UVB-induced Melanogenesis Research Articles

UVB-induced TRPS1 regulates MITF transcription activity to promote skin pigmentation

Hyperpigmented dermatoses are characterized by increased skin pigmentation caused by genetic, environmental factors and inflammation, which lasts a long time and is difficult to treat. Ultraviolet (UV), especially ultraviolet B (UVB), is the primary external factor inducing skin pigmentation. However, the specific regulatory mechanisms are not fully understood. Through analysis of GEO datasets from four UV-exposed skin cell/tissue samples, we found that TRPS1 is the only gene differentially expressed in multiple datasets (GSE22083, GSE67098 and GSE70280) and highly positively correlated with the expression of key melanogenesis genes. Consistently, we observed that TRPS1 is highly expressed in sun-exposed skin tissues compared to non-exposed skin. Additionally, the expression of TRPS1 was also significantly upregulated after UVB irradiation in isolated skin tissues and melanocytes, while knockdown of TRPS1 expression inhibited the UVB-induced melanogenesis. Further research revealed that overexpression of TRPS1 increased melanin content and tyrosinase activity in MNT1 cells, as well as upregulated the expression levels of key melanogenesis genes (MITF, TYR, TYRP1, DCT). In contrast, inhibition of TRPS1 expression showed the opposite effect. Moreover, we found that TRPS1 can bind to the promoter region of MITF, inhibiting the expression of MITF can antagonize the melanogenesis induced by TRPS1. In conclusion, UVB-induced TRPS1 promotes melanogenesis by activating the transcriptional activity of MITF.

UVB promotes melanogenesis by regulating METTL3.

Ultraviolet (UV) radiation is the primary exogenous inducer of skin pigmentation, although the mechanism has not been fully elucidated. N6-methyladenosine (m6 A) modification is one of the key epigenetic form of gene regulation that affects multiple biological processes. The aim of this study was to explore the role and underlying mechanisms of m6 A modification in UVB-induced melanogenesis. Low-dose UVB increased global m6 A modification in melanocytes (MCs) and MNT1 melanoma cell line. The GEPIA database predicted that methyltransferase METTL3 is positively correlated with the melanogenic transcription factor MITF in the sun-exposed skin tissues. After METTL3 respectively overexpressed and knocked down in the MNT1, the melanin content and melanogenesis-related genes were significantly upregulated after overexpression of METTL3, especially with UVB irradiation, and downregulated after METTL3 knockdown. METTL3 levels were also higher in melanocytic nevi with high melanin content. METTL3 overexpression and knockdown also altered the protein level of YAP1. SRAMP analysis predicted four high-potential m6 A modification sites on YAP1 mRNA, of which three were confirmed by methylated RNA immunoprecipitation. Inhibition of YAP1 expression can partially reverse melanogenesis induced by overexpression of METTL3. In conclusion, UVB irradiation promotes global m6 A modification in MCs and upregulates METTL3, which increases the expression level of YAP1 through m6 A modification, thereby activating the co-transcription factor TEAD1 and promoting melanogenesis.

Solamargine Alleviated UVB-Induced Inflammation and Melanogenesis in Human Keratinocytes and Melanocytes via the p38 MAPK Signaling Pathway, a Promising Agent for Post-inflammatory Hyperpigmentation.

Post-inflammatory hyperpigmentation (PIH) is a common acquired pigmentary disorder occurring after skin inflammation or injury. Ultraviolet B irradiation could exaggerate PIH clinically due to its effect on promoting cutaneous inflammation and melanogenesis in keratinocytes and melanocytes, respectively. Solamargine (SM), a steroidal alkaloid glycoside extracted from Solanum undatum, significantly inhibits Ultraviolet B (UVB)-induced pro-inflammatory cytokines IL-1α, IL-1β, IL-8, and IFN-γ, as well as paracrine melanogenic factors ET-1, α-MSH, and bFGF in human keratinocytes. Additionally, SM significantly attenuated UVB-induced melanin synthesis in human epidermal melanocytes through down-regulation of tyrosinase activity and expression of MITF, TRP-1, TRP-2, and tyrosinase. SM exerted an anti-inflammatory effect in UVB-irradiated keratinocytes through the p38 MAPK/Nrf2/HO-1 signaling pathway. With its anti-inflammatory and whitening effect, SM may improve PIH through paracrine regulations of keratinocytes and direct action on melanocytes, making it a promising agent for PIH.

