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Research Article(Z)-5-(24-Dihydroxybenzylidene)thiazolidine-24-dionePrevents UVB-Induced Melanogenesis and Wrinkle Formationthrough Suppressing Oxidative Stress in HRM-2 Hairless Mice

Bonggi Lee1 Kyoung Mi Moon1 Seong Jin Kim1 So Hee Kim1 Dae Hyun Kim12

Hye Jin An1 Ji Won Jeong1 Ye Ra Kim1 Sujin Son1 Min Jo Kim1 Ki Wung Chung1

Eun Kyeong Lee1 Pusoon Chun3 Young Mi Ha4 Min-Sun Kim5 Sang Hyun Mo6

Hyung Ryong Moon12 and Hae Young Chung12

1College of Pharmacy Pusan National University Busan 609-735 Republic of Korea2Molecular Inflammation Research Center for Ageing Intervention (MRCA) Pusan National University Republic of Korea3College of Pharmacy Inje University Busan 609-735 Republic of Korea4Department of Chemistry Dong-A University Busan 609-735 Republic of Korea5College of Pharmacy Sunchon National University Sunchon Republic of Korea6Bio-FDampC Songdo Mirae-ro 30 Incheon Republic of Korea

Correspondence should be addressed to Hyung Ryong Moon mhr108pusanackr and Hae Young Chung hyjungpusanackr

Received 12 January 2016 Revised 1 April 2016 Accepted 5 April 2016

Academic Editor Rosa Tundis

Copyright copy 2016 Bonggi Lee et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Uncontrolled melanogenesis and wrinkle formation are an indication of photoaging Our previous studies demon-strated that (Z)-5-(24-dihydroxybenzylidene)thiazolidine-24-dione (MHY498) inhibited tyrosinase activity and melanogenesisin vitro Objective To examine in vivo effects of MHY498 as an antiaging compound on UVB-induced melanogenesis and wrinkleformation we topically appliedMHY498 on dorsal skin of HRM-2 hairless miceMethods Using histological analysis we evaluatedeffects ofMHY498 onmelanogenesis andwrinkle formation after UVB exposure In addition relatedmolecular signaling pathwayswere examined using western blotting fluorometric assay and enzyme-linked immunosorbent assay Results MHY498 suppressedUVB-inducedmelanogenesis by inhibiting phosphorylation of CREB and translocation ofMITF protein into the nucleus which arekey factors for tyrosinase expression Consistently tyrosinase protein levels were notably reduced in the dorsal skin of the hairlessmice by MHY498 treatment Furthermore MHY498 inhibited UVB-induced wrinkle formation and collagen fiber destructionby increasing type 1 procollagen concentration and decreasing protein expression levels of MMPs which play an essential role incollagen fiber degradation As a mechanism MHY498 notably ameliorated UVB-induced oxidative stress and NF-120581B activation inthe dermal skin of the hairless mice Conclusion Our study suggests that MHY498 can be used as a therapeutic or cosmetic agentfor preventing uncontrolled melanogenesis and wrinkle formation

1 Introduction

Skin plays an important role as protective barrier againstultraviolet (UV) irradiation and environmental pollutionBecause human skin is continuously exposed to variousenvironmental factors skin aging may be an inevitable nat-ural process However preventing UV-induced skin ageingis important because short-term UV exposure can cause

sunburn inflammation immune suppression and dermalconnective tissue damage [1] and chronic exposure to UVcan ultimately cause photoaging and skin cancer throughincreasing oxidative stress [2 3] Uncontrolledmelanogenesisand collagen fiber destruction followed by wrinkle formationare a hallmark of photoaging Wrinkle formation is closelyassociated with collagen fibrils [1] It is characterized bydecreased skin elasticity and degeneration of the extracellular

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 2761463 9 pageshttpdxdoiorg10115520162761463

2 Oxidative Medicine and Cellular Longevity

Table 1 Treatment scheme for UVB and MHY498

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28UVB 150 (mJcm2) x x x x x x x x x x x x x xMHY498 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘

matrix (ECM) such as collagen which is produced by fibrob-lasts in the dermis [4] Therefore preventing collagen fiberdestruction is essential for inhibiting wrinkle formation

On the other hand melanogenesis is a protective processof skin against UV-induced damage [5] but chronic andrepeated exposures to UV can lead to premature aging of theskin [5] Furthermoremelanin accumulation is directly asso-ciatedwith pigmentation disorders such asmelasma frecklesand solar lentigo [6 7] Melanocyte is located in the basallayer of the epidermis and amain cell type to secretemelaninTyrosinase is an important enzyme responsible for skin pig-mentation in mammals [8] which catalyses two rate-limitingsteps in melanogenesis the hydroxylation of tyrosine to 34-dihydroxyphenylalanine (DOPA) and the oxidation ofDOPAtoDOPAquinoneTherefore tyrosinase plays an essential rolein melanin production in melanocytes and inhibiting tyrosi-nase is an attractive target for ameliorating pigmentationdisorders and various cosmetics

Oxidative stress plays a synergistic role in UV-inducedskin damage Reactive oxygen species (ROS) such as ∙O

2

minus∙OH and NO can be induced by UV and the accumulatedROS eventually causes wrinkle formation and melanogenesisin skin [9] As a mechanism ROS activate NF-120581B signalingwhich is responsible for inflammation in skin [10] In addi-tion it has been shown that ROS and NF-120581B activation stim-ulates expression and activation of matrix metalloproteinases(MMPs) that stimulate collagen degradation and inhibitcollagen synthesis [9 11] Therefore preventing ROS gen-eration and thereby reducing oxidative stress are importanttargets for inhibiting UV-induced skin damage

Our previous in vitro study reported that (Z)-5-(24-dihydroxybenzylidene)thiazolidine-24-dione (MHY498)suppressed melanogenesis by directly inhibiting tyrosinase[12] and interfering with tyrosinase expression [13] Howeverin vivo effects of MHY498 on skin pigmentation and wrinkleformation have not been examined Although variouscompounds for inhibiting pigmentation orwrinkle formationhave been screened in cell models they are not effectivein animal models possibly due to low stability and lowselectivity Here our current study examined in vivo effects ofMHY498 on UVB-mediated skin aging including melano-genesis and wrinkle formation using HRM-2 hairless miceThe data showed that MHY498 notably reduced UVB-induced pigmentation collagen fiber destruction and wrin-kle formation in the dorsal skin of hairless mice at leastpartially through reducingUVB-induced oxidative stress andrelated signaling

