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BACKGROUND: The specific role of oxidative stress (OS) in vitiligo and alopecia areata (AA) remains unclear. The aim of this study was to analyze and identify the key markers of OS in vitiligo and AA by bioinformatics. METHODS: We obtained vitiligo and AA datasets from gene expression omnibus (GEO) database. The difference-expressed genes of vitiligo and AA were identified by differential analysis, and the functions of difference-expressed genes were identified by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) enrichment analysis. Subsequently, Veen package was used to obtain the intersection genes of OS-related genes with vitiligo and AA. Finally, we used CIBERSORT to assess the infiltration of immune cells in vitiligo and AA. RESULTS: Through enrichment analysis, we found that vitiligo and AA were mainly enriched in cell cycle and cell adhesion molecular channels. We identified KLB and EIF3C as key genes in OS regulation of vitiligo and AA, and found that KLB and EIF3C participate in disease progression by regulating T cells and neutrophils. CONCLUSIONS: According to our findings, KLB and EIF3C play a crucial role in the progression and development of vitiligo and AA, which have been identified as biomarkers and target for early diagnosis of patients.
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Alopecia Areata , Estrés Oxidativo , Vitíligo , Vitíligo/genética , Alopecia Areata/genética , Humanos , Estrés Oxidativo/genética , Biomarcadores/metabolismo , Biología Computacional , Perfilación de la Expresión Génica , Ontología de Genes , Bases de Datos GenéticasRESUMEN
The decreased adhesion ability of melanocytes to the neighboring keratinocytes prompts melanocytes to lose from the epidermis, comprising the critical step in vitiligo pathogenesis. The repigmentation process involves the migration of melanocytes to the lesional area. This study aims to investigate the role and mechanism of microRNA (miR)-9 in the adhesion and migration of melanocytes during vitiligo repigmentation induced by UVB treatment. The HaCaT keratinocytes were used to mimic lesional condition and the PIG1 melanocytes as perilesional condition. Human lesional vitiligo specimens showed increased miR-9 and decreased adhesion molecules such as E-cadherin and ß1 integrin. Furthermore, UVB exposure upregulated IL-10, E-cadherin, and ß1 integrin, downregulated miR-9 in HaCaT cells. Moreover, the increased IL-10 by UVB exposure decreased miR-9 level by inducing miR-9 methylation via methyltransferase DNMT3A in HaCaT cells. Additionally, miR-9 targeted and inhibited E-cadherin and ß1 integrin in HaCaT cells, and suppressed migration of PIG1 cells to UVB-exposed HaCaT cells. In conclusion, miR-9 was suppressed by IL-10 and inhibited migration of PIG1 cells to HaCaT cells during UVB-mediated vitiligo repigmentation.
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Adhesión Celular/genética , Movimiento Celular/genética , Melanocitos/patología , MicroARNs/genética , Pigmentación de la Piel/genética , Vitíligo/genética , Cadherinas/genética , Estudios de Casos y Controles , Línea Celular , Regulación hacia Abajo/genética , Humanos , Cadenas beta de Integrinas/genética , Interleucina-10/genética , Queratinocitos/patología , Rayos UltravioletaRESUMEN
BACKGROUND: Vitiligo is an autoimmune skin disorder primarily characterized by the absence of melanocytes, leading to the development of white patches on the patient's skin. Narrowband Ultraviolet B (NB-UVB) therapy is among the most effective approaches for stimulating the reformation of hyperpigmentation. This treatment utilizes a narrow spectrum of NBUVB wavelengths ranging from 311 to 313 nm to irradiate the affected area, thereby preventing the destruction of migrating and proliferating melanocytes. Nevertheless, the molecular alterations occurring in both the hair follicle and the interfollicular epidermis during NB-UVB treatment remain unknown. METHODS: In this study, we conducted a comprehensive analysis of the consistency of differentially expressed genes (DEGs) within the enrichment pathways both before and after NB-UVB treatment, utilizing a bioinformatics approach. Furthermore, we employed CYTOHUBBA and Random Forest algorithms to identify and sequence hub genes from the pool of DEGs. Following validation of these hub genes through ROC curve analysis, we proceeded to construct an interaction network between these hub genes, miRNA, and drugs. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was used to further verify the difference in the expression of hub genes between the disease group and the control group. RESULTS: Gene Set Enrichment Analysis of DEGs indicated strong associations with vitiligo in most pathways. Subsequently, we conducted Gene Ontology and Metascape enrichment analyses on the overlapping genes from DEGs. We identified key genes (COL11A1, IGFBP7, LOX, NTRK2, SDC2, SEMA4D, and VEGFA) within the Protein-Protein Interaction (PPI) network. We further explored potential drugs that could be used for the clinical treatment of vitiligo through the drug-hub gene interaction network. Finally, the results of RT-qPCR experiments demonstrated that the expression levels of the identified hub genes in both groups were consistent with the bioinformatics analysis results. CONCLUSION: The hub genes obtained in this study may be a biomarker related to the development of vitiligo pigmentation. Our research not only contributes to a better understanding of the treatment mechanisms of vitiligo but also provides valuable insights for future personalized medical approaches and targeted therapies for vitiligo.
