Rev. bras. farmacogn. vol.26 número2

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Ethanol

extract

of

Prunus

mume

fruit

attenuates

hydrogen

peroxide-induced

oxidative

stress

and

apoptosis

involving

Nrf2/HO-1

activation

in

C2C12

myoblasts

Ji

Sook

Kang

a

_,

_Dong

_Joo

_Kim

b

_,

_Gi-Young

_Kim

c

_,

_Hee-Jae

_Cha

d

_,

_Suhkmann

_Kim

e

_,

_Heui-Soo

_Kim

f

_,

Cheol

Park

g

_,

_Hye

_Jin

_Hwang

a,h

,

Byung

Woo

Kim

a,i

,

Cheol

Min

Kim

j

,

Yung

Hyun

Choi

a,k,∗

a_Blue-Bio_Industry_RIC_and_Anti-Aging_Research_Center,_Dongeui_University,_Busan,_Republic_of_Korea b_Institute_of_Hukyong_Food,_Busan,_Republic_of_Korea

c_Laboratory_of_{Immunobiology,}_Department_of_Marine_Life_Sciences,_Jeju_National_University,_Jeju,_Republic_of_Korea d_Department_of_Parasitology_and_Genetics,_Kosin_University_College_of_Medicine,_Busan,_Republic_of_Korea e_Department_of_Chemistry_and_Center_for_Proteom_Biophysics,_Pusan_National_University,_Busan,_Republic_of_Korea f_Department_of_Biological_Sciences_and_Genetic_Engineering_Institute,_Pusan_National_University,_Busan,_Republic_of_Korea g_Department_of_Molecular_Biology,_College_of_Natural_Sciences_&_Human_Ecology,_Dongeui_University,_Republic_of_Korea h_Department_of_Food_and_Nutrition,_College_of_Natural_Sciences_&_Human_Ecology,_Dongeui_University,_Busan,_Republic_of_Korea i_Department_of_Life_Science_and_{Biotechnology,}_College_of_Natural_Sciences_&_Human_Ecology,_Dongeui_University,_Busan,_Republic_of_Korea j_Department_of_{Biochemistry,}_Busan_National_University_College_of_Medicine,_Yangsan,_Republic_of_Korea

k_Department_of_{Biochemistry,}_Dongeui_University_College_of_Korean_Medicine,_Busan,_Republic_of_Korea

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5April2015 Accepted7June2015

Availableonline29January2016

Keywords: Prunusmume

Oxidative stress DNA damage Apoptosis Nrf2/HO-1

a

b

s

t

r

a

c

t

ThefruitofthePrunusmume(Siebold)Siebold&Zucc.,Rosaceae(Koreanname:Maesil)haslong

beenusedasahealthfoodorvaluablemedicinalmaterialintraditionalherbmedicineinSoutheastAsian countries.Inthisstudy,wedeterminedthepotentialtherapeuticefficacyoftheethanolextractofP.mume fruits(EEPM)againstH2O2-inducedoxidativestressandapoptosisinthemurineskeletalmusclemyoblast

celllineC2C12,andsoughttounderstandtheassociatedmolecularmechanisms.Theresultsindicatedthat exposureofC2C12cellstoH2O2causedareductionincellviabilitybyincreasingthegenerationof

intra-cellularreactiveoxygenspeciesandbydisruptingmitochondrialmembranepermeability,leadingtoDNA

damageandapoptosis.However,pretreatmentofthecellswithEEPMbeforeH2O2exposureeffectively

attenuatedthesechanges,suggestingthatEEPMpreventedH2O2-inducedmitochondria-dependent

apo-ptosis.Furthermore,theincreasedex-pressionandphosphorylationofnuclearfactorerythroid2-related

factor2(Nrf2)andup-regulationofhemeoxygenase-1(HO-1),aphaseIIantioxidantenzyme,were

detectedinEEPM-treatedC2C12cells.WealsofoundthatzincprotoporphyrinIX,anHO-1inhibitor,

attenuatedtheprotectiveeffectsofEEPMagainstH2O2-inducedreactiveoxygenspeciesaccumulation

andcytotoxicity.Therefore,theseresultsindicatethattheactivationoftheNrf2/HO-1pathwaymight

beinvolvedintheprotectionofEEPMagainstH2O2-inducedcellularoxidativedamage.Inconclusion,

theseresultsshowthatEEPMcontributestothepreventionofoxidativedamageandcouldbeusedasa

nutritionalagentforoxidativestress-relateddiseases.