2 Materials and Methods

21 Mice HRM-2 hairless mice (6-week-old males) wereobtained from Hoshino Laboratory Animals (Yashio

Saitama Japan) The mice were maintained with 12 h12 hlightdark cycle and received ad libitum access to standardlaboratory diet and water MHY498 (200120583L of 05 120583M and5 120583M)was prepared in a solution containing propylene glycoland ethanol (3 7) MHY498 solution or the sham solutionincluding propylene glycol and ethanol was topically appliedto a designated site (3 cm times 3 cm) on dorsal skin of hairlessmice daily over the whole study period SpecificallyMHY498or the sham solution was pretreated for 3 days before UVB(302 nm) exposure From day 4 to day 25 2 h after MHY498or the sham solution treatment mice were exposed to UVBevery other day for 1 h in an UVB exposure chamber at150mJcm2 (see Table 1 for treatment scheme) From day 26UVB was exposed every day to maximize the effect of UVBon skin pigmentation and wrinkle formation based on ourpreliminary experiments to pick up the best UVB treatmentcondition (Table 1) After 28 days of the treatments themice were sacrificed and dorsal skin was excised and quicklyfrozen in a nitrogen tank for western blotting and measuringoxidative stress For staining purpose the excised skinwas fixed in 4 paraformaldehyde All animal studies wereapproved by the Institutional Animal Care Committee ofPusanNationalUniversity andwere performed in accordancewith the guidelines for animal experiments issued by PusanNational University

22 UVB Exposure We used a commercially availableUV exposure chamber (Ultraviolet Crosslinker UVP LLCUpland CA USA) for chronic UVB exposure of mice [1415] The UV exposure chamber is designed to measure andcontrol the ultraviolet (UV) radiation within the exposurechamber We used UVB specific tube and sensor provided bythe company that continually measures the UVB energy andautomatically adjusts to variations in UVB intensity thatoccur as the UVB tubes age Therefore we could minimizepotential contamination of UVA UVC and infrared radia-tion

23 Evaluation of Depigmenting Activities In Vivo Aftertreatment of MHY498 followed by UVB exposure darknessof skin sites was measured on day 28 of the treatment usinga CR-10 spectrophotometer (Konica Minolta Sensing IncSakai Osaka Japan) which describes colors using 119871lowast (higherand lower valuesmeanwhiter and blacker resp) as describedby the Commission Internationale de lrsquoEclairage color sys-tem When we compared nontreated mice with the controlmice treated with the sham solution containing propyleneglycol and ethanol there was no difference in skin brightnessvalues after UVB exposure Therefore we only used micetreated with the sham solution as a control group

Oxidative Medicine and Cellular Longevity 3

24 Fontana-Masson Staining Fontana-Masson staining wasperformed to detect melanin formation in skin of hairlessmice Fresh skin samples were fixed in 4 paraformaldehydeovernight at room temperature and stained for detectingmelanin using a Fontana-Masson staining kit (AmericanMastertech Inc Lodi CA USA) Briefly sliced skin sampleswere stained with ammoniacal silver solution for 60min at60∘C The samples were incubated in 01 gold chloridefollowed by 5 sodium thiosulfate Melanin spots wereobserved using an AE-31 light microscopy (Motic HongKong)

25 Massonrsquos Trichrome Staining Massonrsquos trichrome stain-ing was performed as previously described [16] Fresh skinsamples were fixed in 4 paraformaldehyde overnight atroom temperature and paraffin-embedded skin specimenswere sectioned at 5 120583m deparaffinized and stained withMassonrsquos trichrome to visualize collagenfibers Staining tissuesections were examined under an opticalmicroscope (EclipseTS100 Nikon Instruments Inc Melville NY USA)

26 Western Blotting Protein samples from skin lysates(30 120583g) were separated by sodium dodecyl sulfate-poly-acrylamide gel and transferred to polyvinylidene fluoride(PVDF) membranes which were immediately placed in 5nonfat milk blocking buffer containing 10mM Tris (pH75) 100mM NaCl and 01 Tween 20 The membrane waswashed in TBS-Tween buffer for 30min and then incubatedwith specific primary antibodies indicated in the figurelegends (dilution 1 1000) at 4∘C overnight After washingwith TBS-Tween buffer the membrane was incubated witha horseradish peroxidase-conjugated anti-mouse antibody(Santa Cruz 1 10000) an anti-rabbit antibody (Santa Cruz1 10000) or an anti-goat antibody (Santa Cruz 1 10000) at25∘C for 1 hThe immunoblots were visualized usingWesternBright Peroxide solution (Advansta CA USA) and Davinch-Chemi CAS-400 (Davinch-K Korea) according to the man-ufacturerrsquos instructions Antibodies used in this study are thefollowing p-CREB (SC-101663) tyrosinase (SC-15341)MITF(SC-11002) MMP1 (SC-12348) MMP9 (SC-6840) MMP12(SC-30072) MMP13 (SC-12363) type 1 procollagen (SC-25973) type 3 procollagen (SC-8779) p-p65 (S536) (SC-33020) iNOS (SC-8310) 120573-actin (SC-47778) and TFIIB (SC-225)

27 Measurement of ROS ROS generation was measured aspreviously described [17] Based on the oxidation of non-fluorescent 2101584071015840-dichlorofluorescein diacetate (DCF-DA) tohighly fluorescent 2101584071015840-dichlorofluorescein (DCF) in thepresence of esterases and ROS including lipid peroxides afluorometric assaywas used to examine ROS levels in the dor-sal skin of hairless mice Briefly DCF-DA (50 120583M)wasmixedwith skin homogenates (10 120583L) to a final volume of 250120583LFluorescence intensity was recorded every 5min for 30minusing a fluorescence plate reader (GENios Tecan Instru-ments Salzburg Austria) at emission and excitation wave-lengths of 530 and 485 nm respectively

