Tyrosinase-related Protein Research Articles

The active site of human Tyrosinase-related Protein: can it be inhibited by plants?

Tyrosinase and tyrosinase-related proteins (TYRPs) play an important role in melanogenesis. TYRPs present in the membrane of melanosome, are known to function in the activation and stabilization of tyrosinase, melanosome synthesis, increase eumelanin/pheomelanin ratio, and in reducing oxidative stress due to its peroxidase effect. Besides, the tyrosinase enzyme is the main enzyme that catalyzes the rate of melanin synthesis, whereas the downregulation of tyrosinase is the most prominent way of d

Analysis of how obtaining melanocytes by magnetic cell separation contributes to autoepidermal transplantation technology in treating leucoderma.

Leucoderma, a depigmentation of the skin, is a common disease in humans, and has been observed in cattle, horses and buffalo as well. To analyze the correlation between melanin stem cells and the differentiation and proliferation of melanocytes (MCs). Magnetic cell separation was used to separate melan-A+ cells and PAX3+ cells, which were cultured in vitro. The L-DOPA staining was used to observe cell morphology; Cell Counting Kit-8 (CCK8) was used to determine the cell proliferation rate; and flow cytometry (FCM) was used to determine cell cycle changes. The relative mRNA levels of melanocyte-inducing transcription factor (MITF), dopachrome tautomerase (DCT) and melan-A in cells were determined with reverse-transcription polymerase chain reaction (RT-PCR). The number of MC dendrites increased and extended continually during in vitro culture following magnetic cell separation. The proportion of positive L-DOPA staining cells increased from a baseline 40.70% to 82.03%, and the cell proliferation rate increased from 335.0% at D3 to 1577.4% at D20. The results of FCM showed that the cell proportion at the G1 stage in the D20 group was significantly lower than the D3 group; the cell proportion at the G2/M stage also decreased significantly. The expression of MITF and melan-A increased as the culture time increased, while the expression of DCT decreased. The number of MC stem cells decreased and mature MCs increased gradually, indicating that MC stem cells can gradually differentiate into mature MCs during in vitro culture following magnetic cell separation.

Evaluation of the effect of narrow band-ultraviolet B on the expression of tyrosinase, TYRP-1, and TYRP-2 mRNA in vitiligo skin and their correlations with clinical improvement: A retrospective study.

Narrowband-ultraviolet B (NB-UVB) is considered one of the main therapeutic tools in vitiligo, which is able to induce repigmentation and halt depigmentation. However, little remains known about the effect of NB-UVB on TYR gene family, the main pigmentary genes, in vitiligo patients. To assess the effect of NB-UVB on expression of some genes related to the pigmentary problem of vitiligo; tyrosinase (TYR), tyrosinase related protein 1 (TYRP1) and tyrosinase related protein 2 (TYRP2), mRNA levels of those genes were quantitatively evaluated by Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) in skin biopsies obtained from 30 patients with nonsegmental vitiligo and five healthy controls. Vitiligo patients were classified into two groups; group 1, involving 12 untreated vitiligo patients and group 2, including 18 vitiligo patients treated by NB-UVB. The levels of TYR, TYRP-1, and TYRP-2 mRNAs in untreated group were significantly lower than in control subjects (P < .001). In NB-UVB treated group, the three genes were significantly higher than in group 1 (P < .001), however, they were still significantly lower than in the control subjects (P < .001). A significant positive correlation was detected between TYR and TYRP-2 genes in group 2 (P = .03). This study demonstrated that mRNA level of TYR, TYRP-1, and TYRP-2, which decreased in vitiligo, was significantly increased upon treatment with NB-UVB. Accordingly, the mechanism of depigmentation in vitiligo disease and repigmentation by NB-UVB treatment may be related to the changes in the expression of these genes.

Phoenix dactylifera L. Seed Extract Exhibits Antioxidant Effects and Attenuates Melanogenesis in B16F10 Murine Melanoma Cells by Downregulating PKA Signaling.

