Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

BACKGROUND: Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. METHODS AND RESULTS: PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

Study on the Skincare Effects of Red Rice Fermented by Aspergillus oryzae In Vitro.

Red rice, a variety of pigmented grain, serves dual purposes as both a food and medicinal resource. In recent years, we have witnessed an increasing interest in the dermatological benefits of fermented rice extracts, particularly their whitening and hydrating effects. However, data on the skincare advantages derived from fermenting red rice with Aspergillus oryzae remain sparse. This study utilized red rice as a substrate for fermentation by Aspergillus oryzae, producing a substance known as red rice Aspergillus oryzae fermentation (RRFA). We conducted a preliminary analysis of RRFA's composition followed by an evaluation of its skincare potential through various in vitro tests. Our objective was to develop a safe and highly effective skincare component for potential cosmetic applications. RRFA's constituents were assessed using high-performance liquid chromatography (HPLC), Kjeldahl nitrogen determination, the phenol-sulfuric acid method, and enzyme-linked immunosorbent assay (ELISA). We employed human dermal fibroblasts (FB) to assess RRFA's anti-aging and antioxidative properties, immortalized keratinocytes (HaCaT cells) and 3D epidermal models to examine its moisturizing and reparative capabilities, and human primary melanocytes (MCs) to study its effects on skin lightening. Our findings revealed that RRFA encompasses several bioactive compounds beneficial for skin health. RRFA can significantly promote the proliferation of FB cells. And it markedly enhances the mRNA expression of ECM-related anti-aging genes and reduces reactive oxygen species production. Furthermore, RRFA significantly boosts the expression of Aquaporin 3 (AQP3), Filaggrin (FLG), and Hyaluronan Synthase 1 (HAS1) mRNA, alongside elevating moisture levels in a 3D epidermal model. Increases were also observed in the mRNA expression of Claudin 1 (CLDN1), Involucrin (IVL), and Zonula Occludens-1 (ZO-1) in keratinocytes. Additionally, RRFA demonstrated an inhibitory effect on melanin synthesis. Collectively, RRFA contains diverse ingredients which are beneficial for skin health and showcases multifaceted skincare effects in terms of anti-aging, antioxidant, moisturizing, repairing, and whitening capabilities in vitro, highlighting its potential for future cosmetic applications.

Comparative Analysis of Olive-Derived Phenolic Compounds' Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes.

Olive leaf contains plenty of phenolic compounds, among which oleuropein (OP) is the main component and belongs to the group of secoiridoids. Additionally, phenolic compounds such as oleocanthal (OL) and oleacein (OC), which share a structural similarity with OP and two aldehyde groups, are also present in olive leaves. These compounds have been studied for several health benefits, such as anti-cancer and antioxidant effects. However, their impact on the skin remains unknown. Therefore, this study aims to compare the effects of these three compounds on melanogenesis using B16F10 cells and human epidermal cells. Thousands of gene expressions were measured by global gene expression profiling with B16F10 cells. We found that glutaraldehyde compounds derived from olive leaves have a potential effect on the activation of the melanogenesis pathway and inducing differentiation in B16F10 cells. Accordingly, the pro-melanogenesis effect was investigated by means of melanin quantification, mRNA, and protein expression using human epidermal melanocytes (HEM). This study suggests that secoiridoid and its derivates have an impact on skin protection by promoting melanin production in both human and mouse cell lines.

Combination of 308-nm excimer laser and piperine promotes melanocyte proliferation, migration, and melanin content production via the miR-328/SFRP1 axis.

OBJECTIVE: Both piperine and a 308-nm excimer laser have significant curative effects on vitiligo. This study mainly explored the molecular mechanism of a 308-nm excimer combined with piperine in regulating melanocyte proliferation. METHODS: Epidermal melanocytes were cultured in piperine solution, and the cells were irradiated by an XTRAC excimer laser treatment system at 308-nm output monochromatic light. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were for detecting the expression levels of genes or proteins. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Transwell method was for assessing cell viability and migration capacity. The content of melanin was also detected. RESULTS: The combination of the 308-nm excimer laser and piperine enhanced the cell proliferation, migration, and melanin production of melanocytes and upregulated the level of miR-328, and restraint of miR-328 reversed the influence of the 308-nm excimer laser and piperine. Secreted frizzled-related protein 1 (SFRP1) is a direct target gene of miR-328, and miR-328 can inhibit the expression of SFRP1 and elevate the protein level of the Wnt/ß-catenin signaling pathway. CONCLUSION: The 308-nm excimer laser combined with piperine may be more efficient than piperine alone in the remedy of vitiligo, and the miR-328/SFRP1 and Wnt/ß-catenin pathways are participated in the proliferation, migration, and melanin synthesis of melanocytes.

