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Published:February05,2014DOI:10.
1371/journal.
pone.
0088282TheRegulationofAlfalfaSaponinExtractonKeyGenesInvolvedinHepaticCholesterolMetabolisminHyperlipidemicRatsYinghuaShi,RuiGuo,XiankeWang,DediYuan,SenhaoZhang,JieWang,XuebingYan,ChengzhangWangAbstractToinvestigatethecholesterol-loweringeffectsofalfalfasaponinextract(ASE)anditsregulationmechanismonsomekeygenesinvolvedincholesterolmetabolism,40healthy7weeksoldmaleSpragueDawley(SD)ratswererandomlydividedintofourgroupswith10ratsineachgroup:controlgroup,hyperlipidemicgroup,ASEtreatmentgroup,ASEpreventiongroup.
Thebodyweightgain,relativeliverweightandserumlipid1evelsofratsweredetermined.
Totalcholesterol(TC)andtotalbileacids(TBA)levelsinliverandfeceswerealsomeasured.
Furthermore,theactivityandmRNAexpressionsofHmgcr,Acat2,Cyp7a1andLdlrwereinvestigated.
Theresultsshowedthefollowing:(1)TheabnormalserumlipidlevelsinhyperlipidemicratswereamelioratedbyASEadministration(bothASEpreventiongroupandtreatmentgroup)(P15mmol/L,andTG>1.
2mmol/L)wereidentifiedashyperlipidemicrats[25].
Toidentifyinductionofhyperlipidemia,attheendof3and4weekafterfeedinghigh-lipiddiet,bloodsamplewascollectedfromtailveinoftherats,andthenassayedforserumTCandTGlevelsusingastandardenzymaticassaykit(BioSinoBio-technologyandScienceInc.
,China).
Afterserumlipiddetermination,20ratswhichwerefedwithhigh-lipiddietfor4weeksallshowedhyperlipidemicsymptom.
Alfalfasaponinextract(ASE)wasprovidedbyHebeiBao'enBiotechnologyCo.
,Ltd(Shijiazhuang,China),whichwasextractedfromtheleavesandstemofalfalfa,andthepuritywas62%.
ThepreparationofASEwasasfollows:Thepowdereddriedstemsandleavesofalfalfaweredefattedbysoxhletextractionwithpetroleumether(2*24h),andthedefattedpowerwasextractedwith75%ethanol(10ml/g)for3hwithconstantstirring.
Aftersuctionfiltration,theextractionwasrepeated.
Theextractswerecombinedandevaporatedundervacuum.
Thedriedextractswasdissolvedindistilledwaterataconcentrationof100g/landthenfractionatedonamacroporousadsorptionresinAB-8columnwithdistilledwater,50%ethanolrespectively.
Theethanolextractedsaponinswereobtainedfromthe50%ethanolfractions,andevaporatedundervacuum.
ToevaluateeffectsofASEonhyperlipidemicrats,10identifiedhyperlipidemicrats(oraladministrationwith2mldistilledwaterat9:00ameverymorningduringtheperiodofhyperlipidemicmodelestablishment)werecontinuouslyfedwithahigh-lipiddiet,andatthesametimeonceeverymorningtreatedwith240mg/kg/dayASEin2mldistilledwaterbyoralgavagefromthebeginningof5week.
Thetreatmentslastedfor4weeks.
Furthermore,toinvestigatepreventioneffectsofASEonhyperlipidemicrats,fromthebeginningofhyperlipidemicmodelestablishment,10ratswerefedwithahigh-lipiddiet,andatthesametimeonceeverymorningtreatedwith240mg/kg/dayASEin2mldistilledwaterbyoralgavagefor8weeks.
Thedoseof240mg/kgwasdecidedbasedonanearlierstudy[21].
Hyperlipidemicratswerefedwithhigh-lipiddietandorallyadministratedwith2mldistilledwateratthesametimeonceeverymorningfor8weeks.
Thecontrolratswerefedwithstandardlabchowandorallyadministratedwith2mldistilledwateratthesametimeonceeverymorningfor8weeks.
Sothereare4groupswith10ratsineachgroup:controlgroup,hyperlipidemicgroup,ASEtreatmentgroupandASEpreventiongroup.
SamplingDatacollection.
Theratsweremonitoreddailyforgeneralhealthandweighedindividuallyatthebeginningandendoftheexperiment.
Thedailyfeedintakeandweightgainwererecordedduringtheexperimentalperiod.
Bloodsampling.
Attheendof8week,ratswerefastedovernightandkilledunderetheranesthesia.