Background: The mode of action of Phoenix dactylifera seed extract in skin care has never been explored. Methods: P. dactylifera L. seeds were extracted by ultrasonic extraction. The antioxidant characteristics of the extract were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS+) assays and scavenging methods. The total phenolic content, reducing capacity, iron (II) ion-chelation, and intracellular reactive oxygen species (ROS)-scavenging capacities were also investigated. The effects of P. dactylifera L. seed extract on melanogenesis were evaluated spectrophotometrically by a mushroom tyrosinase activity assay, determination of intracellular tyrosinase activity, and melanin content. The expression levels of melanogenesis-related proteins were analyzed by Western blotting. Results: The results revealed that the P. dactylifera L. seed extract exerted apparent antioxidant capacity and significantly decreased intracellular ROS content at concentrations of 0.245 and 0.49 (mg/mL). Furthermore, the extract decreased the expression of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2), and inhibited melanogenesis in B16F10 cells. Conclusions: Our results revealed that P. dactylifera L. seed extract attenuated melanogenesis in B16F10 cells by downregulating protein kinase A (PKA) signaling pathways. Hence, the extract could be used as a type of skin-whitening agent in skin care products.

Schisandrin B inhibits α-melanocyte-stimulating hormone-induced melanogenesis in B16F10 cells via downregulation of MAPK and CREB signaling pathways.

Schisandrin B (Sch B), a lignan compound in Schisandra, possesses antioxidant, anti-inflammatory, and antiobesity activities. The effect of Sch B on melanogenesis and molecular mechanisms are still unknown. Therefore, we aimed to investigate the antimelanogenic effects of Sch B on α-melanocyte-stimulating hormone-induced B16F10 cells and elucidate the underlying molecular mechanisms. We found that Sch B significantly suppressed melanin content and mushroom tyrosinase (TYR) activity. Sch B treatment decreased the expression of TYR, melanocyte-inducing transcription factor (MITF), tyrosinase-related protein (TRP) 1, and TRP2. Moreover, Sch B modulated the phosphorylation of p38, extracellular-regulated protein kinase, c-Jun N-terminal kinase, and cAMP-response element binding protein (CREB), implying that these pathways may be involved in suppressing melanogenesis. Furthermore, we found that Sch B decreased melanogenesis by downregulating MITF and melanogenic enzymes via MAPK and CREB pathways. Overall, these findings indicate that Sch B has the potential use in whitening.

Anti-melanogenic property of ginsenoside Rf from Panax ginseng via inhibition of CREB/MITF pathway in melanocytes and ex vivo human skin

BackgroundGinsenosides of Panax ginseng are used to enhance skin health and beauty. The present study aimed to investigate the potential use of ginsenoside Rf (Rf) from Panax ginseng as a new anti-pigmentation agent. MethodsThe anti-melanogenic effects of Rf were explored. The transcriptional activity of the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and the expression levels of tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related proteins (Tyrps) were evaluated in melanocytes and UV-irradiated ex vivo human skin. ResultsRf significantly inhibited Forskolin (FSK) or UV-stimulated melanogenesis. Consistently, cellular tyrosinase activity and levels of MITF, tyrosinase, and Tyrps were downregulated. Furthermore, Rf suppressed MITF promoter activity, which was stimulated by FSK or CREB-regulated transcription coactivator 3 (CRTC3) overexpression. Increased CREB phosphorylation and protein kinase A (PKA) activity induced by FSK were also mitigated in the presence of Rf. ConclusionRf can be used as a reliable anti-pigmentation agent, which has a scientifically confirmed and reproducible action mechanism, via inhibition of CREB/MITF pathway.

Capsaicin Inhibits the Expression of Melanogenic Proteins in Melanocyte via Activation of TRPV1 Channel: Identifying an Inhibitor of Skin Melanogenesis.

Chili pepper belongs to the genus Capsicum of Solanaceae family. Capsaicin is the primary capsaicinoid in placenta and flesh of chili pepper fruit, which has been shown to have various pharmacological functions, including gastric protection, anti-inflammation, and obesity treatment. Here, we revealed that capsaicin as well as chilli extract was able to inhibit synthesis of melanin in melanocytes. In cultured melanocytes, the melanin content was reduced to 54 ± 6.55% and 42 ± 7.41% with p < 0.001 under treatment of 50 μM capsaicin for 24 and 72 h, respectively. In parallel, the protein levels of tyrosinase and tyrosinase-related protein-1 were reduced to 62 ± 8.35% and 48 ± 8.92% with p < 0.001. Such an inhibitory effect of capsaicin was mediated by activation of transient receptor potential vanilloid 1-induced phosphorylation of extracellular signal-regulated kinase. This resulted in a degradation of microphthalmia-associated transcription factor, leading to reduction of melanogenic enzymes and melanin. These results revealed that capsaicin could be an effective inhibitor for skin melanogenesis. Hence, chili pepper, as our daily food, has potential in dermatological application, and capsaicin should be considered as a safe agent in treating hyperpigmentation problems.

