Jia-Hui Jin,Yu-Yan Jiang,Yan Wang?,Zhao-Wei Meng?,Di-Hua Li,Lei Zhang,Hao Wang,Yan-Jun Zhang

1Graduate School,Tianjin University of Traditional Chinese Medicine,Tianjin301617,China;2Department of Nuclear General,Tianjin Medical University General Hospital,Tianjin300052,China;3College of Pharmaceutical Engineering of Traditional Chinese Medicine,Tianjin University of Traditional Chinese Medicine,Tianjin301617,China;4Research Institute,Tianjin NanKai Hospital,Tianjin300100,China;5Research and Development Department,Tianjin ShangMei Cosmetics Co.,Ltd.,Tianjin300380,China;6School of Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine,Tianjin301617,China.

Abstract

Keywords:melanin,Nrf2/HO-1,oxyresveratrol,reactive oxygen species,resveratrol,tyrosinase

Introduction

UVB irradiation induces melanogenesis through different mechanisms or factors.The skin is in a“self-protection”state when it absorbs physical light waves,and it protects itself from UV radiation damage by enhancing the activity of tyrosinase(TYR)and producing melanin from hydroxylation.1-2

Previous studies have suggested that the antioxidant defense system of the body aids greatly in reducing oxidative stress.3Nrf2,expressed in melanocytes,is an important transcription factor in the cellular antioxidant system.Nrf2can activate cytoprotective genes and regulate antioxidant response elements(ARE)by inducing the expression of antioxidants and phase II detoxifying enzymes.4-5Jian et al.6found that HO-1is an antioxidant gene that can be induced via the Nrf2/ARE pathway and reduce hydrogen peroxide-induced oxidative damage in human melanocytes.In order to further explore the relationship between the body’s antioxidation defense system and melanin production and confirm that the Nrf2/HO-1signaling pathway and melanin generation can aid in effectively eliminating UVB-induced ROS,we using PIG1cells conducted this study.

Morus alba L.is the primary species among mulberries.It has been commonly used in cosmetic products,functional foods and folk medicines.Morus alba contains phenolic compounds such as oxyresveratrol(OXYR)and resveratrol(RES).7These two kind of naturally occurring phenolic compounds with various bioactivities,including extending the life of caenorhabditis elegans,8antioxidant and antityrosinase activity,9-10inhibitting cellular melanogenesis.11In our previous studies,12it was found that both OXYR and RES have antioxidant and TYR inhibitory effects.However,their ability to resist oxidation and inhibit TYR varies greatly.When L-tyrosine is used as a substrate,OXYR has a stronger ability to inhibit tyrosinase than RES;when L-dopa is used as a substrate,RES has a stronger ability to inhibit tyrosinase than OXYR.Based on the existing research,we further study OXYR and RES inhibit TYR activity and melanin production whether have a synergistic effect in this study.

To the best of our knowledge,this study is the first to confirm the internal connection between the Nrf2/HO-1 signaling pathway and UVB-induced stress responses of melanocytes.At the same time,it was confirmed that OXYR and RES can synergistically inhibit TYR activity and melanin production.We anticipated that the results would facilitate the development of a novel synergistic strategy for treatment of skin injury using antioxidants and TYR inhibitors.We also aim to provide novel insight for future studies on TYR inhibitors.

Materials and methods

Chemicals and reagents

The immortalized human epidermal melanocyte cell line PIG1(CRL-2208TM)was purchased from Guandao Engineering(Shanghai,China).Fetal bovine serum was purchased from Gibco(Grand Island,NY,USA).Human melanocyte growth supplement-2and Medium-254-500 were purchased from Thermo Fisher Scientific(Waltham,MA,USA).The following antibodies were applied:β-actin(K101527P;Solarbio,Beijing,China);Nrf2(#12721),TYR(#11873),and HO-1(#5853)(Cell Signaling Technology,Danvers,MA,USA);melanocyte-inducing transcription factor(MITF)(ab20663),TYR-related protein1(TRP1)(ab249974),and TYR-related protein 2(TRP2)(ab103463)(Abcam,Cambridge,UK);and goat anti-rabbit IgG(BA1054;Boster,Wuhan,China).pCMV6-XL5and pCMV6-XL5-Nrf2(10μg)were obtained from OriGene Technologies(Rockwell,MD,USA).The protein extraction kit(P0033)was purchased from Beyotime Institute of Biotechnology(Shanghai,China).OXYR(≥98%,AZ71G001)and RES(≥98%,100401)were acquired from Sabinsa Corporation(Piscataway,NJ,USA).All other purchased chemicals were of high purity and reagent grade.