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Clinical studies have proven that ultraviolet B (UVB) based phototherapy can induce perifollicular and marginal repigmentation patterns in the skin of vitiligo patients. It is, however, difficult to conceive how melanocytes can easily exit from their tightly interconnected epidermal microenvironment to reenter a different location in the skin to establish a new network with neighboring keratinocytes. While it is known that matrix metalloprotease 9 (MMP9) is involved in the degradation of the extracellular matrix in physiological or pathological processes, little is known about whether MMP9 affects melanocyte migration in vitiligo repigmentation. To investigate the effects of the p53 transient receptor potential cation channel subfamily M member 1 (TRPM1)/microRNA (miR/miRNA)211MMP9 axis to regulate melanocyte migration following exposure to UVB, the expression profile of MMP9 in cultured human melanocytes transfected with or without the miR211mimic and p53GFP lentiviral vector, respectively were determined. Quantitative polymerase chain reaction and western blotting were used to examine p53, TRPM1 and MMP9 mRNA and protein levels in UVBexposed and unexposed cells. The capacity of melanocytes to migrate on collagen IV substrate was estimated using a Transwell migration assay. Interestingly, the upregulation of p53 and MMP9 at the mRNA and protein levels was evident in melanocytes treated with single or repeat exposures to UVB, whereas levels of TRPM1 and miR211 were significantly suppressed in UVBexposed melanocytes compared with the UVBunexposed control cells. These results indicate that the p53TRPM1/miR211MMP9 axis is significantly activated in melanocytes exposed to UVB. Notably, the ability of melanocyte migration was altered by the overexpression of p53 using a lentiviral vector and by the upregulation of miR211 using an miRNA mimic. That altered migration could be neutralized by cotreatment with GM6001 (a broadspectrum MMP inhibitor). Overall, these results show that the MMP9mediated migration of melanocytes is regulated by a novel mechanism driven by the p53TRPM1/miR211MMP9 axis. Activation of the p53TRPM1/miR211MMP9 axis potentially represents an attractive therapeutic target to improve repigmentation outcomes in vitiligo patients.
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Metaloproteinasa 9 de la Matriz/metabolismo , Melanocitos/metabolismo , Melanocitos/efectos de la radiación , MicroARNs/metabolismo , Canales Catiónicos TRPM/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta , Adolescente , Adulto , Western Blotting , Movimiento Celular/efectos de la radiación , Células Cultivadas , Humanos , Adulto JovenRESUMEN
Although vitamin C (VC, L-ascorbic acid) has been widely used as a skin lightening agent for a long time, the mechanism by which it inhibits melanogenesis remains poorly understood. It is well-documented that the intramelanocytic pH is an important factor in regulating tyrosinase function and melanosome maturation. The activity of tyrosinase, the rate-limiting enzyme required for melanin synthesis, is generally minimal in an acidic environment. Given that VC is an acidic compound, we might speculate that the intracellular acidification of melanocytes induced by VC likely reduces melanin content through the suppression of tyrosinase activity. The results of this study reveal that treatment of melanocytes with VC or its derivatives, magnesium ascorbyl phosphate (MAP) and 3-O-ethyl-L-ascorbic acid (AAE), resulted in significant decreases in the tyrosinase activity and melanin content and in the levels of intracellular reactive oxygen species (ROS), indicating that VC and its derivatives possess antimelanogenic and antioxidative activities. Western blotting analysis indicated that VC, MAP, and AAE exert their antimelanogenic activity by inhibiting the tyrosinase activity rather than by downregulating the expression of melanogenic proteins such as tyrosinase, premelanosome protein 17 (Pmel17) and microphthalmia-associated transcription factor (MITF). Further, we found that the reduced tyrosinase activity of melanocytes treated with VC or its derivatives could be reactivated following intracellular neutralization induced by ammonium chloride (NH4Cl) or concanamycin A (Con A). Finally, we examined the expression of sodium-dependent VC transporter-2 (SVCT-2) using western blotting and qPCR, which revealed that there was a significant increase in the expression of SVCT-2 in melanocytes following treatment with VC. VC-mediated intracellular acidification was neutralized by phloretin (a putative SVCT-2 inhibitor) in a dose-dependent manner. Taken together, these data show that VC and its derivatives suppress tyrosinase activity through cytoplasmic acidification that potentially results from enhanced VC transmembrane transport via the VC transporter SVCT-2.