7,3',4'-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses α-Melanocyte-Stimulating Hormone-Induced Melanogenesis by Targeting Melanocortin 1 Receptor.

7,3′,4′-Trihydroxyisoflavone (7,3′,4′-THIF) is a metabolite of daidzein which is a representative isoflavone found in soybean. Recent studies suggested that 7,3′,4′-THIF exerts a hypopigmentary effect in B16F10 cells, however, its underlying molecular mechanisms and specific target protein remain unknown. Here, we found that 7,3′,4′-THIF, but not daidzein, inhibited α-melanocyte-stimulating hormone (MSH)-induced intracellular and extracellular melanin production in B16F10 cells by directly targeting melanocortin 1 receptor (MC1R). Western blot data showed that 7,3′,4′-THIF inhibited α-MSH-induced tyrosinase, tyrosinase-related protein-1 (TYRP-1), and tyrosinase-related protein-2 (TYRP-2) expressions through the inhibition of Microphthalmia-associated transcription factor (MITF) expression and cAMP response element-binding (CREB) phosphorylation. 7,3′,4′-THIF also inhibited α-MSH-induced dephosphorylation of AKT and phosphorylation of p38 and cAMP-dependent protein kinase (PKA). cAMP and Pull-down assays indicated that 7,3′,4′-THIF strongly inhibited forskolin-induced intracellular cAMP production and bound MC1R directly by competing with α-MSH. Moreover, 7,3′,4′-THIF inhibited α-MSH-induced intracellular melanin production in human epidermal melanocytes (HEMs). Collectively, these results demonstrate that 7,3′,4′-THIF targets MC1R, resulting in the suppression of melanin production, suggesting a protective role for 7,3′,4′-THIF against melanogenesis.

Melanogenesis Effect of 7-acetoxy-4-methylcoumarin in B16F10 Melanoma Cells

The increased interest in anti-whitening dyes has enhanced the research interest to identify efficient melanogenic activators. Melanogenesis is the process of melanin production by melanocytes in the hair follicles and skin, which is mediated by several enzymes, such as microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein (TRP)-1, and TRP-2. This study investigated the melanogenesis-stimulating effect of 4-Methylumbelliferone (4MUMB) and its synthetic derivatives, 7-acetoxy-4-methylcoumarin (7A4MC) and 4-methylheriniarin (4MH) in B16F10 melanoma cells. The cytotoxicity of these compounds was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, followed by the assessment of the melanin content and the intracellular TYR activity. Finally, the expression levels of the key enzymes involved in melanogenesis were investigated. 7A4MC increased melanin production in B16F10 cells relative to that by 4MUMB and 4MH treated cells in a dose-dependent manner without significant cytotoxicity. Concomitantly, 7A4MC significantly increased TYR activity and enhanced the expression of MITF, which significantly induced the expression of TRP-1, TRP-2, and TYR. Furthermore, 7A4MC stimulated melanogenesis via increased phosphorylation of c-Jun N-terminal kinases (JNK) and reduced phosphorylation of protein kinase B (AKT). These results confirmed the melanogenesis-inducing effects of 7A4MC and indicated its potential use as an anti-hair bleaching agent in cosmetics industries.

Essential Oils of Alpinia nantoensis Retard Forskolin-Induced Melanogenesis via ERK1/2-Mediated Proteasomal Degradation of MITF

The anti-melanogenic activity of essential oils of Alpinia nantoensis and their bioactive ingredients were investigated in vitro. Treatment with leaf (LEO) and rhizome (REO) essential oils of A. nantoensis, significantly reduced forskolin-induced melanin production followed by down-regulation of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1) expression at both transcriptional and translational levels. Further studies revealed that down-regulation TYR and TRP-1 were caused by LEO/REO-mediated suppression of Microphthalmia-associated transcription factor (MITF), as evidenced by reduced nuclear translocation of MITF. Also, we found that LEO/REO induce the sustained activation of ERK1/2, which facilitate subsequent proteasomal degradation of MITF, as confirmed by that LEO/REO failed to inhibits MITF activity in ERK1/2 inhibitor treated cells. In addition, a significant increase of ubiquitinated MITF was observed after treatment with LEO and REO. Furthermore, the chemical composition of LEO and REO were characterized by gas chromatography-mass spectrometry (GC-MS) resulted that camphor, camphene, α-pinene, β-pinene, isoborneol and D-limonene were the major compounds in both LEO and REO. Further studies revealed that α-pinene and D-limonene were the active components responsible for the anti-melanogenic properties of LEO and REO. Based on the results, this study provided a strong evidence that LEO and REO could be promising natural sources for the development of novel skin-whitening agents for the cosmetic purposes.

