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.

山羊皮肤组织miRNA测序与miR-129-5p调控黑色素生成的功能研究

旨在筛选对黑色素生成起调节作用的小RNA,并探究其对山羊肤色及毛色的调控机制。本研究采集了健康酉州乌羊(Youzhou dark goat, YZDG)、川东白山羊(Chuandong white goat, CDWG)100日龄胎羊皮肤样本(n=3),和健康2~3周岁大足黑山羊(Dazu black goat, DBG)、内蒙古绒山羊(Inner Mongolia cashmere goat, IMCG)个体皮肤样本(n=3),利用组织切片染色技术观察皮肤中黑色素沉积情况;通过小RNA测序技术筛选差异miRNAs;培养B16-F10皮肤黑色素瘤细胞,利用细胞转染、qPCR、Western Blot、黑色素含量检测等技术验证miR-129-5p对黑色素生成的影响。结果显示,黑色素颗粒明显在YZDG胎羊皮肤和DBG毛囊的毛球、毛干、外根鞘等部位沉积,而在CDWG胎羊皮肤和IMCG表皮、毛囊中没有被观察到。经测序分析,在肤色差异的YZDG和CDWG中筛选到62个差异表达miRNAs,其中31个在乌皮山羊中上调,31个下调。在毛色差异的DBG和IMCG中,筛选到38个差异表达miRNAs,其中10个在黑色被毛山羊中表达上调,28个表达下调。两组测序结果均显示miR-129-5p在乌皮和黑色被毛山羊皮肤中高表达(P<0.05)。在细胞中过表达miR-129-5p后,相比于对照组,mimics组细胞黑色素沉积量提高了18.9%(P<0.05),TYR、TYRP1基因表达量分别上调57.3%和16.5%(P<0.05),蛋白表达量分别显著上调49.2%和40.2%(P<0.05);但MITF基因及其蛋白表达量无显著变化(P>0.05)。在抑制miR-129-5p后,inhibitor组TYR基因mRNA表达下调38.9%、蛋白表达水平下调21.1%(P<0.05);TYRP1、MITF蛋白表达水平分别下调25.3%及28.4%(P<0.05)。本研究发现,miR-129-5p在不同肤色及毛色的山羊皮肤中差异表达,且可通过调控TYR、TYRP1等关键基因的表达影响黑色素的生成,是山羊肤色和毛色形成过程的重要调节因子。

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.

来源于裂壶藻渣的外泌体制备、鉴定及其抗黑色素生成功效初探

[目的]初步探究来源于海洋微藻裂壶藻(Schizochytrium limacinum)的外泌体的医美功能.[方法]从提取藻油后的裂壶藻废渣中提取外泌体,通过透射电子显微镜(TEM)和纳米流式分析仪(NanoFCM)对其进行结构鉴定,采用二喹啉甲酸(BCA)法测定其中的蛋白浓度,并建立黑点青鳉(Oryzias melastigma)模型测试外泌体抗黑色素生成功效.[结果]TEM结果可见裂壶藻源外泌体呈典型的圆形或椭圆形杯状形态,NanoFCM测得外泌体浓度2.96×10^(12)mL^(-1),粒径为(90.8±19.9)nm;黑点青鳉实验证实裂壶藻源外泌体导致黑色素斑数量减少,具有显著的抗黑色素生成功效,且抑制率与浓度呈正相关.[结论]本研究建立了裂壶藻渣源外泌体的制备方法和抗黑色素生成功效测试模型,为其高值化利用及美白功效评价提供了新的方向和策略.

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.

二氢杨梅素通过α-MSH/MITF通路抑制黑色素生成的研究

目的:评价二氢杨梅素(DMY)对小鼠黑色素瘤细胞B16的影响,并揭示其抑制黑色素产生的作用机制。方法:CCK8法筛选DMY对B16细胞增殖活性影响的最佳浓度。分为对照组、模型组(α-MSH)阳性药组(α-MSH+熊果苷)、给药组(α-MSH+DMY),采用CCK8法测定不同组别B16细胞的增殖活性,ELISA法检测B16细胞中黑色素和酪氨酸酶的含量,透射电镜观察细胞中黑素小体数量,分子对接法预测DMY降低黑色素含量作用的蛋白,免疫荧光法和Western blot分别测α-MSH和MITF蛋白的表达。结果:DMY抑制B16细胞增殖的最佳浓度为62.5μmol·L^(-1)。最佳浓度下,与模型组相比,DMY可以抑制B16细胞的增殖,降低黑色素和酪氨酸酶含量,减少黑素小体数目,抑制α-MSH和MITF蛋白表达。结论:二氢杨梅素通过调节α-MSH/MITF通路抑制B16细胞产生黑色素。

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.

