补肾抗衰汤对氧化应激所致的秀丽隐杆线虫生育缺陷的影响-其它医学论文

补肾抗衰汤对氧化应激所致的秀丽隐杆线虫生育缺陷的影响-其它医学论文 作者:曹新国,侯莉莉,陈军霞,陆勤 【摘要】目的:探讨补肾抗衰汤对秀丽隐杆线虫因氧化应激诱导所致 生育缺陷的作用。方法:将终浓度为0.33g/mL的补肾抗衰汤按25%、 50%、75%和100%梯度稀释后对L4期线虫培养。应激方式采用紫外 线照射,20J/(m2?min),、热休克(36?,2h)和百草枯(2mmol/L,2h) 处理。通过子宫内卵数、卵的大小和传代时间来评价秀丽隐杆线虫的 生育能力。结果:野生型N2线虫经过紫外线照射、热休克和百草枯 处理后，子宫内卵数、卵的大小均显著减少，传代时间明显增加;过 氧化氢酶和过氧化物歧化酶活性被抑制。补肾抗衰汤对野生型线虫没 有毒性或其他副作用。高浓度补肾抗衰汤不仅能够减轻由紫外线照 射、热休克和百草枯所导致的生育缺陷，而且可以增加过氧化氢酶和 超氧化物歧化酶被各种应激抑制的活性。此外，高浓度补肾抗衰汤可 以明显恢复mev1突变体线虫受损的生育能力。结论:氧化应激 对生育能力有负面影响，高浓度补肾抗衰汤可明显逆转氧化应激所致 的线虫生育缺陷。 【关键词】补肾抗衰汤;氧化应激;生育;线虫 Thereproductivesuccessesofanimalsandhumansdependonaseriesofcarefulneuroendocrineevents,andthereproductionfailurewilloccurifthetimingforanyoneoftheseeventsisdisrupted ,1,.Undersuchconditions,animalreproductionwillbeatriskduetotheeffectsofstress,andthereproductiveprocesscontrolledbytheneuroendocrinesystemappearstobethemostvulnerable ,1,.Forexample,whendairycattlearesubjectedtoheatstress,reproductiveefficiencydeclines.Cowsunderheatstresshavereduceddurationandintensityofestrus,alteredfolliculardevelopment,andimpairedembryonicdevelopment ,2,.Stresscanbedefinedasanevent(physical,environmental,psychological,etc.)whichsignificantlychallengesthehomeostasisoftheanimals.Inmammals,stressimpactsonthereproductiveaxisatthehypothalamusandthepituitarygland ,3,.Variousstressfulconditionshaveadverseeffectsonreproductionofanimals,andthestressconditionsincludeenvironmentalfactors(i.e.,temperature,humidity,metalsandirradiation),physicalfactors(i.e.,transport,shearing,restraintandstrenuousexercise),metabolicfactors(i.e.,insulin inducedhypoglycaemia),immunologicalfactors(i.e.,infectionandendotoxinsadministration),andpsychologicalfactors(i.e.,isolation,socialinteractionsandmentalarithmetictasks),15,. Caenorhabditiselegans,aclassicalmodelorganism,isafreelivingnematodewithanervoussystem,specializedmuscles,anddigestiveandreproductivesystems,6,.ThereproductiveprocesshasbeeninvestigatedextensivelyinC.elegans,79,.C.eleganshermaphroditesareself fertile,andthEirreproductiveprocessismodulatedbydiverseenvironmentalcues,7 9,.Amongtheseenvironmentalcues,environmentalstresscanaffecttherepro ductiveprocessofnematodessignificantly.ThedatafromCoohilletal,10, showedtheeffectsofultraviolet(UV)irradiationonlarvaldevelopmentandfertility.Thereproductiveprocesscanalsobealteredbyheatstressinnematodes,11,.ElectromagneticnanopulseexposurewillberesultedinthedecreasedfertilityofC.elegansbyinterferingwiththefertilizationordevelopment,12,.Inaddition,exposuretoheavymetalsoflead,silver,nickelandzincwillcauseseveredefectsofbroodsizeandgenerationtimeinexposednematodes,13 16,. BushenKangshuaiTang(BKT),acompoundtraditionalChineseherbalmedicine,hasbeenwidelyusedforclinicaltreatmentofprematureovarianfailure(POF).Thismedicinecanalleviatetheinhibitoryactionofexcessiveandrogenonovariangranulosacellsandregulatetheovarianfunctionbypromotingfolliculardevelopment,increasingthelevelsofestrogenandprogestogen,andimprovingtheratioofestrogen/androgen.Inpresentstudy,weprovidedevidencesfortheretrievaleffectofthismedicineonoxidativestress inducedreproductivedefectsusingaC.elegansmodel.Threeparametershadbeenselectedtoevaluatethereproductiveprocess,whichwereeggsinuterus,broodsize,andgenerationtime. 