Carvedilol, an Adrenergic Blocker, Suppresses Melanin Synthesis by Inhibiting the cAMP/CREB Signaling Pathway in Human Melanocytes and Ex Vivo Human Skin Culture.

Catecholamines function via G protein-coupled receptors, triggering an increase in intracellular levels of 3′,5′-cyclic adenosine monophosphate (cAMP) in various cells. Catecholamine biosynthesis and the β-adrenergic receptor exist in melanocytes; thus, catecholamines may play critical roles in skin pigmentation. However, their action and mechanisms mediating melanogenesis in human skin have not yet been investigated. Therefore, we examined the potential anti-melanogenetic effect of carvedilol, a nonselective β-blocker with weak α1-blocking activities. Carvedilol reduced melanin content and cellular tyrosinase activity without compromising cellular viability in normal human melanocytes as well as in mel-Ab immortalized mouse melanocytes. Carvedilol downregulated microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Carvedilol treatment led to the downregulation of phosphor-cAMP response element-binding protein (CREB). Moreover, the increase in cAMP levels upon treatment with forskolin reversed the anti-melanogenic action of carvedilol. In addition, carvedilol remarkably reduced the melanin index in ultraviolet-irradiated human skin cultures. Taken together, our results indicate that carvedilol effectively suppresses melanogenesis in human melanocytes and ex vivo human skin by inhibiting cAMP/protein kinase A/CREB signaling. The anti-melanogenic effects of carvedilol have potential significance for skin whitening agents.

Anti-Melanogenesis Effects of Lotus Seedpod In Vitro and In Vivo.

Melanogenesis has many important physiological functions. However, abnormal melanin production causes various pigmentation disorders. Melanin synthesis is stimulated by α-melanocyte stimulating hormone (α-MSH) and ultraviolet (UV) irradiation. Lotus seedpod extract (LSE) has been reported as possessing antioxidative, anti-aging, and anticancer activities. The present study examined the effect of LSE on melanogenesis and the involved signaling pathways in vitro and in vivo. Results showed that non-cytotoxic doses of LSE and its main component epigallocatechin (EGC) reduced both tyrosinase activity and melanin production in the α-MSH-induced melanoma cells. Western blotting data revealed that LSE and EGC inhibited expressions of tyrosinase and tyrosinase-related protein 1 (TRP-1). Phosphorylation of p38 and protein kinase A (PKA) stimulated by α-MSH was efficiently blocked by LSE treatment. Furthermore, LSE suppressed the nuclear level of cAMP-response element binding protein (CREB) and disturbed the activation of melanocyte inducing transcription factor (MITF) in the α-MSH-stimulated B16F0 cells. The in vivo study revealed that LSE inhibited melanin production in the ear skin of C57BL/6 mice after exposure to UVB. These findings suggested that the anti-melanogenesis of LSE involved both PKA and p38 signaling pathways. LSE is a potent novo natural depigmenting agent for cosmetics or pharmaceutical applications.

Abstract 14834: Protective Role of Cardiomyocyte-derived Ddt in Ischemic Cardiomyopathy

