PIG1 Cells Research Articles

NAcM-OPT protects keratinocytes from H2O2-induced cell damage by promoting autophagy.

This study aimed to investigate the protective effect of NAcM-OPT, a small molecule inhibitor of defective in cullin neddylation 1 (DCN1), on H2O2-induced oxidative damage in keratinocytes. Immortalized human keratinocytes (HaCaT cells) were treated with NAcM-OPT and exposed to oxidative stress. CCK-8 assays were used to measure cell viability. The mGFP-RFP-LC3 dual fluorescent autophagy indicator system was utilized to evaluate changes in autophagic flux. Western blotting was used to measure the expression of the autophagy-related proteins LC3 and Beclin 1. Keratinocytes were treated with the autophagy activator rapamycin, and HaCaT cell supernatant was added to PIG1 cells (immortalized human melanocytes), followed by evaluation of tyrosinase (TYR) expression via qRT-PCR. NAcM-OPT increased cell viability and cell proliferation. Furthermore, this molecule promoted autophagic flux through increased expression of autophagy-related proteins under H2O2-induced oxidative stress. Additionally, rapamycin increased the mRNA levels of TYR in PIG1 cells. Moreover, NAcM-OPT alleviated mitochondrial damage, restored mitochondrial function, and upregulated the expression of NFE2L2, HO1, NQO1, and GCLM. Importantly, NAcM-OPT also increased epidermal thickness, follicle length, and melanin synthesis under oxidative stress in vivo. These findings suggest that NAcM-OPT may be a promising small molecule antioxidant drug for the treatment of vitiligo.

Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

Molecular mechanisms underlying Tao-Hong-Si-Wu decoction treating hyperpigmentation based on network pharmacology, Mendelian randomization analysis, and experimental verification

Context Hyperpigmentation, a common skin condition marked by excessive melanin production, currently has limited effective treatment options. Objective This study explores the effects of Tao-Hong-Si-Wu decoction (THSWD) on hyperpigmentation and to elucidate the underlying mechanisms. Materials and methods We employed network pharmacology, Mendelian randomization, and molecular docking to identify THSWD’s hub targets and mechanisms against hyperpigmentation. The Cell Counting Kit-8 (CCK-8) assay determined suitable THSWD treatment concentrations for PIG1 cells. These cells were exposed to graded concentrations of THSWD-containing serum (2.5%, 5%, 10%, 15%, 20%, 30%, 40%, and 50%) and treated with α-MSH (100 nM) to induce an in vitro hyperpigmentation model. Assessments included melanin content, tyrosinase activity, and Western blotting. Results ALB, IL6, and MAPK3 emerged as primary targets, while quercetin, apigenin, and luteolin were the core active ingredients. The CCK-8 assay indicated that concentrations between 2.5% and 20% were suitable for PIG1 cells, with a 50% cytotoxicity concentration (CC50) of 32.14%. THSWD treatment significantly reduced melanin content and tyrosinase activity in α-MSH-induced PIG1 cells, along with downregulating MC1R and MITF expression. THSWD increased ALB and p-MAPK3/MAPK3 levels and decreased IL6 expression in the model cells. Discussion and conclusion THSWD mitigates hyperpigmentation by targeting ALB, IL6, and MAPK3. This study paves the way for clinical applications of THSWD as a novel treatment for hyperpigmentation and offers new targeted therapeutic strategies.

