The mechanisms of melanogenesis inhibition by glabridin:molecular docking,PKA/MITF and MAPK/MITF pathways

Glabridin is the main ingredient of hydrophobic fraction in licorice extract and has been shown to have anti-melanogenesis activity in skins.However,the underlying mechanism(s)remain not completely understood.The aim of this study is thus to elucidate the possible mechanisms related to the melanogenesis suppression by glabridin in cultured B16 murine melanoma cells and in UVA radiation induced hyperpigmentation model of BALB/c mice as well.Molecular docking simulations revealed that between catalytic core residues and the compound.The treatment by glabridin significantly downregulated both transcriptional and/or protein expression of melanogenesis-related factors including melanocyte stimulating hormone receptor(MC1R),microphthalmia-associated transcription factor(MITF),tyrosinase(TYR),TYR-related protein-1(TRP-1)and TRP-2 in B16 cells.Both PKA/MITF and MAPK/MITF signaling pathways were found to be involved in the suppression of melanogenesis by glabridin in B16 cells.Also in vivo glabridin therapy significantly reduced hyperpigmentation,epidermal thickening,roughness and inflammation induced by frequent UVA exposure in mice skins,thus beneficial for skin healthcare.These data further look insights into the molecular mechanisms of melanogenesis suppression by glabridin,rationalizing the application of the natural compound for skin healthcare.

Anti-Melanogenesis Activity of Supercritical Carbon Dioxide Extract from Perilla frutescens Seeds

Perilla frutescens seed (PFS) oil is reported to inhibit skin photoaging;however, its effect on melanogenesis has not yet been investigated. Herein, we tested the anti-melanogenesis activity of an oil-based extract from PFS with supercritical carbon dioxide (scCO2). In a cell culture system, B16 mouse melanoma cells were treated with the PFS scCO2 extract and other samples. The PFS scCO2 extract decreased melanin production by approximately 90% in B16 mouse melanoma cells without cytotoxicity at 100 μg/mL. This effect was greater than that of the well-known melanogenesis inhibitor, kojic acid. Although a hexane-extracted PFS oil and a squeezed PFS oil also decreased melanin production in the B16 cells, the inhibitory effect of the PFS scCO2 extract was higher than both of these. Chemical analysis of the PFS scCO2 extract and squeezed PFS oil showed that almost 90% of the components of both oils were α-linolenic acid, linoleic acid, and oleic acid. Furthermore, the ratio of those three fatty acids across both samples was almost the same. When the three fatty acids were mixed in the same ratio as in the PFS scCO2 extract, the IC50 of the mixture for melanin production in B16 melanoma cells was identical to that of the PFS scCO2 extract. However, the IC50 of the squeezed PFS oil was approximately 6.6 times higher than that of the mixture. Although those fatty acids are the main inhibitory ingredients against melanin production in all of the extracts, some factor(s) in the squeezed PFS reduce their affinity with the cells. These results indicated that the PFS scCO2 extract could be a superior melanogenesis inhibitor. Although its main ingredients are probably the same as those of the squeezed PFS oil, it is necessary to extract with scCO2 for stronger anti-melanogenesis activity.

Inhibitory effect of Cyrtomium falcatum on melanogenesis in α-MSH-stimulated B16F10 murine melanoma cells

Objective:To explore the anti-melanogenic potential of Cyrtomium falcatum.Methods:The effects of Cyrtomium falcatum crude extract and its solvent fractions on tyrosinase activity,melanin content,and the expressions of melanogenesis-related genes and proteins were analyzed inα-melanocyte-stimulating hormone(α-MSH)-stimulated B16F10 cells.Results:α-MSH treatment significantly increased tyrosinase activity,and extracellular and intracellular melanin content,as well as the expression levels of tyrosinase,microphthalmia-associated transcription factor(MITF),tyrosinase-related protein(TRP)-1,and TRP-2 in B16F10 cells.Treatment with Cyrtomium falcatum crude extract and its solvent fractions reduced tyrosinase activity and extracellular and intracellular melanin content and downregulated the expression levels of tyrosinase,MITF,TRP-1,and TRP-2 in a dose-dependent manner.Conclusions:Cyrtomium falcatum has potential anti-melanogenesis effects and can be used as a potential source material in cosmeceutical industry for the research and development of novel lead molecules with whitening properties.