UVB irradiation and sample treatment

PIG1cells were cultured in complete medium254(M254),including44.5%(v/v)M254,5%(v/v)fetal bovine serum,and0.5%(v/v)human melanocyte growth supplement-2.All cells were maintained at37°C in a5%carbon dioxide incubator.After a24-hours period of culture,the number of cells accounted for approximately70%.We removed the supernatant and superadded phosphate-buffered saline(PBS).The cells were then exposed to UVB at a dose of100,200,300,400,500,and600mJ/cm2with a UVB source(peak of311nm)(SH4B;Shanghai Sigma High Technology Co.,Ltd.,Shanghai,China).A UVB radiometer with a UVB304sensor(UV-B;Shenzhen XRC Electronics Co.,Ltd.,Shenzhen,China)was used to measure the exposed energy.Immediately after UVB exposure,the PBS was replaced by complete M254and incubated with5%carbon dioxide at37°C.The control group was unirradiated,whereas the other setup was treated with the same dose as the UVB group.For western blot and realtime quantitative PCR(qPCR),the cells were harvested 24hours after UVB irradiation.

MTT assay

Cell proliferation was detected based on the methods described by Liu et al.13In brief,the cells were incubated for4h in culture medium with10μL of MTT(5mg/mL).After removing the supernatant,100μL of dimethyl sulfoxide(DMSO)was added to the complete M254.Finally,an infinite200?PRO multifunctional microplate detector(Kendi Trade Co.,Ltd.,Shanghai,China)was used to measure its absorbance at490nm.

Cell apoptosis assay

PIG1cells(6×105cells/well)were seeded in60-mm plates andincubatedfor24hoursincarbondioxidefor70%to80%confluency.ThecompleteM254waslaterreplacedby1mLof PBS.The cells were exposed to UVB irradiation at a dose ranging from0to600mJ/cm2.The PBS was then immediately replaced by complete M254,and the cells were incubated for 24hours to assay the apoptosis rates.Apoptotic and necrotic cells were identified using the Annexin V-FITC Apoptosis Detection Kit(abs50001;Absin Bioscience,Shanghai,China)in accordance with the manufacturer’s instructions.13Cell apoptosis was detected by Annexin V/propidium iodide staining as described by Liu et al.13

Nrf2transient transfection

PIG1cells(1×105cells/well for6-well plates and1×104cells/well for96-well plates)were plated into each well and cultured for24hours.For Nrf2overexpression,the cells were transfected with pCMV6-XL5and pCMV6-XL5-Nrf2(3μg/well for6-well plates and0.15μg/well for96-well plates)at60% to75% confluency using Lipofectamine2000(L-7003;Invitrogen,Carlsbad,CA,USA).After24hours of incubation,the cells were recovered in normal media after removal of the transfection reagents and then incubated for an additional 48hours after transfection to assess the changes in mRNA and protein.Control cells(no transfection),pCMV6-XL5 transfection(no Nrf2),and pCMV6-XL5-Nrf2transfection were included in each experiment.

Melanin content

The melanin content assay from the study by Zeng et al.14was adopted.Cells were exposed to UVB irradiation at a dose of300mJ/cm2,then immediately replaced by either complete M254or90μmol/L OXYR,10μmol/L RES,and90μmol/L OXYR+10μmol/L RES and incubated for48hours.Next,the cells were dissolved in1mol/L NaOH/10% DMSO solution and incubated at90°C to solubilize the melanin.The control group was treated similarly to the UVB group but did not receive UVB stimulation.The total melanin in each cell suspension was determined by measuring the absorbance at405nm.