Background: D-dopachrome tautomerase (DDT), the only homolog of macrophage migration inhibitory factor (MIF), is a cytokine highly expressed in cardiomyocytes and exerts autocrine-paracrine effects by signaling through the CD74 receptor. Endogenous DDT and MIF prevent acute ischemia-reperfusion injury and pressure overload-induced heart failure in mice. This study investigated whether endogenous cardiomyocyte DDT has a role in ischemic cardiomyopathy (ICM). Methods: LV tissue was obtained from patients with ICM during heart transplantation and from non-transplanted donor hearts. Plasma DDT concentrations were measured in heart failure outpatients with ICM. Cardiomyocyte-specific DDT knockout (cKO) and littermate control (CON) mice underwent MI or sham surgery. Serial echocardiography was performed to assess LV remodeling after MI or sham surgery. Tissue from the non-infarct region was analyzed 3 days and 4 weeks after MI or sham surgery for histology and molecular studies. Results: Cardiac DDT mRNA and protein expression were reduced in LV from patients transplanted for ICM (n=8). Plasma DDT concentrations below the median value were associated with worse survival in ICM outpatients (p&lt;0.05, n=32). In mice, baseline LV function was similar in DDT cKO and CON after sham surgery and 3 days post-MI. However, DDT cKO mice developed more rapid LV dilatation and decreased LV ejection fraction and stroke volume as early as 1-week post-MI (n=4-6/group, all P&lt;0.05). The DDT cKO mice had smaller cardiomyocyte cross-sectional area 4 weeks after MI (p &lt;0.05), as well as early diminished phosphorylation of mTOR and S6-kinase (3 days post-MI). They also showed increased apoptosis 3 days post-MI and an early increase in p38 MAP kinase activation. Conclusion: Cardiomyocyte-derived DDT prevents adverse cardiac remodeling in ICM, potentially through modulating mTOR/S6 kinase (adaptive hypertrophy) and p38 MAP kinase (limiting apoptosis). Down-regulation of DDT in patients with ICM may contribute to the pathogenesis of advanced heart failure.

5-hydroxymethyl-2-furaldehyde purified from Japanese pear (Pyrus pyrifolia Nakai cv. Nijisseiki) juice concentrate inhibits melanogenesis in B16 mouse melanoma cells

Pear juice concentrate prepared by boiling Japanese pear (Pyrus pyrifolia Nakai cv. Nijisseiki) juice can significantly inhibit the activity of tyrosinase, a key enzyme in melanin synthesis in human skin. Using the ethanol extract of pear juice concentrate, we homogeneously purified an active compound that was identified as 5-hydroxymethyl-2-furaldehyde (5-HMF) through 1H- and 13C-NMR and mass spectroscopy. We observed that 5-HMF inhibited the monophenolase and diphenolase activities of mushroom tyrosinase as a mixed-type inhibitor (K i values of 3.81 and 3.70mmol/L, respectively). In B16 mouse melanoma cells, treatment with 170µmol/L of 5-HMF significantly reduced α-melanocyte-stimulated melanin synthesis by suppressing the cyclic adenosine monophosphate-dependent signaling pathway involved in melanogenesis. The results of our study indicated that 5-HMF can be potentially used as a skin-lightening agent in the cosmetic industry. Abbreviations: AC: adenylate cyclase; CREB: cAMP response element-binding protein; dhFAME: S-(-)-10,11-Dihydroxyfarnesoic acid methyl ester; DMEM: dulbecco's modified eagle medium; l-DOPA: 3-(3,4-Dihydroxyphenyl)- l-alanine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HEPES: 4-(2-Hydroxyethyl)-1-piperazine ethane sulfonic acid; 5-HMF: 5-Hydroxymethyl-2-furaldehyde; MITF: microphthalmia-associated transcription factor; α-MSH: α-Melanocyte-stimulating hormone; PKA: protein kinase A; PVDF: polyvinylidene difluoride; SDS: sodium dodecyl sulfate; TRP1: tyrosinase-related protein 1; TRP2: tyrosinase-related protein 2.

Autoimmune response against tyrosinase induces depigmentation in C57BL/6 black mice

Regulation of melanogenesis by tyrosinase has now become an attractive approach for treatment of vitiligo but still the role of tyrosinase in the induction of depigmentation remains largely unexplored. This study was explored the role of tyrosinase in the induction of autoimmune depigmentation in C57BL/6 mice. Depigmentation was induced in C57BL/6 mice by tyrosinase immunization. Induced depigmentation was characterized by visual detection and was verified by histopathological analysis of lesional and non-lesinal skin biopsies. Moreover, induced depigmentation was re-validated by gene expression analysis of vitiligo-relevant genes by Taqman assays. Immunization of C57BL/6 mice by tyrosinase induces depigmentation on hairs as well as on skin. Immunoassays with Protein A-purified immune IgGs showed high titre antibodies against tyrosinase. Histopathological analysis showed that the total melanocytes were depleted from the basal layer of the epidermis and also from the dermis of depigmented lesions. The gene expression of vitiligo-relevant genes TYRP1, DCT, MLANA, MCIR, POMC, FOXJ2, CSNK1G3, SOX10, PMEL and KIT was significantly low in lesional skin as compared with non-lesional skin (p < .05). In contrast, the mRNA expression of CASP3 and NFκB1 was significantly high in lesional skin of depigmented mice as compared with non-lesional skin (p < .05). Furthermore, involvement of cellular immunity in depigmentation was confirmed by the reduction of CD4+:CD8+ lymphocytes ratio. In conclusion, this study shows that the autoimmune response against tyrosinase induces depigmentation in black C57BL/6 mice. The data obtained from the lesional and non-lesional skin biopsies showed the same features as were reported in human vitiligo patients.