Melanin Inhibitory Effect of Tuber himalayense Isolated in Incheon, Korea.

There has been a growing interest in skin beauty and antimelanogenic products. Melanogenesis is the process of melanin synthesis whereby melanocytes are activated by UV light or hormone stimulation to produce melanin. Melanogenesis is mediated by several enzymes, such as tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), and TRP-2. In this study, we investigated the effect of Tuber himalayense extract on melanin synthesis in α-melanocyte-stimulating hormone (α-MSH)-treated B16F10 melanoma cells. We confirmed that T. himalayense extract was not toxic to α-MSH-treated B16F10 melanoma cells and exhibited a significant inhibitory effect on melanin synthesis at concentrations of 25, 50, and 100 µg/ml. Additionally, the T. himalayense extract inhibited melanin, TRP-1, TRP-2, tyrosinase, and MITF, which are enzymes involved in melanin synthesis, in a concentration-dependent manner. Furthermore, T. himalayense extract inhibited the mitogen-activated protein kinase (MAPK) pathways, such as extracellular signal-regulated kinase-1/2 (ERK), c-Jun N-terminal kinase (JNK), and p38. Therefore, we hypothesized that various components of T. himalayense extract affect multiple factors involved in melanogenesis in B16F10 cells. Our results indicate that T. himalayense extract could potentially be used as a new material for preparing whitening cosmetics.

Multiple gene-drug prediction tool reveals Rosiglitazone based treatment pathway for non-segmental vitiligo.

Vitiligo is a skin disease characterized by selective loss of melanocytes, which seriously affects the appearance and causes great psychological stress to patients. In this study, we performed a comprehensive analysis of two vitiligo microarray datasets from the GEO database using bioinformatics tools to identify 297 up-regulated mRNAs and 186 down-regulated mRNAs, revealing important roles for pathways related to melanin synthesis, tyrosine metabolism, and inflammatory factors, such as "PPAR signaling pathway", "tyrosine metabolism", "nonalcoholic fatty liver disease (NAFLD) pathway", "melanogenesis", and "IL-17 signaling pathway". Combining the Search Tool for Interacting Chemicals (STITCH) database 5.0 and the drug-gene interaction database 3.0 (DGIdb), we identified that the PPAR-γ agonist rosiglitazone may promote melanin synthesis via EDNRB. Next, we investigated the mechanism of rosiglitazone and PPAR-γ pathway in promoting melanin production. Consistent with the results of bioinformatics analysis, the expression levels of PPAR-γ, EDNRB, and TYR were significantly reduced in human non-segmental vitiligo skin along with the reduction of MITF, a key gene for epidermal melanogenesis. Meanwhile, rosiglitazone increased melanin synthesis capacity in melanocytes and zebrafish by activating PPAR-γ and upregulating TYR, TYRP-1, and TYRP-2. Conversely, treatment of melanocytes with the PPAR-γ antagonist GW resulted in inhibition of melanin synthesis and expression of melanin-related factors. At the same time, simultaneous treatment of rosiglitazone with GW reversed the inhibitory effect of GW on melanin synthesis. In this study, we identified that rosiglitazone, an important insulin sensitizer, promotes melanin synthesis in melanocytes by increasing PPAR-γ activity and upregulating the expression levels of EDNRB and TYR. These findings may provide new ideas for exploring the pathogenesis and potential therapeutic targets of non-segmental vitiligo.

Neurog1-Derived Peptides RMNE1 and DualPep-Shine Penetrate the Skin and Inhibit Melanin Synthesis by Regulating MITF Transcription.

Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, "DualPep-Shine", which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.