Platycladus orientalis shells promote melanogenesis via p38,AKT and Wnt/β-catenin signaling pathways

Background:Platycladus orientalis,which has been employed in traditional Chinese medicine for cool blood,antibacterial,promotion of hair growth and therapy of poliosis for centuries.However,there have been few reports focusing on Platycladus orientalis shells treating pigmentary disorders.Methods:In present study,gas chromatography–mass spectrometry was applied to analyzing the volatile composition of Platycladus orientalis shells and cellular metabolism.Experiments in vivo were carried out to evaluate the effect of Platycladus orientalis shells treating pigmentation disorders in C57BL/6J mice.Results:Our results indicated that cedrol occupied the largest percentage in Platycladus orientalis shells(17.1%).Meanwhile,Platycladus orientalis shells up-regulated the content of palmitic acid and increased melanin content and tyrosinase activity.Its mechanism possibly involved in the inhibiting phosphorylation of AKT and β-catenin,increasing phosphorylation of p38 to promote microphthalmia-associated transcription factor expression.The animal experiment also proved that Platycladus orientalis shells promoted melanogensis and hair blacken in C57BL/6J mice.Conclusion:All in all,Platycladus orientalis shells potentially promoted melanogenesis in vitro and in vivo.Cedrol was regarded as the main active substance in Platycladus orientalis shells.Therefore,it could be used for treatment of pigmentary disorders under safe concentration in the prospective application.

Unripe Fruit Extracts of <i>Mangifera indica L.</i>Protect against AGEs Formation, Melanogenesis and UVA-Induced Cell Damage

In this study, we explored the effects of unripe fruit extracts of Mangifera indica L. on the anti-aging activity in skin cells. Mangifera indica L. is a popular economical and medicinal plant with numerous health-beneficial properties. The aqueous extracts of unripe fruit of Mangifera indica L. were obtained and subjected to HPLC and NMR analyses for the identification of bioactive compounds. The anti-glycation effect of Mango unripe fruit extracts was monitored by in vitro model system of AGEs (Advanced glycation end products) formation. Mango unripe fruit extracts significantly inhibited the AGEs formation in a dose-dependent manner. Meanwhile, Mango unripe fruit extracts possessed a comparable efficiency to commercialized Kojic acids in the inhibition of melanogenesis in B16-F10 melanoma cells. The UVA-induced cell damages can be prevented and repaired by Mango unripe fruit extracts in skin fibroblast CCD-966SK. Compared to the untreated control, Mango unripe fruit extracts significantly increased the cell viability while being applied before (36%) or after (43%) UVA irradiation. These results verified the potential application of Mango unripe fruit extracts in the skin protection and recovery from UVA irradiation, as well as the suppression of AGEs formation and melanogenesis.

Conjugation of Magnetite Nanoparticles with Melanogenesis Substrate, NPrCAP Provides Melanoma Targeted, <i>in Situ</i>Peptide Vaccine Immunotherapy through HSP Production by Chemo-Thermotherapy

In order to develop melanoma-targeted in situ peptide vaccine immunotherapy, magnetite nanoparticles were conjugated with a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP). Magnetite nanoparticles introduced thermotherapy which caused non-apoptotic cell death and generation of heat shock protein (HSP) upon exposure to alternating magnetic field (AMF). NPrCAP was expected to develop a melanoma-targeted therapeutic drug because of its selective incorporation into melanoma cells and production of highly reactive free radicals, that result in not only oxidative stress but also apoptotic cell death by reacting with tyrosinase.

Anti-Melanogenesis Effect of Daniellic Acid Isolated from <i>Daniellia oliveri</i>(Rolfe) Hutch. &Dalziel (Leguminosae) Oleoresin of Burkina Faso