Silencing of Rab23 by siRNA inhibits ultraviolet B-induced melanogenesis via downregulation of PKA/CREB/MITF.

Recent investigations have shown that the Rab family of GTPases is associated with all aspects of melanogenesis. However, the effect of Rab23, which localizes to the plasma membrane and regulates the endocytic pathway within eukaryotic cells, in melanogenesis has not been reported. To understand the role of Rab23 in UVB-induced melanogenesis, we evaluated changes in the level of melanin, activity of tyrosinase and levels of melanogenesis-related proteins such as microphthalmia transcription factor and tyrosinase-related protein-1 (TRP-1) and the melanosome transport-related protein complex Rab27a-melanophilin-myosin Va after the downregulation of Rab23 in B16F10 and SK-MEL-2 cells with or without UVB irradiation. Our results showed that downregulating Rab23 reduced the melanin level and tyrosinase activity and inhibited the expression of proteins involved in UVB-induced melanogenesis. Rab23 colocalized with mature melanosomes marked with TRP-1. Furthermore, downregulating Rab23 induced the abnormal accumulation of melanosomes around the nucleus. We demonstrated that the downregulation of Rab23 inhibited melanin synthesis and melanosome transport by decreasing the PKA/CREB/MITF pathway, which is the key regulator of UVB-induced melanogenesis.

UVB-Induced Secretion of IL-1β Promotes Melanogenesis by Upregulating TYR/TRP-1 Expression In Vitro.

Purpose Ultraviolet radiation (UVR) is one of the exogenous stimuli increasing melanogenesis. UV light, especially UVB, is also a potent inducer of epidermal cytokine release. This study is aimed at determining the underlying mechanisms by which UVB-induced cytokines in keratinocytes regulate melanin production in vitro. Methods Expression levels of mRNA for interleukin- (IL-) 1, IL-1β, IL-6, IL-10, IL-17, and tumor necrosis factor-alpha (TNF-α) were measured using RT-qPCR at various time points after UVB irradiation in C57BL/6 mice and HaCaT cells. NaOH lysis and L-dihydroxyphenylalanine (L-DOPA) oxidation method were used to measure melanin content and tyrosinase (TYR) activity, respectively, in melanoma B16 cells. RT-qPCR and Western blot were used to assess mRNA and protein levels of microphthalmia-associated transcription factor (MITF), TYR, tyrosine-related protein-1 (TRP-1), and tyrosine-related protein-2 (TRP-2) in B16 cells. Finally, expression levels of cyclooxygenase-2 (COX-2) mRNA and stem cell factor (SCF) in HaCaT cells were measured following knockdown of IL-1β using siRNA (siIL-1β). Results UVB irradiation increased IL-1β mRNA expression levels in both C57BL/6 mice and HaCaT cells. The melanin content, TYR activity, and expression levels of TYR and TRP-1 were all raised when B16 cells were treated with 4 pg/l of IL-1. Moreover, IL-1β also upregulated the expression levels of SCF and COX-2 in nonirradiated HaCaT cells. Conversely, knockdown of IL-1β attenuated UVB irradiation-induced upregulation of SCF and COX-2 expression in keratinocytes. Conclusions UVB-induced melanogenesis is mediated in part by IL-1β, leading to upregulation of the TYR/TRP1 expression in melanoma B16 cells. IL-1β can also stimulate the expression of COX-2 and SCF in HaCaT cells, which in turn increase melanin synthesis in melanocytes. These results suggest that anti-inflammatory approaches could possibly mitigate UVB-induced hyperpigmentation.

Platelet-rich plasma protects human melanocytes from oxidative stress and ameliorates melanogenesis induced by UVB irradiation.

To investigate the role of platelet-rich plasma (PRP) from different sources in alleviating oxidative stress and ameliorating melanogenesis in UVB-irradiated PIG1 cells, PIG1 cells were irradiated with 80 mJ/cm2 UVB prior to 1% PRP application and the following experiments were taken: the viability of UVB-irradiated PIG1 cells, cellular malondialdehyde (MDA) and reactive oxygen species (ROS) content, and activities of antioxidant enzymes. Western blotting was utilized to detect the expression level of proteins associated with melanin synthesis, apoptosis, and DNA lesions. We found that PRP intervention promoted cell proliferation, reduced MDA and ROS content, increased the activities of series of antioxidant enzymes, and alleviated DNA damages in UVB-damaged PIG1 cells. It is important to note that PRP treatment inhibited UVB-induced melanogenesis via the PI3K/Akt/GSK3β signal pathway. Therefore, we suppose PRP treatment exerts a protective role through their antioxidation effect on UVB-damaged PIG1 cells and hinders melanogenesis induced by UVB irradiation.