Eumelanin level determination by enzyme-linked immunosorbent assay

Eumelanin was assayed using a commercially available human enzyme-linked immunosorbent assay kit(JL45240-96T;Jianglai Biological Technology Co.,Ltd.,Shanghai,China)in accordance with the manufacturer’s instructions.The absorbance(A450)was measured using a plate reader(Tecan,Zürich,Switzerland)with a detection sensitivity of≥1.0pg/mL.The eumelanin content was calculated by interpolating the results onto a standard curve that was generated using the absorbance measurements of synthetic eumelanin with a correction for the total protein amounts present in the cell lysate supernatants.

Detection of intracellular ROS

At24hours or30minutes after UVB irradiation,the cells were incubated in a serum-free medium with10mmol/L DCFH-DA for30minutes.The cells were washed two times with M254,and the green fluorescence corresponding to the intracellular ROS levels was detected under a fluorescence microscope.

TYR inhibition assay

Mushroom TYR inhibition assay

The mushroom TYR inhibition assay was conducted using the methods described below.OXYR and RES were dissolved in suitable DMSO and diluted in0.1mmol/L PBS(pH of6.8).Experiments were performed in triplicate in96-well plates.The reaction mixture was mixed with 80μL of0.1mmol/L PBS,40μL of5mmol/L levodopa(LDOPA)(L8220;Solarbio),and40μL of OXYR,RES,and OXYR+RES at different concentrations.The reaction mixture was incubated at37°C for15minutes.Next,40μL of100units/mL mushroom TYR(33K34411;TCI,Shanghai,China)was added to the reaction mixtures and incubated at37°C for20minutes.Dopachrome formation was monitored at475nm with an infinite 200?PRO multifunctional microplate detector.The rate of TYR activity inhibition(I%)was determined according to the following formula:

where A1and A2are the absorbance values of the blank at 475nm at0and20minutes,respectively,and B1and B2are the absorbance levels of the test sample at475nm at0 and20minutes,respectively.

Intracellular TYR inhibition assay

Intracellular TYR activity in PIG1cells was estimated by measuring the L-DOPA oxidation capacity as described by Rong et al.,15with slight modifications.PIG1cells(1×104cells/well)were seeded into96-well plates and incubated overnight.The complete M254was then replaced by 100μL of PBS,and the cells were exposed to300mJ/cm2UVB irradiation.The PBS was immediately replaced by complete M254and OXYR,RES,or OXYR+RES for 24hours.The PIG1cells were washed with PBS and lysed in50μL of1% Triton X-100.The cells’lysates were stored at-80°C for30minutes and then allowed to thaw for10minutes at room temperature.Next,10μL of freshly prepared L-DOPA(0.25%)was added and incubated for2hours at37°C.Absorbance was measured at475nm using an infinite200?PRO multifunctional microplate detector.

RNA extraction and real-time qPCR

Total RNA was extracted using the Eastep?Super Total RNA Extraction Kit(LS1040;Promega,Shanghai,China)according to the manufacturer’s instructions.After purification and quality checking,the extracted total RNA was reverse-transcribed to cDNA using the total mRNA as a template according to the M-MLV operation instructions(R123-01;Vazyme,Nanjing,China).cDNA was stored at-80°C for later use.qPCR was performed to detect the expression of MITF,TYR,TRP1,TRP2,Nrf2,and HO-1according to the SYBR method.Three wells were set for each sample and indexed using GAPDH as the internal reference.The relative expression content of the target RNA was calculated using the2-△△Ctmethod.The primer sequences used are listed in Table1.

Western blot

The protein extracted from PIG1cells was obtained using a protein extraction kit in accordance with the manufacturer’s protocols.Protein samples were subjected to electrophoresis and then transferred to polyvinylidene difluoride membranes.The blot was incubated with the following primary antibodies at4°C overnight:primary antibodies to β-actin(1:1,500),MITF(1:900),TYR(1:1,000),TRP1(1:1,000),TRP2(1:1,000),Nrf2(1:1,000),and HO-1(1:1,000).The membrane was then incubated with goat anti-rabbit IgG(1:4,000).The relative density of each band was analyzed using an imaging densitometer.The densitometry values were normalized using β-actin.

