綠茶能夠延長壽命是通過促進(jìn)氧化應(yīng)激實(shí)現(xiàn)的

綠茶中的兒茶素使得機(jī)體更長壽、更健康中國是茶葉的故鄉(xiāng)，也是茶文化的發(fā)源地。自從4700多年前第一杯茶水誕生以來，茶飲已成為全球最常見的飲料之一，僅次于水。無論是古籍還是現(xiàn)代醫(yī)學(xué)研究，都認(rèn)為飲用茶水有益健康。雖然所有的茶葉都來自同一種植物——茶樹，但因氧化程度的不同而決定了茶葉的品種。例如，綠茶是未經(jīng)氧化的，這也就意味著，綠茶含有最豐富的多酚類化合物兒茶素，它被認(rèn)為是一種強(qiáng)大的抗氧化劑，可以中和或防止由氧自由基引起的體內(nèi)氧化應(yīng)激，從而防止對細(xì)胞或DNA造成損傷。因此，綠茶具有延緩衰老的作用。然而，近日發(fā)表在《Aging》上的一項(xiàng)研究中，來自德國耶拿大學(xué)、瑞士蘇黎世聯(lián)邦理工學(xué)院（ETH）和華中農(nóng)業(yè)大學(xué)的研究人員得出了一個(gè)顛覆性的結(jié)論：綠茶中的兒茶素非但沒有抑制氧化應(yīng)激，反而在短期內(nèi)促進(jìn)了氧化應(yīng)激。愛喝綠茶的先別慌，讓我們來看看到底是怎么回事。此前的臨床試驗(yàn)和流行病學(xué)研究都表明，飲用綠茶對健康有益，包括降血壓、降血糖、降血脂和減肥等。綠茶葉中含量最豐富的多酚類化合物是表沒食子兒茶素沒食子酸酯（EGCG）、表兒茶素沒食子酸酯（ECG），它們占綠茶兒茶素總量的70%。在各種模式生物中進(jìn)行的實(shí)驗(yàn)表明，由于代謝適應(yīng)和增強(qiáng)對活性氧(ROS)的抵抗力，綠茶兒茶素有助于延長壽命。然而，綠茶兒茶素在哺乳動(dòng)物中的生物利用度較差，使得人類口服后不太可能達(dá)到這個(gè)濃度。盡管如此，一些獨(dú)立的臨床試驗(yàn)證實(shí)，飲用綠茶可以改善各種健康生理指標(biāo)。在服用4.5克無咖啡因的綠茶固體后，人體內(nèi)EGCG、ECG和表兒茶素（EC）的最大血漿濃度達(dá)到了2.5μM。在這項(xiàng)研究中，研究人員就測試了2.5μM的濃度是否足以通過誘導(dǎo)秀麗隱桿線蟲（Caenorhabditiselegans）的有絲分裂反應(yīng)來促進(jìn)其壽命。研究人員發(fā)現(xiàn)，EGCG和ECG在2.5μM的濃度下就能增強(qiáng)線蟲的體能并延長其壽命；而且，這個(gè)相對較低的劑量足以抑制線蟲的線粒體呼吸鏈活性。隨后，在分離的小鼠肝線粒體中的實(shí)驗(yàn)表明，EGCG和ECG抑制了線粒體復(fù)合物I的活性。EGCG給予6小時(shí)和ECG給予12小時(shí)后，復(fù)合物I被抑制的同時(shí)，還出現(xiàn)短暫的活性氧（ROS）形成和ATP下降。事實(shí)證明，EGCG和ECG延長線蟲的壽命取決于能量代謝物AMP激活的激酶AAK-2和NAD依賴的蛋白去乙?；窼IR-2的存在。這些數(shù)據(jù)表明，由于瞬時(shí)AMP下降引起的后續(xù)能量缺乏觸發(fā)了線蟲的能量傳感器AAK-2和SIR-2.1。此外，ROS水平的暫時(shí)增加可能會促進(jìn)分裂原激活的蛋白激酶同源物PMK-1的活性，從而促進(jìn)相應(yīng)的信號級聯(lián)。