28 Measurement of ONOOminus Peroxynitrite (ONOOminus) gen-eration was measured as previously described [17] Per-oxynitrite (ONOOminus) generation was examined by mea-suring the oxidation of DHR-123 Briefly dorsal skinhomogenate (10 120583L) was mixed with the rhodamine solution(50mM sodium phosphate buffer 90mM sodium chloride5mM diethylenetriaminepentaacetate [DTPA] and DHR-123) Changes in fluorescence intensity were observed every5min for 30min using a fluorescence plate reader withexcitation and emission wavelengths at 485 and 535 nmrespectively

29 Histological Analysis of Skin We performed histologicalanalysis of the dorsal skin using a commercially availablesilicone impression material (SILFLO) to mold the wrinkleon dorsal skin in silicone We followed the manufacturerrsquosinstruction for preparing skin samples We used a commer-cially available institution to get images of wrinkle using themolded skin samples (Oriental Medicine Industry SupportCenter South Korea)

210 Statistical Analysis All results are expressed as mean plusmnSEM Treatments were compared using one-way ANOVAfollowed by Bonferroni test 119875 values lt005 were consideredstatistically significant

3 Results and Discussion

31 Effect of MHY498 on UV-Induced Skin Pigmentation ofHRM-2 Hairless Mice We first examined whether MHY498has cytotoxic effects on Hs27 human dermal fibroblastsand B16F10 mouse skin melanoma cells Data showed thatMHY498 has no cytotoxic effects on both cell lines upto 10 120583m (Supplementary Figures 1(a) and 1(b) in Sup-plementary Material available online at httpdxdoiorg10115520162761463) Also when we treated the sham orMHY498 solution on dorsal skin of hairless mice for 28 daysno visible evidence of skin irritation was found (Figure 1(a))To examine in vivo effects of MHY498 on skin pigmentationwe topically applied the shamorMHY498 solution to the dor-sal skin of the hairless mice for 3 days From day 4 UVB wasexposed to the skin 2 h after MHY498 treatment RepeatedUVB exposure (150mJcm2) for 4 weeks darkened skin ofmice as expected (Figure 1(a)) However MHY498 treatmentat 05 120583M or 5 120583M markedly ameliorated the UVB-induceddarkening of the skin (Figure 1(a)) To confirm the bright-ening effect of MHY498 on the skin we measured darknessof the skin using CR-10 spectrophotometer UVB exposurereduced 119871lowast values in which higher values represent whitercolor (Figure 1(b)) However MHY498 treatment recov-ered UVB-induced pigmentation of the skin (Figure 1(b))To investigate whether the MHY498-mediated brighteningeffect of the skin is due to inhibition ofmelanogenesis we per-formed Fontana-Masson staining of the dorsal skin sectionsCompared to the control group (Figure 1(c)) UVB exposureinducedmelanogenesis evidenced by black spots of epidermis(Figure 1(d)) However MHY498 treatment at 05 120583M wasenough to reduce melanogenesis (Figure 1(e)) and MHY498


UVB(sham solution)

CON MHY498(05 120583M)

MHY498(5120583M)

(a)

UVBCON

UVB

minus4

minus3

minus2

minus1

0

1

2 MHY498(05 120583M)

MHY498

ΔLlowast

valu

e

lowastlowastlowast

(5120583M)

(b)

CON

(c)

UVB (150mJcm2)

(d)

MHY498 (05 120583M) + UVB (150mJcm2)

(e)

MHY498 (5120583M) + UVB (150mJcm2)

(f)

Figure 1 MHY498 ameliorates UVB-induced skin pigmentation MHY498 was pretreated for 3 days From day 4 2 h after MHY498application to the dorsal skin of the hairless mice UVBwas exposed to the mice as shown in Table 1 (119899 = 11group) After 4 weeks pictures ofthe dorsal skin were taken (a) Darkness of the skin sites was measured on day 28 of the treatment using a CR-10 spectrophotometer (KonicaMinolta Sensing Inc Sakai Osaka Japan) Higher 119871lowast values represent whiter colors (119899 = 11group) (b) AfterMHY498 application followedby UVB exposure skin samples were excised and Fontana-Masson staining was performed as described in Materials and Methods (cndashf)Microscopic analysis of dorsal skin sections from (c) control mice without UVB exposure (d) mice with UVB exposure and (e) MHY498(05 120583M) or (f) MHY498 (5120583M) treated mice Arrows represent where melanogenesis was induced by UVB Data are represented as mean plusmnSEM 119875 lt 001 and

119875 lt 0001 compared to a control group without UVB exposure lowastlowastlowast119875 lt 0001 compared to control group with UVBexposure

at 5 120583M fully recovered UVB-induced melanogenesis (Fig-ure 1(f)) These data suggest that MHY498 ameliorates UVB-induced skin darkening in mice probably through inhibitingmelanogenesis

32 Effect of MHY498 on UV-Induced Wrinkle Formationand Collagen Fiber Destruction of HRM-2 Hairless Mice Weexamined whether MHY498 has an inhibitory effect on UV-mediated wrinkle formation by histological analysis of thedorsal skin samples UVB exposure visibly increased wrinkle

formation compared to the control group whereas MHY498treatment markedly reduced it (Figures 2(a)ndash2(d)) Becausewrinkle formation is closely associated with impairment ofskin structure and collagen fiber we performed Massonrsquostrichrome staining of the dorsal skin sections Comparedto the control group showing dense collagen fiber structure(Figure 2(e)) UVB exposure induced collagen fiber destruc-tion with hyperkeratosis a hallmark of chronic UV expo-sure (Figure 2(f)) However MHY498 treatment markedlydecreased these features (Figures 2(g)-2(h)) Consistently


CON

(a)

UVB

(b)

MHY498 (05 120583M)

(c)

MHY498 (5120583M)

(d)

CON

200x

(e)

UVB

200x

(f)

200x

MHY498 (05 120583M)

(g)

200x

MHY498 (5120583M)

(h)

Collagen area

0

50000

100000

150000

200000

CON UVB

(120583m

2 )

lowastlowast

MHY498MHY498(05 120583M) (5120583M)

(i)