Activation of VEGF receptors in response to UVB promotes cell proliferation and melanogenesis of normal human melanocytes

VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and signaling via VEGFRs extends beyond the classical roles in blood vessel formation. We previously showed VEGFRs were also expressed in epidermal keratinocytes and activation of VEGFR-2 by ultraviolet B (UVB) was involved in the pro-survival mechanism. Here, we show that both VEGF165 and UVB enhanced the expression of VEGFRs (including VEGFR-1, VEGFR-2 and NRP-1) in normal human melanocytes, and increased expression of VEGFRs by UVB was mediated through hypoxia and oxidative stress. Also, VEGF165 and UVB promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2, and UVB-induced phosphorylation of VEGFR-1 and VEGFR-2 required PKA but not P38 MAPK. In addition, UVB and VEGF165 contributed to the over-expression of melanogenic proteins in melanocytes, which could be reduced by neutralization of VEGFR-1 and/or VEGFR-2. UVB, but not VEGF165 promoted cell proliferation, while neutralization of VEGFR-1 and/or VEGFR-2 abolished this effect. UVB showed stronger than VEGF165 in promoting tyrosinase activity and melanin production, while neutralization of VEGFR-2 was stronger in reducing these effects than that of VEGFR-1. Furthermore, tranexamic acid (TA) decreased tyrosinase activity and melanin production via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that VEGFRs are functionally involved in UVB-induced melanogenesis, and TA can inhibit melanogenesis at least in part by targeting VEGFRs in melanocytes.

Downregulation of TUG1 promotes melanogenesis and UVB-induced melanogenesis.

Studies have revealed that taurine upregulated gene 1 (TUG1), an important member of the long non-coding RNA family, is involved in the regulation of cell growth, tumorigenesis and invasion, insulin secretion and so on. However, its role in melanogenesis has not been explored. This study attempts to explore the effects of TUG1 on melanogenesis and its regulatory mechanisms. We evaluated the expression changes in melanogenesis-related genes and detected phosphorylation levels of ERK, JNK and P38 in TUG1 downregulated melanocytes. After exposure of melanocytes to UVB irradiation, the expression of TUG1 and melanogenesis-related genes was detected. We found that the expression of tyrosinase (TYR), tyrosine-related protein 1 (TYRP1) and tyrosine-related protein 2 (TYRP2) was upregulated and that the phosphorylation level of ERK was downregulated by downregulating TUG1. Inhibition of TUG1 could further upregulate the expression of UVB-induced melanogenesis-related genes. In conclusion, TUG1 negatively regulates melanocyte melanogenesis via the ERK pathway and plays a negative role in UVB-induced melanogenesis.

Spinosin is a flavonoid in the seed of Ziziphus jujuba that prevents skin pigmentation in a human skin model

Abstract Because tyrosinase catalyzes essential steps in melanin synthesis, tyrosinase inhibitors are widely used for skin–lightening. To reduce the side effects of synthetic compounds, safer tyrosinase inhibitors isorated from natural products have gained attention. Here, we examined the anti-melanogenic effect of the seed of Ziziphus jujuba and identified eight major compounds, of which spinosin exhibited the strongest tyrosinase inhibitory activity (IC50 = 47 µM). When further examined, spinosin suppressed αMSH- or UVB-induced melanogenesis in B16F10 cells without cytotoxicity. The anti-melanogenic effect was also shown in a human skin model. As an underlying mechanism, in silico analysis showed that spinosin may bind to and suppress tyrosinase activity by forming multiple hydrogen bonds and hydrophobic interactions with the binding pocket of tyrosinase. Lineweaver-Burk and Dixon plots further revealed that spinosin acts as a competitive inhibitor of tyrosinase (Vmax, 11.61 × 103, 11.63 × 103, and 11.62 × 103, Km, 0.139, 0.185, and 0.239 for the con, 10 µM, 50 µM of spinosin, respectively). In conclusion, spinosin, a major compound in the seed of Ziziphus jujuba, could be a novel additive for treating pigmentation disorders or skin-lightening cosmetics.