這些信號通路通過增強(qiáng)ROS防御超氧化物歧化酶(SOD)和過氧化氫酶(CTL)的活性、增加了氧化應(yīng)激抵抗力，從而促進(jìn)健康和壽命來激發(fā)適應(yīng)性反應(yīng)。綜上所述，抑制線粒體復(fù)合體I再次被證明是促進(jìn)壽命延長途徑的有力工具。該研究通訊作者、ETH健康科學(xué)與技術(shù)系能量代謝中心MichaelRistow教授說：“這意味著綠茶兒茶素實(shí)際上不是抗氧化劑，而是一種促氧化劑，可以激發(fā)機(jī)體提高自我保護(hù)能力，這個(gè)作用與接種疫苗類似?！盧istow對這種機(jī)制的作用并不感到驚訝。他的研究團(tuán)隊(duì)早在2009年就表明，運(yùn)動(dòng)促進(jìn)健康的原因在于短期內(nèi)增加氧化應(yīng)激，從而提高身體的防御能力。減少熱量攝入也有同樣的效果，這已經(jīng)在動(dòng)物實(shí)驗(yàn)中多次得到證實(shí)。喂食低熱量食物的小鼠比喂食正常高熱量食物的小鼠活得更長。Ristow解釋說：“所以綠茶中的兒茶素也會以類似的方式發(fā)揮作用，這對我來說是有道理的?！彼硎荆@項(xiàng)研究結(jié)果同樣適用于人類。生物中和氧自由基的基本生化過程在進(jìn)化史上是保守的，存在于從單細(xì)胞酵母到人類的一切生物中。Ristow自己每天都喝綠茶，但他建議不要服用綠茶提取物或濃縮物。他說：“達(dá)到一定的濃度后，它就會變成有毒物質(zhì)。高劑量兒茶素會抑制線粒體，導(dǎo)致細(xì)胞死亡，這在肝臟中尤其危險(xiǎn)。任何過量攝入這些多酚的人都有損傷器官的風(fēng)險(xiǎn)。”抗氧化？綠茶中的兒茶素竟是通過氧化應(yīng)激起效Aging：綠茶中的兒茶素單體ECG和EGCG被認(rèn)為是抗氧化劑，但最新研究發(fā)現(xiàn)它們不但不抗氧化，反而會在短時(shí)間內(nèi)提高氧化應(yīng)激水平，導(dǎo)致細(xì)胞和生物體的防御能力增強(qiáng)，使得機(jī)體更長壽、更健康EGCGandECGpromotelifespan,fitness,andstressresistancewhenappliedatlowdosesOralabsorptionandabsolutebioavailabilityofgreenteacatechinsarelowinmammals[12],reachingtotalmaximumplasmaconcentrationsof2.5μMinhumansafteradministrationofmaximal4.5gofdecaffeinatedgreenteasolids[14].However,severalindependentclinicaltrialsreportedbeneficialeffectsofEGCGandECGregardinghealthparameters[1–4].Therefore,wehypothesizedthatlowerconcentrationsofEGCGandECGthanthosestudiedpreviously[11]arestilleffectiveandimprovelifespanandstressresistancein

