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1.
Oxid Med Cell Longev ; 2019: 2084805, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31214276

RESUMEN

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.


Asunto(s)
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 Arriba
2.
Sci Rep ; 9(1): 2769, 2019 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-30808963

RESUMEN

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.


Asunto(s)
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/genética
3.
Cell Cycle ; 17(7): 844-857, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29623762

RESUMEN

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.


Asunto(s)
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 Ultravioleta
4.
Int J Mol Med ; 41(4): 2079-2085, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29336472

RESUMEN

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.


Asunto(s)
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/metabolismo
5.
Cell Prolif ; 50(6)2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28833830

RESUMEN

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.


Asunto(s)
Calcio/metabolismo , Melanocitos/metabolismo , Melanosomas/metabolismo , Rayos Ultravioleta , Células Cultivadas , Técnicas de Cocultivo/métodos , Humanos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Melaninas/biosíntesis , Melanocitos/efectos de la radiación , Melanosomas/efectos de la radiación , Rayos Ultravioleta/efectos adversos
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