Figure 2 MHY498 ameliorates UVB-induced collagen fiber destruction MHY498 was pretreated for 3 days From day 4 2 h after MHY498application to the dorsal skin of the hairless mice UVB was exposed to the mice every other day for 1 h After 4 weeks skin samples wereexcised and Massonrsquos trichrome staining was performed as described in Materials and Methods (andashd) Control mice without UVB exposure(a) mice with UVB exposure (b) and MHY498 (05 120583M) (c) or MHY498 (5120583M) treated mice (d) Collagen staining appears blue Collagenarea was calculated based on microscopic analysis of the dorsal skin staining (119899 = 11group) (e) Data are represented as mean plusmn SEM119875 lt 001 compared to a control group without UVB exposure and lowast119875 lt 005 compared to the group with UVB exposure

reduced collagen area after UVB exposure was recovered byMHY498 treatment (Figure 2(i)) These data indicate thatinhibiting collagen fiber destruction and hyperkeratosis maycontribute to the antiwrinkle effect of MHY498 after UVBexposure

33 MHY498 Inhibits UVB-Induced CREB PhosphorylationandMITFTranslocation into theNucleus Our previous studyreported that MHY498 inhibits nitric oxide-induced tyrosi-nase expression in vitro [13] To examine molecular path-way(s) underlying the antimelanogenic effect of MHY498on the skin of mice we investigated protein levels of keymolecules that play an important role in melanogenesisAdenosine 3101584051015840-cyclicmonophosphate (cAMP) response ele-ment binding protein (CREB) andmicrophthalmia transcrip-tion factor (MITF) which induce transcription of tyrosinaseare key transcription factors for melanogenesis [18] Our

data showed that UVB exposure markedly increased phos-phorylated CREB an active form of CREB in the nucleusbut MHY498 treatment reduced phosphorylated CREB tothe level comparable to the control group without UVBexposure (Figures 3(a) and 3(b)) Furthermore increasedMITF translocation into the nucleus by UVB exposure wassignificantly decreased by MHY498 treatment (Figures 3(a)and 3(c)) These data suggest that MHY498 efficiently blocksCREB activation andMITF translocation into the nucleus Inparallel with this UVB-induced increase in the protein levelof tyrosinase was notably reduced by MHY498 treatment(Figures 3(d) and 3(e)) Our previous in vitro study showedthat MHY498 scavenges NO suppresses a NO-mediated sig-naling pathway and thus subsequently downregulated tyrosi-nase expression andmelanogenesis in B16F10melanoma cells[13]The current study confirmed thatMHY498 is effective inpreventing UVB-induced melanogenesis in vivo


MITF

p-CREB

CREB

TFIIB

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

(a)

0

1

2

3

4

p-CR

EB (a

rbitr

ary

unit)

UVBCON

lowastlowast

MHY498(05 120583M)

MHY498(5120583M)

(b)

0

1

2

3

MIT

F (a

rbitr

ary

unit)

UVBCON

lowast

MHY498(05 120583M)

MHY498(5120583M)

(c)

Tyrosinase

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(d)

0

1

2

3

4

5

Tyro

sinas

e (ar

bitr

ary

unit)

UVBCON

lowast

lowast

MHY498(05 120583M)

MHY498(5120583M)

(e)

Figure 3 MHY498 reduces CREB phosphorylation and MITF translocation into nucleus MHY498 was pretreated for 3 days From day 42 h after MHY498 application to the dorsal skin of the hairless mice UVB was exposed to the mice every other day for 1 h After 4 weeksskin samples were excised and homogenated Western blotting was performed using skin tissues for detecting (a) p-CREB CREB MITFand TFIIB in nucleus fraction and (d) tyrosinase and 120573-actin in cytosol fraction (119899 = 4group) The protein levels of (b) p-CREB (c) MITFand (e) tyrosinase were semiquantified using Image J software p-CREB and MITF protein levels were normalized by TFIIB (119899 = 4group)Tyrosinase protein levels were normalized by 120573-actin A representative blot is shown from four experiments that yielded similar results Dataare represented as mean plusmn SEM lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control group without UVB exposure


MMP1

MMP13

MMP12

MMP9

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(a)

Type 1 procollagen

Type 3 procollagen

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(b)

Type 1 procollagen ELISA

0

50000

100000

150000

200000

(ng

mL)

UVBCON

lowastlowastlowastlowastlowastlowast

MHY498(05 120583M)

MHY498(5120583M)

(c)

Figure 4 MHY498 reduces protein levels of MMPs and procollagens MHY498 was pretreated for 3 days From day 4 2 h after MHY498application to the dorsal skin of the hairless mice UVB was exposed to the mice every other day for 1 h After 4 weeks skin samples wereexcised and homogenated Western blotting was performed to detect protein levels of MMP1 MMP9 MMP12 MMP13 and 120573-actin (119899 =4group) (a) Protein levels of type 1 and type 3 procollagen and 120573-actin (119899 = 4group) (b) For western blotting results a representativeblot is shown from four experiments that yielded similar results Hs27 human dermal fibroblasts were cultured in DMEM until reaching85 confluence Cultured human fibroblasts were pretreated with MHY498 for 2 h and exposed to UVB (30mJcm2) Type 1 procollagenconcentration was measured using a commercially available ELISA kit (119899 = 5group) (c) Data are represented as mean plusmn SEM 119875 lt 001compared to a control group without UVB exposure and lowastlowastlowast119875 lt 0001 compared to the group with UVB exposure

It has been reported that melanogenesis may be a protec-tive mechanism against UV-induced DNA damage [19 20]To examine whether the MHY498-mediated inhibition ofmelanogenesis increases DNA damage we measured proteinlevels of DNA damage markers such as phospho-P53 ATRand phosphohistone H2AX using the nucleus fraction of skinhomogenate As expected UVB exposure increased proteinlevels of phospho-P53 (Ser15) and phosphohistone H2AX(Ser139) whereas MHY498 treatment appears to reversethem (Supplementary Figure 2) There was no clear changeof ATR protein levels in response to UVB or MHY498(Supplementary Figure 2)These data indicate thatMHY498-mediated inhibition of melanogenesis may not increase DNAdamage in skin

34 MHY498 Inhibits UVB-Induced MMP Protein Expres-sion MMPs are responsible for ROS-induced collagen fiberdegradation and inhibition of collagen synthesis [21] To

examine a molecular signaling pathway responsible for colla-gen recovery by MHY498 after UVB exposure we measuredprotein levels of MMP family by western blotting using thedorsal skin homogenate of the hairless mice UVB exposureincreased protein levels of MMP1 MMP9 MMP12 andMMP13 butMHY498 treatment reduced them (Figure 4(a))In parallel with this MHY498 treatment recovered the UVB-induced decrease in type 1 and type 3 procollagen proteinlevels in the skin (Figure 4(b)) indicating that the protectiveeffect of collagen fiber by MHY498 is mediated at leastpartially through reducing MMP protein levels