C.elegans.

Indeed,EGCGandECGappliedataconcentrationof2.5μMweresufficienttosignificantlyextendthemediumlifespan(Table1)of

C.elegans

from28.8±0.3to30.8±0.1days(Figure1A)andfrom28.8±0.3to30.6±0.3days(Figure1B),respectively,causinganextensionof6.9%forEGCGand6.2%forECGtreatment.Themaximumlifespan(Table1)wasextendedfrom35.7±0.6to36.9±0.1daysbyEGCGtreatment(Figure1A)andfrom35.7±0.6to37.1±0.3daysbyECGtreatment(Figure1B),reachinganextensionof3.4%forEGCGand3.9%forECG.Next,wetestedwhetherprolongedlifespanalsocorrelateswithimprovedfitnessandstressresistance.Locomotionisdependentonfunctionalmusclemass,connectivetissues,andneuronalsignaling.Consequently,motilityisasuitablemarkerforhealth[15].EGCGandECGtreatmentimprovedthenematodes’motilityafter7daysofincubation(Figure1C).Moreover,treatmentof

C.elegans

withECGC(Figure1D)andECG(Figure1E)for7dayssignificantlyincreasedstressresistance(Table2)tothefreeradicalgeneratorparaquat.Consequently,EGCGandECGenhancedfitnessandstressresistance,bothcrucialparametersforhealth.Table1.Lifespanresultsandstatisticalanalysis.Strains,CompoundsMaxlifespanindays±SD(10th

percentile)Mediumlifespanindays±SD(50th

percentile)Numberofexperiments(n)P

valueversuscontrolNumberofnematodesN2DMSO35.7±0.628.8±0.3182831N2EGCG36.9±0.130.8±0.118<0.00012842N2ECG37.1±0.330.6±0.315<0.00012777N2BHA36.4±0.628.9±0.490.38381548N2BHA+EGCG36.0±0.329.2±0.490.31141581N2BHA+ECG35.9±0.529.6±0.460.66821451aak-2(ok524)

DMSO24.220.9±0.23462aak-2(ok524)

EGCG24.4±0.220.5±0.330.1015465aak-2(ok524)

ECG24.6±0.720.4±0.430.9876452sir-2.1(ok434)

DMSO28.6±0.124.3±0.43400sir-2.1(ok434)

EGCG28.9±1.224.5±0.330.1858355sir-2.1(ok434)

ECG28.2±0.523.7±0.130.24436pmk-1(km25)

DMSO34.9±0.627.9±0.43548pmk-1(km25)

EGCG35.5±1.528.2±1.130.7759567pmk-1(km25)

ECG35.3±0.728.0±0.330.7363581skn-1(zu67)

DMSO16.5±0.514.1±0.16424skn-1(zu67)

EGCG17.0±0.214.2±0.160.5286432skn-1(zu67)

ECG16.8±0.214.360.4823440daf-16(mgDF47)

DMSO22.5±0.320.1±0.13660daf-16(mgDF47)

EGCG22.2±0.219.830.029774daf-16(mgDF47)

ECG21.7±0.619.4±0.23<0.0001707sod-2(gk257)

DMSO33.3±0.427.2±0.43730sod-2(gk257)

EGCG33.1±0.427.5±0.430.9525788ctl-2(ok1137)

DMSO32.5±0.426.8±0.73602ctl-2(ok1137)

ECG32.9±0.527.2±0.430.1718614Figure1.

Increasedlifespan,locomotionactivity,andstressresistanceafterEGCGandECGtreatment.

TherepresentativeoutcomeoflifespanassayofN2wild-typenematodesinthepresenceof2.5μMEGCGversusDMSO.(A)TherepresentativeoutcomeoflifespanassayofN2wild-typenematodesinthepresenceof2.5μMECGversuscontrol.(B)LocomotionquantificationforN2wild-typenematodesafter7daysexposuretoDMSO,2.5μMEGCG,or2.5μMECG.(C)Therepresentativeoutcomeofthesurvivalanalysis(h)ofN2nematodesin50mMparaquatsolutionafter7daysofpretreatmentwithEGCG(D)orECG(E)incomparisontowormspretreatedwithDMSO.

P-valuesareasindicatedinthegraphs.See

Table1

and

Table2

forcorrespondingdetaileddataandstatisticalanalysesoflifespanassaysandofparaquatstressassay,respectively.Table2.Paraquatstressassayresultsandstatisticalanalysis.TreatmentsNumberofexperiments(n)P

valueversuscontrolNumberofnematodesN2DMSO694N2EGCG6<0.000192N2ECG6<0.000193ComplexIinhibitionbyEGCGandECGhampersmitochondrialrespirationtemporarilyandinducesatransientROSsignalPreviousreportshavesuggestedthatgreenteacatechinsinduceSIRT1/SIR-2.1andFOXO/DAF-16signalingbyaninitialincreaseinROSlevels.However,theROSsourcehasremainedunidentifiedinpreviousreports[11].WecouldconfirmthatROSisessentialforlifespanextensionprovokedbycatechins,showingthattheantioxidantbutylatedhydroxyanisole(BHA)preventsthelife-prolongingeffectofECGC(Figure2A)andECG(Figure2B).Moreover,wefoundthat25μMofEGCGandECGsignificantlyhampertheactivityofcomplexIinmurinelivermitochondria(Figure2C)andthemitochondrialrespirationinmitochondriaisolatedfromratliver(Figure2D).Thesefindingsareinlinewithreducedmitochondrialrespirationin

C.elegans

after6–12hoursexposureto2.5μMEGCG(Figure2E)orECG(Figure2F).Notably,mitochondrialrespirationrecoveredafter24hand120hoftreatmentwithEGCG(Figure2E)andECG(Figure2F),pointingtocompensationofaninitiallyimpairedmitochondrialfunction.ThetimecourseofinitialdiminutionandthesubsequentrecoveryofmitochondrialrespirationcorrelateswithROSlevels,whichincreasedsignificantlyafter6hofECGC(Figure2G)and12hofECG(Figure2H)administrationanddroppedsignificantlyafter24hand120hofcatechintreatment(Figure2G,

2H).Figure2.