Statistical analysis

Statistical analysis was performed using SPSS Statistics for Windows,Version17.0(SPSS Inc.,Chicago,IL,USA).Data are presented as mean±standard deviation.Oneway analysis of variance was performed to compare the differences between groups.A two-independent-samples test was performed to assess differences between two groups.A P value of＜0.05was considered statistically significant.Isobologram analysis was used to analyze the interaction between OXYR and RES in the mushroom TYR inhibition experiment.

Results

UVB phototoxicity on PIG1cells

Apoptosis was assessed in PIG1cells.Compared with the control cells,the number of apoptotic and necrotic cells increased in a dose-dependent manner after UVB irradia-tion over24hours(Fig.1A).MTT assays showed an insignificant inhibitory effect on PIG1cells regardless of the UVB dose(100,200,or300mJ/cm2).However,UVB doses of≥400mJ/cm2resulted in a high number of dead cells.The cell proliferative activity in the300-mJ/cm2group was95% of that in the control group,but the difference was not statistically significant(Fig.1B).

Table1 Primers used for qPCR.

Nrf2/HO-1pathway activated by UVB irradiation in PIG1cells

Figure1.Effects of different UVB doses on cell proliferation.(A)UVB irradiation caused apoptosis in PIGI cells,which were stained with Annexin V-FITC and propidium iodide for15minutes and analyzed by flow cytometry.Representative flow cytometry graphs are shown.(B)Effects of different UVB irradiation doses on the proliferative activity of PIG1cells.???P＜0.001vs.control group;n=5.UVB:ultraviolet B.

The changes in Nrf2and HO-1mRNA and protein expression in the susceptibility of melanocytes to UVB irradiation were investigated.After irradiation with 300mJ/cm2of UVB for24hours,the mRNA expression levels of Nrf2and HO-1were higher than those in the control group(Fig.2A).After UVB irradiation of PIG1 cells,the protein expression levels of Nrf2and HO-1were higher than those in the control group(Fig.2B).

Activation of MITF,TYR,and other melanogenesisrelated enzymes by UVB irradiation in PIG1cells

After UVB irradiation with300mJ/cm2of UVB for 24hours,the MITF,TYR,TRP1,and TRP2mRNA levels were higher than those in the control group(Fig.2C).The protein expression of MITF,TYR,TRP1,and TRP2was higher than that in the control group(Fig.2D).

Reduction of melanin/eumelanin formation by Nrf2 upregulation

To investigate the relationships among ROS induced by UVB irradiation,melanogenesis,and the Nrf2pathway,we used pCMV6-XL5-Nrf2to upregulate Nrf2expression.Increased Nrf2protein levels were observed48hours after transfection,indicating the success of Nrf2upregulation.β-actin was used as the internal control.The protein expression levels of Nrf2were significantly increased.PIG1cells transfected with pCMV6-XL5-Nrf2for 48hours had the same mRNA level as that of Nrf2,while these changes were absent in the mock-treated cells(Fig.3A–3B).

The melanin content of PIG1cells was significantly increased after irradiation with300mJ/cm2of UVB.This increase was also seen in PIG1cells pretreated with pCMV6-XL5-Nrf2-UV(i.e.,the cells were transfected with pCMV6-XL5-Nrf2and then irradiated with UVB),but at a significantly lower degree than in the UV group(Fig.3C).The same trend was observed in the eumelanin content of the cells(Fig.3D).

The ROS content of the PIG1cells was measured by flow cytometry.The ROS content of the PIG1cells was significantly increased30minutes and24hours after irradiation with300mJ/cm2of UVB,indicating that UVB irradiation can significantly increase ROS generation.The ROS content of the PIG1cells pretreated with pCMV6-XL5-Nrf2-UV was also significantly increased.However,the increase in the latter was significantly lower than that in the former(Fig.3E).These results suggest that Nrf2upregulation can eliminate the ROS produced by UVB and reduce the melanin and eumelanin production.