Additive Effect of a Combination of Artocarpus lakoocha and Glycyrrhiza glabra Extracts on Tyrosinase Inhibition in Melanoma B16 Cells.

Artocarpus lakoocha (Al) and Glycyrrhiza glabra (Gg) extracts have been reported to show tyrosinase inhibitory activity and melanin pigment reduction. This is the first study to assess the combination of Al and Gg extracts in enhancing inhibition of tyrosinase and reduction of melanin pigments. Al and Gg extracted by maceration in 70% and 95% ethanol were analyzed for oxyresveratrol and glabridin using Ultra High Performance Liquid Chromatography. Extracts of Al and Gg singly and combinations of Al95 and Gg95 were tested for cytotoxicity, tyrosinase inhibitory activity, and reduction of melanin pigments in melanoma B16 cells. Al95 had higher antioxidant, tyrosinase inhibitory activity and reduced more melanin pigments in B16 cells compared to Al70, and exhibited higher levels of oxyresveratrol. Gg95 inhibited oxidative stress and mushroom tyrosinase better than Gg70, and exhibited higher levels of glabridin. Combinations of Al95 and Gg95 at various ratios (concentration of 0.1 mg/mL) were not cytotoxic to B16 cells. Interestingly, Al95 and Gg95 combined at a ratio 9:1 reduced melanin pigment up to 53% in B16 cells. This combination of Al95 and Gg95 extracts exhibited the additive effect of reducing melanin pigments by suppressing the expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein-2 (TRP-2) in B16 cells. The combination of Al and Gg extracts could be developed as skin care products for hyperpigmentation treatment.

Anti-skin aging activities of green tea (Camelliasinensis (L) Kuntze) in B16F10 melanoma cells and human skin fibroblasts

IntroductionSkin aging is a multifactorial process caused by chronological changes and environmental factors. Green tea, a processed tea plant (Camellia sinensis (L) Kuntze), possesses health benefits and has been used in traditional medicines and natural products. However, the anti-skin aging effect of green tea in clinical trials has been a controversial issue. To clarify this without the interference of systemic involvement, the objectives of study were to investigate the activities of green tea against skin aging in B16F10 melanoma cells and human skin fibroblasts. MethodsGreen tea leaves were extracted to standardise the phenolic profile. The cytotoxicity and the anti-skin aging activities, including melanogenesis assay, antioxidant activity, collagen content analysis, and matrix metalloproteinase-2 inhibitory assay, were evaluated in cell culture. ResultsGreen tea in this study composed mainly of epigallocatechin gallate (1,903.77 ± 33.59 μg/mL), epigallocatechin (930.24 ± 27.04 μg/mL), and epicatechin gallate (607.84 ± 22.65 μg/mL). The cell viabilities of 0.5 % (0.0875 mg/mL) green tea, the noncytotoxic concentration, were 96.04 ± 6.76 and 93.44 ± 0.95 % in B16F10 melanoma cells and human skin fibroblasts, respectively. Green tea exhibited the activities against skin aging, including the significant suppression of melanin production via inhibition of tyrosinase and tyrosinase-related protein-2 activities, the potent antioxidant, and the significant matrix metalloproteinase-2 inhibition (p < 0.001). ConclusionsThe study results have shown that green tea may be a functional processed plant which could be used as an anti-skin aging agent in natural remedies, including food, health, and cosmetic products.

New insight into the role of exosomes in vitiligo.