Introduction

Oxidativestressisimplicatedinnumerousdiseasescausedby theoverproductionofreactiveoxygenspecies(ROS).ROS, deriva-tivesof cellularmetabolicreactions,modulate thefundamental physiological functions of aerobic life. Excess amounts of ROS

∗ Correspondingauthor.

E-mail:choiyh@deu.ac.kr(Y.H.Choi).

damagevariouscellularmolecules,suchasproteins,lipids,nucleic acids,andothermacromolecularsubstances,resultingincellular dysfunctionandapoptosis(Wangetal.,2013;Wu etal.,2010). Therefore,supplementationwithantioxidants,includingsynthetic andnaturalantioxidants,couldreduceoxidativestressand ame-liorateoxidativestress-relateddiseasesthroughinductionofphase IIantioxidantenzymesaswellassuperoxidedismutase(SOD)and catalase(Wojciketal.,2010;Guerra-Araizaetal.,2013).

AmongphaseIIantioxidantenzymes,whichareregulatedby antioxidantresponsiveelements(AREs)atthetranscriptionlevel,

http://dx.doi.org/10.1016/j.bjp.2015.06.012

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J.S.Kangetal./RevistaBrasileiradeFarmacognosia26(2016)184–190

hemeoxygenase-1(HO-1)is arate-limitingenzymeinvolvedin theconversionofhemetobiliverdinandcarbonmonoxide,among othercellularreactions(Martinet al., 2004;Chen etal., 2003). Bilirubinfunctionsasapotentantioxidantandcarbonmonoxide hasbeenreportedto mediatetheanti-apoptoticeffects of HO-1inresponsetoinflammatorycytokinestimulation(Surhetal., 2008;Sonetal.,2013).Nuclearfactorerythroid2-relatedfactor 2(Nrf2),aleucinezipperredox-sensitivetranscriptionfactor,isa keyregulatorofantioxidantanddetoxificationgeneexpression. Undernormalconditions,Nrf2bindstokelch-likeECH-associated protein1(Keap1)inthecytoplasmandislatersubjectedto protea-somaldegradation.However,Nrf2translocatesfromthecytoplasm tothenucleus following a varietyof stimuli, and subsequently bindstoAREpresentwithinthepromoterregionofgenes encod-ingforphaseIIenzymes,resultingintheup-regulationof their transcription(Terazawaetal.,2013;Nitureetal.,2014).Moreover, recentevidenceindicatesthatNrf2promotescellsurvivalby pre-ventinganincreaseinROSinvariousconditionsofoxidativestress (Hybertsonetal.,2011;Nitureetal.,2014).Therefore,theNrf2-ARE pathwayiscurrentlythemostimportantendogenousantioxidant signalingpathway.

Prunusmume(Siebold)Siebold&Zucc.(Koreanname:Maesil) isadeciduoustreeoftheFamilyRosaceae,whichisnowwidely cultivatedinSoutheastAsiacountries,includingKorea,China,and Japan(Matsudaetal.,2003;WenandShi,2012).Thevariousparts ofthisplanthavebeenusedashealthfoodsormedicinal mate-rialsintraditionalmedicineforgenerations(WenandShi,2012; Yanetal.,2014).Inparticular,itsfruithasbeeneatensinceancient timesinAsiancountriesasatraditionalherbalmedicineforrelief offatigue,diarrhea,fever,dyspepsia,andintestinalandskin disor-dersforthousandsofyears(Yanetal.,2014;WenandShi,2012; Zhanget al.,2011;Jeongetal.,2006).The fruitoftheP.mume

contains abundantphenolic compounds,suchasphenolic acids andflavonoids(Jeongetal.,2006;Kitaetal.,2007;Mitanietal., 2013),whichmaybeinvolvedinthebiologicaleffectsofanti-viral, anti-inflammatory,immunoenhancing,andantineoplastic activi-ties(Zhangetal.,2011;Yingsakmongkonetal.,2008;Parketal., 2011;Enomotoetal.,2010;Tsujietal.,2011;Jungetal.,2010;Tada etal.,2012;Leeetal.,2013;Jeongetal.,2006).Althoughthereare severalreportsontheantioxidantactivityandfreeradical scaveng-ingactivitiesofP.mume(Yanetal.,2014;Sangetal.,2002;Leeetal.,