ALDH2 is a novel biomarker and exerts an inhibitory effect on melanoma

Melanoma is a malignant skin tumor. This study aimed to explore and assess the effect of novel biomarkers on the progression of melanoma. Differently expressed genes (DEGs) were screened from GSE3189 and GSE46517 datasets of Gene Expression Omnibus database using GEO2R. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted based on the identified DEGs. Hub genes were identified and assessed using protein–protein interaction networks, principal component analysis, and receiver operating characteristic curves. Quantitative real-time polymerase chain reaction was employed to measure the mRNA expression levels. TIMER revealed the association between aldehyde dehydrogenase 2 (ALDH2) and tumor immune microenvironment. The viability, proliferation, migration, and invasion were detected by cell counting kit-8, 5-ethynyl-2′-deoxyuridine, wound healing, and transwell assays. Total 241 common DEGs were screened out from GSE3189 and GSE46517 datasets. We determined 6 hub genes with high prediction values for melanoma, which could distinguish tumor samples from normal samples. ALDH2, ADH1B, ALDH3A2, DPT, EPHX2, and GATM were down-regulated in A375 and SK-MEL-2 cells, compared with the human normal melanin cell line (PIG1 cells). ALDH2 was selected as the candidate gene in this research, presenting a high diagnostic and predictive value for melanoma. ALDH2 had a positive correlation with the infiltrating levels of immune cells in melanoma microenvironment. Overexpression of ALDH2 inhibited cell viability, proliferation, migration, and invasion of A375/SK-MEL-2 cells. ALDH2 is a new gene biomarker of melanoma, which exerts an inhibitory effect on melanoma.

Kaempferol promotes melanogenesis and reduces oxidative stress in PIG1 normal human skin melanocytes

Vitiligo is an autoimmune disease characterized by depigmentation. Kaempferol is a flavonoid compound with broad anti-inflammatory and antioxidant properties. The purpose of this study was to investigate the effect of kaempferol on melanogenesis in PIG1 normal human skin melanocytes and its response to oxidative stress. The effect of kaempferol on melanin synthesis in PIG1 normal human skin melanocytes was explored by measuring tyrosinase activity, melanin content, mRNA and protein expression of key enzymes and expression of related pathway proteins. The effects of kaempferol pretreatment on cell viability, apoptosis, ROS level and HO-1 protein level under H2 O2 stimulation were explored. When treated with kaempferol, the tyrosinase activity and melanin content of PIG1 cells increased, the mRNA and protein expressions of TYR, TRP1, TRP2 and MITF increased, and the phosphorylation level of ERK1/2 increased. Upon the stimulation of H2 O2 , kaempferol reduced the production of ROS, decreased apoptosis and increased the protein expression of HO-1 in PIG1 cells. In addition, kaempferol inhibited oxidative stress-induced melanin reduction and promoted melanin synthesis in PIG1 cells and protected against H2 O2 -induced oxidative stress damage.

Mechanism of Action of a Chinese Herbal Compound Containing Quercetin, Luteolin, and Kaempferol in the Treatment of Vitiligo Based on Network Pharmacology and Experimental Verification.

This study aimed to explore the mechanisms of Baishi tablets (BSTs) in the treatment of vitiligo through network pharmacology-based identification and experimental validation. In brief, the compounds and related targets of BST were extracted from the TCMSP database, and disease information was obtained from the OMIM, GeneCards, PharmGkb, TTD, and DrugBank databases. A Venn diagram was generated to visualize the common targets of BST and vitiligo. GO and KEGG analyses were performed to explore the potential biological processes and signaling pathways. The PPI network and core gene subnetwork were constructed using STRING and Cytoscape software. In addition, the measurement of apoptosis in PIG1 cells and intracellular reactive oxygen species were measured using quercetin (QU), luteolin (LU), and kaempferol (KA) to protect melanocytes from oxidative stress. A total of 55 compounds with 236 targets and 1205 vitiligo-related genes were obtained from the TCMSP database. GO and KEGG analyses were performed to explore the potential biological processes and signaling pathways, revealing that BST may cure vitiligo by influencing the biological processes of cellular oxidative stress and related signaling pathways. A critical subnetwork was obtained with 13 core genes by analyzing the PPI network, which includes HMOX1, CXCL8, CCL2, IL6, MAPK8, CASP3, PTGS2, AKT1, IL1B, MYC, TP53, IFNG, and IL2. Furthermore, a molecular docking analysis was conducted to simulate the combination of compounds and gene proteins, reflecting that QU, LU, and KA can strongly bind the core genes. Through a series of experimental validations, we found that QU, LU, and KA could attenuate H2O2-induced apoptosis in melanocytes. Further evidence revealed that QU, LU, and KA could enhance the scavenging of intracellular reactive oxygen species (ROS). Based on the results of network pharmacology analysis and experimental verification, QA, LU, and KA can be utilized to protect PIG1 cells by inhibiting oxidative stress and reducing the intracellular level of ROS. This may explain the underlying mechanism of BST therapy and provide a novel strategy for the treatment of vitiligo.