Adipose tissue-derived Muse cells promote autophagy and oxidative stress tolerance in human epidermal melanocytes.

To investigate the effect of human adipose tissue-derived multilineage-differentiating stress-enduring (Muse) cells on the oxidative stress injury of human epidermal melanocytes (HEMs) in vitro. HEMs were treated with H2O2 to establish an oxidative stress injury model and then were co-cultured with adipose tissue-derived Muse cells. Immunohistochemistry, flow cytometry and Western blotting were used to assess changes in autophagy flux, apoptosis, expression of melanin synthesis related proteins and proliferation of melanocytes. Our findings demonstrate that co-culture with Muse cells significantly increased the tolerance of HEMs to oxidative stress, enhanced autophagy flux and reduced apoptosis. The expression of proteins related to the formation of melanin increased as did cell proliferation. Treatment with the autophagy inhibitor, 3-methyladenine (3MA), partially counteracted the improvement of oxidative stress tolerance in melanocytes elicited by co-culture with Muse cells. Muse cells promote autophagy and oxidative stress tolerance of melanocytes.

Deoxyvasicinone with Anti-Melanogenic Activity from Marine-Derived Streptomyces sp. CNQ-617.

The tricyclic quinazoline alkaloid deoxyvasicinone (DOV, 1) was isolated from a marine-derived Streptomyces sp. CNQ-617, and its anti-melanogenic effects were investigated. Deoxyvasicinone was shown to decrease the melanin content of B16F10 and MNT-1 cells that have been stimulated by α-melanocyte-stimulating hormone (α-MSH). In addition, microscopic images of the cells showed that deoxyvasicinone attenuated melanocyte activation. Although, deoxyvasicinone did not directly inhibit tyrosinase (TYR) enzymatic activity, real-time PCR showed that it inhibited the mRNA expression of TYR, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). In the artificial 3D pigmented skin model MelanodermTM, deoxyvasicinone brightened the skin significantly, as confirmed by histological examination. In conclusion, this study demonstrated that the marine microbial natural product deoxyvascinone has an anti-melanogenic effect through downregulation of melanogenic enzymes.

In vivo melanin 3D quantification and z-epidermal distribution by multiphoton FLIM, phasor and Pseudo-FLIM analyses.

Characterizing melanins in situ and determining their 3D z-epidermal distribution is paramount for understanding physiological/pathological processes of melanin neosynthesis, transfer, degradation or modulation with external UV exposure or cosmetic/pharmaceutical products. Multiphoton fluorescence intensity- and lifetime-based approaches have been shown to afford melanin detection, but how can one quantify melanin in vivo in 3D from multiphoton fluorescence lifetime (FLIM) data, especially since FLIM imaging requires long image acquisition times not compatible with 3D imaging in a clinical setup? We propose an approach combining (i) multiphoton FLIM, (ii) fast image acquisition times, and (iii) a melanin detection method called Pseudo-FLIM, based on slope analysis of autofluorescence intensity decays from temporally binned data. We compare Pseudo-FLIM to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes/keratinocytes coculture and in vivo human skin. Using parameters of global 3D epidermal melanin density and z-epidermal distribution profile, we provide first insights into the in vivo knowledge of 3D melanin modulations with constitutive pigmentation versus ethnicity, with seasonality over 1 year and with topical application of retinoic acid or retinol on human skin. Applications of Pseudo-FLIM based melanin detection encompass physiological, pathological, or environmental factors-induced pigmentation modulations up to whitening, anti-photoaging, or photoprotection products evaluation.

Human immunocompetent choroid-on-chip: a novel tool for studying ocular effects of biological drugs.

Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies.

Intragenic recombination within the pun allele of the pink-eyed dilution locus in pre-melanocytes and primordial germ cells of embryonic mice treated with N-ethyl-N-nitrosourea.