Figure2.Effects of UVB irradiation on antioxidant genes and melanogenic genes in PIG1cells.(A)Irradiation with300mJ/cm2of UVB increased the mRNA expression levels of Nrf2and HO-1compared with the control group.(B)Western blot analysis demonstrated that after UVB irradiation,the protein expression of Nrf2and HO-1was higher than that in the control group.(C)The mRNA levels of MITF,TYR,TRP1,and TRP2were higher than those in the control group.(D)The protein expression levels of MITF,TYR,TRP1,and TRP2were determined through western blot analysis.??P＜0.01,?P＜0.05vs.control group;n=3.UVB:ultraviolet B;TYR:tyrosinase;TRP:TYR-related protein;MITF:melanocyte-inducing transcription factor.

Figure3.Analysis of related indicators after transfection with pCMV6-XL5-Nrf2in PIG1cells.Nrf2protein level(A)and Nrf2mRNA(B)were measured by Western blot analysis and RT-PCR,respectively,after transfection with pCMV6-XL5-Nrf2.β-actin was used as an internal control.The melanin(C),eumelanin(D),and ROS(E)contents of the PIG1cells were tested.???P＜0.001,??P＜0.01,?P＜0.05vs.control group;###P＜0.001,##P＜0.01vs.UVB group;n=3.UVB:ultraviolet B;ROS:reactive oxygen species.

The ROS content was lower after irradiation for24 hours than after irradiation for30minutes,indicating that the large amount of ROS generated by the UVB irradiation activated the intracellular self-protection system to gradually remove ROS from the body and approach the normal level(Fig.3E).

Effects of Nrf2upregulation on TYR,Nrf2,and HO-1

To investigate the existence of a trade-off between the Nrf2/HO-1antioxidant system and melanogenesis(melanin/eumelanin),we analyzed the protein expression levels of Nrf2,HO-1,and TYR together with their mRNA expression levels in the PIG1cells pretreated with pCMV6-XL5-Nrf2-UV or UVB irradiation.The protein expression levels of Nrf2and HO-1in the PIG1cells pretreated with pCMV6-XL5-Nrf2-UV were significantly increased.In contrast,the protein levels of TYR in the same PIG1cells were significantly decreased(Fig.4A and4B).The same trend was observed in the mRNA levels(Fig.4C).

Effects of OXYR,RES,and OXYR+RES on TYR activity

The half maximal inhibitory concentration(IC50)is the concentration at which50% of a compound’s activity is lost.Two compounds were measured at different concentrations.The IC50of mushroom TYR inhibited by OXYR and RES solutions was16μmol/L and15μmol/L,respectively.

The effect points(IC50values)of the various compounding ratios of OXYR and RES fell below the addition line and the95% confidence limit,indicating that the compounding ratios within the study range had synergistic effects(Fig.5A).OXYR and RES were most effective when the ratio was9:1and the combination index was0.2077(Table2).

Experiments were carried out with a fixed ratio of cells.When the concentration of OXYR and RES was90μmol/L and10μmol/L,respectively,the inhibition rate at this dose was1.68times higher than the sum of the inhibition rates of OXYR and RES alone.The effect of synergistic inhibition of TYR was strongest under these circumstances;therefore,this concentration was selected for subsequent experiments(Fig.5B).

Effects of OXYR,RES,and OXYR+RES on MITF,TYR,and other melanogenesis-related enzymes after UVB irradiation of melanocytes

UVB radiation of PIG1cells can influence melanogenesisrelated enzymes and the Nrf2/HO-1pathway.To evaluate the effect of OXYR,RES,and OXYR+RES in PIG1cells,we measured the protein levels of MITF,TYR,TRP1,TRP2,Nrf2,and HO-1with western blotting.Based on previous results,we already knew that exposure of PIG1 cells to UVB significantly increases the MITF,TYR,TRP1,TRP2,Nrf2,and HO-1levels compared with control cells(Fig.2A and3A).However,the group treated with OXYR,RES,and OXYR+RES showed decreases in UVB-induced MITF,TYR,TRP1,TRP2,Nrf2,and HO-1(Fig.6A–6D).The degree of reduction of the protein expression levels in ascending order was OXYR,RES,and OXYR+RES.