Collagen precursors are produced by fibroblasts in skinand play an important role in maintaining collagen structure[22] To examine whether MHY498 has protective effect ona collagen precursor in fibroblasts after UVB exposure wemeasured intracellular type 1 procollagen (a major collagenprecursor in skin) concentration in human dermal fibroblastscultured primarily fromhuman fetal skin [23] using anELISA


ROS

0

20

40

60FL

Um

inm

g pr

otei

n

UVCON

lowastlowastlowastlowast

MHY498(05 120583M)

MHY498(5120583M)

(a)

0

100

200

300

400

FLU

min

mg

prot

ein

UVCON

ONOOminus


MHY498(05 120583M)

MHY498(5120583M)

(b)

TF2B

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

p-p65 (S536)NF-120581B

p65NF-120581B

(c)

iNOS

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

120573-actin

(d)

Figure 5 MHY498 reduces UVB-induced oxidative stress and related signaling MHY498 was pretreated for 3 days From day 4 2 h afterMHY498 application to the dorsal skin of the hairless mice UVB was exposed to the mice every other day for 1 h (119899 = 6group) After4 weeks skin samples were excised and ROS (a) and ONOOminus (b) generation were measured using DCF-DA and DHR-123 as fluorescentprobes (119899 = 6group) (c) Western blotting images of p-p65 (S536) p65 and TFIIB in the nucleus extracts (d) Protein levels of NF-120581B targetgene iNOS in the cytosol extracts For western blotting results a representative blot is shown from four experiments that yielded similarresults NF-120581B nuclear factor-120581B TFIIB transcription factor II B Each value was expressed as mean plusmn SEM 119875 lt 005 and

119875 lt 001 versuscontrol without UVB exposure lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus the UVB-exposed group

kit UVB exposure significantly decreased type 1 procollagenconcentration in the fibroblasts (Figure 4(c)) On the otherhand MHY498 treatment fully recovered UVB-inducedreduction in type 1 procollagen concentration to the levelcomparable to the control groupwithout UVB exposure (Fig-ure 4(c)) These data suggest that MHY498-mediated type 1procollagen recovery in the fibroblasts at least partially con-tributes to the MHY498-mediated protection against UVB-induced collagen destruction

35 MHY498 Decreases UVB-Induced Oxidative Stress andRelated Signaling UV exposure induces oxidative stress thatis responsible for inducing MMP expression followed bycollagen fiber degradation and tyrosinase expression followedby melanogenesis To examine whether MHY498 decreasesoxidative stress after UVB exposure we measured ROS andONOOminus levels in the skin homogenate of the hairless miceUVBexposure dramatically increasedROS andONOOminus con-centration whereas MHY498 treatment reduced them to thelevel comparable to the control group without UVB exposure(Figures 5(a) and 5(b)) suggesting that MHY498 may reduceoxidative stress as an antioxidant Because UV-induced ROS

can activate and translocate NF-120581B into the nucleus which isclosely associated with skin aging including collagen degra-dation and excessive melanogenesis [9 11 24] we investi-gated whether MHY498 inhibits NF-120581B activation using skinfibroblasts It has been reported that phosphorylation of NF-120581B p65 on Ser 536 is essential for its capacity to transactivategenes [25] Therefore protein levels of p-p65 (S536) and p65were examined in the nucleus fraction by western blottingUVB increased the protein levels of p-p65 (S536) in thenucleus whereas MHY498 reduced it (Figure 5(c)) Consis-tently protein level of NF-120581B target gene iNOS was increasedby UVB treatment but reduced by MHY498 treatment (Fig-ure 5(d)) suggesting that inhibition of NF-120581B signaling byMHY498 may contribute to the protective effects on skinpigmentation and collagen destruction against UVB

Together in the dermal skin of the HRM-2 hairlessmice MHY498 reduced UVB-induced skin pigmentationby suppressing melanogenesis at least partially throughdecreases in CREB phosphorylation and MITF translocationto nucleus In addition MHY498 inhibited UVB-inducedwrinkle formation and collagen destruction possibly due toinhibiting MMP expression As a potential mechanism we


assumed that the reduced oxidative stress and NF-120581B sig-naling contribute to the antimelanogenic and antiwrinkleeffects of MHY498 Therefore MHY498 may be applied as apharmaceutical agent for protecting UV-induced skin dam-age

Competing Interests

The authors have no conflict of interests to declare

Authorsrsquo Contributions

Bonggi Lee and KyoungMiMoon contributed equally to thiswork

Acknowledgments

This work was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korean gov-ernment (MSIP) (Grant no 2009-0083538) and by theBasic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education Science and Technology (Grant no NRF-2013R1A1A2009949) The authors thank Aging Tissue Bankfor providing materials and research information

References

[1] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

[2] T Quan Z Qin W Xia Y Shao J J Voorhees and G J FisherldquoMatrix-degrading metalloproteinases in photoagingrdquo Journalof Investigative Dermatology Symposium Proceedings vol 14 no1 pp 20ndash24 2009

[3] JWenk P Brenneisen CMeewes et al ldquoUV-induced oxidativestress and photoagingrdquo Current problems in dermatology vol29 pp 83ndash94 2001

[4] M Egbert M Ruetze M Sattler et al ldquoThe matricellularprotein periostin contributes to proper collagen function andis downregulated during skin agingrdquo Journal of DermatologicalScience vol 73 no 1 pp 40ndash48 2014

[5] M Brenner and V J Hearing ldquoThe protective role of melaninagainst UV damage in human skinrdquo Photochemistry and Photo-biology vol 84 no 3 pp 539ndash549 2008

[6] M T H Khan ldquoNovel tyrosinase inhibitors from naturalresourcesmdashtheir computational studiesrdquo Current MedicinalChemistry vol 19 no 14 pp 2262ndash2272 2012

[7] C Liang J-H Lim S-H Kim and D-S Kim ldquoDioscin asynergistic tyrosinase inhibitor from the roots of Smilax chinardquoFood Chemistry vol 134 no 2 pp 1146ndash1148 2012