1Materialsandmethods 1.1Materials 1.1.1PreparationofBKTandchemicalsBKTconsistsofninemedicinalherbs:SemenCuscutae(Tusizi)15g,RhizomaPolygonatiSibirici(Huangjing)15g,pre paredRadixRehmanniae(Shudihuang)15g,HerbaCynomoriiSongarici(Roucongrong)10g,RadixMorindaeOfficinalis(Bajitian)10g,RadixAngelicaeSinensis(Danggui)10g,RhizomaChuanxiong(Chuanxiong)6g,Fluoritum(Zishiying)15g,andFructusSchisandraeChinensis(WuwEIzi)6g,whichwerepurchasedfromPharmacyofNanjingMaternityandChildHealthCareHospital.Theherbsweresoakedin2000mLdistilledwater(DW)for2h,andthenboiledfor60minforthefirstdecoction;thesecondandthirddecoctionswereobtainedbyaddingDWto1000mLeachandthenboiledfor1h.Afterbeingmixed,thedecoctionwascondensedwithwaterbathtocrudedrugcontent0.33g/mLandstoredat4? untiluse.AllotherchemicalswereobtainedfromSigma Aldrich(St.Louis,MO,USA). 1.1.2C.elegansmodelAllexperimentswereperformedonhermaphroditeC.elegans.Thestrainsusedinthecurrentstudywerewild typeN2,andmutantofmev 1(kn1),originallyobtainedfromtheCaenorhabditisGeneticsCenter(Minneapolis,MN,USA).Theyweremaintainedonnematodegrowthmedium(NGM)platesandseededwithEscherichiacoliOP50at20?asdescribedbyBrenner ,17,.AgesynchronouspopulationsofL2orL4 larvastagenematodeswereobtainedbycollectionasdescribed,18,.TheL2 orL4larvastagenematodeswerewashedwithdouble distilledwatertwice,followedbywashingwithmodifiedKmediumonce(50mmol/LNaCl,30mmol/LKCl,10mmol/LNaOAc,pH5.5) ,19,.Exposureswereperformedinsterilecultureplates(glass)for2honL4larvastagenematodeswith25%,50%,75%,and100%ofexaminedBKTdilutedwithM9buffer.Experimentsofallconcentrationswererepeated3timesrespectively.Approximately100nematodesweretransferredin100μ Ltoeachexposuresolutioninthemicropipetter.Allexposureswerecarriedoutin20?incubatorfor2hintheabsentoffood.AllthecontrolsarewildtypeC.eleganswithoutBKTadministration. 1.2Methods 1.2.1BroodsizeassayTheprocedurewasperformedaspreviouslydescribed,15,.Briefly,broodsizewasassayedbyplacingexposedorcontrolnematodesontoindividualcultureplates.Theexaminednematodesweretransferredfourtimestoanewwellevery1.5days,andthetotalnumberofeggsreleasedontheplateswasscored.Atleast20replicateswereperformedforstatisticalpurposes. 1.2.2EggsinuterusToscorethenumberofeggsintheuterus,thetreatedorcontroladultnematodeswereindividuallytransferredtoadropofsolutioncontainingcommercialbleachand1mol/LNaOHonaglassslidecoveredwithanagarpad.Thebleachsolutiondissolvedthebodyoftheadultnematodes,andeggswerescoredimmediatelyunderdigitopticswithaZeissmicroscope. 1.2.3GenerationtimeassayTheexperimentwasperformedaspreviouslydescribed ,15,.Thegenerationtimereferstothetimefromdayofbirth(P0)eggtothefirs tfilialgeneration(F1)egg.Atleast20replicateswereperformedforstatisticalpurposes. 