Over the past years, natural products have been used as useful candidates for prevention and treatment of skin disorders such as skin darkening. In this current research, Daniellia oliveri which was a potential source of cosmeceutical agent was selected to investigate its active components. Daniellic acid isolated from the oleoresin was characterized by using data from 1H-NMR, 13C-NMR, HSQC, IR, and online chemo-informatic analysis. The daniellic acid antioxidant, anti-proliferative, and tyrosinase inhibition capabilities were evaluated. This compound possessed an anti-DPPH and iron (III) reducing effect compared to quercetin. It was able to inhibit 9 tumor cells with IC50 going from 0.03 mM (U373) to 0.14 mM (Malme-3M). Interestingly daniellic acid inhibits tyrosinase activity with 1.20 mM as IC50. The tyrosinase inhibition mechanism was noncompetitive mixed-type with un-significant effect on cell melanogenesis. Daniellic acids induced a half-reduction of melanin production in B16F10 cell in IBMX stimulation (p < 0.05). The same observation was effective in Malme-3M melanin production with a significant daniellic acid action than kojic acid (p < 0.05) without reducing cell viabilities. This bioactive daniellic acid could explain the traditional uses of oleoresins from Daniellia oliveri for genitor-urinary tract diseases treatments, wound healing, and skin ailments in Burkina Faso.

Effect of Hoechunyangkyeok-San Extract on Melanogenesis

Forsythia fructus has been shown to have antioxidative, anti-inflammatory, antibacterial, anti-aging and whitening effects. Hoechunyangkyeok-san (Forsythia viridissima-prescription) is a traditional herbal medicine, which has been clinically used for treating febrile and inflammatory disorders. This work was carried out to investigate the skin whitening effects of Forsythia viridissima-prescription extract (a hydrolyzed extract of Hoechunyangkyeok-san: SID White HYC) on skin. The effects of SID White HYC were assessed the melanin contents in B161 melanoma cells and the pigmented equivalent with HMB45 and Fontana Masson staining in 3D skin model. Then, we examined the expression of major pigment enzymes regulating melanin synthesis and melanosome transport related proteins in B16F1 cells. SID White HYC significantly inhibited the melanin synthesis (56.7% and 30.6% inhibition at 100 μg/mL, intracellular and secreted, respectively) in B16F1 cells and 3D skin model. In addition, western blotting analysis showed that SID White HYC reduced the expression of melanin synthesis and melanosome transport related proteins in B16F1 cells. In clinical trials, the cream containing 0.05% SID White HYC showed skin depigmentation effect without any irritation. These results suggest that SID White HYC may be useful inhibition of melanogenesis and melanosome transport. Therefore, SID White HYC may have potential as a skin-whitening ingredient in cosmetics.

Epimedin B exhibits pigmentation by increasing tyrosinase family proteins expression,activity,and stability

Epimedin B(EB)is one of the main flavonoid ingredients present in Epimedium brevicornum Maxim.,a traditional herb widely used in China.Our previous study showed that EB was a stronger inducer of melanogenesis and an activator of tyrosinase(TYR).However,the role of EB in melanogenesis and the mechanism underlying the regulation remain unclear.Herein,as an extension to our previous investigation,we provide comprehensive evidence of EB-induced pigmentation in vivo and in vitro and elucidate the melanogenesis mechanism by assessing its effects on the TYR family of proteins(TYRs)in terms of expression,activity,and stability.The results showed that EB increased TYRs expression through microphthalmia-associated transcription factor-mediated p-Akt(referred to as protein kinase B(PKB))/glycogen synthase kinase 3β(GSK3β)/β-catenin,p-p70 S6 kinase cascades,and protein 38(p38)/mitogen-activated protein(MAP)kinase(MAPK)and extracellular regulated protein kinases(ERK)/MAPK pathways,after which EB increased the number of melanosomes and promoted their maturation for melanogenesis in melanoma cells and human primary melanocytes/skin tissues.Furthermore,EB exerted repigmentation by stimulating TYR activity in hydroquinone-and N-phenylthiourea-induced TYR inhibitive models,including melanoma cells,zebrafish,and mice.Finally,EB ameliorated monobenzone-induced depigmentation in vitro and in vivo through the enhancement of TYRs stability by inhibiting TYR misfolding,TYR-related protein 1 formation,and retention in the endoplasmic reticulum and then by downregulating the ubiquitination and proteolysis processes.These data conclude that EB can target TYRs and alter their expression,activity,and stability,thus stimulating their pigmentation function,which might provide a novel rational strategy for hypopigmentation treatment in the pharmaceutical and cosmetic industries.