[8] K U Schallreuter S Kothari B Chavan and J D SpencerldquoRegulation of melanogenesismdashcontroversies and new con-ceptsrdquo Experimental Dermatology vol 17 no 5 pp 395ndash4042008

[9] R Pandel B Poljsak A Godic and R Dahmane ldquoSkinphotoaging and the role of antioxidants in its preventionrdquo ISRNDermatology vol 2013 Article ID 930164 11 pages 2013

[10] S Bell K Degitz M Quirling N Jilg S Page and K BrandldquoInvolvement of NF-120581B signalling in skin physiology anddiseaserdquo Cellular Signalling vol 15 no 1 pp 1ndash7 2003

[11] G J Fisher S C Datta H S Talwar et al ldquoMolecular basis ofsun-induced premature skin ageing and retinoid antagonismrdquoNature vol 379 no 6563 pp 335ndash339 1996

[12] S H Kim YMHa KMMoon et al ldquoAnti-melanogenic effectof (Z)-5-(24-dihydroxybenzylidene) thiazolidine-24-dione anovel tyrosinase inhibitorrdquo Archives of Pharmacal Research vol36 no 10 pp 1189ndash1197 2013

[13] S H Kim Y J Choi K M Moon et al ldquoThe inhibitory effectof a synthetic compound (Z)-5-(24-dihydroxybenzylidene)thiazolidine-24-dione (MHY498) on nitric oxide-inducedmelanogenesisrdquo Bioorganic amp Medicinal Chemistry Letters vol23 no 15 pp 4332ndash4335 2013

[14] S R Kim Y R Jung H J An et al ldquoAnti-wrinkle andanti-inflammatory effects of active garlic components and theinhibition ofMMPs via NF-120581B signalingrdquo PLoS ONE vol 8 no9 Article ID e73877 2013

[15] B Lee K M Moon S Son et al ldquo(2RS4R)-2-(24-Dihydrox-yphenyl)thiazolidine-4-carboxylic acid prevents UV-inducedwrinkle formation through inhibiting NF-120581B-mediated inflam-mationrdquo Journal of Dermatological Science vol 79 no 3 pp313ndash316 2015

[16] M H Park J Y Park H J Lee et al ldquoThe novel PPAR 120572120574 dualagonist MHY 966 modulates UVB-induced skin inflammationby inhibiting NF-120581B activityrdquo PLoS ONE vol 8 no 10 ArticleID e76820 2013

[17] Y J Choi Y Uehara J Y Park et al ldquoSuppression of melano-genesis by a newly synthesized compound MHY966 via thenitric oxideprotein kinaseG signaling pathway inmurine skinrdquoJournal of Dermatological Science vol 68 no 3 pp 164ndash1712012

[18] J P Ebanks R R Wickett and R E Boissy ldquoMechanisms reg-ulating skin pigmentation the rise and fall of complexion col-orationrdquo International Journal of Molecular Sciences vol 10 no9 pp 4066ndash4087 2009

[19] N Agar and A R Young ldquoMelanogenesis a photoprotectiveresponse to DNA damagerdquoMutation Research vol 571 no 1-2pp 121ndash132 2005

[20] M S Eller M Yaar and B A Gilchrest ldquoDNA damage andmelanogenesisrdquo Nature vol 372 no 6505 pp 413ndash414 1994

[21] Y R Jung D H Kim S R Kim et al ldquoAnti-wrinkle effect ofmagnesium lithospermate B from Salvia miltiorrhiza BUNGEinhibition of MMPs via NF-kB signalingrdquo PLoS ONE vol 9 no8 Article ID e102689 2014

[22] B Goldberg E H Epstein Jr and C J Sherr ldquoPrecursors ofcollagen secreted by cultured human fibroblastsrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 69 no 12 pp 3655ndash3659 1972

[23] Y-B Chae J S Lee H-J Park et al ldquoAdvanced adipose-derivedstem cell protein extracts with antioxidant activity modulatesmatrix metalloproteinases in human dermal fibroblastsrdquo Envi-ronmental Toxicology and Pharmacology vol 34 no 2 pp 263ndash271 2012

[24] Y J Kim and T Yokozawa ldquoModulation of oxidative stress andmelanogenesis by proanthocyanidinsrdquo Biological and Pharma-ceutical Bulletin vol 32 no 7 pp 1155ndash1159 2009

[25] F Yang E Tang K Guan and C-Y Wang ldquoIKK120573 plays anessential role in the phosphorylation of RelAp65 on serine 536induced by lipopolysacchariderdquo Journal of Immunology vol 170no 11 pp 5630ndash5635 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Treatment scheme for UVB and MHY498

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28UVB 150 (mJcm2) x x x x x x x x x x x x x xMHY498 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘

matrix (ECM) such as collagen which is produced by fibrob-lasts in the dermis [4] Therefore preventing collagen fiberdestruction is essential for inhibiting wrinkle formation

On the other hand melanogenesis is a protective processof skin against UV-induced damage [5] but chronic andrepeated exposures to UV can lead to premature aging of theskin [5] Furthermoremelanin accumulation is directly asso-ciatedwith pigmentation disorders such asmelasma frecklesand solar lentigo [6 7] Melanocyte is located in the basallayer of the epidermis and amain cell type to secretemelaninTyrosinase is an important enzyme responsible for skin pig-mentation in mammals [8] which catalyses two rate-limitingsteps in melanogenesis the hydroxylation of tyrosine to 34-dihydroxyphenylalanine (DOPA) and the oxidation ofDOPAtoDOPAquinoneTherefore tyrosinase plays an essential rolein melanin production in melanocytes and inhibiting tyrosi-nase is an attractive target for ameliorating pigmentationdisorders and various cosmetics

Oxidative stress plays a synergistic role in UV-inducedskin damage Reactive oxygen species (ROS) such as ∙O

2

minus∙OH and NO can be induced by UV and the accumulatedROS eventually causes wrinkle formation and melanogenesisin skin [9] As a mechanism ROS activate NF-120581B signalingwhich is responsible for inflammation in skin [10] In addi-tion it has been shown that ROS and NF-120581B activation stim-ulates expression and activation of matrix metalloproteinases(MMPs) that stimulate collagen degradation and inhibitcollagen synthesis [9 11] Therefore preventing ROS gen-eration and thereby reducing oxidative stress are importanttargets for inhibiting UV-induced skin damage