1.2.4UVirradiationexperimentsApproximately30L2 stagelarvaewereirradiatedonNGMplateswithoutfoodat20J/(m2?min)byus ingagermicidalbulb(254nm)for2hoursasdescribedbyMurakamietal,20,.AllUVirradiationassayswereperformedat20?,andafterwardsfurth ermaintainedat20?.Allassayswerereplicatedmorethanthreetimes. 1.2.5HeatshockexperimentsApproximately30L2 stagelarvaegrownat20?wereheatstressedfor2hat36? andafterwardsfurthermaintainedat20?.Allassayswerereplicatedmorethant hreetimes. 1.2.6ParaquattreatmentApproximately30L2 stagelarvaeweretreatedwith2mmol/Lparaquatsolutionfor2hat20?.Allassa yswerereplicatedmorethanthreetimes. 1.2.7SuperoxidedismutaseandcatalaseactivitiesThetreatedorcontroladultnematodeswereusedfortheassayofsuperoxidedismutase(SOD)andcatalase(CAT)activitiesaspreviouslydescribed ,21,.TheCATactivitywasestimatedfollowingthemethodofAbei,22,.Thedecreaseinabsorptionwasmeasuredspectrophotometricallyat240nm.Anextinctioncoefficientof43.6/(mol?cm)wasusedtodeterminetheenz ymeactivity.TheSODactivitywasmeasuredbyusingthekitfromRandoxLaboratoriesfollowingthemanufacturer’sprotocol.Thedatawerethesummaryofth reetrials. 1.3StatisticalanalysisAlldatainthisarticlewereexpressedasx?s.One wayanalysisofvariance(ANOVA)followedbyaDunnett’ sttestwasusedtodeterminethesignificantdifferencesbetweenthegroups.Pvalue,0.05wasconsideredstatisticaldifference,Pvalue, 0.01wasconsideredstatisticalsignificantdifference. 2Results 2.1AdministrationofBKTdidnotaffectthereproductionofwild typeN2nematodesWefirstinvestigatedtheeffectsofBKTadministrationonthereproductiveprocessofwild typeN2nematodes.Theeggnumberinuterus,broodsize,andgenerationtimewereexaminedafter2hadministrationof25%,50%,75%,and100%BKTonL4larvae.AsshowninFigure1,nosignificantincreasesordecreasesofeggnumberinuterus,broodsize,andgenerationtimecouldbeobservedinnematodesafterexposedtodifferentconcentrationsofBKTascomparedwiththecontrol.Usually,eggnumberinuterusandbroodsizereflectthereproductivecapacity,whereasalongergenerationtimeindicatesalowreproductivespeed.Therefore,administrationofBKTdidnotenhanceorsuppressthereproductiveprocessunderwildtypebackgroundinC.elegans.Thatis,ourdatasuggestthatadministrationofBKTdidnotcausetoxicoralteredeffectsonanimals’reproduction. 2.2EffectsofadministrationofBKTonreproductivedefectsinducedbyUVirra diationinC.elegansInC.elegans,UVirradiation,heat shock,andparaquattreatmentcancauseoxidativestress,23 26,.Consideringthepossibilitythattheoxidativestressmaynegativelyregulatethereproductiveprocess,wenextexaminedwhethertheadministrationofBKTcouldretrievethereproductivedefectsinducedbyUVirradiation.AsshowninFigure2,afterUVirradiationat20J/(m2?min),theeggnumberinuterusandbroodsizeofexposednematodesweresignificantlydecreasedascomparedwiththecontrol(P<0.01),whilethegenerationtimewassignificantlyincreasedascomparedwiththecontrol(P<0.01).Again,administrationof75%and100%ofBKTwouldmarkedlyincreasetheeggnumberinuterus(75%,P<0.05;100%,P<0.01)andbroodsize(P<0.01),andreducethegenerationtime(75%,P<0.05;100%,P<0.01)ascomparedwithUVirradiation.