GATA3 ameliorates melanocyte injuries in vitiligo through SIRT3-mediated HMGB1 deacetylation.

Vitiligo is a skin depigmentation disorder. GATA3 expression is downregulated in vitiligo patients, and its role and regulatory mechanism in vitiligo are unclear. GATA3 and HMGB1 levels were detected by qRT-PCR in peripheral blood cells of vitiligo patients and healthy controls, as well as H2 O2 -treated PIG1 cells. Their expression correlation was assessed by Pearson analysis. qRT-PCR, MTT assay, Ki67 immunostaining, flow cytometry, ELISA and Western blot were applied to determine GATA3 expression, cell survival, cell proliferation, cell apoptosis, melanin contents, and melanin-related protein expressions. The cellular distributions of HMGB1 and its deacetylation levels were detected by Western blot. The binding of GATA3 to SIRT3 promoter and effects on SIRT3 expression and HMGB1 deacetylation was determined by dual-luciferase assay, ChIP assay, and Western blot. GATA3 was decreased, and HMGB1 was increased in vitiligo. Pearson correlation assay showed that they were negatively correlated. H2 O2 significantly inhibited cell survival, proliferation, melanin secretion, and melanin-related protein expressions but remarkably increased cell apoptosis. GATA3 overexpression could distinctly reverse the effects of H2 O2 through decreasing HMGB1 expression and retained HMGB1 in nuclear due to the decreased HMGB1 acetylation. GATA3 bound to the SIRT3 and subsequently decreased H2 O2 -induced HMGB1 acetylation. Overexpressing HMGB1 or knockdown of SIRT3 could reverse the effects of GATA3 overexpression. GATA3 inhibited H2 O2 -induced injury in PIG1 cells and enhanced melanin secretion by SIRT3-regulated HMGB1 deacetylation, which might provide new evidence to treat vitiligo.

Study on the Mechanism of miR-125b-5p Affecting Melanocyte Biological Behavior and Melanogenesis in Vitiligo through Regulation of MITF.

The goal was to confirm the mechanism by which miR-125b-5p influences melanocyte biological behavior and melanogenesis in vitiligo by regulating MITF. oe-MITF, sh-MITF, miR-125b-5p mimic, NC-mimic, NC-inhibitor, and miR-125b-5p inhibitor were transfected into cells by cell transfection. Western blotting was used to detect the related protein expression, qRT-PCR was used to detect miR-125b-5p and MITF expression, immunohistochemistry was used to detect the MITF-positive cells in vitiligo patients tissues, and a dual-luciferase reporter system was used to detect the target of miR-125b-5p and MITF. PIG1 and PIG3V cell proliferation by the CCK-8 method, cell cycle progression and apoptosis by flow cytometry, apoptosis was detected by TUNEL, Tyr activity and melanin content were measured using Tyr and melanin content assay kits. Compared with the healthy control group, the expression of miR-125b-5p in the tissues and serum of vitiligo patients was upregulated, and the expression of MITF was downregulated; compared with PIG1 cells, the expression of miR-125b-5p and MITF in the PIG3V group was consistent with the above. Compared with the NC-minic group, the cell proliferation activity of the miR-125b-5p mimic group decreased, apoptosis increased, and the expression levels of melanogenesis-related proteins Tyr, Tyrp1, Tyrp2, and DCT were downregulated. Compared with the NC-inhibitor group, the above indices in the miR-125b-5p inhibitor group were all opposite to those in the miR-125b-5p mimic group. Transfection of oe-MITF into the miR-125b-5p mimic group reversed the effect of the miR-125b-5p mimic, while transfection of sh-MITF enhanced the effect of the miR-125b-5p mimic. miR-125b-5p affects vitiligo melanocyte biological behavior and melanogenesis by downregulating MITF expression.