Ganoderma lucidum polysaccharide inhibits UVB-induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways.

Ultraviolet (UV)-induced pigmentation is very common in clinical practice, but the current treatments are rarely effective, accompanied by some side effects. Ganoderma lucidum polysaccharide (GLP) is a natural antioxidant with no toxic side effects, which can antagonize UVB-induced fibroblast photo aging. The study aims to explore the role of GLP in inhibiting UVB-induced melanogenesis and its possible mechanism. The expression of melanogenesis genes such as microphthalmia-associated transcription factor (MITF), tyrosine (TYR), tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2), ras-related protein Rab-27A (Rab27A), and Myosin shows an upward trend after exposure of B16F10 and PIG1 cells to UVB irradiation, but GLP can downregulate the expression of genes related to UVB-induced melanogenesis. GLP can inhibit UVB-activated protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signaling pathways. Besides, GLP protects mitochondria from UVB damage and inhibits reactive oxygen species (ROS) production. Also, UVB-induced cyclic adenosine monophosphate (cAMP) can be inhibited. It has been found in the experiments of UVB-induced skin pigmentation in zebrafish that GLP is capable of inhibiting UVB-induced skin pigmentation. Meanwhile, it can greatly relieve erythema reaction in guinea pig skin caused by high-dosage UVB irradiation. In conclusion, this study shows that GLP can inhibit UVB-induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways and is a potential natural safe whitening sunscreen additive.

Anti-melanogenic activity of steamed and freeze-dried mature silkworm powder

Silkworms, Bombyx mori L., have been traditionally used as an oriental medicine to manage diverse pathological conditions including dermatological problems in East Asia, in addition to the use of their cocoons for making fabrics. The “mature” silkworms of B. mori have recently been regarded as a potential health supplement due to gaining their edibility for humans by a newly-developed steaming method. Therefore, the present study was conducted to investigate the effects of steamed and freeze-dried mature silkworm powder (SMSP) on skin pigmentation and melanogenesis. For elucidating the hypo-pigmenting activity, lightness of a designated site on the murine dorsal skin was measured in vivo. During the experiment, hyper-pigmentation was induced on the skin by cumulative exposure to ultraviolet B (UVB) irradiation. At the end of the experiment, melanin production on the skin was visualized by Fontana-Masson staining. Orally administered SMSP significantly and dose-dependently reduced abnormal pigmentation caused by UVB on the murine dorsal skin. SMSP also showed a potential anti-melanogenic efficacy in modulating UVB-induced melanogenesis. Taken together, SMSP was identified for the first time as a potential candidate for a novel anti-melanogenic agent, which showed hypo-pigmenting efficacy against UVB-induced melanogenesis in vivo when administered orally.

Inhibitory effect of 660‐nm LED on melanin synthesis in in vitro and in vivo

Skin hyperpigmentary disorders including postinflammatory hyperpigmentation, melasma, solar lentigines, and conditions like freckles are common. The light-emitting diodes (LEDs) are the latest category of nonthermal and noninvasive phototherapy to be considered in skin pigmentation disorder treatment. The purpose of this study was to investigate the effects of 660-nm LED on inhibition of melanogenesis. We investigated whether a 660-nm LED affected melanin synthesis in invitro and invivo models, and we explored the mechanisms involved. The inhibitory effect of 660-nm LED on melanin synthesis was evaluated in B16F10 cells and HRM-2 melanin-possessing hairless mice were used to evaluate the antimelanogenic effects of 660-nm LED. Interestingly, 660-nm LED inhibited alpha-melanocyte-stimulating hormone-induced tyrosinase activity in B16F10 cells. We also found that 660-nm LED decreased MITF and tyrosinase expression and induced the activation of ERK. These findings suggest that the depigmenting effects of 660-nm LED result from downregulation of MITF and tyrosinase expression due to increased ERK activity. The 660-nm LED reduced UVB-induced melanogenesis in the skin of HRM-2 via downregulation of tyrosinase and MITF. These findings suggest 660-nm LED is a potentially depigmentation strategy.