Effects of OXYR,RES,and OXYR+RES on melanogenesis

The melanin content of PIG1cells was significantly lower after incubation with90μmol/L OXYR,10μmol/L RES,or90μmol/L OXYR+10μmol/L RES in complete medium after300mJ/cm2of UVB than that in the UV group irradiated with300mJ/cm2of UVB(Fig.6E).Furthermore,the melanin content in the OXYR+RES group was lower than that in the OXYR group and RES group.

Discussion

UV irradiation stimulates melanin synthesis as a form of protection against damage.UVB light is mostly absorbed by the epidermis because of its low penetration into the dermis.However,an excessive stress response and damage induced by ROS generation from UVB can lead to further cell damage and harmful reactions such as pigmentation,erythema,immunosuppression,loss of hair,and premature aging.16-17In this study,we found that UVB irradiation can significantly inhibit PIG1cell growth regardless of the UVB dose.However,when the UVB dose was≥400mJ/cm2,cell viability significantly decreased.Based on these results,the UVB dose of300mJ/cm2was applied in subsequent experiments.

Upon exposure to UV irradiation,epidermal melanocytes produce melanin that is transferred to neighboring keratinocytes,thereby allowing UV radiation absorption to protect the cells from UV irradiation-induced damage and displaying photo-protective and thermoregulatory roles.18Melanin can be classified into two types,eumelanin and pheomelanin,19that have different chemical and biological reactions upon exposure to light because of their different structures and physical properties.For example,eumelanin acts as an effective sunscreen that protects skin cells against damage from UV radiation.In contrast,pheomelanin is more sensitive to radiation than eumelanin,generates ROS after UV exposure,and has harmful effects.20-21The color of skin is mainly determined by the ratio of eumelanin and pheomelanin.The physiological function of eumelanin is superior to that of pheomelanin.Therefore,we measured the content of melanin and eumelanin.

Melanogenesis is stimulated by α-melanocyte-stimulating hormone,which binds to the melanocortin1receptor and transduces a signal to increase the expression of MITF by activating cyclic adenosine monophosphate.22-23MITF controls the expression of melanogenesis-related enzymes such as TYR,TRP1,and TRP2.24ROS are also associated with melanogenesis.25-26Previous studies have shown that MITF is phosphorylated and then degraded under ROS stimulation.27In the present study,when UVB irradiation was performed for30minutes,the ROS content,melanin and eumelanin production,and melanogenesis-associated proteins MITF,TYR,TPR1,and TPR2were increased.These results indicate that after UVB irradiation,a large amount of ROS was produced in PIG1cells,which stimulated the ROS-MITF signaling pathway in vivo.This in turn activated proteins related to melanogenesis and triggered melanin and eumelanin production in order to scavenge ROS and protect melanocytes from ROS.After 24hours,the ROS in the body that have been exacerbated by UVB exposure were cleared by the spontaneous clearance mechanism of melanocytes and tended toward normal levels.

Figure4.Analysis of Nrf2,HO-1,and TYR protein and mRNA expression levels after transfection with Nrf2and irradiation with300mJ/cm2of UVB in PIG1cells.Nrf2,HO-1,and TYR proteins(A)and mRNA(B)levels were measured by Western blot analysis and RT-PCR,respectively,after transfection with pCMV6-XL5-Nrf2.###P＜0.001,##P＜0.01,#P＜0.05vs.UV group;n=3.UVB:ultraviolet B;TYR:tyrosinase.

Figure5.TYR inhibitory effects of drugs were tested according to the L-DOPA oxidation method.(A)Isobologram plots of various compound ratios of OXYR and RES.-□-represents the theoretical IC50value(IC50add)after compounding,and-?-represents the actual IC50value after compounding(IC50mix).(B)Inhibitory effect of OXYR and RES at a ratio of9:1on intracellular TYR.The OXYR:RES(μmol/L)ratios of54:6,72:8,90:10,108:12,and126:14are shown in order1,2,3,4,and5.TYR:tyrosine;OXYR:oxyresveratrol;RES:resveratrol,IC50:half maximal inhibitory concentration.