Exosomal miR-22-3p from Mesenchymal Stem Cells Inhibits the Epithelial-Mesenchymal Transition (EMT) of Melanoma Cells by Regulating LGALS1.

The mortality rate from melanoma has been rising and hence new therapeutic approaches for this disease have received extensive attention, especially the search for novel therapeutic targets. The aim of this study was to find new targets for the treatment of melanoma through a bioinformatics and experimental approach. First, we screened for differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) between melanoma and normal tissues using the TCGA-SKCM, GTEX, and GSE24996 datasets. Next, we identified epithelial-mesenchymal transition (EMT)-related DEGs and analyzed their expression levels and association with patient survival. The expression level of DEGs was then confirmed in normal human melanocytes and melanoma cells. Bioinformatics analysis was used to identify miRNAs that targeted the most highly expressed DEG, LGALS1, and their binding confirmed using dual luciferase. Enriched pathways for the LGALS1 target miR-22-3p were also analyzed. miR-22-3p was overexpressed in cells in order to investigate changes in cell activity and in related genes and proteins. Exosomes from human bone marrow mesenchymal stem cells (MSCs) were coated with miR-22-3p to examine its effect on EMT. The expression levels of LGALS1, CPXM1, and APLNR were higher in melanoma than in normal tissues and were associated with worse patient survival. The differential expression of these genes was confirmed using normal human skin melanocytes (PIG1) and human melanoma cells (WM-266-4). LGALS1 was the most differentially expressed gene between WM-266-4 and PIG1 cells, and was also predicted to be a target for miR-22-3p. The results of dual luciferase experiments confirmed that miR-22-3p could bind to LGALS1. Following the overexpression of miR-22-3p in WM-266-4 cells, the cell viability decreased, the expression levels of LGALS1, VIM and SNAI2 decreased, the expression level of CDH1 increased, and cell apoptosis increased. Transfection of miR-22-3p using exosomes resulted in similar effects. We identified three genes (LGALS1, CPXM1, APLNR) that showed a high level of differential expression in melanoma. LGALS1 is a target for miR-22-3p binding and this can inhibit the EMT of melanoma cells, thereby preventing the development of melanoma. Moreover, exosomes secreted by MSCs can be loaded with miR-22-3p, thus regulating the EMT process in melanoma cells.

Lycium barbarum polysaccharide promotes proliferation of human melanocytes via activating the Nrf2/p62 signaling pathway by inducing autophagy in vitro.

Vitiligo is a skin disease characterized by lack of functional melanocytes. Lycium barbarum polysaccharide (LBP) has been demonstrated to preserve keratinocytes and fibroblasts against oxidative stress. This study aimed to explore the efficacy and underlying mechanisms of LBP on autophagy in H2 O2 -damaged human melanocytes. Cellular viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin V-fluorescein isothiocyanate/propidium iodide double staining. Reverse transcription-polymerase chain reaction, western blotting and electron microscopy were performed to detect autophagy. The protein expression level of Nrf2 and p62 were assessed by western blotting. Plasmid transfection and lentiviral infection were used to overexpress and silence Nrf2 in PIG1 cells. LBP promoted the proliferation and inhibited apoptosis of H2 O2 -damaged PIG1 cells. LBP increased the proliferation of H2 O2 -damaged PIG1 cells via induction of autophagy, and Nrf2 shRNA experiment confirmed that LBP activated the Nrf2/p62 signal pathway. These results suggest that LBP may be used for the treatment of vitiligo. PRACTICAL APPLICATIONS: Goji berry is the mature and dried fruit of Lycium barbarum L., which is a common food with a long history in China, as well as a Traditional Chinese Medicine. Our previous research found that LBP could activated the Nrf2/ARE pathway in an ultraviolet (UV)-induced photodamage model of keratinocytes, and increase the levels of phase II detoxification and antioxidant enzymes. We firstly confirmed the anti-vitiligo effects of L. barbarum polysaccharide (LBP) by inducing autophagy and promoted proliferation of human melanocytes, and LBP induced autophagy via activating the Nrf2/p62 signaling pathway in this study. These results proved that LBP can be an effective therapy for vitiligo treatment.