EGCGandECGinhibitcomplexI,whichresultsinatemporaryhamperingofmitochondrialrespirationandaboostinROSproduction.

TherepresentativeoutcomeoflifespanassayofN2wildtypenematodesinthepresenceof2.5μMEGCGco-appliedwithanantioxidant;DMSOvs.2.5μMEGCGvs.10μMBHAvs.2.5μMEGCGincombinationwith10μMBHA.(A)TherepresentativeoutcomeoflifespanassayofN2wildtypenematodesinthepresenceof2.5μMECGco-appliedwithanantioxidant;DMSOvs.2.5μMECGvs.10μMBHAvs.2.5μMECGincombinationwith10μMBHA.(B)ComplexIactivityinmurinelivermitochondriaaftertreatmentwithDMSO,25μMEGCGor25μMECG.(C)MitochondrialrespirationofratlivermitochondriaaftertreatmentwithDMSO,25μMEGCGor25μMECG.(D)MitochondrialrespirationofN2wild-typenematodesaftertreatmentwithDMSOor2.5μMEGCGfor6h,24h,or120hmeasuredasoxygenconsumptionrateandnormalizedtoproteincontent.(E)MitochondrialrespirationofN2wild-typenematodesaftertreatmentwithDMSOor2.5μMECGfor12h,24h,or120h,measuredasoxygenconsumptionrateandnormalizedtoproteincontent.(F)ROSproductionofN2wild-typenematodesaftertreatmentfor6h,24h,or120hwith0.1%DMSOor2.5μMEGCG.(G)ROSproductionofN2wild-typenematodesaftertreatmentfor6h,24h,or120hwith0.1%DMSOor2.5μMECG.(H)

P-valuesareasindicatedinthegraphs.See

Table1

forcorrespondingdetaileddataandstatisticalanalysesoflifespanassays.AMPKandSIRT1areessentialforcatechin-inducedlifespanextensionInhibitionofcomplexIreducesNADH’soxidationtoNAD+,necessaryforglyceraldehyde3-phosphateconversionto1,3-bisphosphoglycerateduringglycolysis.Consequently,reducedlevelsofNAD+hamperglycolysisandtheproductionofpyruvate,whichenterstheKrebscycletobeconvertedintowaterandCO2

[16].Inlinewiththesereports,ECGCreducedtheoxidationofradioactivelylabeledglucoseby20%,asshownbyimpairedproductionofthe

14C-labeledCO2

(Figure3A).ECGtreatmentalsotendedtoreducetheglucoseturnover.However,theeffectsremainednon-significant(Figure3A).ThetimecourseofmetabolicmanipulationbyEGCGandECGwasalsoreflectedinoverallATPlevels.Inlinewithcatechin-inducedinhibitionofmitochondrialrespiration(Figure2E,

2F)andglycolysis(Figure3A),overallATPlevelsdroppedafter6hofEGCG(Figure3B)and12hofECG(Figure3C)treatmentinnematodesbeforerecoveringafter24h.AlackofATP,resultinginahigherAMPtoATPratio,iswell-knowntoactivatetheAMP-dependentkinaseAMPK[17].The

C.elegans

homologofAMPK,AAK-2,isinvolvedinlifespanextensioninresponsetoimpairedglycolysis[18]andinsulin/IGF-1signaling[19].Indeed,EGCG(Figure3D)andECG(Figure3E)failedtoextendlifespanin

aak-2

deficientmutants.Notably,AMPKenhancesNAD+-dependenttypeIIIdeacetylasesirtuin1activitybyincreasingcellularNAD+levels[20].In

sir-2.1

defectivemutants,EGCG(Figure3F)andECG(Figure3G)didnotachievealifespanextension,provingthatEGCGandECGprolonglifespaninanAMPK-andSIRT1-dependentmanner.Thesefindingsalignwithpreviousreportsshowingthatcatechins’lifespanextensiondependsonAMPK,SIRT1,andFOXO[11].Figure3.