ROS generated from UVB irradiation can also stimulate antioxidant and/or phase II detoxifying enzymes as a protective measure.Antioxidant activity is essential in the prevention and reparation of UV-induced skin damage and photoaging.Nrf2is a key transcription factor expressed in melanocytes and is enhanced during cellular defenses.28-29UV radiation,particularly UVB radiation,significantly increases the expression levels of the transcription factor Nrf230and subsequently its main target antioxidant gene,HO-1.6Our discovery is consistent with previous studies,showing that Nrf2and its correspondingbiphasic protein HO-1were activated when cells were exposed to UVB irradiation.

Table2 Inhibitory effect of OXYR and RES on mushroom tyrosinase(n=3).

To provethe hypothesis of involvement of theNrf2/HO-1 pathway in cell protection against UVB light,we upregulated Nrf2in subsequent experiments followed by UVB irradiation.As a result,the protein and mRNA levels related to the antioxidant system(Nrf2/HO-1)were significantly increased.However,theprotein andmRNA levels relatedto melanogenesis(TYR)were significantly decreased.Combined with the findings from previous studies,we concluded that both the Nrf2/HO-1pathway and melanogenesis are self-protection methods that protect PIG1cells against UVB radiation damage.

Our findings from this study revealed a microbalance between melanogenesis and the direct presence of Nrf2/HO-1,whereby melanogenesis and TYR are reduced but ROS scavenging effects are increased by high expression of Nrf2.TYR inhibition is currently the main focus in the treatment of pigmentation disorders.Because TYR only exists in melanocytes in the basal layer of the epidermis,there are challenges in administration and limitations in the therapeutic effects of such treatments.

The effects of OXYR and RES on TYR activity in vitro and cellular melanin synthesis are different.11This may be due to the fact that many signal pathways affect melanogenesis,such as MITF,31and RES has a certain inhibitory effect on these various pathways.Therefore,the inhibition of melanogenesis should be determined by the comprehensive effect of compounds on multiple signal pathways.

RES reportedly has mushroom TYR inhibitor activity.32In addition to direct inhibition,RES is also known to inhibit melanocyte-stimulating hormone signaling in melanoma cells and to reduce the tyrosinases TRP1,TRP2,and MITF.33-35According to the results reported by Park et al.,11the inhibitory effects of RES and OXYR on the key proteins related to above melanin production are different.Our study clearly showed that OXYR and RES have a synergistic effect on melanogenesis in PIG1cells through downregulating MITF,TRP1,and HO-1.

Our previous research suggested that an effective antioxidant can improve the activities of TYR inhibitors.In combination with these findings,Nrf2is believed to be a promising target for future treatments of pigmentation disorders because TYR inhibition and antioxidant defense simultaneously occur.To the best of our knowledge,this study is the first to clarify the relationship between the Nrf2/HO-1antioxidant system and melanogenesis(melanin/eumelanin),thereby providing a new target for such basic treatments,facilitating comprehensive improvement in the basic therapeutic effects,and providing a new research idea for the cosmetics industry.Although this manuscript has clarified the microbalance between the antioxidant system and melanogenesis,the skin does not only contain melanocytes.We used only PIG1cells for this study;therefore,there may be slight differences between the experimental results and the actual clinical situation.

Figure6.Effect of OXYR,RES,and OXYR+RES on MITF,TYR,TRP1,TRP2and melanogenesis after UVB irradiation of melanocytes.(A,B)The protein expression of MITF,TYR,TRP1,TRP2,Nrf2,and HO-1was higher than that in the control group.Protein optical density relative to β-actin.(C)Effect of OXYR,RES,and OXYR+RES on melanogenesis.???P＜0.001,??P＜0.01,?P＜0.05vs.UV group;###P＜0.001,##P＜0.01,#P＜0.05vs.OXYR group;n=3.OXYR:oxyresveratrol;RES:resveratrol;MITF:Microphthalmia-associated transcription factor;TYR:tyrosine;TRP:TYR-related protein;UVB:ultraviolet B.

Source of funding

The study was supported by the National Natural Science Foundation of China(No.81602773).