However,administrationofBKTat25%and50%couldonlymoderatelyalterthereproductiveprocessinUVirradiatednematodes.Inaddition,thedefectsofeggnumberinuterus,broodsizeandgenerationtimeinducedbyUVirradiationcouldbeeffectivelyalleviatedbyadministrationof75%ofBKT.Thedefectsofeggnumberinuterus,broodsize,andgenerationtimeinducedbyUVirradiationcouldbeeffectivelyalleviatedbyadministrationof100%ofBKT.Therefore,administrationofBKTathigherconcentrationscouldlargelyretrievethereproductivedefectsinducedbyUVirradiationinC.elegans. 2.3EffectsofadministrationofBKTinreproductivedefectsinducedbyheatshockinC.elegansWefurtherinvestigatedwhethertheadministrationofBKTc ouldalsolargelyretrievethereproductivedefectsinducedbyheatshock.AsshowninFigure3,heatshocktreatmentat36?for2honL2 stagelarvaedecreasedtheeggnumberinuterusandbroodsize,andincreasedgenerationtimeascomparedwiththecontrol(P<0.01).Followingheatshocktreatment,administrationofhighconcentrations(75%and100%)ofBKTtoC.elegansresultedinsignificantdecreasesofeggnumberinuterusandbroodsize,andincreaseofgenerationtimeascomparedwithheat shocktreatmentonly(75%,P<0.05;100%,P<0.01).Incontrasttothis,administrationof25%and50%ofBKTwouldnotsignificantlyaltertheeggnumber,broodsize,andgenerationtimeofheatshock treatednematodes.Therefore,administrationof75%and100%ofBKTcouldalsolargelyrecoverthereproductivedefectsformedinheat shocktreatednematodes. 2.4EffectsofadministrationofBKTonreproductivedefectsinducedbyparaquattreatmentinC.elegansParaquattreatmentcausesoxidativestressthroughametabolicallycatalyzedreactiontoresultindepletionofcellularnicotinamideadeninedinucleotidephosphate(NADPH)andproductionofROS,primarilysuperoxideanions ,27,.AsshowninFigure4,treatmentwith2mmol/Lparaquatsolutioncausedobviousreductionofeggnumberandbroodsizeandelongationofgenerationtimeascomparedwiththecontrol(P<0.01).Followingparaquattreatment,administrationof75%and100%ofBKTcouldsignificantlyalleviatethedefectsofeg gnumberinuterus(75%,P<0.05;100%,P<0.01),broodsize(P<0.01),andgenerationtime(P<0.01)inducedbyparaquatexposure,whereasadministrationof25%and50%ofBKTwouldnothaveobviouseffectsonthereproductiveprocessinparaquat treatednematodes.Moreover,thedefectsofeggnumberinuterus,broodsize,andgenerationtimeinducedbyparaquattreatmentcouldbeeffectivelyalleviatedbyadministrationof75%ofBKTbyapproximately65.2%,55.4%,and62.5%,respectively.Inaddition,thedefectsofeggnumberinuterus,broodsize,andgenerationtimeinducedbyparaquattreatmentcouldbeeffectivelyalleviatedbyadministrationof100%ofBKTbyapproximately83.5%,77.2%,and81.6%,respectively.Therefore,administrationofhighconcentrationsofBKTwouldlargelyretrievethereproductivedefectsformedinparaquattreatednematodes. 