Hyaluronan degrading enzymes in fungi

Silkworms, Bombyx mori L., have been traditionally used as an oriental medicine to manage diverse pathological conditions including dermatological problems in East Asia, in addition to the use of their cocoons for making fabrics. The “mature” silkworms of B. mori have recently been regarded as a potential health supplement due to gaining their edibility for humans by a newly-developed steaming method. Therefore, the present study was conducted to investigate the effects of steamed and freeze-dried mature silkworm powder (SMSP) on skin pigmentation and melanogenesis. For elucidating the hypo-pigmenting activity, lightness of a designated site on the murine dorsal skin was measured in vivo. During the experiment, hyper-pigmentation was induced on the skin by cumulative exposure to ultraviolet B (UVB) irradiation. At the end of the experiment, melanin production on the skin was visualized by Fontana-Masson staining. Orally administered SMSP significantly and dose-dependently reduced abnormal pigmentation caused by UVB on the murine dorsal skin. SMSP also showed a potential anti-melanogenic efficacy in modulating UVB-induced melanogenesis. Taken together, SMSP was identified for the first time as a potential candidate for a novel anti-melanogenic agent, which showed hypo-pigmenting efficacy against UVB-induced melanogenesis in vivo when administered orally.

(Z)-5-(2,4-Dihydroxybenzylidene)thiazolidine-2,4-dione Prevents UVB-Induced Melanogenesis and Wrinkle Formation through Suppressing Oxidative Stress in HRM-2 Hairless Mice.

Background. Uncontrolled melanogenesis and wrinkle formation are an indication of photoaging. Our previous studies demonstrated that (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) inhibited tyrosinase activity and melanogenesis in vitro. Objective. To examine in vivo effects of MHY498 as an antiaging compound on UVB-induced melanogenesis and wrinkle formation, we topically applied MHY498 on dorsal skin of HRM-2 hairless mice. Methods. Using histological analysis, we evaluated effects of MHY498 on melanogenesis and wrinkle formation after UVB exposure. In addition, related molecular signaling pathways were examined using western blotting, fluorometric assay, and enzyme-linked immunosorbent assay. Results. MHY498 suppressed UVB-induced melanogenesis by inhibiting phosphorylation of CREB and translocation of MITF protein into the nucleus, which are key factors for tyrosinase expression. Consistently, tyrosinase protein levels were notably reduced in the dorsal skin of the hairless mice by MHY498 treatment. Furthermore, MHY498 inhibited UVB-induced wrinkle formation and collagen fiber destruction by increasing type 1 procollagen concentration and decreasing protein expression levels of MMPs, which play an essential role in collagen fiber degradation. As a mechanism, MHY498 notably ameliorated UVB-induced oxidative stress and NF-κB activation in the dermal skin of the hairless mice. Conclusion. Our study suggests that MHY498 can be used as a therapeutic or cosmetic agent for preventing uncontrolled melanogenesis and wrinkle formation.

P21-Activated Kinase 4 Critically Regulates Melanogenesis via Activation of the CREB/MITF and β-Catenin/MITF Pathways

p21-activated kinase 4 (PAK4) regulates a wide range of cellular events, including cytoskeletal remodeling, cell growth, and survival. Our previous study identified PAK4 as a key regulator of cAMP-response element-binding protein (CREB) that acts upstream of microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. We therefore investigated the role of PAK4 in melanogenesis. Melanocytes express both PAK2 and PAK4 isoforms, but only RNA interference knockdown of PAK4 significantly influenced α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in B16 melanoma cells. Consistent with this result, PAK4 inhibition by PF3758309, a potent ATP-competitive inhibitor of PAKs, suppressed not only α-MSH-induced melanogenesis in B16 melanoma and human epithelial melanocyte cells but also UVB-induced melanogenesis in the skin of melanin-possessing hairless mice (HRM-2) in a dose-dependent manner. Inhibition of PAK4 over several days markedly decreased the levels of CREB, MITF, and tyrosinase in both HRM-2 mice and B16 melanoma cells. Moreover, PAK4 knockdown and inhibition suppressed α-MSH-stimulated β-catenin phosphorylation at serine 675 (S675) but enhanced phosphorylation at S33/37, an indicator for ubiquitination-dependent proteolysis. Together, our results provide evidence that PAK4 promotes α-MSH/UVB-induced melanogenesis via the CREB and Wnt/β-catenin signaling pathways and suggest that PAK4 may be a potential therapeutic target in pigmentation disorders.

