed the study. The SCORAD value decreased dramatically after 4 weeks of moisturizer application (p = 0.000). The general symptoms of atopic dermatitis were also greatly improved. At baseline, most patients reported their symptoms as mild and moderate, but after 4 weeks 20 of the patients (69%) had no symptoms. The TEWL was not changed, but the SC hydration increased significantly (p = 0.000). No significant adverse effects were observed. CONCLUSIONS: Moisturizer containing lipid granules effectively controlled atopic dermatitis.

Impact of NAD(P)H:quinone oxidoreductase-1 on pigmentation.

We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription-PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process.

Assembly of Coenzyme Q10 nanostructure resembling nascent discoidal high density lipoprotein particle.

There are tremendous drug candidates that suffer from insolubility in water. In the present study, it is shown that Coenzyme Q10 (CoQ10), a model water-insoluble compound, can be nanoparticulated into a water-soluble form using apolipoprotein A-I (apoA-I). Similar to the way that apoA-I forms nascent discoidal high density lipoprotein (ndHDL) particles by bordering acyl chain tails of phospholipids, CoQ10 could be enclosed into the circle of a disk made of apoA-Is. The resulting nanostructure of CoQ10 and apoA-I was water-soluble with a size of approximately 12 nm in diameter and was physically more robust than liposome. We expect that the strategy suggested in this study can be exploited to assemble nano-sized, water-soluble structures of various water-insoluble drug candidates.

Fructose 1,6-diphosphate alleviates UV-induced oxidative skin damage in hairless mice.

Reactive oxygen species (ROS) are involved in the deleterious effects of UV light on skin. The antioxidant defense system is considered to be crucial for protecting skin from ROS. Recently, we showed that fructose 1,6-diphosphate (FDP), a glycolytic metabolite, reduced oxidative stress in UVB-irradiated keratinocytes. This study set out to determine whether topically applied FDP could exert protective effects against UV-induced skin damage in hairless mice. An in vitro skin permeation study using Franz-type diffusion cells showed that the amount of [14C]-FDP that diffused through the skin increased in a time-dependent manner, and about 3.5% of the applied FDP penetrated the skin after 24 h. Topical application of FDP (1%) preserved the endogenous antioxidant capacity of skin such as catalase and glutathione, which were significantly reduced after UVB irradiation without FDP. FDP also reversed the loss of catalase protein and prevented the accumulation of carbonylated proteins induced by UVB irradiation. These results provide evidence that topically administered FDP could penetrate into the skin and attenuate UVB-induced oxidative skin damage in hairless mice.

Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds.

Although many hypo-pigmenting agents are currently available, the demand for novel whitening agents is increasing, in part due to the weak effectiveness and unwanted side effects of currently available compounds. To screen for novel hypo-pigmenting agents, many methodologies such as cell culture and enzymatic assays are routinely used. However, these models have disadvantages in terms of physiological and economic relevance. In this study, we validated zebrafish as a whole-animal model for phenotype-based screening of melanogenic inhibitors or stimulators. We used both the well-known melanogenic inhibitors (1-phenyl-2-thiourea, arbutin, kojic acid, 2-mercaptobenzothiazole) and newly developed small molecule compounds (haginin, YT16i). All the tested compounds produced inhibitory effects on the pigmentation of zebrafish, most likely due to their inhibitory potential on tyrosinase activity. In simultaneous in vivo toxicity tests, a newly developed melanogenic inhibitor YT16i showed massive abnormalities in terms of deformed morphologies and cardiac function. Together, these results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. We suggest that the zebrafish system is a novel alternative to mammalian models, with several advantages including the rapidity, cost-effectiveness, and physiological relevance.

Deoxypodophyllotoxin reduces skin pigmentation of brown Guinea pigs.

In this report, we have demonstrated that deoxypodophyllotoxin from Anthriscus sylvestris (L.) Hoffm decreases UV-induced skin pigmentation of brown guinea pigs. Deoxypodophyllotoxin (0.05 % in propylene glycol: ethanol: water = 5 : 3:2) was topically applied twice daily for two weeks to dorsal skin of brown guinea pigs that were exposed to UV irradiation using a solar simulator. Visual inspection and Fontana-Masson staining both demonstrated that deoxypodophyllotoxin reduced skin pigmentation and total epidermal melanin when compared to that of vehicle-treated areas, suggesting that deoxypodophyllotoxin maybe applicable to treat hyperpigmentation.

Development of 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent.

A stable derivative of kojic acid, 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one (Kojyl-APPA), was synthesized in good yield. The effects of Kojyl-APPA on tyrosinase activity and melanin synthesis were investigated. Kojyl-APPA showed tyrosinase inhibition effect (30%) in situ, but not in vitro. Kojyl-APPA inhibited tyrosinase activity significantly at 24 h after treatment in normal human melanocytes. It means that Kojyl-APPA is not a direct inhibitor of tyrosinase itself, but it is converted to a potential inhibitor kojic acid enzymatically in cells. In addition, Kojyl-APPA decreased melanin content to 75% of control in melanoma cells and decreased neomelanin synthesis to 43% of control in normal human melanocytes. Its permeation through skin increased by about 8 times as compared with kojic acid.