2.5AdministrationofBKTafterstressexposurealteredtheCATandSODactivitiesinC.elegansCAT,actinginconcertwithSOD,belongstothemajordefenseenzymesagainstsuperoxideradicals,andactivitiesofCATandSODaredirectlylinkedtooxidativestress ,28,29,.ToexaminewhethertheretrievaleffectsofBKTonreproductivedefectsinducedbyUVirradiation,heat shock,andparaquattreatmentarethroughalleviatingthedamagefromoxidativestress,wefurtherinvestigatedtheactivitychangesofCATandSODinstressexposedandBKT administratednematodes.AsshowninFigure5,UVirradiation,heat shock,andparaquattreatmentallcausedsignificantdecreasesofCATandSODactivitiesascomparedwiththecontrol,suggestingtheoccurrenceofseveredamagefromoxidativestress.Moreover,administrationof50%,75%,and100%ofBKTcouldsignificantlyincreasetheCATandSODactivitiesofUVirradiated(CAT:50%,P<0.05;75%and100%,P<0.01.SOD:P<0.01),heatshock treated(CAT:P<0.01.SOD:50%,P<0.05;75%and100%,P<0.01),andparaquat treated(CAT:P<0.01;SOD:P<0.01)nematodesascomparedwiththoseinstress exposednematodeswithoutBKTadministration.Furthermore,thedecreasesofCATactivityinUVirradiated,heatshocktreatedandparaquat treatednematodescouldbeeffectivelyalleviatedbyadministrationof50%BKTbyapproximately27.1%,41.5%,and38.2%,respectively,andthedecreasesofSODactivityinUVirradiated,heatshocktreated,andparaquat treatednematodescouldbeeffectivelyrecoveredbyadministrationof50%BKTbyapproximately44.6%,39.3%,and42.6%,respectively.ThedecreasesofCATactivityinUVirradiated,heatshockandparaquat treatednematodescouldbeeffectivelyalleviatedbyadministrationof75%ofBKTbyapproximately47.9%,56.9%,and58.3%,respectively,andthedecreasesofSODactivityinUVirradiated,heatshocktreated,andparaquat treatednematodescouldbeeffectivelyrecoveredbyadministrationof75%BKTbyapproximately64.6%,66.4%,and65.9%,respectively.Inaddition,thedecreasesofCATactivityinUVirradiated,heatshock treated,andparaquat treatednematodescouldbeeffectivelyalleviatedbyadministrationof100%ofBKTbyapproximately63.5%,71.3%,and79.4%,respectively,andthedecreasesofSODactivityinUVirradiated,heatshocktreated,andparaquat treatednematodescouldbeeffectivelyrecoveredbyadministrationof100%ofBKTbyapproximately83.6%,84.2%,and82.2%,respectively.Therefore,administrationofhighconcentrationsofBKTcouldlargelyrecoverthereproductivedefectsinducedbyUVirradiation,heat shockandparaquattreatmentbyalleviatingthedamagefromtheoxidativestress. 2.6AdministrationofBKTlargelyrescuedthereproductivedefectsformedinmev1mutantnematodesmev1encodesasubunitofsuccinate coenzymeQoxidoreductaseincomplex? oftheelectrontransportchain,andmutationofthisgenewillcauseelevatedlevelsofsuperoxideandshortlifespan,30,.Mutationofmev 1resultsinseveralphenotypesconsistentwithelevatedoxidativestresssuchassensitivitytothesuperoxidegeneratorparaquatorhighoxygen,whichprovidesausefultoolfortestingcompoundsormedicinesforantioxidantproperties ,31,32,.AsshowninFigure6,mutationofmev 1alsocausedseverereproductivedefectswithsignificantreductionofeggnumberinuterusandbroodsize,andelongationofgenerationtimeascomparedwithwild typeN2nematodes(P<0.01),indicatingtheimportantroleofelevatedlevelsofsuperoxideininducingreproductivedefects.Inaddition,weobservedthatadministrationof50%,75%,and100%ofBKTallwouldnoticeablyincreasetheeggnumberinuterusandbroodsizeorshortenthegenerationtimeofmev1mutantnematodesascomparedwiththoseinmev 