Our previous in vitro study reported that (Z)-5-(24-dihydroxybenzylidene)thiazolidine-24-dione (MHY498)suppressed melanogenesis by directly inhibiting tyrosinase[12] and interfering with tyrosinase expression [13] Howeverin vivo effects of MHY498 on skin pigmentation and wrinkleformation have not been examined Although variouscompounds for inhibiting pigmentation orwrinkle formationhave been screened in cell models they are not effectivein animal models possibly due to low stability and lowselectivity Here our current study examined in vivo effects ofMHY498 on UVB-mediated skin aging including melano-genesis and wrinkle formation using HRM-2 hairless miceThe data showed that MHY498 notably reduced UVB-induced pigmentation collagen fiber destruction and wrin-kle formation in the dorsal skin of hairless mice at leastpartially through reducingUVB-induced oxidative stress andrelated signaling

2 Materials and Methods

21 Mice HRM-2 hairless mice (6-week-old males) wereobtained from Hoshino Laboratory Animals (Yashio

Saitama Japan) The mice were maintained with 12 h12 hlightdark cycle and received ad libitum access to standardlaboratory diet and water MHY498 (200120583L of 05 120583M and5 120583M)was prepared in a solution containing propylene glycoland ethanol (3 7) MHY498 solution or the sham solutionincluding propylene glycol and ethanol was topically appliedto a designated site (3 cm times 3 cm) on dorsal skin of hairlessmice daily over the whole study period SpecificallyMHY498or the sham solution was pretreated for 3 days before UVB(302 nm) exposure From day 4 to day 25 2 h after MHY498or the sham solution treatment mice were exposed to UVBevery other day for 1 h in an UVB exposure chamber at150mJcm2 (see Table 1 for treatment scheme) From day 26UVB was exposed every day to maximize the effect of UVBon skin pigmentation and wrinkle formation based on ourpreliminary experiments to pick up the best UVB treatmentcondition (Table 1) After 28 days of the treatments themice were sacrificed and dorsal skin was excised and quicklyfrozen in a nitrogen tank for western blotting and measuringoxidative stress For staining purpose the excised skinwas fixed in 4 paraformaldehyde All animal studies wereapproved by the Institutional Animal Care Committee ofPusanNationalUniversity andwere performed in accordancewith the guidelines for animal experiments issued by PusanNational University

22 UVB Exposure We used a commercially availableUV exposure chamber (Ultraviolet Crosslinker UVP LLCUpland CA USA) for chronic UVB exposure of mice [1415] The UV exposure chamber is designed to measure andcontrol the ultraviolet (UV) radiation within the exposurechamber We used UVB specific tube and sensor provided bythe company that continually measures the UVB energy andautomatically adjusts to variations in UVB intensity thatoccur as the UVB tubes age Therefore we could minimizepotential contamination of UVA UVC and infrared radia-tion

23 Evaluation of Depigmenting Activities In Vivo Aftertreatment of MHY498 followed by UVB exposure darknessof skin sites was measured on day 28 of the treatment usinga CR-10 spectrophotometer (Konica Minolta Sensing IncSakai Osaka Japan) which describes colors using 119871lowast (higherand lower valuesmeanwhiter and blacker resp) as describedby the Commission Internationale de lrsquoEclairage color sys-tem When we compared nontreated mice with the controlmice treated with the sham solution containing propyleneglycol and ethanol there was no difference in skin brightnessvalues after UVB exposure Therefore we only used micetreated with the sham solution as a control group












UVB(sham solution)

CON MHY498(05 120583M)

MHY498(5120583M)

(a)

UVBCON

UVB

minus4

minus3

minus2

minus1

0

1

2 MHY498(05 120583M)

MHY498

ΔLlowast

valu

e

lowastlowastlowast

(5120583M)

(b)

CON

(c)

UVB (150mJcm2)

(d)

MHY498 (05 120583M) + UVB (150mJcm2)

(e)

MHY498 (5120583M) + UVB (150mJcm2)

(f)







CON

(a)

UVB

(b)

MHY498 (05 120583M)

(c)

MHY498 (5120583M)

(d)

CON

200x

(e)

UVB

200x

(f)

200x

MHY498 (05 120583M)

(g)

200x

MHY498 (5120583M)

(h)

Collagen area

0

50000

100000

150000

200000

CON UVB

(120583m

2 )

lowastlowast

MHY498MHY498(05 120583M) (5120583M)

(i)






MITF

p-CREB

CREB

TFIIB

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

(a)

0

1

2

3

4

p-CR

EB (a

rbitr

ary

unit)

UVBCON

lowastlowast

MHY498(05 120583M)

MHY498(5120583M)

(b)

0

1

2

3

MIT

F (a

rbitr

ary

unit)

UVBCON

lowast

MHY498(05 120583M)

MHY498(5120583M)

(c)

Tyrosinase

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(d)

0

1

2

3

4

5

Tyro

sinas

e (ar

bitr

ary

unit)

UVBCON

lowast

lowast

MHY498(05 120583M)

MHY498(5120583M)

(e)



MMP1

MMP13

MMP12

MMP9

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(a)

Type 1 procollagen

Type 3 procollagen

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(b)


0

50000

100000

150000

200000

(ng

mL)

UVBCON


MHY498(05 120583M)

MHY498(5120583M)

(c)







ROS

0

20

40

60FL

Um

inm

g pr

otei

n

UVCON


MHY498(05 120583M)

MHY498(5120583M)

(a)

0

100

200

300

400

FLU

min

mg

prot

ein

UVCON

ONOOminus


MHY498(05 120583M)

MHY498(5120583M)

(b)

TF2B

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

p-p65 (S536)NF-120581B

p65NF-120581B

(c)

iNOS

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

120573-actin

(d)









Competing Interests




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























UVB(sham solution)

CON MHY498(05 120583M)

MHY498(5120583M)

(a)

UVBCON

UVB

minus4

minus3

minus2

minus1

0

1

2 MHY498(05 120583M)

MHY498

ΔLlowast

valu

e

lowastlowastlowast

(5120583M)

(b)

CON

(c)

UVB (150mJcm2)

(d)

MHY498 (05 120583M) + UVB (150mJcm2)

(e)

MHY498 (5120583M) + UVB (150mJcm2)

(f)







CON

(a)

UVB

(b)