Exosomes are nanosized extracellular vesicles that originate from endosomes and are secreted by most cells into the extracellular space. They serve as mediators of intercellular communication and have been implicated in the regulation of several physiological and pathological processes. Vitiligo is a depigmentation skin disease caused by progressive destruction of autologous epidermal melanocytes. Autoimmune intolerance is one of the leading theories proposed for melanocyte destruction in vitiligo via CD8+, regulatory T (Treg) and T helper 17 (Th17) cell imbalance in adaptive immunity. In this review, we investigate the association of exosomes with vitiligo and emphasize the role of exosomes in immune regulation, melanocyte-keratinocyte interactions, and melanogenesis. The exosomal pathway is necessary for the regulation of CD8+, Treg and Th17 cells in both pathological and physiological conditions. Exosomes derived under pathological conditions can influence CD8+, Treg and Th17 cell balance in the disease microenvironment, which may contribute to disruption of autoimmune tolerance in vitiligo. In addition, exosomes serve as mediators of communication between keratinocytes and melanocytes in the melanogenesis pathway and may also be involved in melanosome transport. They also regulate melanocyte survival and the protein expression of enzymes such as tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein-2 (TYRP2) and microphthalmia-associated transcription factor (MITF) in melanogenesis, which suggests that melanin production is associated with exosomes. An improved understanding of the role of exosomes in immune regulation and melanogenesis may help to elucidate the pathogenesis of vitiligo and lead to the development of potential diagnostic markers and therapeutic options.

Protoporphyrin IX Stimulates Melanogenesis, Melanocyte Dendricity, and Melanosome Transport Through the cGMP/PKG Pathway.

Protoporphyrin IX (PPIX) is a heterocyclic organic compound that is the last intermediate in the heme biosynthetic pathway. PPIX, due to its photodynamic effects, is utilized in the treatment of skin diseases. Furthermore, PPIX has been utilized as a melanogenesis-stimulating agent in various studies. However, the exact function and mechanism underlying PPIX action in melanocytes remain to be elucidated. In the present study, we sought to further investigate how PPIX affects melanocyte melanogenesis, and whether PPIX is involved in melanin transport. Our findings demonstrated that PPIX increased melanocyte dendricity and melanosome transport, in addition to increasing melanogenesis. PPIX functions primarily by activating the guanylate cyclase (GC) and cyclic guanosine 3’, 5’-monophosphate/protein kinase G (cGMP/PKG) signaling pathways. Once activated, these pathways increase tyrosinase activity and the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2), myosin Va, melanophinin, Ras-related protein Rab-27A (Rab27a), and cell division cycle 42 (Cdc42), promoting melanogenesis, melanocyte dendricity, and melanosome transport. Furthermore, the melanogenic effects of PPIX were confirmed in vivo in a zebrafish model system. Our results indicate that PPIX is not cytotoxic and may, thus, be utilized as a pigmentation enhancer.

Adoptive T Cell Therapy with IL-12-Preconditioned Low-Avidity T Cells Prevents Exhaustion and Results in Enhanced T Cell Activation, Enhanced Tumor Clearance, and Decreased Risk for Autoimmunity.

Optimal ex vivo expansion protocols of tumor-specific T cells followed by adoptive cell therapy must yield T cells able to home to tumors and effectively kill them. Our previous study demonstrated ex vivo activation in the presence of IL-12-induced optimal CD8+ T cell expansion and melanoma regression; however, adverse side effects, including autoimmunity, can occur. This may be due to transfer of high-avidity self-specific T cells. In this study, we compared mouse low- and high-avidity T cells targeting the tumor Ag tyrosinase-related protein 2 (TRP2). Not surprisingly, high-avidity T cells provide superior tumor control, yet low-avidity T cells can promote tumor regression. The addition of IL-12 during in vitro expansion boosts low-avidity T cell responsiveness, tumor regression, and prevents T cell exhaustion. In this study, we demonstrate that IL-12-primed T cells are resistant to PD-1/PD-L1-mediated suppression and retain effector function. Importantly, IL-12 preconditioning prevented exhaustion as LAG-3, PD-1, and TOX were decreased while simultaneously increasing KLRG1. Using intravital imaging, we also determined that high-avidity T cells have sustained contacts with intratumoral dendritic cells and tumor targets compared with low-avidity T cells. However, with Ag overexpression, this defect is overcome, and low-avidity T cells control tumor growth. Taken together, these data illustrate that low-avidity T cells can be therapeutically beneficial if cocultured with IL-12 cytokine during in vitro expansion and highly effective in vivo if Ag is not limiting. Clinically, low-avidity T cells provide a safer alternative to high-avidity, TCR-engineered T cells, as IL-12-primed, low-avidity T cells cause less autoimmune vitiligo.