EGCGandECGinduceadropincellularATPlevelsandrequireAMPK/SIRT1signalingtoextendlifespan.14CO2

productionofN2wild-typenematodesaftertreatmentwith0.1%DMSO,2.5μMEGCGor2.5μMECGfortheindicatedtime.(A)ATPcontentforvariousincubationperiodsofN2wild-typenematodeswith0.1%DMSOor2.5μMEGCG.(B)ATPcontentfordifferentincubationperiodsofN2wild-typenematodeswith0.1%DMSOor2.5μMEGCG.(C)Therepresentativeoutcomeoflifespanassayof

aak-2

mutantstreatedwith0.1%DMSOversus2.5μMEGCG(D)or2.5μMECG.(E)Therepresentativeoutcomeoflifespanassayof

sir-2.1

mutantstreatedwith0.1%DMSOversus2.5μMEGCG(F)or2.5μMECG.(G)

P-valuesareasindicatedinthegraphs.See

Table1

forcorrespondingdetaileddataandstatisticalanalysesoflifespanassays.p38MAPK,NRF2,andFOXOarerequiredforthelifespanextensioninducedbycatechinsAsshownin

Figure2,EGCGandECGblockcomplexIactivityand,thus,induceatransientriseinROSlevels.ROS[21]andAMPK[22]arepotentialmediatorsofthep38MAPkinasepathways.Thehomologofthemammalianp38MAPK,PMK-1,hasbeenidentifiedasacrucialcomponentinthelifespanextensionof

C.elegans

[23,

24].Inlinewiththesepreviousreports,wefoundthatneitherEGCG(Figure4A)norECG(Figure4B)treatmentextendslifespanin

pmk-1

deficientmutants.Next,wetestedtheimpactofwhetherthetranscriptionfactorSKN1,thewormhomologofNRF2andadownstreamtargetofPMK1underconditionsofoxidativestress[25–27],isinvolvedinthelifespanextensionprovokedbycatechins.Again,noEGCG-(Figure4C)orECG-induced(Figure4D)lifespanextensioncouldbeobservedin

skn-1

mutantworms.DAF-16isthehomologofamammalianFOXOandisreportedtorespondtophysicalandenvironmentalstress[28].

daf-16

mutantwormsaresensitivetooxidativestressandhaveshortenedlifespans.Moreover,DAF-16canactivateorrepressthetranscriptionoftargetgenesinvolvedindauerformation,lifespan,stressresistance,andfatstorageof

C.elegans

[29].EGCGandECGdecreasedmeanlifespanin

daf-16

deficientnematodesfrom20.1±0.1to19.8days(Figure4E)andfrom20.1±0.1to19.4±0.2days(Figure4F),respectively.Themaximumlifespanwasdecreasedfrom22.5±0.3to22.2±0.2daysbyEGCGtreatment(Figure4E)andfrom22.5±0.3to21.7±0.6daysbyECGtreatment(Figure4F).TheseresultssuggestthatDAF-16isindispensableforEGCG’sandECG’slifespanextensionandshowthat

daf-16

deficientnematodesareespeciallypronetoaROSlevelriseinducedbycatechins.Figure4.

EGCGandECGmediatelifespanextensiondependentonPMK-1/p38MAPK,SKN-1/NRF2,andDAF-16/FOXO.

Therepresentativeoutcomeoflifespanassayof

pmk-1

mutantstreatedwith0.1%DMSOversus2.5μMEGCG(A)or2.5μMECG.(B)Therepresentativeoutcomeoflifespanassayof

skn-1

mutantstreatedwith0.1%DMSOversus2.5μMEGCG(C)or2.5μMECG.(D)Therepresentativeoutcomeoflifespanassayof

daf-16

mutantstreatedwith0.1%DMSOversus2.5μMEGCG(E)or2.5μMECG.(F)