1mutantnematodeswithoutBKTadministration(P<0.01),whereasadministrationof25%ofBKTwouldnotobviouslyaffectthereproductivephenotypesofmev1mutantnematodes.Moreover,theeggnumberinuterusofmev1mutantnematodescouldberecoveredby41.2%,70.1%,and89.3%,respectively,afteradministrationof50%BKT,andthebroodsizeofmev 1mutantnematodescouldbealleviatedby38.4%,69.4%,and90.7%,respectively,afteradministrationof75%BKT,andthegenerationtimeofmev1mutantnematodescouldberetrievedby52.4%,72.1%,and85.7%,respectively,afteradministrationof100%BKTascomparedwithwild typeN2nematodes.Therefore,administrationofhighconcentrationsofBKTlargelyrescuedthereproductivedefectsformedinmev 1mutantnematodeswithelevatedoxidativestress. Figure1EffectsofBKTadministrationoneggnumberinuterus,broodsizeandg enerationtimeinwildtypeN2nematodes Exposureswereperformedfor2honL4 larvastagenematodeswith25%,50%,75%,and100%ofexaminedBKTdilutedwithM9buffer.Atleast20replicateswereperformedforassayofeggnumberinuterus,broodsizeandgenerationtimeforstatisticalpurposes.OnewayANOVAfollowedbyaDunnett’ sttestwasusedtodeterminethesignificanceofthedifferencesbetweenthegroups.Dataareexpressedasx?s,n=30. Figure2RetrievaleffectsofBKTadministrationoneggnumber inuterus,broodsizeandgenerationtimeinUVirradiatedwild typeN2nematodes Approximately30L2 stagelarvaewereirradiatedonNGMplateswithoutfoodat20J/(m2?min).All UVirradiationassayswereperformedat20?,andafterwardsfurthermaintaine dat20?.Exposureswereperformedfor2honL4 larvastagenematodeswith25%,50%,75%,and100%ofexaminedBKTdilutedwithM9buffer.Allassayswerereplicatedmorethanthreetimes.Dataareexpressedasx?s,n=30.*P<0.05,**P<0.01,vscontrolgroup;?P<0.05,?? P<0.01,vsUVgroup. Figure3RetrievaleffectsofBKTadministrationoneggnumberinuterus,broodsizeandgenerationtime inheatshocktreatedwildtypeN2nematodes HS,heatshock.Approximately30L2stagelarvaegrownon20? wereheatstressedfor2hat36? andafterwardsfurthermaintainedat20?.Exposureswereperformedfor2hon L4 larvastagenematodeswith25%,50%,75%,and100%ofexaminedBKT.Allassayswerereplicatedmorethanthreetimes.Dataareexpressedasx? s,n=30.*P<0.05,**P<0.01,vscontrolgroup;?P<0.05,?? P<0.01,vsHSgroup. Figure4RetrievaleffectsofBKTadministrationoneggnumberinuterus, broodsizeandgenerationtimeinparaquattreatedwild typeN2nematodes Approximately30L2 stagelarvaeweretreatedwith2mmol/Lparaquatsolutionfor2handsurvivalat20?.Exposureswereperformedfor2honL4 larvastagenematodeswith25%,50%,75%,and100%ofexaminedBKTdilutedwithM9buffer.Allassayswerereplicatedmorethanthreetimes.Dataareexpressedasx?s,n=30.*P<0.05,**P<0.01,vscontrolgroup;?P<0.05,?? P<0.01,vsparaquatgroup. Figure5EffectsofBKTadministrationonCATandSODactivitiesofUVirradiated, heatshockandparaquattreatedwildtypeN2nematodes HS,heatshock.Approximately30L2 stagelarvaewereirradiatedonNGMplateswithoutfoodat20J/(m2?min)at20 ?,orheatstressedfor2hat36?,ortreatedwith2mmol/Lparaquatsolutionfor2 handsurvivalat20?.Exposureswereperformedfor2honL4 larvastagenematodeswith25%,50%,75%,and100%ofexaminedBKTdilutedwithM9buffer.Allassayswerereplicatedmorethanthreetimes.Dataareexpressedasx?s,n=30.