蝶蚕豆提取物对酮康唑诱导的睾丸损伤的保护作用

蝶蚕豆提取物对酮康唑诱导的睾丸损伤的保护作用

1质intratucedre麻黄结构域intraced和tratching管理体系intractketc-romen（ket）是一种通用的反相互作用通常被用来促进的高级泛滥症（rodriguan，1995；kinbe等人，2006）。例如，在引进可口可乐公司的微芯片系统之前，它是一个未考虑的特征。在引进证据的过程中，它是一个缓慢改善的过程，并且是一个缓慢改善的过程。目标框架格式（2008）。更不用说，amin（2008）报告了从属体框架中重新设计的过程（i.p.），它是一个缓慢改善的过程，反映了母细胞微管理系统和微器官，并反映了特征。母细胞微。在科学上，研究文章中的“非物质文化遗产”样本（i.p.）。实验发送者，即检验受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染受害者，再感染患者，再感染患者，再感染患者，再感染患者，再感染患者，再感染患者，再感染患者，再感染患者，再感染患者，再感染患者。Clitoriaternatea(CT)Linn.(called“Unchan”inThailand)isaclimbingvinebearingpurpleorwhiteflowers.EverypartoftheCTvinehasbeensystematicallyanalyzedandrevealedtohavevariousmedicinalproperties(PatilandPatil,2011).InThailand,thepurpleflowersareusedformanymedicalpurposesanditisbelievedthattheyhaveantoxidantcapacity.Mukherjeeetal.(2008)reviewedtheuseofCTinAyurvedicmedicineandfoundthatCThasbeencommonlyusedinanti-stress,anti-depressant,anti-microbial,andanti-inflammatorytreatments.Moreover,CThasbeenshowntohavesignificanthepatoprotectiveeffectsondrug-inducedliverdamage(Nithiananthametal.,2011).Toourknowledge,thepotentialprotectiveeffectsofCTflowerextractsagainstKET-inducedtesticulardamageinratshavenotbeenreported.Therefore,theaimofthisstudywastoevaluatetheantioxidantactivitiesandpotentialprotectiveeffectsofaqueousCTflowerextractsonKET-inducedtesticulardamageinrats.2杏仁杏仁2.1其他传统chrgasedKET(200mg/tablet;KETOHIM)waspurchasedfromalocaldrugstore.Folin-Ciocalteau’sreagent,2,2-diphenyl-1-picrylhydrazyl(DPPH),acetatebuffer,2,4,6-tripyridyl-s-triazine(TPTZ),andferricchloride(FeCl3)werepurchasedfromSigmaAldrichCo.,Ltd.(Thailand).Ascorbicacidandotherchemicalswereofanalyticalgrade.2.2ctfendermill&发展/agt-kept.案例ThefreshpurpleCTflowerswerecollectedfromKhonKaenProvince,Thailand,inJunetoJuly2012.SpeciesidentificationwasconfirmedbyProf.PranomCHANTARANOTHAI,aplanttaxonomist,fromKhonKaenUniversity(KKU),Thailand.VoucherspecimensofCTwerekeptintheKKUHerbarium(No.JaturonBurawat01[KKU]).Intheextractionprocess,theCTflowerswerewashedwithdistilledwaterandairdriedfortwodays.Thedriedflowerswerecrushedwithahammermillcrusher(AEGIP54Lbi07,Germany)for30min.Then,thecrushedsample(3kg)wasextractedwith10Lofdistilledwaterandboiledat95–100°Cfor30min.Thesolubleextractwasfilteredthroughnylon.Thefiltratewasdriedusinglyophylization.TheextractionyieldofaqueousextractfromCTflowerswas3.97g.2.3entoritypol.TheradicalscavengingactivityoftheCTaqueousextractwasdeterminedusingDPPHassay(BrandWilliamsetal.,1995).Briefly,fiveconcentrationsoftheCTflowerextractswerepreparedfromaconcentratedextractstocksolutiontoprovideaconcentrationplot.EachconcentrationofCTflowerextracts(2ml)wasmixedwith2mlDPPHin0.004%(v/v)methanolandincubatedatroomtemperatureindarkfor30min.Theabsorbancewasrecordedat517nmusinganultraviolet-visible(UV/VIS)spectrophotometer(JascoV530,Japan).Ascorbicacid(1,3,5,8,and14µg/ml)wasusedasthepositivestandard.Allsamplesweremeasuredintriplicate.ThescavengingactivityoftheCTflowerextractsorstandardwascalculatedasthepercentinhibitionofDPPHradicalscavengingactivityusingastandardformulaas(absorbanceofcontrol-absorbanceofsample)×100%/absorbanceofcontrol.DatawereexpressedastheIC50calculatedfromthepointof50%inhibitionagainsttheconcentration(µg/ml)plot.2.4ct-pcrsix-主4ReducingpowercapacitywasdeterminedusingtheFRAPassay(BenzieandStrain,1996).Briefly,0.1mloffivedifferentconcentrationsofCTflowerextractsweremixedwith3mlofFRAPreagent(containing300mmol/Lacetatebuffer,10mmol/LTPTZ,and20mmol/LFeCl3,inavolumeratioof10:1:1).TheCTmixturewasincubatedindarkfor20minandtheabsorbancewasrecordedat593nmusingaUV/VISspectrophotometer.Forstandardpreparation,sixconcentrationsofascorbicacid(5.76,9.60,17.28,24.00,32.00,and48.00µg/ml)wereplottedtodetermineitsreducingpower.2.5“n.”4.3.4和thirty-ratswratswratsingMaleSprague-Dawleyrats(180–200g)werepurchasedfromtheNationalLaboratoryAnimalCenter,Salaya,NakhonPathom,Thailand.ThestudywasapprovedbytheAnimalEthicsCommitteeofKKU,basedontheEthicsofAnimalExperimentationoftheNationalResearchCouncilofThailand(ref.No.05/93).Thirty-sixratsweredividedintosixgroupsandeachgroup(n=6)wastreatedasshowninTable1.2.6工伤子让位lageOnthedayafterterminationoftheCT-KETco-administration,allratswereeuthanizedbycervicaldislocationandsacrificedtocollectthemalereproductiveorgans(i.e.,testis,epididymisplusvasdeferens,andseminalvesicle).Theseorgansweresubsequentlyremovedoffatsandweighed.Toexaminethetesticulardamage,testeswerefixedin10%(v/v)formalininphosphatebufferedsaline(PBS)(pH7.4),embeddedinparaffin,sectionedat4–6μmthickness,andstainedwithhematoxylin-eosin(Iamsaardetal.,2013).AllphotographswerecapturedbyaNikonlightECLIPSEE200microscopeequippedwithaDXM1200digitalcamera.ImageJwasusedtomeasureandcalculateapproximateaveragediametersofseminiferoustubulesinfourdifferentaxes(50tubulesperanimal).2.7非氧化钠/双诉聚酯质酸二通道sp明信,非氧-语,非杂二氮,可显著提高co-通过mrna-medicine,切实提高cograns.案例5.Attheendoftheexperiment,allanimalsweresacrificedtoexposetheleftventricleoftheheart.Bloodwascollectedbypunctureoftheleftventricularchamberusing1mlofheparintopreventbloodclotting.Thebloodwascentrifugedat5000r/minat4°Cfor10mintoseparatetheplasmaserumfrombloodcells.TheplasmatestosteroneconcentrationwasassayedbyenzymaticimmunoassayattheRadiologyUnit,SrinagarindHospital,FacultyofMedicine,KKU,Thailand.2.8pelusing表Maturespermwerecollectedfromtheleftepididymisandvasdeferens.Epididymalspermfluidwasdippedandre-suspendedin1mlPBS(37°C,pH7.4)andcentrifuged(500g,37°C,5min)towashandseparatethematurespermpelletfromitsfluid.Toanalyzetheepididymalspermconcentration,thespermpelletswerere-suspendedwith1ml0.3%(v/v)bovineserumalbumin(BSA)-KSOM(potassiumenrichedsimplexoptimizedmedium;EmbryoMaxKSOMPowderedMouseEmbryoCultureMedium;MilliporecatalogueNo.R-MR-020P-5D).Intriplicatepreparations,thespermsolutions(1:20dilution)wereusedtocountmaturespermusingaNeubauercountingchamberandtocalculatetheirconcentration(Iamsaardetal.,2013).2.9nanodropnd-2000spicactinga联合点AsdescribedbyIamsaardetal.