Four-Octyl Itaconate Attenuates UVB-Induced Melanocytes and Keratinocytes Apoptosis by Nrf2 Activation-Dependent ROS Inhibition.

Vitiligo is an acquired skin depigmentation disease in which excessive reactive oxygen species (ROS) play a critical pathogenic role in melanocyte destruction. The complex crosstalk between melanocytes and keratinocytes in vitiligo suggests that treatments aimed at protecting both the cells might be meaningful. In this study, we investigated the effect of 4-octyl itaconate (4-OI), an itaconate derivative, on ultraviolet B- (UVB-) induced apoptosis in HaCaT and PIG1 cells and the underlying mechanisms. HaCaT and PIG1 cells were pretreated with 4-OI (50 or 100 μM) for 24 h and then exposed to 300 mJ/cm2 UVB (emission range 290–320 nm, emission peak 310 nm). ROS levels and cell apoptosis were investigated using fluorescence microscopy and flow cytometry 24 h after irradiation. In addition, nuclear translocation and the expression of pathway-related proteins and mRNAs were detected using confocal microscopy, western blotting, and qRT-PCR, respectively. Our results demonstrated that UVB induced apoptosis in HaCaT and PIG1 cells, whereas inhibition of ROS production could reverse this effect. Furthermore, 4-OI attenuated UVB-induced apoptosis in HaCaT and PIG1 cells in a concentration-dependent manner by reducing the ROS levels. Moreover, 4-OI induced nuclear translocation and activation of nuclear factor erythroid 2-related factor 2 (Nrf2), and Nrf2 silencing reversed the inhibitory effect of 4-OI on the UVB-induced increase in ROS production and apoptosis in HaCaT and PIG1 cells. In addition, in vivo experiments using the Institute of Cancer Research mouse model showed that 4-OI via tail vein injection (10 mg/kg/day for six consecutive days) could reduce skin damage induced by UVB (400 mJ/cm2/day for five consecutive days). In conclusion, 4-OI can protect melanocytes and keratinocytes from UVB-induced apoptosis by Nrf2 activation-dependent ROS inhibition and can potentially treat skin disorders associated with oxidative stress, such as vitiligo.

Ginsenoside Rk1 protects human melanocytes from H2O2‑induced oxidative injury via regulation of the PI3K/AKT/Nrf2/HO‑1 pathway.

Vitiligo is a cutaneous depigmentation disorder caused by melanocyte injury or aberrant functioning. Oxidative stress (OS) is considered to be a major cause of the onset and progression of vitiligo. Ginsenoside Rk1 (RK1), a major compound isolated from ginseng, has antioxidant activity. However, whether RK1 can protect melanocytes against oxidative injury remains unknown. The aim of the present study was to investigate the potential protective effect of RK1 against OS in the human PIG1 melanocyte cell line induced with hydrogen peroxide (H2O2), and to explore its underlying mechanism. PIG1 cells were pretreated with RK1 (0, 0.1, 0.2 and 0.4 mM) for 2 h followed by exposure to 1.0 mM H2O2 for 24 h. Cell viability and apoptosis were determined with Cell Counting Kit-8 and flow cytometry assays, respectively. The activity levels of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were analyzed using ELISA kits. Protein expression levels, including Bax, caspase-3, Bcl-2, phosphorylated-AKT, AKT, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), cytosolic Nrf2 and nuclear Nrf2, were analyzed using western blot analysis. In addition, the expression and localization of Nrf2 were detected by immunofluorescence. RK1 treatment significantly improved cell viability, reduced the apoptotic rate and increased the activity levels of SOD, CAT and GSH-Px in the PIG1 cell line exposed to H2O2. In addition, RK1 treatment notably induced Nrf2 nuclear translocation, increased the protein expression levels of Nrf2 and HO-1, and the ratio of phosphorylated-AKT to AKT in the PIG1 cells exposed to H2O2. Furthermore, LY294002 could reverse the protective effect of RK1 in melanocytes against oxidative injury. These data demonstrated that RK1 protected melanocytes from H2O2-induced OS by regulating Nrf2/HO-1 protein expression, which may provide evidence for the application of RK1 for the treatment of vitiligo.