The forward or reverse processes of intragenic recombination (IGR), which occur through the addition or deletion of duplicated homologous exons of the pun allele in Pun mice, was observed in vivo, after introducing an homozygous pun allele in a C57BL/6 background. We assessed the frequency of IGR upon N-ethyl-N-nitrosourea (ENU) treatment of pre-melanocytes (PMCs: somatic cells) and primordial germ cells (PGCs: germ cells) of embryonic mice at 10.5 days of development (E10.5). We simultaneously examined IGR and other mutations at the p locus of PMCs responsible for coat color in the offspring obtained by crossing pun/pun with pun/P mice. The frequencies of both spontaneous and ENU-induced IGR were markedly higher than that of the recessive mutation (RM) in PMCs obtained from crossing C57BL/6 and PW strains (Shibuya et al., 1982). ENU also induces IGR at a higher frequency in PGCs at E10.5, which was observed in the next generation. These results indicate that ENU, which preferentially induces gene mutations through base substitution, also induces IGR at a high frequency in the pun allele in both somatic and germ cells of embryonic mice at the E10.5 developmental stage.

The combination of sucrose dilaurate and sucrose laurate suppresses HMGB1: an enhancer of melanocyte dendricity and melanosome transfer to keratinocytes.

BACKGROUND: Hyperpigmented spots are common issues in all ethnicities, involving multiple intrinsic and extrinsic factors such as UVB exposure, hormone balance, inflammatory status and ageing. OBJECTIVES: To determine (i) melanocyte dendricity in multiple facial spot types, (ii) impact of High Mobility Group Box 1 (HMGB1), and the combination of sucrose dilaurate and sucrose laurate (SDL) on melanogenesis and melanocyte dendricity, and (iii) SDL effect on facial spots in a human use test. METHODS: Facial spot and adjacent non-spot skin biopsies were collected from Chinese women (age 20-70). Histological assessment of melanocyte dendricity was performed for 3 spot types (solar lentigo, melasma and postinflammatory hyperpigmentation) by immunofluorescent staining for c-kit/MITF. Keratinocyte, melanocyte and melanocyte-keratinocyte co-culture models were used to assess HMGB1 release by UVB radiation, the effects of HMGB1 and SDL on melanin production, melanocyte dendricity and melanosome transfer. The effect of an SDL-containing moisturizer on appearance of facial hyperpigmented spots was assessed against a vehicle control in an 8-week human use test. RESULTS: Melanocytes in spot areas are more dendritic than melanocytes in adjacent non-spot skin across three investigated spot types. In cell culture models, a moderate UVB-radiation exposure caused release of HMGB1 from keratinocytes. HMGB1 did not alter melanin production in melanocytes, but enhanced melanocyte dendricity and melanosome transfer. SDL reduced HMGB1 release from keratinocytes, inhibited melanin production, reversibly suppressed melanocyte dendricity and reduced melanosome transfer. In the human use test, SDL-containing moisturizer reduced appearance of spots versus vehicle. CONCLUSION: Increased melanocyte dendricity was observed in multiple types of facial spots. Addition of HMGB1 protein increased melanocyte dendricity and melanosome transfer in cell cultures, implicating potential involvement in spot formation. SDL suppressed melanin production, melanocyte dendricity and melanosome transfer in vitro and reduced appearance of spots in the use test, suggesting SDL is an effective solution to address hyperpigmented spot concerns.

Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

7-desacetoxy-6,7-dehydrogedunin discovered by high-throughput screening system suppresses melanogenesis through ATP-P2X7 signaling inhibition.

BACKGROUND: Hyperpigmented skin disorders such as melasma and lentigo are common photoaging diseases that cause cosmetic problems. The pigmentation is usually exacerbated by ultraviolet (UV) radiation, and various factors and pathways are involved in UV-mediated melanogenesis. Adenosine 5'-triphosphate (ATP), a well-known molecular unit of intracellular energy, is also regarded as a mediator of UV-mediated melanogenesis via the P2X7 purinergic receptor. OBJECTIVE: To discover natural substances with an anti-melanogenic effect through inhibition of ATP-P2X7 axis by high-throughput screening (HTS). METHODS: Among natural compounds provided by the Korea Chemical Bank, chemical compounds with a P2X7 inhibiting effect were screened through an HTS system. Then the selected compounds were verified for their anti-melanogenic effect after treating primary human epidermal melanocytes (PHEMs) with and without ATP. The expression of MITF, tyrosinase, and PMEL/gp100 was analyzed by Western blot, and melanin content was measured as 405 nm absorbance. RESULTS: Among 962 natural compounds, 58 showed greater than 80% suppression of YO-PRO-1 fluorescence, representing P2X7 activity. Among them, considering cell viability, chemical stability, and availability, 7-desaxacetoxy-6,7-dehydrogedunin (7DG), a limonoid natural compound, was selected. The expression of MITF, tyrosinase, and PMEL/gp100; tyrosinase enzyme activity; and melanin content, which were increased by ATP treatment were abrogated by 7DG. Even when 7DG was treated in PHEMs without addition of ATP, tyrosinase expression and melanin content were significantly decreased. Hypopigmenting effect of 7DG was confirmed in ex vivo culture of human skins. CONCLUSIONS: 7DG has an anti-melanogenic effect through ATP-P2X7 pathway inhibition and could be a potential skin whitening material.