小分子化合物I942在UVB照射诱导人黑素细胞树突形成及黑色素合成的促进作用

目的:探究小分子化合物I942在UVB照射诱导人黑素细胞树突形成及黑色素合成的促进作用。方法:使用浓度为0、3.125、6.25、12.5、25、50μmol/L的小分子化合物I942处理正常永生化人表皮黑素细胞系PIG1,采用CCK-8法检测各组24、48、72 h时的细胞活力。将PIG1细胞分为对照组、UVB组、UVB+2.5μmol/L I942组、UVB+5μmol/L I942组、UVB+10μmol/LI942组,按照分组名称进行相应处理。多巴染色观察各组细胞形态,免疫荧光染色观察黑素小体及骨架蛋白F-actin分布情况,氢氧化钠裂解法测定黑色素含量,多巴氧化反应法测定酪氨酸酶活性。qRT-PCR、WB检测树突形态相关、黑色素合成相关mRNA与蛋白表达水平。结果:浓度为0、3.125、6.25、12.5、25、50μmol/L的小分子化合物I942对PIG1细胞活力无明显影响(P>0.05)。与对照组相比,UVB组、UVB+2.5μmol/L I942组、UVB+5μmol/L I942组、UVB+10μmol/L I942组细胞树突数量、gp100、F-actin、黑色素含量、酪氨酸酶活性、MITF、TYR、TRP-1、TRP-2、Rac1mRNA与蛋白水平均升高(P<0.05),RhoAmRNA与蛋白水平降低(P<0.05)。结论:小分子化合物I942结合UVB照射诱导能够增加黑素细胞的树突数量,提高细胞黑色素含量,提升酪氨酸酶活性,促进黑色素合成。

黑色素的生成代谢机制及研究方法进展

随着时代的发展,“以白为美”思潮在近几年迎来了高涨期,美白市场空前繁荣。黑色素合成代谢过程在很大程度上调控着皮肤的颜色,因此美白剂的开发主要依赖于活性物质对黑色素的调节作用。本文将黑色素的合成、转运以及代谢过程细分为以下五个步骤:1)黑素细胞内黑素小体的装配;2)黑素小体的成熟及黑素的合成;3)成熟的黑素小体向黑素细胞树突状远端移动;4)黑素小体转移至角质形成细胞;5)黑素小体在角质形成细胞中的再分布或降解、排出。结合以上五个步骤的最新研究进展对其关键机制、重要标志物以及检测方法进行总结阐述,为美白剂的开发提供更加具体且科学的理论基础及开发思路。

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.

调节黑色素生成的主要信号通路

介绍了皮肤中的黑色素和血红素等色素,综述了黑色素生成的信号通路及其相关的信号分子,提出了美白原料的筛选策略。黑色素细胞生成的黑色素是影响皮肤颜色最大的色素。在黑色素细胞中,多个信号通路如cAMP/PKA、SCF/c-Kit/ERK、Wnt/Frizzled/GSK-3/β-catenin、p38/MSK1、PI3K/Akt、OPNs/CAMKII/PKC、AMPK、JNK、PLC/PKC-β、GC/PKG、PPARs/RXR等调控黑色素的生成。这些信号通路通过调控转录因子MITF的表达调节黑色素合成所需的酶,以及黑色素小体结构和转运相关的蛋白的表达,从而控制黑色素的生成。皮肤细胞合成分泌的多种信号分子如α-MSH,endothelin-1,ACTH,Activin A,TNF-α,IL-1α,KGF,IL-17,TNF-α等,以及人体分泌的性激素等通过结合黑色素细胞受体调控上述影响黑色素生成的信号通路。