MHY498 (05 120583M)

(c)

MHY498 (5120583M)

(d)

CON

200x

(e)

UVB

200x

(f)

200x

MHY498 (05 120583M)

(g)

200x

MHY498 (5120583M)

(h)

Collagen area

0

50000

100000

150000

200000

CON UVB

(120583m

2 )

lowastlowast

MHY498MHY498(05 120583M) (5120583M)

(i)






MITF

p-CREB

CREB

TFIIB

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

(a)

0

1

2

3

4

p-CR

EB (a

rbitr

ary

unit)

UVBCON

lowastlowast

MHY498(05 120583M)

MHY498(5120583M)

(b)

0

1

2

3

MIT

F (a

rbitr

ary

unit)

UVBCON

lowast

MHY498(05 120583M)

MHY498(5120583M)

(c)

Tyrosinase

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(d)

0

1

2

3

4

5

Tyro

sinas

e (ar

bitr

ary

unit)

UVBCON

lowast

lowast

MHY498(05 120583M)

MHY498(5120583M)

(e)



MMP1

MMP13

MMP12

MMP9

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(a)

Type 1 procollagen

Type 3 procollagen

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(b)


0

50000

100000

150000

200000

(ng

mL)

UVBCON


MHY498(05 120583M)

MHY498(5120583M)

(c)







ROS

0

20

40

60FL

Um

inm

g pr

otei

n

UVCON


MHY498(05 120583M)

MHY498(5120583M)

(a)

0

100

200

300

400

FLU

min

mg

prot

ein

UVCON

ONOOminus


MHY498(05 120583M)

MHY498(5120583M)

(b)

TF2B

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

p-p65 (S536)NF-120581B

p65NF-120581B

(c)

iNOS

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

120573-actin

(d)









Competing Interests




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CON

(a)

UVB

(b)

MHY498 (05 120583M)

(c)

MHY498 (5120583M)

(d)

CON

200x

(e)

UVB

200x

(f)

200x

MHY498 (05 120583M)

(g)

200x

MHY498 (5120583M)

(h)

Collagen area

0

50000

100000

150000

200000

CON UVB

(120583m

2 )

lowastlowast

MHY498MHY498(05 120583M) (5120583M)

(i)






MITF

p-CREB

CREB

TFIIB

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

(a)

0

1

2

3

4

p-CR

EB (a

rbitr

ary

unit)

UVBCON

lowastlowast

MHY498(05 120583M)

MHY498(5120583M)

(b)

0

1

2

3

MIT

F (a

rbitr

ary

unit)

UVBCON

lowast

MHY498(05 120583M)

MHY498(5120583M)

(c)

Tyrosinase

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(d)

0

1

2

3

4

5

Tyro

sinas

e (ar

bitr

ary

unit)

UVBCON

lowast

lowast

MHY498(05 120583M)

MHY498(5120583M)

(e)



MMP1

MMP13

MMP12

MMP9

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(a)

Type 1 procollagen

Type 3 procollagen

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(b)


0

50000

100000

150000

200000

(ng

mL)

UVBCON


MHY498(05 120583M)

MHY498(5120583M)

(c)







ROS

0

20

40

60FL

Um

inm

g pr

otei

n

UVCON


MHY498(05 120583M)

MHY498(5120583M)

(a)

0

100

200

300

400

FLU

min

mg

prot

ein

UVCON

ONOOminus


MHY498(05 120583M)

MHY498(5120583M)

(b)

TF2B

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

p-p65 (S536)NF-120581B

p65NF-120581B

(c)

iNOS

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

120573-actin

(d)









Competing Interests




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MITF

p-CREB

CREB

TFIIB

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

(a)

0

1

2

3

4

p-CR

EB (a

rbitr

ary

unit)

UVBCON

lowastlowast

MHY498(05 120583M)

MHY498(5120583M)

(b)

0

1

2

3

MIT

F (a

rbitr

ary

unit)

UVBCON

lowast

MHY498(05 120583M)

MHY498(5120583M)

(c)

Tyrosinase

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(d)

0

1

2

3

4

5

Tyro

sinas

e (ar

bitr

ary

unit)

UVBCON

lowast

lowast

MHY498(05 120583M)

MHY498(5120583M)

(e)



MMP1

MMP13

MMP12

MMP9

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(a)

Type 1 procollagen

Type 3 procollagen

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(b)


0

50000

100000

150000

200000

(ng

mL)

UVBCON


MHY498(05 120583M)

MHY498(5120583M)

(c)







ROS

0

20

40

60FL

Um

inm

g pr

otei

n

UVCON


MHY498(05 120583M)

MHY498(5120583M)

(a)

0

100

200

300

400

FLU

min

mg

prot

ein

UVCON

ONOOminus


MHY498(05 120583M)

MHY498(5120583M)

(b)

TF2B

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

p-p65 (S536)NF-120581B

p65NF-120581B

(c)

iNOS

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

120573-actin

(d)









Competing Interests




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MMP1

MMP13

MMP12

MMP9

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(a)

Type 1 procollagen

Type 3 procollagen

UVB (150mJcm2)MHY498 (120583M)

minus

minus minus

+ + +

05 5

120573-actin

(b)


0

50000

100000

150000

200000

(ng

mL)

UVBCON


MHY498(05 120583M)

MHY498(5120583M)

(c)







ROS

0

20

40

60FL

Um

inm

g pr

otei

n

UVCON


MHY498(05 120583M)

MHY498(5120583M)

(a)

0

100

200

300

400

FLU

min

mg

prot

ein

UVCON

ONOOminus


MHY498(05 120583M)

MHY498(5120583M)

(b)

TF2B

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

p-p65 (S536)NF-120581B

p65NF-120581B

(c)

iNOS

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

120573-actin

(d)









Competing Interests




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ROS

0

20

40

60FL

Um

inm

g pr

otei

n

UVCON


MHY498(05 120583M)

MHY498(5120583M)

(a)

0

100

200

300

400

FLU

min

mg

prot

ein

UVCON

ONOOminus


MHY498(05 120583M)

MHY498(5120583M)

(b)

TF2B

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

p-p65 (S536)NF-120581B

p65NF-120581B

(c)

iNOS

MHY498 (120583M)

minus

minus minus

+ + +

05 5

150mJ UVB

120573-actin

(d)









Competing Interests




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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