*P<0.05,**P<0.01,vscontrolgroup;?P<0.05,?? P<0.01,vsUVgroup;?P<0.05,??P<0.01,vsHSgroup;?P<0.05,?? P<0.01,vsparaquatgroup. Figure6AdministrationofBKTcouldretrievethedefectsofeggnumber inuterus,broodsizeandgenerationtimeinmev1mutantnematodes N2,wildtypeN2nematodes.Exposureswereperformedfor2honL4 larvastagenematodeswith25%,50%,75%,and100%ofexaminedBKTdilutedwithM9buffer.Allassayswerereplicatedmorethanthreetimes.Dataareexpressedasx?s,n=30.*P<0.05,**P<0.01,vscontrolgroup;?P<0.05,?? P<0.01,vsmev1group. 3Discussion 3.1OccurrenceofoxidativestressisoneoftheimportantreasonstoinducereproductivedefectsinC.elegansPreviousstudieshavesuggestedthatstressexposurewillinduceseverereproductivedefectsinexposedanimals.Forexample,thechemicalof2 bromopropaneisselectivelytoxictoovarianfolliclesinanimalmodels,andexposuretothistoxinattheworkplaceislinkedcloselytoanincreaseintheincidence ofearlymenopauseandinfertilityinfemaleworkers ,33,.Exposuretopolycyclicaromatichydrocarbons,majortoxiccomponentsofcigarettesmoke,willcausefollicledestructioninexperimentalanimals,34,.Amongtheinvestigatedstressescapableofinducingreproductivedefects,oxidativestressmayplayanimportantroleinnegativelyregulatingthereproductiveprocess.Oxidativestresshasbeensupposedtobeoneofthepossiblemechanismsofovariandamage,35,. Inthepresentstudy,weprovideevidencetosupporttheimportantroleofoxidativestressinnegativelyregulatingreproductiveprocess.UVirradiationandheat shocktreatmentcancauseoxidativestressinnematodeC.elegans,23 25,.Inaddition,paraquatwasexploredtofurtherinvestigatetheroleofoxidativestressinreproductioncontrolasitisknowntogeneratesuperoxideanionsinvivo,andtobetoxictoC.elegans ,26,.AsindicatedbythedatafromFigure2toFigure4,UVirradiation,20J/(m2?min),,heat shocktreatment(36?,2h),andparaquattreatment(2mmol/L,2h)allsignificantlyreducedtheeggnumberinuterusandbroodsize,andincreasedthegenerationtimeofthetreatedwild typeN2nematodesascomparedwiththecontrol.Atthesametime,UVirradiation,heat shocktreatment,andparaquattreatmentsignificantlydecreasedtheactivitiesofCATandSODinthetreatedwild typeN2nematodes(Figure5).Especially,mutationofmev 1couldmarkedlydecreasetheeggnumberinuterusandbroodsize,andprolongthegenerationtimeascomparedwithwild typenematodes(Figure6).Moreover,mutationofmev 1alsoreducedtheactivitiesofCATandSODinmutantnematodes(datanotshown).Therefore,ourdatasuggestthattheoxidativestresscannegativelyregulatethereproductiveprocessinexposednematodes. InC.elegans,someotherevidencecanfurthersupportourconclusiontodifferentdegrees.Exposuretoheavymetalofbariumwouldcauseseveredefectsofbroodsizeandgenerationtime,aswellasobviousdecreaseofCATandSODactivities,21,.KnockdownofK10C2.4,whichencodesahomologoffumarylacetoacetatehydrolase,producesthephenotypesofimpairedfertilityandactivationofoxidativestress,36,.Oxidativeandheat shockstresscaninducegermcellapoptosisthroughap53andEGL 1independentpathwayinnematodes ,37,.Moreover,mutationoftheSaccharomycescerevisiaeNDI1genewillincreasetheproductionofdamagingreactiveoxygenspecies,andexpressionofthisgeneinC.eleganscanproducemarkedimprovementsinanimalreproduction,38,. 