Anti-melanogenic properties of FBCC-EP850 derived from Carex pumila Thunb

Objective:To elucidate the anti-melanogenic potential of Carex pumila Thunb.extract(FBCC-EP850).Methods:A collection of 180 plant extracts was tested for inhibition of mushroom tyrosinase activity using an in vitro assay.Among them,FBCC-EP850 exhibited the most promising inhibitory activity.Further analysis was conducted to investigate its mechanisms and therapeutic potential in reducing melanogenesis in B16F10 melanoma cells and zebrafish larvae.Results:FBCC-EP850 inhibited mushroom tyrosinase activity in a dose-dependent manner,with a half-maximal inhibitory concentration of 45.83μg/mL.FBCC-EP850 at concentrations up to 50μg/mL demonstrated minimal cytotoxicity against B16F10 melanoma cells and no adverse effects on zebrafish larvae.Treatment with 50μg/mL of FBCC-EP850 significantly reducedα-melanocyte stimulating hormone-induced melanin production and suppressed cellular tyrosinase activity in B16F10 melanoma cells.Additionally,FBCC-EP850 at 25 and 50μg/mL effectively diminished hyperpigmentation inα-melanocyte stimulating hormone-stimulated zebrafish larvae.Its anti-melanogenic action could be attributed to modulation of the cAMP-CREB-MITF signaling pathway.Conclusions:Carex pumila extract can inhibit melanogenesis by modulating the cAMP-CREB-MITF signaling pathway,which can be used as a promising candidate for treating hyperpigmentation disorders.

Advances in the function of traditional medicine for vitiligo treatment

Vitiligo has a significant impact on a substantial number of individuals worldwide.Traditional Chinese medicine has a long history of serving as a therapeutic treatment for vitiligo.Nevertheless,given the increasing volume of research on the utilization of traditional Chinese medicine for vitiligo treatment,it is imperative to conduct a comprehensive review that elucidates the efficacy of Chinese traditional medicine and other active ingredients in the treatment of vitiligo.This paper presents a comprehensive overview of the clinical preparations used to treat vitiligo,while also highlighting the potential monomers and extracts derived from traditional Chinese medicine for vitiligo treatment.A thorough analysis of the pharmacological effects of traditional Chinese medicine on vitiligo treatment will provide valuable insights and reliable information for the development of new treatment strategies.

Effect and mechanism of tacrolimus on melanogenesis on A375 human melanoma cells

Background Topical tacrolimus has been used for vitiligo as a common treatment option for more than ten years while the underlying mechanism is still uncertain.The aim of this study was to investigate the direct effects of tacrolimus on the melanogenesis and migration on human A375 melanoma cells.The expression of c-KIT mRNA and protein of human A375 cells were also investigated.Methods The cultured A375 human melanoma cells were randomly assigned to control and tacrolimus treatment groups （10,102,103and 104 nmol/L）.The cell proliferation was measured with Cell Counting Kit-8 assays.Melanin content was measured with NaOH method.Transwell migration assay was used to measure cell migration.The expression of c-KIT mRNA and protein were measured with real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry respectively.Results The cell proliferation of the 103 and 104 nmol/L tacrolimus groups were significantly lower （0.666±0.062 and 0.496±0.038） as compared with the control （0.841±0.110,P 〈0.05）.The mean melanin content in all groups treated with different concentration of tacrolimus （10,102,103,104 nmol/L） increased compared with the control group （P 〈0.05）.Dosedependent increase in cell migration were seen in all tacrolimus-treated groups （P 〈0.01）.The expression of c-KIT mRNA level in A375 cells exposed to tacrolimus （103and 104 nmol/L） had significantly increased by 3.03-fold and 3.19-fold respectively compared with the control （P 〈0.05）.Conclusions Although tacrolimus had no effects on cell proliferation on A375 human melanoma cells,it could increase the melanin content and cell migration.The expression of c-KIT mRNA and protein increased dose-dependently in tacrolimus-treated groups as compared with the control.Our study demonstrated that tacrolimus could enhance the melanogenesis and cell migration on A375 cells.