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.

Paeonol protects melanocytes against hydrogen peroxide-induced oxidative stress through activation of Nrf2 signaling pathway.

Oxidative stress plays a pivotal role in the pathogenesis of vitiligo. Paeonol, a phenolic compound found in the root bark of Cortex Moutan, has been suggested to have therapeutic effects on various diseases through nuclear factor Nrf2 mediated antioxidant pathways. However, the therapeutic effects of paeonol on vitiligo have not been investigated. In this study, we investigated whether paeonol could protect melanocytes against oxidative stress through Nrf2 activation. Hydrogen peroxide (H2 O2 ) was used to mimic the oxidative stress. PIG1 cells were pretreated with paeonol for 24 h, and then were treated with H2 O2 for 24 h. To detect the role of Nrf2, siRNA method was used to knockdown the Nrf2 expression. After that, cell viability, melanin content, tyrosinase activity, intracellular reactive oxygen species, antioxidant enzymes activities, nuclear translocation of Nrf2, and mRNA expression of Nrf2 downstream antioxidant genes were detected. Paeonol improved cell viability and melanogenesis in PIG1 cells treated with H2 O2 . Paeonol also alleviated oxidative stress by restoring the activities of superoxide dismutase, catalase, and glutathione peroxidase. Paeonol promoted Nrf2 nuclear translocation and the expression of its downstream-regulated antioxidative genes under oxidative stress. Furthermore, Nrf2 knockdown by siRNA abolished the protective effects of paeonol on PIG1 cells against oxidative damage. Paeonol protected melanocytes against H2 O2 -induced oxidative stress by Nrf2 mediated antioxidant pathways. Paeonol is a potential therapeutic drug for vitiligo.

Oxyresveratrol-induced Activation of Nrf2/HO-1 Signaling Pathway Enhances Ability of Resveratrol to Inhibit UVB-induced Melanin

Abstract Objective: This study was performed to investigate the relationship between the human melanogenesis and antioxidant systems and to further confirm the synergistic effect of oxyresveratrol (OXYR) and resveratrol (RES) in human epidermal melanocyte cell line. Methods: The human epidermal melanocyte line PIG1 cells were divided into the UV groups and control group, treated with different doses of UVB and without UVB, respectively. MTT assay and flow cytometry were used to detect cell viability and apoptosis. The expression of Nrf2/HO-1 and melanogenesis-associated proteins/genes was measured by Western blotting and real-time qPCR (RT-qPCR). pCMV6-XL5-Nrf2 was used to upregulate the expression of Nrf2. Subsequently, the proteins/genes levels of Nrf2/HO-1 and tyrosinase (TYR), melanin/eumelanin content, and reactive oxygen species (ROS) were analyzed. Isobologram analysis and cell experiment were used to analyze whether OXYR and RES inhibit TYR synergistically. Western blotting, RT-qPCR, and NaOH splitting method were used to determine the Nrf2/HO-1 and melanogenesis-associated proteins/genes expression and melanin content to evaluate the efficacy of OXYR and RES. Results: The activated Nrf2 and HO-1 eliminated ROS produced by UVB irradiation. The melanogenesis-associated proteins/genes of melanocyte-inducing transcription factor (MITF, P < 0.01 on protein expression), TYR (both P < 0.01), TYR-related protein (TRP)-1 (both P < 0.05), and TRP2 (P < 0.05 on mRNA expression) were activated in PIG1 cells by UVB irradiation. Simultaneously, the upregulation of Nrf2 significantly reduced melanogenesis formation (P < 0.001) and TYR level (P < 0.01 on protein expression). Moreover, OXYR and RES synergistically inhibited TYR activity (P < 0.001) and reduced melanin content (P < 0.001). Conclusions: A microbalance exists between Nrf2/HO-1 signaling and melanogenesis production in the UVB-induced responses of melanocytes. Simultaneously, OXYR enhances the ability of RES to inhibit melanin production.