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Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Citoplasma/metabolismo , Melaninas/metabolismo , Melanocitos/fisiología , Melanosomas/metabolismo , Monofenol Monooxigenasa/metabolismo , Preparaciones para Aclaramiento de la Piel/metabolismo , Animales , Ácido Ascórbico/análogos & derivados , Diferenciación Celular , Línea Celular , Citoplasma/química , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Pigmentación de la Piel , Transportadores de Sodio Acoplados a la Vitamina C/genética , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Regulación hacia ArribaRESUMEN
Accumulating evidence suggests a potential role of transient receptor potential vanilloid 1 (TRPV1) channels in inflammatory and cancer-related pain. However, the role of TRPV1 in the maintenance of neuropathic pain remains elusive. The current study investigated the effects of transient Trpv1 gene silencing using a small interference RNA (siRNA) on neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. Seven days after CCI, the TRPV1 siRNA was intrathecally administered (5 µg/15 µl, once daily for 2 days). TRPV1 and Ca2+/calmodulin-dependent protein kinase II (CAMKII) expression and extracellular signal-regulated kinase (ERK) phosphorylation in the spinal cord were detected using western blotting. The thresholds to mechanical and thermal stimuli were determined before and after intrathecal TRPV1 siRNA administration. TRPV1 and CAMKII expression and ERK2 phosphorylation in the spinal cord were upregulated after CCI. Intrathecal administration of the TRPV1 siRNA not only attenuated behavioural hyperalgesia but also reduced the expression of TRPV1 and CAMKII, as well as ERK2 phosphorylation. Based on these results, silencing of the TRPV1 gene in the spinal cord attenuates the maintenance of neuropathic pain by inhibiting CAMKII/ERK2 activation and suggests that TRPV1 represents a potential target in pain therapy.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Neuralgia/patología , Médula Espinal/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Constricción Patológica , Masculino , Neuralgia/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genéticaRESUMEN
Baicalin is a traditional Chinese herbal medicine commonly used for hair loss, the precise molecular mechanism of which is unknown. In the present study, the mechanism of baicalin was investigated via the topical application of baicalin to reconstituted hair follicles on mice dorsa and evaluating the effect on canonical Wnt/ßcatenin signaling in the hair follicles and the activity of dermal papillar cells. The results indicate that baicalin stimulates the expression of Wnt3a, Wnt5a, frizzled 7 and disheveled 2 whilst inhibiting the Axin/casein kinase 1α/adenomatous polyposis coli/glycogen synthase kinase 3ß degradation complex, leading to accumulation of ßcatenin and activation of Wnt/ßcatenin signaling. In addition, baicalin was observed to increase the alkaline phosphatase levels in dermal papillar cells, a process which was dependent on Wnt pathway activation. Given its nontoxicity and ease of topical application, baicalin represents a promising treatment for alopecia and other forms of hair loss. Further studies of baicalin using human hair follicle transplants are warranted in preparation for future clinical use.