Superoxide dismutase 1 inhibits alpha-melanocyte stimulating hormone and ultraviolet B-induced melanogenesis in murine skin.

BackgroundOver the last decade, the incidence of ultraviolet B (UVB)-related skin problems has increased. Oxidative stress caused by UVB induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyperpigmentation. Therefore, increasing the antioxidant abilities of skin cells is thought to be a beneficial strategy for the development of sunscreen agents. Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that is known to exhibit antioxidant properties.ObjectiveThe purpose of this study was to investigate the effect of SOD1 on alpha-melanocyte stimulating hormone (α-MSH) and UVB-induced melanogenesis in B16F10 melanoma cells and HRM-2 melanin-possessing hairless mice.MethodsThe inhibitory effect of SOD1 on tyrosinase activity was evaluated in a cell-free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of SOD1 in vitro, and HRM-2 melanin-possessing hairless mice were used to evaluate the antimelanogenic effects of SOD1 in vivo.ResultsWe found that SOD1 inhibited melanin production in a dose-dependent manner without causing cytotoxicity in B16F10 melanoma cells. SOD1 did not inhibit tyrosinase activity under cell-free conditions. The results indicate that SOD1 may reduce pigmentation by an indirect, nonenzymatic mechanism. We also found that SOD1 decreased UVB-induced melanogenesis in HRM-2 melanin-possessing hairless mice, as visualized through hematoxylin and eosin staining and Fontana-Masson staining.ConclusionOur results indicate that SOD1 has an inhibitory effect on α-MSH and UVB-induced melanogenesis, indicating that SOD1 may be a promising sunscreen agent.

Inhibition of c-Kit signaling by diosmetin isolated from Chrysanthemum morifolium

The interaction of stem cell factor (SCF) with its cognate receptor c-Kit is closely associated with the survival and maturation of melanocytes. To investigate novel depigmentation agents, we screened 2,000 plant extracts for c-Kit inhibitors to identify active small molecules by using time-resolved fluorescence enzyme assays. For the active extracts identified as inhibitors of c-Kit enzyme, we evaluated the effects of the active extracts and isolated flavonoids on c-Kit phosphorylation in MO7e/melanocytes. Anti-melanogenic activity was also examined in melanocytes and melanoderm model. The flavonoids such as diosmetin, apigenin, acacetin and luteolin isolated from Chrysanthemum morifolium were found to be active in inhibiting c-Kit both at enzyme and cellular levels. In addition, these flavonoids attenuated SCF-induced proliferation of human primary melanocytes without toxicity and suppressed ultraviolet (UV) B irradiation-mediated melanin synthesis significantly. Among the active flavonoids, diosmetin was found to inhibit SCF-induced melanogenesis in a human melanoderm model. These results strongly suggest that C. morifolium extract and diosmetin have potential to suppress SCF-/UVB-induced melanogenesis, and could be developed as anti-pigmentation agents.

Effect of a Garcinia gardneriana (Planchon and Triana) Zappi hydroalcoholic extract on melanogenesis in B16F10 melanoma cells

Ethnopharmacology relevanceGarcinia gardneriana (Planchon and Triana) Zappi (Clusiaceae) is popularly called “bacopari” in southern Brazil. The leaves of this plant are traditionally used to treat skin disorders. Aim of studyThis study evaluated the effects of a hydroalcoholic extract of Garcinia gardneriana leaves (HEGG) on B16F10 murine melanoma cells in order to search for new depigmenting agents. Materials and methodsThe effects of HEGG were assessed in melanin content assays in B16F10 melanoma cells compared with the reference drug kojic acid (500mM). Melanin content was measured after spontaneous melanogenesis, UVB-induced melanogenesis and melanogenesis induced by α-MSH. At the same time, cell viability assays were conducted. Intracellular and mushroom tyrosinase activity assays were employed to evaluate the effect of HEGG on tyrosinase activity. ResultsHEGG decreased the level of melanin under all three experimental conditions of melanin content evaluation without reducing cell viability. In intracellular tyrosinase assays, the enzyme's activity was reduced about 19% with extract concentrations ranging 0.1–10µg/mL. In the mushroom tyrosinase activity assay a maximal inhibition of 35% (1000µg/mL) was observed. ConclusionThese results suggest that HEGG inhibition relates to its tyrosinase activity. Therefore, the hydroalcoholic extract of Garcinia gardneriana shows great potential for use as a depigmenting agent in hyperpigmentation disorders.