MicroRNA-340 is involved in ultraviolet B-induced pigmentation by regulating the MITF/TYRP1 axis.

ObjectiveThere is growing evidence that ultraviolet B (UVB) irradiation can change the expression profile of microRNAs (miRNAs) in immortalized human epidermal melanocytes (Pig-1). We aimed to investigate the effect of miR-340 on regulating UVB-induced pigmentation.MethodsReal-time quantitative PCR (qRT-PCR) was used to evaluate the expression of miR-340 in Pig-1 cells. Immunoblotting analysis, qRT-PCR, and luciferase reporter assays were used to detect the potential target of miR-340. The sodium hydroxide dissolution assay was used to assess the effect of miR-340 on changes in melanin content.ResultsExpression of miR-340 was reduced in human Pig-1 cells after UVB irradiation. We found a negative correlation between miR-340 and melanocyte inducing transcription factor (MITF) in Pig-1 cells after UVB irradiation. Knockdown and overexpression of MITF in Pig-1 cells down- and upregulated melanogenesis, respectively. Overexpression of miR-340 inhibited MITF expression, reduced the amount of melanin, and suppressed expression of multiple key molecules involved in the pigment synthesis pathway, whereas knockdown of miR-340 showed the opposite results.ConclusionsOur results showed that miR-340 inhibited melanogenesis by regulating the downstream molecules of MITF and its signaling pathways, suggested that miRNA-340 may be a new target for the clinical treatment of UVB-induced pigmentation.

287 Secreted frizzled-related protein 5 (SFRP5 ) inhibits the melanin synthesis of melanocytes via Wnt/β-catenin signaling pathway in vitiligo

Vitiligo is an acquired chronic depigmentation disorder characterized by progressive depigmentation due to the destruction of epidermal melanocytes. The dysfunction of melanocytes plays an important role in the pathogenesis of vitiligo. In this study, we found that secreted frizzled-related protein 5 (SFRP5) is overexpressed in the skin lesions of vitiligo patients. Compared with normal epidermal melanocytes (PIG1), the expression of SFRP5 is increased in vitiligo melanocytes (PIG3V). To investigate the effect of SFRP5 on melanin synthesis, PIG1 cells were infected with recombinant SFRP5 adenovirus (AdSFRP5), and PIG3V cells were infected with recombinant siSFRP5 adenovirus (AdsiSFRP5).

721 Abnormal autophagosome formation increased melanocyte sensitivity to H2O2-induced oxidative stress in vitiligo

Autophagy is a controlled self-digestion process which can protect cells against oxidative damage. Dysregulated autophagy has been demonstrated to increase melanocyte sensitivity to oxidative stress in vitiligo. However, the exact mechanism is still not clear. This study aimed to determine the implications of autophagy for melanocyte survival in response to oxidative stress. Our results demonstrated that the autophagic flux in PIG1 exposure to H2O2 was significantly enhanced compared with that in PIG3V, which were accompanied by high level of ROS accumulation, membrane potential changes, and increased apoptosis. It indicates that vitiligo melanocytes exhibited hypersensitivity to H2O2-induced oxidative injury due to dysregulated autophagy. To further explore the mechanism, we performed RNA sequencing to compare the RNA expression in PIG1 and PIG3V cells exposed to H2O2, the bioinformatic analysis indicate that autophagosome formation was impaired in vitiligo melanocytes, our in vitro study also showed that inhibition of autolysosome degradation can not lead to autophagosome accumulation in vitiligo melanocytes, confirming that the impairment of autophagosome formation is responsible for the defects of autophagy in vitiligo melanocytes, the further study also showed that overexpression of HSF1, the main transcription factor for Atg5 and Atg12, could reduce H2O2-induced oxidative damage of vitiligo melanocytes. Our data demonstrated that owing to the deficiency of HSF1 expression, the autophagosome formation was blocked, which resulted in autophagy impairment and further increase the sensitivity of vitiligo melanocytes to oxidative stress. Our results indicating that targeting autophagy may be a potential therapy option.