2013),theexact molecularmechanism(s)of actionsofP.mume

extractagainstoxidativestressinvolvedintheNrf2/HO-1signaling pathwayareyettobedescribed.Therefore,inthepresentstudy,we examinedtheabilityoftheethanolextractofP.mumefruits(EEPM) toprotectcellsfromhydrogenperoxide(H2O2)-inducedcell dam-ageandelucidatedthemechanismunderlyingtheprotectiveaction inamouse-derivedC2C12myoblastmodel.

Materialsandmethods

PreparationofEEPM

DriedfruitsofPrunusmume(Siebold)Siebold&Zucc.,Rosaceae, wereobtainedfromInstituteofHukyongFood(Busan,Republic ofKorea),whichwereauthenticatedbyProfessorSuHyunHong, DepartmentofBiochemistry,DongeuiUniversityCollegeofKorean Medicine(Busan,RepublicofKorea).ForthepreparationofEEPM, freeze-driedfruitsofP.mumewereextractedwithethanol(100g

per 1l) at 60◦_C _for _three _days _using _a _blender. _The _extract wascentrifuged at 10,000×g for 20min, and thesupernatants werethen collected and immediatelyfiltered througha What-manfilter(poresize,0.22␮m).Thefiltratewaslyophilizedand

stored at −70◦C. The yield (w/w) of the extract was ∼5.0%. The powder wasdissolved to a 100mg/ml concentration with

dimethylsulfoxide (DMSO,Sigma–AldrichChemicalCo., St.Paul, MN, USA). The voucher specimens (accession number DEU-36) havebeendepositedatapubliclyavailableNaturalResourceBank ofDongeuiUniversityCollegeofOrientalMedicine.

CellcultureandEEPMtreatment

TheC2C12myoblastcelllinewaspurchasedfromtheAmerican TypeCulture Collection(Manassa,VA,USA).Theywerecultured in Dulbecco’s modified Eagle’s medium (DMEM,WelGENE Inc., Daegu,RepublicofKorea)supplementedwith10%heat-inactivated fetal bovine serum (FBS, WelGENE Inc.) and 100␮g/ml

peni-cillin/streptomycinantibiotics(WelGENEInc.)inahumidified5% CO2atmosphereat37◦C.ThestocksolutionofEEPMwasdiluted withDMEMbeforeeveryexperiment.

Measurementofcellviability

Toinvestigatethecytotoxicity,cellswereseededinto6-well plates and exposed to various concentrations of EEPM in the absenceorpresenceofH₂O₂and/orzincprotoporphyrinIX,a HO-1 inhibitor(ZnPP, Sigma–Aldrich) forthe indicatedtimes. After completionofthetreatments,5mg/mlof 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT,Sigma–Aldrich)was addedtoeachwellcontinuouslyat37◦_C._After₃_h_incubation,_the culturesupernatantwasremovedfromthewellsandtheformazan complexwasdissolvedinDMSO.Theabsorbanceofeachwellwas detectedat540nmwithamicroplatereader(MolecularDevices, PaloAlto,CA,USA).Cellviabilityisexpressedasapercentageof untreatedcells.

MeasurementofintracellularROSlevels

The production of intracellular ROS was quantified using the oxidation-sensitive fluorescent probes 2′_,7′ -dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Eugene, OR, USA). After treatment, the cells were har-vested, suspendedin phosphate-bufferedsaline(PBS), andthen incubatedwith10␮MH2DCFDAfor20minatroomtemperature

inthedark.Thefluorescenceintensitywasmeasuredbyaflow cytometer(BectonDickinson,SanJose,CA,USA)atanexcitation wavelength of 488nm and an emission wavelength of 530nm (Kimetal.,2014).