Bloodwascollectedbycardiacpunctureandleftatroomtemperatureforcoagulation.
Theserumwasobtainedbycentrifugationat3000*g,4°Cfor10minandstoredat-70°Cforlateruse.
Theliverwasremovedandwashedwithnormalsaline,blotteddryonfilterpaper,weighed,thenimmediatelyfrozeninliquidnitrogenandstoredat70°Cforfurtheranalysis.
Liversampling.
0.
5gliverofeachratwashomogenizedinPhosphateBufferedSaline(PBS,pH7.
2)(0.
25g/ml)at4°C.
Thesupernatantwasthencentrifugedat4000*g,4°Cfor10min.
Thepreparationwasadjustedtoindicatedconcentrationandstoredat70°Cforfutureuse.
ProteinconcentrationinsupernatantwasmeasuredbyBradfordmethod[26].
Fecessampling.
Fecesofeachratwerecollectedduringthelast3daysexperimentalperiodanddriedat60°C.
Feceswereweighedandgrindedinto0.
5mdiameterpowder.
0.
5gFecespowderofeachratwereextracted3timeswith10mlof95%ethanolat60°Candthenfilteredaswellasevacuatedthoroughly.
TheresiduewasdissolvedinPBSbysonication.
Thepreparationwasadjustedtoindicatedconcentrationandstoredat70°Cforlateranalysis.
BioassaysBiochemicalassayofserumlipid,cholesterolandbileacidsintheliverandfeces.
Levelsoftriglyceride(TG),totalcholesterol(TC),high-densitylipoproteincholesterol(HDL-C)andlow-densitylipoproteincholesterol(LDL-C)inserumweredeterminedusingtheHitachi911analyzer(Roche)withcommercialkits(BioSinoBio-technologyandScienceInc.
)accordingtothemanufacturer'sinstruction.
Levelsoftotalcholesterol(TC)andtotalbileacids(TBA)intheratliverorfecalpreparationweredeterminedusingtheHitachi911analyzer(Roche)withcommercialkits(BioSinoBio-technologyandScienceInc.
)accordingtothemanufacturer'sinstruction.
Enzymaticactivityassay.
EnzymaticactivityofHMGCR,ACAT2andCYP7A1aswellasconcentrationofLDLRinratliverweredeterminedrespectivelyusingRatELISAKits(GENMED)[27],[28]accordingtothemanufacturer'sinstructionusingBertholdLB940microplatereader(BertholdTechnologies).
RNApreparation,cDNAsynthesisandreal-timeRT-PCR.
TotalRNAwaspreparedusingTrizolreagent(Invitrogen)accordingtothemanufacturer'sprotocol.
OnemicrogramRNAwastranscribedintocDNAusingOmniscriptreversetranscriptase(QIAGEN)accordingtomanufacturer'sprotocol.
Real-timequantitativePCR(QPCR)wasusedtodetecttheexpressiondifferenceofHmgcr,Acat2,Cyp7a1andLdlrinhepatictissuesamongtreatments.
β-Actinwasusedasaninternalcontrol.
TheprimersforQPCRwerepresentedinTable1.
Real-timeQPCRwasperformedona96-wellPCRplateintriplicatewithatotalreactionvolumeof10Lcontaining1LcDNA,5uLSYBRGreenMastermix,0.
1uLofeachspecificforwardandreverseprimers,and3.
8Lnuclease-freewater,PCRwascarriedoutinanABIPRISM7700sequencedetectionsystem(AppliedBiosystems)with2minat95°Cforpredegeneration,thenfollowedby40cyclesat95°Cfor15s,60°Cfor20sand72°Cfor30seach.
ThereactionmixturewithnocDNAwasconsideredasthenegativecontroltoconfirmtheabsenceofprimerdimerization.
Thecyclethreshold(Ct)valuesweredeterminedbySequenceDetectionSystemsoftwareversion1.
7a.
QualitativePCRwasperformedtoconfirmformationofasingleproductineachreactionbeforequantitation.
Thetargetgeneexpressionsofthesampleswereexhibitedasfoldchangefromcontrol.
Allgeneswerenormalisedwithβ-Actin.
Table1.
SYBRGreenprimersequencesusedforreal-timeRT-PCR.
doi:10.
1371/journal.
pone.
0088282.
t001页码,2/8(W)w2014/2/27http://www.
plosone.
org/article/info%3Adoi%2F10.
1371%2Fjournal.
pone.
0088282CreatePDFfileswithoutthismessagebypurchasingnovaPDFprinter(http://www.
novapdf.
com)StatisticalanalysisAllresultswereexpressedasmean±SEM.