Effects of six compounds with different chemical structures on melanogenesis

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology.In the present study,we investigated the melanogenic activity of six structurally distinct compounds,namely,scopoletin,kaempferol,chrysin,vitamin D_3,piperine,and 6-benzylaminopurine.We determined their effectiveness,toxicity,and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model.The melanogenic activity of 6-benzylaminopurine,the compound identified as the most potent,was further verified by measuring green fluorescent protein concentration in tyrp1 a:eGFP(tyrosinase-related protein 1)zebrafish and mitfa:eGFP(microphthalmia associated transcription factor)zebrafish and antioxidative activity.All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF.6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20μmol·L^(-1 )in vivo and 100μmol·L^(-1) in vitro,and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1 a:e GFP and mitfa:e GFP zebrafish in vitro.However,its relative anti-oxidative activity was found to be very low.Overall,our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism,by increasing melanin content via positive regulation of tyrosinase activity in vitro,as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.

Triterpenoids and phenolics from the fruiting bodies of Inonotus hispidus and their activations of melanogenesis and tyrosinase

Two new 24-methyl lanostane triterpenoids,hispindic acids A and B（1 and 2）,and a new phenolic compound,hispinine（7）,along with nine known compounds（3-6,and 8-12）,were isolated from the fruiting bodies of Inonotus hispidus.Their structures were elucidated based on the extensive analysis of spectroscopic data（NMR and HRMS）.Hispindic acid A（1） possesses an unusual formyl group at C-30.Compounds 1,3-4,and 8 showed stronger activate abilities of melanogenesis and tyrosinase in B16 melanoma cells than those of positive control,8-methoxypsoralen,at 50 μmol/L

Role of gap junctions between keratinocyte and melanocyte in melanogenesis

The process ofmelanogenesis in melanocytes and the transport of melanin in the form ofmelanosomes to the neighboring keratinocytes are the key steps in human skin pigmentation. Keratinocytes and melanocytes interact in intricate manner to maintain the homeostasis. The present study was designed to understand the role of cell-cell interaction through the gap junctions between melanocytes and keratinocytes on melanogenesis. We show that, inhibition of the gap junctional activity between human keratinocytes and melanocytes in a coculture system using gap junction blocker lowers the expression of key regulatory genes of melanogenesis such as tyrosinase and microphthalmia- associated transcription factor （MITF）. This was followed by concurrent decrease in tyrosinase protein levels and activity. Our results show the preliminary evidence for the regulation of melanogenesis in melanocytes through direct gap junctional communication by keratinocytes. Deciphering the mechanism and factors involved in the process would uncover the significance of gap junctions in melanogenesis.

Identifying a melanogenesis-related candidate gene by a high-quality genome assembly and population diversity analysis in Hypsizygus marmoreus

Hypsizygus marmoreus is one of the most important edible fungi in Basidiomycete division and includes white and gray strains.However,very limited knowledge is known about the genomic structures and the genetic basis for the white/gray diversity of this mushroom.Here,we report the near-complete high-quality H.marmoreus genome at the chromosomal level.Comparative genomics analysis indicates that chromosome structures were relatively conserved,and variations in collinearity and chromosome number were mainly attributed by chromosome split/fusion events in Aragicales,whereas the fungi genome experienced many genomic chromosome fracture,fusion,and genomic replication events after the split of Aragicales from Basidiomycetes.Resequencing of 57 strains allows us to classify the population into four major groups and associate genetic variations with morphological features,indicating that white strains were not originated independently.We further generated genetic populations and identified a cytochrome P450 as the candidate causal gene for the melanogenesis in H.marmoreus based on bulked segregant analysis (BSA)and comparative transcriptome analysis.The high-quality H.marmoreus genome and diversity data compiled in this study provide new knowledge and resources for the molecular breeding of H.marmoreus as well as the evolution of Basidiomycete.

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals,microRNAs(miRNAs)play key roles in multiple biological processes by regulating the expression of target genes.Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors;however,its function remains unclear.In this study,we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation(P<0.01),tyrosinase activity(P=0.001)and significantly reduced(P<0.001)melanin production.Functional prediction revealed that the 3′-untranslated region(UTR)of MAP3K8 has a putative miR-370-5p binding site,and the interaction between these two molecules was confirmed using luciferase reporter assays.In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits(P<0.01)MAP3K8 expression via direct targeting of its 3′UTR.Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition(P<0.01)of melanocyte proliferation and significant reduction(P<0.001)in melanin production,which is consistent with our observations for miR-370-5p.Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA(containing sites for the targeted binding to miR-370-5p)was significantly rescued(P≤0.001),which subsequently promoted significant increases in cell proliferation(P<0.001)and melanin production(P<0.01).Collectively,these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.