Measurementofmitochondrialmembranepotential(MMP)

The mitochondrial transmembrane electrochemical gradi-ent was measured using the mitochondrial potential sensor 5,5′_,6,6′_{-tetrachloro-1,1}′_,3,3′_{-tetraethyl-imidacarbocyanine}_iodide (JC-1,Sigma–Aldrich),acellpermeable,cationic,andlipophilicdye, whichisinternalizedandconcentratedbyrespiringmitochondria, reflectingchangesinMMPinlivecells.Briefly,cellswerecollected, resuspendedinPBS,andthenincubatedwith10␮MJC-1under

darkconditionsfor30minat37◦_C._After_the_JC-1_was_removed, thecellswerewashedwithPBStoremoveunbounddye.The cellu-larfluorescenceintensitywasquantifiedusingaflowcytometryat anexcitationwavelengthof480nmandanemissionwavelength of530nm(Seoetal.,2014).

DAPInuclearstaining

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2.5␮g/mlDAPIsolutionfor10minatroomtemperature.Thecells

werewashedtwicewithPBS,and stainednucleiwereanalyzed using a fluorescence microscope (Carl Zeiss, Oberkochen, Ger-many).

Determinationofapoptoticcellsbyflowcytometry

To assess the induced cell apoptosis rate quantitatively, a fluorescein-conjugatedannexinV(annexinV-FITC)stainingassay wasperformedaccordingtothemanufacturer’sprotocol(BD Bio-sciences,SanJose,CA,USA). Thecells werewashed twicewith ice-coldPBSandstainedwithannexinV-FITCandpropidiumiodide (PI)for15minatroomtemperatureinthedark.Thedegreeof apo-ptosiswasquantifiedasapercentageoftheannexinV-positiveand PI-negative(annexinV+_/PI−_cells)_cells_by_flow_cytometry.

Cometassay(single-cellgelelectrophoresis)

ThedegreeofoxidativeDNAdamagewasassessedinacomet assay.Thecellsuspensionwasmixedwith0.5%lowmeltingagarose (LMA)at37◦_C,_and_the_mixture_was_spread_on_a_fully_frosted micro-scopicslideprecoatedwith1%normalmeltingagarose(NMA).After thesolidificationoftheagarose,theslidewascoveredwith0.5% LMAandthenimmersedinalysissolution[2.5MNaCl,100mM Na-ethylenediaminetetraaceticacid(EDTA),10mMTris,1%Trion X-100,and10%DMSO,pH10]for1hat4◦_C._The_slides_were_then placedinagelelectrophoresisapparatuscontaining300mMNaOH and10mMNa-EDTA(pH13)for40mintoallowforDNAunwinding andtheexpressionofalkali-labiledamage.Anelectricalfieldwas thenapplied(300mA,25V)for20minat25◦_C_to_draw_the neg-ativelychargedDNAtowardtheanode.Theslideswerewashed threetimesfor5minat25◦_C_in_a_neutralizing_buffer_(0.4_M_Tris, pH7.5),followedbystainingwith20␮g/mlpropidiumiodide(PI,

120.0

A

B

100.0 80.0 60.0 Relativ

e cell viability (%)

Relativ

e cell viability (%)

40.0 20.0 0.0 0 * # # 250 500

EEMP (μg/ml)

750 1000 1500

120.0 100.0 80.0 60.0 40.0 20.0 0.0 – – –

EEMP (μg/ml)

+ +

0 250 500 0 250

+ 500

H2O2(1mM)

Fig.1.Effects of EEPM on H2O2-induced growth inhibition in C2C12 cells. Cells weretreatedwithvariousconcentrationsofEEPMfor24h(A)orpretreatedwith 250␮g/mland500␮g/mlofEEPMfor1handthenincubatedwithorwithout1mM H2O2for6h(B).CellviabilitywasassessedusinganMTTreductionassay.Theresults arethemean±SDvaluesobtainedinthreeindependentexperiments(*p<0.05 compared with control group;#_p_<_0.05_compared_with_H

2O2-treated group).

Sigma–Aldrich).Theslides wereexaminedunderafluorescence microscope.