3.2AdministrationofBKTcanlargelyretrievethereproductivedefectsinducedbyoxidativestressPOFmaybecausedbyanyprocesswhichcanreducethenumberofoocyteswithintheovary ,39,.Environmentalfactors,togetherwithautoimmunegeneticaberrations,ovariandamage,andiatrogenicadmagefollowinginterventionsasinmalignancies,inducethePOFinhumanbEings ,40,.Especially,theimportantroleofoxidativestressinregulatingPOFhasbeenalreadysuggestedbysomestudies ,41,42,.LossofNrf2functionmayblockthedetoxificationof4 vinylcyclohexenediepoxideforovariandamageandincreasetheoxidativestress ,41,.Inaddition,progressiveexternalophthalmoplegiapatientswiththemutationofPOLG,whichencodestheDNApolymerase γ,candevelopPOF,andmouseandyeastmodelswiththismutationshowenhancedamountsofoxidativelesionsandincreasedmtDNAdamage,42,. BKTisacompoundtraditionalChineseherbalmedicinenourishingqi,blood,yinofkidneyforclinicalalleviationofPOF.Thismedicinemayplayaregulatoryactiononovarianfunctionbyactivatingendocrineandautocrinepathways.Someusefulcomponentsmaybecontainedinthismedicine,andthesecomponentsmaybefurtheridentifiedandexploredforthefutureclinicaltreatment.TheantioxidativeactivityofflavonoidsfromSemenCuscutaewasconfirmedbyadoptingspectrometerdeterminingthescavengingactionofflavonoidsonthefreeradicals,43,.PreparedRadixRehmanniaecanincreaseactivitiesofSODandnitricoxidesynthase(NOS)inbraintissueagainstoxidationanddelaysenescenceprocess ,44,.Phenylethanoidglycosides(PEG)extractedfromCistanchesalsaexhibitstrongerantioxidantcapacityascomparedwiththepositivecontrolvitaminE,45,.MorindaofficinalisHowwaterextracthadantioxidantactivityinvegetableoil,andastheadditionincreases,antioxidantactivitybecomesbetter,46,.RadixAngelicaeSinensis,RhizomaLigusticiChuanxiongandSchisandrasphenantheraRehd.etWilsextractsallshowedsignificantantioxidativeeffects ,47,48,.WewillfurtherstudythesingleherbextractsfromSemenCuscutae,RhizomaPolygonatiSibirici,preparedRadixRehmanniae,etc.totrytofindoutthemoreusefulactivecomponents. Inthecurrentwork,weprovideseverallinesofevidencetoindicatetheimportantfunctionofBKTinalleviatingoxidativestress inducedreproductivedefectswiththeaidofmodelorganismC.elegans.Firstly,administrationofBKTcouldeffectivelyalleviatethereproductivedefectsinducedbyUVirradiation,heat shock,andparaquattreatment;secondly,administrationofBKTcouldsignificantlyincreasetheCATandSODactivitiessuppressedbyUVirradiation,heatshock,andparaquattreatmentascomparedwiththecontrol;thirdly,administrationofBKTcouldlargelyrecoverthereproductivedefectsformedinmev1mutantnematodes.Therefore,administrationofBKTcanlargelyretrievetheoxidativestress inducedreproductivedefectsinC.elegans.Theseobservationsareusefulforour understandingtheeffectivefunctionofBKTinalleviatingthePOF. 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