Ponatinib-induced eruptive nevi and melanocytic proliferation.

Ponatinib, an oral third-generation tyrosine kinase inhibitor, is indicated for the treatment of imatinib-resistant leukemia. We experienced a case of ponatinib-induced eruptive nevi, and the biologic effects of ponatinib on melanocytes were investigated. Treatment with ponatinib significantly increased the proliferation of normal human melanocyte or melanoma cells through the upregulation of the extracellular signal-regulated kinase and protein kinase B signaling pathways. The downstream molecules of cyclin B1 and D1 were significantly increased in ponatinib-treated melanocytes. These results demonstrate the capacity of ponatinib to induce the proliferation and tumorigenesis of melanocytes.

Folic Acid Protects Melanocytes from Oxidative Stress via Activation of Nrf2 and Inhibition of HMGB1.

Vitiligo is a cutaneous depigmentation disease due to loss of epidermal melanocytes. Accumulating evidence has indicated that oxidative stress plays a vital role in vitiligo via directly destructing melanocytes and triggering inflammatory response that ultimately undermines melanocytes. Folic acid (FA), an oxidized form of folate with high bioavailability, exhibits potent antioxidant properties and shows therapeutic potential in multiple oxidative stress-related diseases. However, whether FA safeguards melanocytes from oxidative damages remains unknown. In this study, we first found that FA relieved melanocytes from H2O2-induced abnormal growth and apoptosis. Furthermore, FA enhanced the activity of antioxidative enzymes and remarkably reduced intracellular ROS levels in melanocytes. Subsequently, FA effectively activated nuclear factor E2-related factor 2 (Nrf2) pathway, and Nrf2 knockdown blocked the protective effects of FA on H2O2-treated melanocytes. Additionally, FA inhibited the production of proinflammatory HMGB1 in melanocytes under oxidative stress. Taken together, our findings support the protective effects of FA on human melanocytes against oxidative injury via the activation of Nrf2 and the inhibition of HMGB1, thus indicating FA as a potential therapeutic agent for the treatment of vitiligo.

Methylcobalamin Protects Melanocytes from H2O2-Induced Oxidative Stress by Activating the Nrf2/HO-1 Pathway.

PURPOSE: Oxidative stress is considered a major determinant in the pathogenesis of vitiligo. Methylcobalamin (MeCbl) is an activated form of vitamin B12 that regulates inflammatory factors, counters oxidative stress, and reduces apoptosis in many disease models. However, the specific mechanism of MeCbl repigmentation against vitiligo is unknown. In this study, we explored the effect of MeCbl on melanocytes following hydrogen peroxide (H2O2)-induced oxidative stress. METHODS: We established an oxidative stress model using the immortalized human normal melanocyte cell line PIG1. We used a Cell Counting Kit-8 (CCK-8) to detect drug cytotoxicity, and we measured the melanin content of cells using the NaOH method. Intracellular oxidative damage was assessed by flow cytometry and antioxidant enzyme detection kits. In addition, we assessed the presence of apoptosis by flow cytometry and Western blots. We explored the underlying mechanisms of MeCbl during oxidative stress in melanocytes by analyzing the results of experiments based on real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and laser scanning confocal immunofluorescence microscopy. Finally, we repeated the experiments after applying an inhibitor to block the Nrf2 pathway. RESULTS: We found that MeCbl treatment enhanced cell viability, increased melanin content, reduced intracellular reactive oxygen species (ROS) accumulation, increased the activities of antioxidant enzyme superoxide dismutase (SOD) and catalase (CAT), reduced melanocyte apoptosis, and up-regulated the expression of the Nrf2/HO-1 pathway. Moreover, the protective effects of MeCbl were significantly weakened after inhibiting the Nrf2/HO-1 pathway. CONCLUSION: Our results indicate that MeCbl attenuated the H2O2-induced oxidative stress in melanocytes by activating the Nrf2/HO-1 pathway, this suggests that MeCbl may be an effective treatment against vitiligo.