Westernblotanalysis

Thetotalcellularproteinwasextractedwithlysisbuffer(20mM of sucrose,1mMof EDTA, 20␮Mof Tris–HCl,pH7.2, 1mM of

dithiothreitol,10mMofKCl,1.5mMofMgCl2and5␮g/mlof

apro-tinin)for30min.Theproteinconcentrationwasmeasuredusing aBio-Radproteinassay(Bio-RadLab.,Hercules,CA,USA) accord-ingto themanufacturer’sinstructions. Aliquotsof each sample wereloadedintodedicatedwellsofSDS-polyacrylamidegels, sep-aratedbyelectrophoresis,andthentransferredtopolyvinylidene difluoridemembranes(Schleicher&Schuell,Keene,NH,USA).After blockingnonspecificbindingwith5%nonfatdrymilkinTBST (Tris-bufferedsaline-Tween),themembraneswerethenprobedwiththe desiredprimaryantibodiespurchasedfromSantaCruz Biotechnol-ogy(SantaCruz,CA,USA)andCellSignalingTechnology(Danvers, MA,USA),andincubatedovernightat4◦_C._The_membranes_were washedwithTris-bufferedsaline-0.01%(v/v)containing Tween-20 at room temperature for 15min and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Co., Arlington Heights, IL, USA) in TBST for 2h at roomtemperature.Proteinswerevisualizedbyusinganenhanced chemiluminescence(ECL, AmershamCo.) detectionmethod fol-lowedbyfilmexposure.

800

A

B

700 600 500 400 300 Relativ

e fluoresence _intensity

200 100 0 – – – – + – – – – + + – – + + – + # # * – +

H2O2(1 mM) EEMP (500 µg/ml)

EEMP (500 µg/ml)

NAC (5 mM)

H2O2(1 mM) H2O2+ EEMP H2O2+ NAC NAC (5 mM) Control FL2(JC-1 agg regates) FL1(JC-1 monomers) 91.73% 8.27% 30.77% 69.23% 68.11% 31.89% 85.79% 14.21% 92.78% 7.22% 94.29% 5.71% 10010 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4

101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

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Statisticalanalysis

Allmeasurementsweremadeintriplicateand allvaluesare presentedasmean±standarddeviation(SD).Theresultswere sub-jectedtoananalysisofvariance(ANOVA)usingtheTukeytestto analyzethedifference.p<0.05wasconsideredtobestatistically significant.

Results

EEPMprotectsC2C12cellsagainstH2O2-inducedcytotoxicity

ToexaminetheprotectiveeffectofEEPMonH2O2-induced cyto-toxicity,theeffectofEEPMontheviabilityofC2C12cellswasfirst performedforprimarydoseselection.AsshowninFig.1A,theEEPM treatmentdidnotresultin anycytotoxiceffects uptothe con-centrationof1500␮g/ml.Therefore,250and500␮g/mlEEPMwas

chosenastheoptimaldosesforstudyingthecytoprotectiveeffect ofEEPMagainstH2O2-inducedcytotoxicity.Inordertodetermine theeffectsofEEPM,thecellswerepre-treatedwiththeEEPMfor 1hpriortoH2O2administration.AsestimatedbyanMTTassay,cell viabilitywasmarkedlydecreased,toapproximately60%,aftera6h exposureto1mMH2O2.However,whencellswerepre-incubated withEEPM,thereductionofcellviabilitywassignificantly attenu-atedinadose-dependentmanner(Fig.2B).

EEPMdecreasesH2O2-inducedROSproductioninC2C12cells

To investigate whether EEPM could prevent H2O2-induced ROSgenerationandtheresultingoxidativestress,wenext mea-sured the ROS production in cells by using H2DCFDA reagent, a fluorescent dyethat visualizesROS. Asshown in Fig.2A, the intensityoftheDCF-liberatedfluorescentsignalfromtheH2O2 -treated cells was significantly increased, and the signal was markedly reduced in the presence of EEECas well as theROS scavengerN-acetyl-l-cysteine(NAC),whichwasusedasapositive

control.

EEPMattenuatesH2O2-inducedlossofMMPinC2C12cells

SinceMMPregulatesmitochondrialpermeability,which may becriticalforinducingorarrestingtheoxidativestress-induced apoptoticpathway,weevaluatedtheeffectofEEPMontheMMP ofC2C12cellsusingflowcytometry.Afterincubationwith1mM H2O2 for6h,theMMPlevelwasmarkedlyreducedascompared totheuntreatedcontrol(Fig.2B),indicatingmitochondrial dam-ageanddysfunction.Incontrast,pretreatmentwithEEPMorNAC effectivelypreventedthelossofMMPinducedbyH2O2.Thedata indicatedthatEEPMprotectedthecellsagainsttheH₂O₂-induced loweringofROSgenerationbyblockingmitochondrialdysfunction inC2C12cells.