Down-regulation of tyrosinase,TRP-1,TRP-2 and MITF expressions by citrus press-cakes in murine B16 F10 melanoma

Objective:To investigate the suitability of citrus-press cakes,by-products of the juice industry as a source for the whitening agents for cosmetic industry.Methods:Ethylacetate extracts of citrus-press cakes(CCE)were examined for their anti-melanogenic potentials in terms of the inhibition of melanin production and mechanisim of melanogenesis by using Western Blot analysis with tyrosinese,tyrosinase-related protein-1(TRP-1),TRP2,and microphthalmia-associated transcription factor(MITF)proteins.To apply the topical agents,citrus-press cakes was investigated the safety in human skin cell line.Finally flavonoid analysis of CCE was also determined by HPLC analysis.Results:Results indicated that CCE were shown to down-regulate melanin content in a dose-dependent pattern.The CCE inhibited tyrosinase,TRP-2,and MITF expressions in a dose-dependent manner.To test the applicability of CCE to human skin,we used MTT assay to assess the cytotoxic effects of CCE on human keratinocyte HaCaT cells.The CCE exhibited low cytotoxicity at 50μg/mL.Characterization of the citrus-press cakes for flavonoid contents using HPLC showed varied quantity of rutin,narirutin,and hesperidin.Conclusions:Considering the anti-melanogenic activity and human safety,CCE is considered as a potential anti-melanogenic agent and may be effective for topical application for treating hyperpigmentation disorders.

A High Throughput Screening Platform for Skin Tuning Properties from Natural Products: Identification of Skin Tanning Compounds

Skin lightening and tanning are two major areas dominating the market of skincare and cosmetic products. Though the demands are originated from two different communities, the two areas share a same goal—skin colour tuning. The known safe compounds with skin colour tuning activities are limited. In contrary, Chinese medicinal herb provides a pool of natural bioactive compounds, which have been used in Asian countries for long time and have been tested for its toxicity. Here, we demonstrate a high throughput screening platform for potential compounds usable for skin colour tuning. From 147 natural compounds, 26 of them showed potential in skin tanning functions by using the high throughput melanogenesis platform based on the melanogenesis assay on B16 melanocytes. Five of them promoted melanogenesis by over 50%. Moreover, apart from 1, 8-dihydroxyanthraquinone, the other four compounds showed enhancement effect on tyrosinase activity. From the result, the compounds increased the Vmax of tyrosinase without changing the Km in a dose-dependent manner. Thus, there should be no irreversible structural change of the enzyme. Definitely, this report contributes to the development of personalization in skincare and cosmetic products.

Examination of wnt signaling mediated melanin transport and shell color formation in Pacific oyster(Crassostrea gigas)

Mollusca exhibit remarkable diversity in shell coloration,attributed to the presence of melanin,a widely distributed pigment with various essential roles,such as mechanical strengthening,antioxidation and thermoregulation.However,the regulatory network governing melanogenesis and melanin transport in molluscs remains poorly understood.In this study,we conducted a systematic analysis of melanin distribution and transport in the Pacific oyster,utilizing light microscopy and high-resolution transmission electron microscopy.In addition,we characterized CgWnt1 and CgWnt2b-a in Crassostrea gigas,and analyzed Wnt signaling in melanocyte formation.Expression analysis revealed that these genes were predominantly expressed in the mantle of black-shelled individuals,particularly in the outer fold of the mantle.Furthermore,we employed RNA interference and inhibitors to specifically inhibit Wnt signaling in both in vivo and in vitro.The results revealed impaired melanogenesis and diminished tyrosinase activity upon Wnt signaling inhibition.These findings suggest the crucial role of Wnt ligands and downstream factors in melanogenesis.In summary,our study provides valuable insights into the regulatory mechanism of shell pigmentation in C.gigas.By demonstrating the promotion of melanogenesis through Wnt signaling modulation,we contribute to a better understanding of the complex processes underlying molluscan melanin production and shell coloration.