SFRP5 inhibits melanin synthesis of melanocytes in vitiligo by suppressing the Wnt/β-catenin signaling

Secreted frizzled-related protein 5 (SFRP5) plays a pivotal role in regulating the development of many tissues and organs, however, as an inhibitor of Wnt signaling, the role of SFRP5 in vitiligo remains unknown. Hence, we speculated that SFRP5 might be associated with melanogenesis in melanocytes by regulating Wnt signaling in vitiligo. In this study, we found that SFRP5 was overexpressed in the skin lesions of patients with vitiligo. Compared with that in normal epidermal melanocytes (PIG1), the expression of SFRP5 was increased in vitiligo melanocytes (PIG3V). To investigate the effect of SFRP5 on melanin synthesis, PIG1 cells were infected with recombinant SFRP5 adenovirus (AdSFRP5), and PIG3V cells were infected with recombinant siSFRP5 adenovirus (AdsiSFRP5). The results showed that SFRP5 overexpression inhibited melanin synthesis in PIG1 cells through downregulation of microphthalmia-associated transcription factor (MITF) and its target proteins via suppression of the Wnt/β-catenin signaling pathway. Accordingly, SFRP5 silencing increased melanin synthesis and activated the Wnt signaling pathway in PIG3V cells. Moreover, SFRP5 overexpression also downregulated the transcriptional activity of T cell factor/lymphoid enhancer factor (TCF/LEF) in PIG1 cells. Furthermore, this inhibitory effect of SFRP5 on melanin synthesis was reversed by treatment with the β-catenin agonist, SKL2001. The inhibitory action of SFRP5 in pigmentation was further confirmed in vivo using a nude mouse model. Hence, our results indicate that SFRP5 can inhibit melanogenesis in melanocytes. Additionally, our findings showed that SFRP5 plays a vital role in the development of vitiligo, and thus may serve as a potential therapeutic target for vitiligo.

ZMIZ1 promotes the proliferation and migration of melanocytes in vitiligo.

Genome wide association studies have revealed that the zinc finger MIZ-type containing 1 (ZMIZ1) is involved in the pathogenesis of vitiligo; however, the underlying mechanism remains unclear. The present study aimed to investigate the effects of ZMIZ1 on the proliferation, apoptosis and migration of the human melanocyte cell lines PIG1 and PIG3V. ZMIZ1 overexpression and knockdown PIG1 and PIG3V cell models were established by lentivirus infection, and the effects of ZMIZ1 on cell proliferation and apoptosis were determined using an MTT assay and flow cytometry, respectively. Furthermore, the expression levels of proliferation- and apoptosis-associated proteins were analyzed using western blotting. Additionally, Transwell assays were performed to determine the effect of ZMIZ1 on the migration of PIG1 and PIG3V cells. Finally, the effect of ZMIZ1 on cytoskeletal remodeling in PIG1 and PIG3V cells was analyzed using immunocytochemistry. The overexpression of ZMIZ1 promoted the proliferation and inhibited the apoptosis of PIG1 and PIG3V cells, whereas the genetic knockdown of ZMIZ1 resulted in the opposite effects. Furthermore, ZMIZ1 overexpression increased the migration, whereas the knockdown of ZMIZ1 inhibited the migration and altered remodeling of the actin cytoskeleton in PIG1 and PIG3V cells. In conclusion, the results of the present study suggest that ZMIZ1 regulates the proliferation, apoptosis and migration of PIG1 and PIG3V cells, and indicate that ZMIZ1 may serve as a potential therapeutic target for vitiligo.