EEMP (500 µg/ml)

A

C

B

D

PI fluorescence intensity

(log)

+ –

EEMP (500 µg/ml)

H

2_O 2_{(1 mM)}

H2O2 (1 mM)

p-YH2AX

YH2AX +

+ –

EEMP (500 µg/ml)

Annexin V-FITC intensity

3.52% 4.42%

27.28% 8.76%

+ –

EEMP (500 µg/ml) +

Actin +

+

+ –

– –

–

H

2_O 2_{(1 mM)}

+

–

H

2_O 2_{(1 mM)}

+

–

10

4

10

3

10

2

10

1

10

0

100 101 102 103 104

10

4

10

3

10

2

10

1

10

0

100 101 102 103 104

10

4

10

3

10

2

10

1

10

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100 101 102 103 104

10

4

10

3

10

2

10

1

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0

100 101 102 103 104

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EEPMprotectsH2O2-inducedC2C12cellapoptosis

WealsoexaminedtheabilityofEEPMtoprotectagainstH2O2 -triggeredC2C12cell apoptosis usingthe DAPI and Annexin/PI-stainingassays. AsillustratedinFig.3A,C2C12cellsexposedto 1mMH2O2alonedemonstratedcharacteristicapoptoticfeatures, includingcell shrinkage and chromatincondensation and frag-mentationinthenucleusdetectedbyDAPIstaining.However,the pre-treatmentwithEEPMonH2O2-treatedC2C12cellssignificantly reducedthese morphologicalchanges. In addition,the percent-ageofapoptoticcellstreatedwith1mMH2O2wasapproximately 24.60%bytheflowcytometryassay(Fig.3B);however,500␮g/ml

EEPMeffectivelydecreasedthecellapoptosisinducedbyH2O2to 7.70%.TheseresultsshowthatpreconditioningbyEEPMprotects againstoxidativestress-inducedC2C12celldeath.

EEPMpreventsH2O2-inducedDNAdamageinC2C12cells

We next examined the effects of EEPM on H2O2-mediated damage to C2C12 cell DNA using the comet assay and West-ernblotting analysis. Asshown in Fig. 3C, exposureof cells to H2O2aloneincreasedthenumberofDNAbreaks,resultinginan increase influorescence intensityin thetails of thecomet-like structures;however,thisadverseeffectwasmarkedlyreducedby EEPMpretreatment.OurresultsalsoshowedthattreatingC2C12 cellswithH2O2 resultedintheup-regulationofthelevelofthe phosphorylatedhistonevariantH2A.Xatserine139(p-␥H2A.X),

asensitivemarkerforDNAdouble-strandbreaks(Rogakouetal., 1998)(Fig.2C);however,pretreatmentwithEEPMresultedina significantdecreasedp-␥H2A.Xexpression.

EEPMpromotestheexpressionofNrf2andHO-1inC2C12cells

ToexaminewhetherornotNrf2activationisassociatedwith EEPM-mediatedcytoprotection, we monitoredthelevelsof the HO-1protein.AsshowninFig.4,treatmentofC2C12cellswith EEPM induced the expression of the HO-1 protein in a dose-andtime-dependentmanner,butthatotherantioxidantenzymes, NADPH-quinoneoxidoreductase1(NQO1)andthioredoxin reduc-tase1(TrxR1),wereunaffectedbyEEPMtreatment, whichwas associatedwiththeinductionofNrf2.Sincethephosphorylation ofNrf2atSer40byseveralkinasesisalsoacriticalprocessinits stabilizationandnucleartranslocation(Surhetal.,2008;Niture etal.,2014),weexaminedthephosphorylationofNrf2underEEPM treatmenttofurtherconfirmtheNrf2-activatingpropertyofEEPM andobservedthattreatmentofcellswithEEPMcauseddose-and time-dependent increases in thelevels of phosphorylated Nrf2 expression.