三肽D-Trp-Arg-Leu-NH2抑制B16黑色素瘤细胞黑色素合成的机制

为了研究三肽D-Trp-Arg-Leu-NH2(dWRL)对小鼠B16细胞的酪氨酸酶(TYR)活性、黑色素合成及相关基因和蛋白表达的影响,探讨dWRL抑制黑色素生成的可能机制,我们体外培养小鼠B16细胞,采用MTT法测定细胞增殖情况、L-多巴氧化法测定TYR活性、氢氧化钠裂解法测定细胞黑色素含量、qRT-PCR和Western Blot法分别检测药物作用后B16细胞中小眼畸形相关转录因子(MITF)、TYR的mRNA和蛋白表达水平。结果显示三肽dWRL在试验浓度范围内可明显抑制α-黑色素细胞刺激素(α-MSH)诱导的B16细胞内TYR活性、黑色素合成量及MITF、TYR基因及蛋白的表达量,且呈浓度依赖性。所以三肽dWRL在体外能抑制B16细胞黑色素的合成,上述变化可能是通过下调MITF和TYR的基因转录和蛋白表达作用来完成。

蜕皮甾酮对皮肤光老化和黑色素生成的影响

目的观察蜕皮甾酮对皮肤光老化和黑色素生成的影响。方法①实验分为正常对照组、1.0μmol/L蜕皮甾酮组、1.5μmol/L蜕皮甾酮组、2.0μmol/L蜕皮甾酮组,各组分别处理角质形成细胞(HaCaT细胞)、黑素细胞(B16F10细胞)和人真皮成纤维细胞(HDF细胞)后,MTT法检测细胞活力。②实验分为正常对照组、UVB组、1.0μmol/L蜕皮甾酮组、1.5μmol/L蜕皮甾酮组、2.0μmol/L蜕皮甾酮组,Annexin V-FITC/PI双标法检测HaCaT细胞凋亡情况,Hoechst 33258染色法检测凋亡细胞的形态,Western blot法检测HaCaT细胞中基质金属蛋白酶-1(MMP-1)、基质金属蛋白酶-9(MMP-9)、环氧化酶-2(COX-2)、沉默调节蛋白1(Sirt-1)表达情况。③实验分为正常对照组、1.0μmol/L蜕皮甾酮组、1.5μmol/L蜕皮甾酮组、2.0μmol/L蜕皮甾酮组,Western blot法检测HaCaT细胞中透明质酸合成酶2(HAS-2)、转谷氨酰胺酶1(TGM1)和HDF细胞中Ⅰ型胶原蛋白Iα1肽链(COL1A1)表达情况。④实验分为正常对照组、黑色细胞刺激素组、1.0μmol/L蜕皮甾酮组、1.5μmol/L蜕皮甾酮组、2.0μmol/L蜕皮甾酮组、熊果苷组,采用分光光度仪检测B16F10细胞黑色素生成及分泌情况。⑤实验分为正常对照组、左旋多巴组、1.0μmol/L蜕皮甾酮组、1.5μmol/L蜕皮甾酮组、2.0μmol/L蜕皮甾酮组,采用分光光度仪检测各组蘑菇酪氨酸酶活性。结果不同浓度的蜕皮甾酮对HaCaT细胞、B16F10细胞、HDF细胞活力均无明显影响。蜕皮甾酮各组HaCaT细胞凋亡率及HaCaT细胞中MMP-1、MMP-9、COX-2蛋白相对表达量均明显低于UVB组(P均<0.05),HaCaT细胞中Sirt-1蛋白相对表达量均明显高于UVB组(P均<0.05)。蜕皮甾酮各组HaCaT细胞中HAS-2、TGM1蛋白相对表达量和HDF细胞中COL1A1蛋白相对表达量均明显高于正常对照组(P均<0.05)。蜕皮甾酮各组和熊果苷组B16F10细胞黑色素生成和分泌吸光度值均明显低于黑色细胞刺激素组(P均<0.05)。蜕皮甾酮各组蘑菇酪氨酸酶活性均明显低于左旋多巴组(P均<0.05)。结论蜕皮甾酮可能具有延缓皮肤光衰老、抗皱和抗黑色素生成作用。