P-valuesareasindicatedinthegraphs.See

Table1

forcorrespondingdetaileddataandstatisticalanalysesoflifespanassays.EGCGandECGinduceadaptiveresponsesinROShomeostasisandcellularmetabolismAMPK/SIRT1andp38MAPK/NRF2/FOXOsignalingcascadesareassociatedwithantioxidantdefensemechanisms[30].Themajorantioxidantenzymesin

C.elegans

includefivedistinctsuperoxidedismutases,convertingsuperoxidetohydrogenperoxide,andtwocatalases,whichensurethesubsequentconversionofhydrogenperoxidetowater[31].EGCGtreatmentsincreasedSODactivityafter24h(Figure5A)andCTLactivityafter7days(Figure5B).Meanwhile,ECGtreatmentsdidnotsignificantlyincreaseSODactivity(Figure5A)butincreasedCTLactivityafter24hand7days(Figure5B).TheenhancedactivityofSODandCTLcorrelateswiththesubsequentdropofROSlevelsafter24hofEGCGandECGtreatment.Notably,thelifespan-extendingeffectofEGCGandECGisdependentonSOD-2(Figure5C)andcatalase2(CTL-2)(Figure5D).Asshownin

Figure3,complexIinhibitionbyEGCGandECGwasalsoaccompaniedbyareductioninglucoseoxidation.Inlinewiththisfinding,thefatcontentwasfoundtobesignificantlylowerafter120hofEGCGorECGtreatment(Figure5E),pointingtoacatechin-inducedlong-termreprogrammingofcellularmetabolism.Figure5.

EGCGandECGinduceSODandCTLactivityandashiftinlipidmetabolisminthelongterm.

SOD(A)orCTL(B)activityaftertreatmentwith0.1%DMSO,2.5μMEGCGor2.5μMECGfor24hor7days.Therepresentativeoutcomeoflifespanassayof

sod-2

mutantstreatedwith0.1%DMSOor2.5μMEGCG.(C)Therepresentativeoutcomeoflifespanassayof

ctl-2

mutantstreatedwith0.1%DMSOor2.5μMECG.(D)TriglyceridecontentinN2wild-typenematodesaftertreatmentwith0.1%DMSO,2.5μMEGCGor2.5μMECGfor5days,normalizedtoproteincontent.(E)

P-valuesareasindicatedinthegraphs.See

Table1

forcorrespondingdetaileddataandstatisticalanalysesoflifespanassays.DiscussionGreenteaisoneofthemostwidelyconsumedbeveragesworldwide[32].Thepopularityofgreenteamakesitcrucialtostudyitsimpactonhealthandaging.AlthoughEGCG’sandECG’sbioavailabilityisrelativelylow[7,

8],consuming4cupsofgreenteadailyfor8weekssignificantlydecreasesbodyweight[33].Previousreportsalreadyreportedalifespanextensionin

C.elegans

aftertreatmentwith50–300μMEGCG[11].Here,weshowthatalready2.5μMofEGCGandECG,aconcentrationalsopotentiallyachievedaftergreenteaconsumption[14],aresufficienttoinduceanextensionoflifespanandincreasestressresistancebyadaptationalmechanisms.Inthismitohormeticresponse,EGCGandECGactinitiallyasprooxidantsbyprovokingaROSrise.SinceatransientROSburstinducesantioxidantdefensemechanisms,EGCGandECGdisplayantioxidantpropertiesinthelongterm.Inhigherconcentrations,EGCGandECGmightshowharmfuleffectsduetoexcessiveROSproduction.Thisphenomenongetsobviousinstudiesperformedoncancercells.Whiletheantioxidantpotentialofgreenteacatechinsinlowconcentrationswassuggestedasapotentialsolutiontopreventtumorigenesis[34,

35],higherdosagesofcatechinsmightserveasantitumoragentsduetotheinductionofoverwhelmingROSformationandapoptosis[36–41].Notably,EGCGwasmorepotentthanECGinhumancancercelllinesininducingcytotoxiceffects[33]andinhibitingcancercellmotility[42].Indeed,ittookjust6hforEGCG,but12hforECGtoaffectmitochondrialrespiration,ROS,andATPlevels.However,theimpactofthesecompoundswassimilarwhenappliedinthelongterm,yieldingsimilareffectsonlifespan,motility,andstressresistance.Besidestriggeringamitohormeticresponsethroughtheireffectsontranscriptionfactorsandenzymeactivities,catechinswerespeculatedtoexertdirectantioxidantpotentialbyscavengingROS[43,