Pseudoalteromone A, a Ubiquinone Derivative from Marine Pseudoalteromonas spp., Suppresses Melanogenesis.

An ubiquinone derivative, pseudoalteromone A (1), has been isolated from two marine-derived Pseudoalteromonas spp., APmarine002 and ROA-050, and its anti-melanogenesis activity was investigated. The anti-melanogenic capacity of pseudoalteromone A was demonstrated by assessing the intracellular and extracellular melanin content and cellular tyrosinase activity in the B16 cell line, Melan-a mouse melanocyte cell line, and MNT-1 human malignant melanoma cell line. Treatment with pseudoalteromone A (40 µg/mL) for 72 h reduced α-melanocyte-stimulating hormone (α-MSH)-induced intracellular melanin production by up to 44.68% in B16 cells and 38.24% in MNT-1 cells. Notably, pseudoalteromone A induced a concentration-dependent reduction in cellular tyrosinase activity in B16 cell, and Western blot analyses showed that this inhibitory activity was associated with a significant decrease in protein levels of tyrosinase and tyrosinase-related protein 1 (Tyrp-1), suggesting that pseudoalteromone A exerts its anti-melanogenesis activity through effects on melanogenic genes. We further evaluated the skin-whitening effect of pseudoalteromone A in the three-dimensional (3D) pigmented-epidermis model, MelanoDerm, and visualized the 3D distribution of melanin by two-photon excited fluorescence imaging in this human skin equivalent. Collectively, our findings suggest that pseudoalteromone A inhibits tyrosinase activity and expression and that this accounts for its anti-melanogenic effects in melanocytes.

CRTC3, a sensor and key regulator for melanogenesis, as a tunable therapeutic target for pigmentary disorders.

Background: Although CREB phosphorylation is known to be essential in UVB/cAMP-stimulated melanogenesis, CREB null mice did not show identifiable pigmentation phenotypes. Here, we show that CREB-regulated transcription co-activator 3 (CRTC3) quantitatively regulates and orchestrates melanogenesis by directly targeting microphthalmia-associated transcription factor (MITF) and regulating the expression of most key melanogenesis-related genes. Methods: We analyzed CRTC3-null, KRT14-SCF transgenic, and their crossover mice. The molecular basis of CRTC3 effects on pigmentation was investigated by histology, melanin/tyrosinase assay, immunoblotting, shRNA, promoter assay, qRT-PCR, and subcellular localization. These analyses were carried out in primary cultured melanocytes, mouse cell lines, normal human cells, co-cultures, and ex vivo human skin. CRTC/CREB activity screening was performed to identify candidate agents for the regulation of melanogenesis. Results: The coat and skin color of CRTC3-null mice was paler due to a reduction in melanin deposition. Melanogenesis-related genes were reduced in CRTC3-deficient cultured melanocytes and tail skin of CRTC3-null mice. Notably, basal levels of MITF present in CRTC3-null mice were sufficient for melanocytic differentiation/survival. Thus CRTC3-null mice showed a comparable number of epidermal melanocytes compared to control mice. Stem cell factor (SCF) introduction by crossing with KRT14-SCF mice increased epidermal melanocytes and melanin deposition in control and CRTC3-null mice, but the skin color remained still light on the CRTC3-null background. Furthermore, we identified the therapeutic potential of altiratinib to inhibit melanogenesis in human melanocytes and human skin effectively and safely. Conclusion: CRTC3 appears to be a key sensor for melanogenesis and can be used as a reversible and tunable tool for selectively regulating melanogenesis without affecting melanocyte integrity. Thus, CRTC3 can also serve as a screening tool for the discovery of ideal melanogenesis-modulating small molecules.