Nrf2/HO-1pathwayisinvolvedinEEPMprotectionagainstH2O2

treatmentinC2C12cells

In order to provide evidence for the involvement of Nrf2-mediated HO-1 induction in EEPM-mediated antioxidant and cytoprotectiveactivitiesagainstoxidativestress,weinhibited HO-1activitybyusingZnPP,aspecificinhibitorofHO-1.Asshownin Fig.5,inthepresenceofZnPP,theprotective effectofEEPMon H2O2-inducedproductionofROSandthereductionofcellviability weresignificantlyattenuated.Takentogether,theseresults sug-gestthatEEPMexertsitsprotectiveeffectsbyinducingthecellular defensemechanismagainstoxidativestressthroughHO-1 induc-tionviaNrf2activation,andthatHO-1playsacrucialroleinthis

protectioninC2C12cells.

EEMP (μg/ml)

0

0 1 2 3 6

125 250 500

Nrf2

p-Nrf2

HO-1

NQO-1

TrxR1

Actin

Nrf2

p-Nrf2

HO-1

NQO-1

TrxR1

Actin

Time (h)

B

A

Fig.4.Induction of Nrf2 and HO-1 expression by EEPM in C2C12 cells. Cells were incubated with the indicated concentrations of EEPM for 6 h (A) or 500 ␮g/ml EEPM for the indicated time periods (B). Total cellular proteins were separated on SDS-polyacrylamidegelsandthentransferredontonitrocellulosemembranes.The membraneswereprobedwiththespecificantibodiesagainstNrf2,p-Nrf2,HO-1, NQO-1,andTrxR1.ProteinswerevisualizedusinganECLdetectionsystem.Actin wasusedasaninternalcontrol.

Discussion

Themitochondrialelectrontransportsysteminthe mitochon-drialmembraneisoneofthemajorsourcesofintracellularROS generation(Fleuryetal.,2002),wherebythemitochondriaplaya pivotalroleintheROS-mediatedcelldeathprocess.Excessive pro-ductionofROSbymitochondrialdysfunctionfollowingoxidative DNAdamageandexposuretoothergenotoxicfactorsultimately leadstothe activationof themitochondrial apoptotic pathway (alsotermedtheintrinsicapoptoticpathway)ratherthan necro-sis(Wangetal.,2013;Wuetal.,2010).Moreover,H2O2directly induces mitochondrial dysfunction followed by a rapid efflux of intracellular ROS, which increases the permeabilization and depolarizationofthemitochondrialmembrane.Thiseventcould facilitatearapiddisruptionofMMPandthereleaseof apoptosis-inducingfactors,whichactivatethecaspase-dependentsignaling cascades(Kimetal.,2006;Fleuryetal.,2002).Inthepresentstudy, treatmentofC2C12cellswithH2O2causedamarkeddecreasein cellsurvival,intracellularaccumulationofROS,andfurtherinduced thelossofMMP,leadingtoapoptosis.However,whenC2C12cells werepretreatedwithEEPM,theH2O2-inducedreductionofcell viability,accumulationofROS,lossofMMP,andapoptosiswere significantlyattenuated.

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800

A

B

*

#

– – –

– + –

+ – –

+ + –

+ – +

+ + + $

*

#

$

Relativ

e fluoresence _intensity

Cell viability (%)

700

600

500

400

300

200

100

H₂O₂ (1 mM) EEMP (500 µg/ml) ZnPP (10 µM)

– – –

– + –

+ – –

+ + –

– – +

+ + +

H₂O₂ (1 mM) EEMP (500 µg/ml) ZnPP (10 µM) 0

120.0

100.0

80.0

60.0

40.0

20.0

0.0

Fig.5.Effects of an inhibitor of HO-1 on EEPM-mediated attenuation of ROS for-mation and growth inhibition by H2O2in C2C12 cells. Cells were pretreated for 1 h with 1500 ␮g/ml EEPM and then treated for 6 h with or without 1 mM H2O2in the absenceorpresenceof10␮MZnPP.Then,ROSgeneration(A)andcellviability(B) wereestimated.Theresultsarethemean±SDvaluesobtainedinthree indepen-dentexperiments(*p<0.05comparedwithcontrolgroup;#_p_<_0.05_compared_with H2O2-treatedgroup;$p<0.05comparedwithH2O2andEEPM-treatedgroup).