44].Whileamodestincreaseintheplasmaantioxidantcapacityfollowinggreenteaconsumptionwasreported[43],thefractionofstructurallyintactcatechinsreachingtargettissuesisinsignificantcomparedtotheantioxidantpotentialduetointracellularglutathioneachievinglevelsof1–11mM[45–47].Besides,EGCGeveninducedhydrogenperoxideformationinthecellcultureandliquidNGMsystem[44–46].Moreover,hydrogenperoxidemimickedtheeffectofEGCGonsignalingpathways,whileantioxidantsabolishedtheimpactofcatechins[37,

41,

48–50].WecouldshowthatBHApreventedlifespanextensionbyEGCGandECG,suggestingthataninitialriseinROSlevelsisnecessarytoinduceadaptationalmechanismscausingimprovedantioxidantproperties.Previousstudiesalreadyrevealedincreasedhydrogenperoxidelevelsandadose-andtime-dependentdecreaseinglutathionelevelsincellculturemodelsafterapplying50μMofEGCG[43,

51].However,themechanismofhowEGCGandECGinduceROSformationwasnotdescribedsofar[11].Inthecurrentstudy,werevealedthatEGCGandECGinhibitcomplexIoftheETC.ExperimentsinratcerebellargranuleneuronshaveshownthatEGCGaccumulatesexplicitlyinmitochondria,reaching90–95%mitochondrialaccumulationofthispolyphenol[52].Thisfindingiswellalignedwiththeplethoraofliteraturedescribingpolyphenolsascompoundstargetingmitochondria[53,

54].Consequently,weisolatedmitochondriatoinvestigatetheimpactofEGCGandECGonthecomplexesofthemitochondrialETC.Isolatedmitochondriaareseparatedfromtheirnaturalenvironmentandsignalingprocesses,andtheisolationprocessbringstheriskofdamagingmitochondrialmembranesduetoshearforces[55].However,druguptakebymitochondriaisdependentontheintegrityoftheouterandinnermitochondrialmembrane,includingthefunctionoftransporterproteinsandcarriers[56].Since25μMofEGCGandECGwerenecessarytoachieveasignificantinhibitionofcomplexIactivityinmitochondriaisolatedfrommurineliversamplesandtohampermitochondrialrespirationinmitochondriaisolatedfromratliver,weassumethattheisolationprocessaffectedtheintegrityofmitochondrialmembranesand,thereby,mitochondria’spotentialtotakeupcatechinsefficiently.Besides,theisolationofmitochondriayieldsarelativelyhomogenouspopulationofsphericalorganelleswithdisorganizedcristaeanddilutedmatrixcontent.ThestructuralalterationsaffectETCactivityandmitochondrialrespirationrate[57].Weassumethatstructuralchangesincristaeorganizationduetotheisolationprocessmightbeanotherreasonwhy25μMofEGCGandECGwerenecessarytosignificantlyblockcomplexIactivityandmitochondrialrespirationrateinisolatedmitochondria.Inaddition,wepresentthatatemporaryhamperedmitochondrialrespirationgoesalongwithatransientriseinROSlevelsandabriefdropinATP,triggeringsignalingpathwaysassociatedwithlifespanextensionin

C.elegans.Ourfindingsalignwithreportsaboutthe

C.elegans

mutant

nuo-6(qm200),carryingamutationinaconservedsubunitofmitochondrialcomplexI(NUDF84).ThisspecificmutanthasreducedcomplexIfunction,increasedROSlevels[58],andaprolongedlifespan[59].ItwasalsospeculatedthatblockageofthecomplexIofthemitochondrialelectrontransportchaindelaysagingduetoslowedembryonicdevelopmentandlarvalgrowth,decreasedpumpinganddefecationrate,orareducedaccumulationofROSdamage[60–62].However,RNAi-inducedknockdownofthemitochondrialelectrontransportchain’scomplexesattheL3/L4stageissufficienttoinitiatelifespanextensionin

C.elegans.Atthisstage,mitochondriaarealreadyundergoingaperiodofdramaticproliferationandmassivemitochondrialDNAexpansion[63].Moreover,inhibitingrespiratorychaincomponentsduringadulthooddidnotprovokelifespanextensionanymo