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Medicamentos Herbarios Chinos/farmacología , Flavonoides/farmacología , Folículo Piloso/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Alopecia/tratamiento farmacológico , Alopecia/metabolismo , Animales , Células Cultivadas , Femenino , Folículo Piloso/citología , Folículo Piloso/metabolismo , Folículo Piloso/ultraestructura , Ratones , Ratones Endogámicos BALB C , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Melanosomes are membrane-bound intracellular organelles that are uniquely generated by melanocytes (MCs) in the basal layer of human epidermis. Highly pigmented mature melanosomes are transferred from MCs to keratinocytes (KCs), and then positioned in the supra-nuclear region to ensure protection against ultraviolet radiation (UVR). However, the molecular mechanism underlying melanosome (or melanin pigment) transfer remains enigmatic. Emerging evidence shows that exo-/endo-cytosis of the melanosome core (termed melanocore) has been considered as the main transfer manner between MCs and KCs. As KCs in the skin migrate up from the basal layer and undergo terminal differentiation, the melanocores they have taken up from MCs are subjected to degradation. In this study, we isolated individual melanocores from human MCs in culture and then induced their destruction/disruption using a physical approach. The results demonstrate that the ultrastructural integrity of melanocores is essential for their antioxidant and photoprotective properties. In addition, we also show that cathepsin V (CTSV), a lysosomal acid protease, is involved in melanocore degradation in calcium-induced differentiated KCs and is also suppressed in KCs following exposure to UVA or UVB radiation. Thus, our study demonstrates that change in the proportion of melanocores in the intact/undegraded state by CTSV-related degradation in KCs affects photoprotection of the skin.
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Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Fibroblastos/efectos de la radiación , Queratinocitos/efectos de la radiación , Melanocitos/efectos de la radiación , Melanosomas/efectos de la radiación , Antioxidantes/metabolismo , Transporte Biológico , Catepsinas/genética , Diferenciación Celular , Fraccionamiento Celular , Cisteína Endopeptidasas/genética , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Prepucio/citología , Prepucio/metabolismo , Expresión Génica , Humanos , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Masculino , Melaninas/química , Melaninas/metabolismo , Melanocitos/metabolismo , Melanocitos/ultraestructura , Melanosomas/química , Melanosomas/metabolismo , Cultivo Primario de Células , Proteolisis , Rayos UltravioletaRESUMEN
OBJECTIVES: The transfer of melanosomes from melanocytes to neighbouring keratinocytes is critical to protect the skin from the deleterious effects of ultraviolet A (UVA) and ultraviolet B (UVB) irradiation; however, the initial factor(s) that stimulates melanosome transfer remains unclear. In this study, we investigated the induction of retinal-dependent calcium (Ca2+ ) influx in melanocytes (MCs) by UVA or UVB irradiation and the effect of transient receptor potential cation channel subfamily M member 1 (TRPM1) (melastatin1)-related Ca2+ influx on melanosome transfer. MATERIALS AND METHODS: Primary human epidermal MCs were exposed to physiological doses of UVB or UVA light and loaded with a calcium indicator Fluo-4 dye. The change of intracellular calcium of MCs was monitored using a two-photon confocal fluorescence microscopy. MCs were co-cultured with human epidermal keratinocytes (KCs) in the absence or presence of voriconazole (a TRPM1 blocker) or calcium chelators. MCs were also transfected with TRPM1 siRNA for silencing the expression of TRPM1 gene. The melanosome transfer in the co-cultured cells was quantitatively analysed using flow cytometry and was further confirmed by immunofluorescent double-staining. The protein levels and distributions of TRPM1, OPN3 and OPN5 in MCs were measured by Western blotting or immunofluorescent staining. RESULTS: The retinal-dependent Ca2+ influx of UVA-exposed melanocytes differed greatly from that of UVB-exposed melanocytes in the timing-phase. The protein expression of TRPM1 in mono- and co-cultured MCs was dose-dependently up-regulated by UVA and UVB. TRPM1 siRNA-mediated knockdown and the blockage of TRPM1 channel using a putative antagonist (voriconazole) significantly inhibited melanosome transfer in co-cultures following UVA or UVB exposure. CONCLUSIONS: The distinct time-phases of Ca2+ influx in MCs induced by UVA or UVB contribute to the consecutive stimulation of melanosome transfer, thereby providing a potent photoprotection against harmful UV radiation.