oxidative stress stemming from ROS production (Ayala-Pe˜na, 2013; Maynard et al., 2009), suggesting a direct link between ROS signaling and oxidative DNA damage. Severe ROS-induced oxidativestressleadstotheinductionofmitochondria-medicated apoptosis in affected cells due to high levels of DNA damage (Deavalletal.,2012;Chistiakovetal.,2014).Thus,wepresumed thatEEPMmightimprovemitochondrialfunctionthrough elim-inating the overproduction of ROS induced by H2O2, thereby reducingtheH2O2-inducedapoptosis.OurstudyshowedthatH2O2 treatmentincreasedDNAtaillengthinthecometassay,aswellas theexpressionofphospho-H2A.X,whicharewidely-used mark-ersforthedetectionofDNAdamage(Rogakouetal.,1998).Both wereattenuatedbyEEPMinthepresentstudy.Theseresults indi-catedthatEEPMprotectedagainsttheH2O2-inducedapoptosisof C2C12cellsbyreducingDNAdamagefromthedestructiveimpact ofoxidativestressinC2C12cells.

Nrf2, atranscription factor thatis partoftheredox homeo-staticgeneregulatorynetwork,isakeyregulatoroftheexpression of anexpansive setof ARE-mediated geneswhich remove ROS byinducingtheactionsofdetoxifyingenzymes(Hybertsonetal., 2011;Nitureetal.,2014).Increasingevidencesuggeststhatthe elevationofNrf2-mediatedtargetgenes,includingHO-1,also pro-motescellsurvivalinoxidizingenvironmentsviaenhancementof

freeradicalmetabolism,inhibitionofcytokine-mediated inflam-mation,andrecognitionofdamagedDNA(Surhetal.,2008;Son etal.,2013).Manyantioxidantagentshavebeenreportedtoreduce ROSproductionbytheactivationofNrf2/HO-1pathwaythrougha varietyofsignalingpathways(Terazawaetal.,2013;Nitureetal., 2014).Moreover,ithasbeenreportedthatNrf2phosphorylationby severalproteinkinasessuchasphosphatidylinositol-3kinase/Akt, mitogen-activatedproteinkinases,andproteinkinaseCfacilitates itstranslocationintothenucleus,whereuponit bindstoAREin thepromoter regions(Surhetal.,2008;Nitureetal.,2014;Lee etal.,2014).Therefore,wedeterminedthepotentialroleofNrf2 andHO-1inH2O2-inducedC2C12celldamageandEEPM-mediated

cytoprotection. The datafrom thepresent study indicated that treatmentofC2C12cellswithEEPMresultedinanincreaseofNrf2 expressionaswellasitsphosphorylatedformwithaconcomitant increaseinHO-1expression.Furthermore,treatmentwithan HO-1inhibitor,ZnPP,significantlyabrogatedtheprotectiveeffectsof EEPMonH2O2-inducedgrowthinhibitionandROSgeneration, pro-vidingevidencefortheinvolvementofHO-1inductionandNrf2 activationinEEPM-mediatedantioxidantpotential.

Insummary,ourresultsclearlyindicatedthatEEPMexhibiteda protectiveabilityagainstH2O2-inducedcytotoxicityandapoptosis inC2C12myoblasts.EEPMalsosuccessfullysuppressedthe forma-tionofintracellularROS,leadingtoasubstantialregainingofMMP through,atleastinpart,activationofNrf2signalingandinduction ofphaseIIantioxidantenzymes,suchasHO-1.Infuturestudies,this molecularmechanismneedstobevalidatedinvivoasapositive

result,ifconfirmed,wouldprovideessentialinformationtoward thedevelopmentofnewapproachesforeffectivestress-responsive antioxidantsagainstassaultstriggeredbyROS.

Authors’contributions

JSK,DJK,GYKandCPcontributedinrunningthelaboratorywork, analysisofthedataanddraftedthepaper.HJC,SKandHSK con-tributedtocriticalreadingofthemanuscript.HJK,BWK,CMKand YHCdesignedthestudy,supervisedthelaboratoryworkand con-tributedtocriticalreadingofthemanuscript.Alltheauthorshave readthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwassupportedbytheHighValue-addedFood Tech-nologyDevelopmentProgram(314043-3),MinistryofAgriculture, Food and Rural Affairs, and the R&D program of MOTIE/KEIT (10040391,DevelopmentofFunctionalFoodMaterialsandDevice forPreventionofAging-associatedMuscleFunctionDecrease).

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