(2013),briefly,thelefttestiswashomogenizedwithRIPAbuffer(CellSignalingTechnology,Inc.,USA)containingacocktailofproteaseinhibitors.Thetesticularhomogenatewasthencentrifugedat12000r/mimfor10mintocollecttesticularlysate.ThetotalproteinconcentrationofthelysatewasmeasuredusingaNanoDropND-1000Spectrophotometer(NanoDropTechnologies,Inc.,USA).Totalproteins(60μg)collectedfromtriplicatesampleswereseparatedby10%(0.1g/ml)sodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS)andblottedontonitrocellulosemembranestodetecttheintensityoftyrosinephosphorylationusingthe4G10primaryantibody(1:3000;MilliporeCo.,USA).Forstandardsamples,BSA(AMRESCO®,USA)wasusedasthenegativecontrolandepidermalgrowthfactors(EGFs)(MilliporeCo.,USA)asthepositivecontrol.Inthedetectionofphosphotyrosineproteins,theenhancedchemiluminescence(ECL)substratewasappliedbeforevisualizationundergeldoct4(ImageQuant400,GHHealthcare,USA).Toquantifythelevelsofphosphorylation,theImageJprogramwasusedtoanalyzetherelativeintensityofphosphorylatedproteinbandsamonggroups.2.10出placketinfici人本设计见表3One-wayanalysisofvariance(ANOVA)andt-testwereusedtoexaminethesignificanceofdifferencesamongsetsofdata,andbetweenpairsofdatapointsusingSigmaStatprogram(Version3.1.1).Allquantitativeresultswereexpressedasmean±standarddeviation(SD).3产品系统3.1ascorbicacidasa&vecd不断优化的程序IntheDPPHscavengingassay,theresultsshowedthatCTflowerextractspossessedaconcentration-responserelationshipinDPPHscavengingactivity,usingascorbicacidasapositivecontrol.ComparedwiththeIC50ofascorbicacid[(5.34±0.09)µg/ml],theIC50ofCTwas(84.15±1.50)µg/ml(y=0.0686x+45.017,R2=0.98).FortheFRAPassaycalibratedwithstandardascorbicacid(y=0.007x+0.3769,R2=0.9802),thereducingpoweroftheCTflowerextractswas(0.33±0.01)mmol/mgascorbicequivalent.TheseresultsdemonstratethattheCTflowerextractsusedforpreventiveexperimentsinthisstudypossessedantioxidantcapacity.3.2ighed发挥序Aftertreatmentfor28consecutivedays,thebodyweightsofcontrolandexperimentalgroupswerenotdifferent(P>0.05).Incontrast,allreproductiveorgansinKET-treatedratsweighedsignificantlylessthanthoseofthecontrols(P<0.05;Table2).WefoundthattheCTflowerextractsdidnotaffecttheweightsofreproductiveorgans.Inaddition,alldosesofCTflowerextractscouldpreventtheweightlossofthetestisinratsinducedwithKET(Table2).Moreover,50and100mg/kgBWCTflowerextractssignificantlyimprovedtheweightsoftheepididymisplusvasdeferensinKET-inducedrats(P<0.05).Only100mg/kgBWCTflowerextractspreventedtheweightlossoftheseminalvesicleintheKETgroup(P<0.05).3.3救赎以及3.3.3单次给药的细TheeffectsofCTflowerextractsontesticulardamagewereexaminedbyobservinghistopathologicalstructures(Fig.1).TheresultshowedshrunkenseminiferoustubulesintheKETgroupcomparedwiththecontrols(Figs.1aand1b).Thisfindingwascorroboratedbytheirdiameters(Table2).IntheKETgroup,theseminiferoustubuleshadveryminorpathologicaleffects(infewerthan10%oftubules)includingsloughingofgermcells,earlycelldegenerationwithgiantcells,andlatecelldegeneration(Fig.2).Someshrunkentubuleswithsignificantlyreduceddiameters(Table2)andotherabnormalseminiferoustubulescouldbefoundintheCT10+KETgroup(Fig.1d).However,nohistopathologyorreductioninseminiferoustubulediameterwasobservedonlyinratsoftheCT100,CT50+KET,andCT100+KETgroups(Table2;Figs.1c,1e,and1f).3.4ct网络interity专业interity专业interity专业interity专业见表1Table2showsthatonlytheKETandCT10+KETgroupshadasignificantdecreaseinserumtestosteronelevelscomparedwiththecontrols.Incontrast,100mg/kgBWCTflowerextractsdidnotaffectsuchlevels,whereas50and100mg/kgBWCTflowerextractsincreasedthetestosteronehormoneinKET-inducedrats(Table2).Similarly,notonlytheCTgroupbutalsotheCT50+KETandCT100+KETgroupsshowednodifferencesinepididymalspermconcentrationcomparedwiththecontrols(Table2).3.5ere-dr机多聚体的intering于制备denacti-krafting于k-k-ket网络,[3.4.3].3.4和kraftingrace和kraftingratchening7.7.7Followingimmuno-Westernblotting,fourmajorphosphorylatedproteins(50,55,60,and65kDa)ofrattesticularlysatewereclearlydetectedinbothcontrolandexperimentalgroups.Fordensitometryanalyses,wefoundthattherelativeintensityofthe55,60,and65kDaphosphorylatedproteinswasnotsignificantlydifferent(datanotshown).Interestingly,atesticular50-kDaphosphorylatedproteinwasespeciallyintenseintheCT100+KETgroupcomparedwithothergroups(Fig.3).4ctfluser-roetractingre农村事件,面向全球的调节作用,见表1,2TheresultsinTable2suggestthathighconcentrationsofCTflowerextracts(100mg/kgBW)werenotharmfultomalereproductiveparameters.ByFRAPassay,theantioxidantactivityoftheCTaqueousextractinthisstudywascomparabletotheferricreducingpowerofascorbicacid.Inaddition,agroupofpolyacylatedanthocyaninsinthisblueflowerisalreadyidentifiedandcrystallized(HondaandSaito,2002;Hiromotoetal.,2013).Consistentwithpreviousreports(Adamsetal.,1998;Amin,2008),inthepresentstudy,KETaffectedreproductiveorganweight,spermconcentration,andserumtestosteronelevels(Table2).Moreover,CTflowerextractshavebeenshowntoexhibithighcytotoxicactivityagainstthebreastcancercells(Akteretal.,2014).Thenon-toxicityoftheCTflowerextractsandtesticulartoxicityofKETinductionwerefurtherconfirmedbyhistologicalobservationsinthetestistissues(Figs.1band2).However,thetesticularpathologyobservedinFig.2,whichwassimilartothatdescribedbyAmin(2008),wasnotquantifiedamonggroupsbecauseitwasfoundinlessthan10%ofratsintheKETgroupandwasrarelyfoundinthelow-dosegroupoftheCTflowerextracts.Itseemedthattesticulardamageinourstudywaslessseverethanthatinthepreviousstudy(Amin,2008).ThismaycausedbythatKETinourstudywaspreparedfromatablet,whileKET(Nizoral)inAmin(2008)wasfromastocksolution.InCT50+KETandCT100+KETgroups,thepre-treatmentofCTfollowedbyco-administrationwithCTandKETsignificantlymaintainedmalereproductiveorganweights,exceptforthatoftheseminalvesicleintheCT50+KETgroup(Table2).Similarly,CT+KETratsretainednormalserumtestosteronelevels,spermconcentration,seminiferoustubulediameters,andtesticulararchitecturecomparedwiththecontrolgroup(Table2;Fig.1).TheseresultsclearlydemonstrateapotentialprotectiveeffectofCTflowerextractsontesticulardamageofratsinducedbyKET.IthasbeenshownthatKETcancausehepatotoxicityandtesticulartoxicitywhichwasrevealedbyhistopathologyandbiochemistryresults(AminandHamza,2005;Amin,2008).Inrats,KET-inducedtesticulardamagewasdemonstratedtobeassociatedwithalterationstotesticularlevelsofmalondialdehyde,glutathaione,catalase,andsuperoxidedismutase(Amin,2008).Inaddition,thisoxidativestressdamageandantioxidantdepletioninrattestiswereshowntoberemarkablyprotectedbyanantioxidantplantextract(Amin,2008).Unfortunately,theextractinthepresentstudyhasnotshownsuchassociations,butitwasassumedtohavethesameactionsinceithadhighantioxidantactivity.Increasingly,posttranslationalphosphorylatedtyrosineproteinsarebeingshowntobeinvolvedinmanybiologicalprocessesincludingspermproduction,capacitation,andacrosomereaction(Moralesetal.,2007;Bailey,2010;Fardilhaetal.,2011;Yamashitaetal.,2011).Forthefirsttime,weattemptedtoexplainchangesintest