Suppression of melanogenesis by a newly synthesized compound, MHY966 via the nitric oxide/protein kinase G signaling pathway in murine skin

Ultraviolet B (UVB) radiation is the main physiological stimulus for skin pigmentation. Nitric oxide (NO) and the NO/PKG signaling pathway play an important role in UVB-induced melanogenesis, which is related to the induction of expression of tyrosinase. In an attempt to find a novel anti-melanogenic agent, we synthesized a new compound, 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl) phenol (MHY966). The purpose of this study was to investigate the action of MHY966 on NO and the NO-mediated signaling pathway using in vitro and in vivo models of melanogenesis. NO generation, melanin synthesis, and the expression of tyrosinase and PKG were measured in B16F10 melanoma cells to verify the anti-melanogenic effect of MHY966 in vitro. Next, melanin-possessing hairless mice were pre-treated with MHY966 and then irradiated with UVB repeatedly. Morphological, histological, and biochemical analyses including the expressions of PKG, tryosinase and nuclear MITF, and productions of nitric oxide, peroxynitrite and ROS were conducted. MHY966 effectively inhibited NO generation and subsequent melanin synthesis induced by sodium nitroprusside, an NO donor, and suppressed the expression of tyrosinase and PKG. Topical application of MHY966 dose-dependently attenuated UVB-induced pigmentation in a mouse model. This hypopigmentation effect induced by MHY966 treatment was mediated by the down-regulation of tyrosinase, PKG, and nuclear MITF, which was accompanied by decreased NO and NO-related oxidative stress. The novel compound, MHY966 had an inhibitory effect on NO generation and the NO-mediated signaling pathway leading to the down-regulation of tyrosinase. The significance of the present study is the finding of a promising anti-melanogenic agent targeting the NO/PKG signaling pathway.

β-Cryptoxanthin suppresses UVB-induced melanogenesis in mouse: involvement of the inhibition of prostaglandin E2 and melanocyte-stimulating hormone pathways

β-cryptoxanthin (β-CPX) is a carotenoid that is widely contained in the fruits of citrus plants. We evaluated the effect of β-CPX on UVB-induced pigmentation and mRNA expression related to melanogenesis in mouse skin. In addition, changes in melanogenic molecules were evaluated in cultured melanocytes stimulated with prostaglandin (PG) E(2), melanocyte-stimulating hormone (MSH) and endothelin (ET)-1. Mice were irradiated with UVB and were given β-CPX (0.1, 1 and 10 mg/kg) orally for 14 days. Pigmentation was evaluated by skin colour change and microscopic observation. Total RNA was obtained from the skin and the expression of melanogenic mRNA was evaluated by RT-PCR. In cell culture studies, human melanocytes were cultured with β-CPX and melanogenic stimulants (PGE(2), MSH and ET-1) for 6-10 days. Melanin contents, dendricity, melanogenic mRNA and phosphorylation of cyclic AMP response element-binding protein (CREB) were evaluated. β-CPX (10 mg/kg) significantly suppressed skin pigmentation and mRNA expression of cyclooxygenase-2, ET-1 receptors, low-affinity neurotrophin receptor, PGE(2) receptor (EP1), melanocortin 1 receptor (MC1R), tyrosinase (Tyr), tyrosinase-related protein (Tyrp) 1 and microphthalmia transcription factor. β-CPX (10 µg/ml) suppressed melanogenesis induced by PGE(2), MSH and ET-1. In the PGE(2)-stimulated melanocytes, mRNA expressions of EP-1, Tyr and Tyrp1 and phosphorylation of CREB protein were suppressed. In the ET-1-stimulated cells, only expression of CREB protein was suppressed. In the MSH-induced cells, mRNA expression of MC1R and Tyrp1 and protein expression of CREB were suppressed. Oral administration of β-CPX was found to suppress UVB-induced melanogenesis. Suppression of melanogenic enzymes, receptors of melanogenic stimulators, expression and phosphorylation of CREB are thought to be involved in the mechanism.