Thedatawereevaluatedbyone-wayANOVA,andthedifferencesbetweenthemeanswereassessedusingDuncan'stest.
P0.
05).
doi:10.
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0088282.
g001EffectsofASEonserumlipidlevelsofratsAssummarizedinFigure2,serumTG,TCandLDL-Clevelsweremarkedlyelevated(P<0.
05),whereasserumHDL-Clevelsweresignificantlydecreasedinhyperlipidemicratscomparedwiththecontrolgroup(P<0.
05).
AdministrationofASE(bothASEpreventiongroupandtreatmentgroup)ledtosignificantreductionofserumTG,TCandLDL-Clevels(P<0.
05),aswellastheriseofserumHDL-Clevelscomparedtohyperlipidemicgroup(P<0.
05)althoughtheydidnotreachthevaluesofcontrolgroup(p<0.
05controlvsbothASEadministrationgroups),whichindicatedthebeneficialeffectsofASEonserumlipidprofilesinhyperlipidemicrats.
Figure2.
Effectsofalfalfasaponinextractonserumlipidlevelsofrats.
A.
SerumTGlevel.
B.
SerumTClevel.
C.
SerumHDL-Clevel.
D.
SerumLDL-Clevel.
n=10.
TG,triglycerides;TC,totalcholesterol;HDL-C,high-densitylipoproteincholesterol;LDL-C,low-densitylipoproteincholesterol;*P<0.
05,HyperlipidemicgroupVS.
controlgroup;#P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
hyperlipidemicgroup;$P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
controlgroup.
doi:10.
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0088282.
g002EffectsofASEontotalcholesterolandtotalbileacidslevelsinliverandfecesofratsAssummarizedinFigure3and4,theratsfedwithhigh-lipiddietshowedmarkedlyhigherlevelsofliverTCandTBAcomparedwiththecontrolgroup(P<0.
05),ASEadministration(bothASEpreventiongroupandtreatmentgroup)significantlyreducedliverTClevel(P<0.
05)althoughtheydidnotreachthevaluesofcontrolgroup(p<0.
05controlvsbothASEadministrationgroups),howeverbothASEadministrationsignificantlyincreasedliverTBAlevel(P<0.
05).
BothTCandTBAlevelsinfecesofratsfedwithhigh-lipiddietweresignificantlyhigherthanthoseincontrolrats(P<0.
05),andfurtherremarkablyelevatedbybothASEadministration(P<0.
05).
页码,3/8(W)w2014/2/27http://www.
plosone.
org/article/info%3Adoi%2F10.
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pone.
0088282CreatePDFfileswithoutthismessagebypurchasingnovaPDFprinter(http://www.
novapdf.
com)Figure3.
Effectsofalfalfasaponinextractontotalcholesterolandtotalbileacidslevelsinliverofrats.
A.
TClevelinliver.
B.
TBAlevelinliver.
n=10.
TC,totalcholesterol;TBA,totalbileacids;*P<0.
05,HyperlipidemicgroupVS.
controlgroup;#P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
hyperlipidemicgroup;$P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
controlgroup.
doi:10.
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0088282.
g003Figure4.
Effectsofalfalfasaponinextractontotalcholesterolandtotalbileacidslevelsinfecesofrats.
A.
TClevelinfeces.
B.
TBAlevelinfeces.
n=10.
TC,totalcholesterol;TBA,totalbileacids;*P<0.
05,HyperlipidemicgroupVS.
controlgroup;#P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
hyperlipidemicgroup;$P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
controlgroup.
doi:10.
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pone.
0088282.
g004EffectsofASEongeneexpressionandenzymaticactivityinliverofratsRT-PCRdatapresentedinFigure5showedthatgeneexpressionofHmgcrinliverofhyperlipidemicratswasdown-regulatedascomparedwiththecontrolgroup(P<0.
05),andfurthermarkedlyreducedinhyperlipidemicratswithASEadministration(bothASEpreventiongroupandtreatmentgroup)(P<0.
05).
Onthecontrary,geneexpressionofCyp7a1inliverofhyperlipidemicratswasup-regulatedascomparedwiththecontrolgroup(P<0.
05),andfurtherdramaticallyelevatedinhyperlipidemicratswithbothASEadministration(P<0.
05).
GeneexpressionofAcat2inliverofhyperlipidemicratswasup-regulatedascomparedtothecontrolgroup(P<0.
05),andremarkablydecreasedinhyperlipidemicratswithbothASEadministration(P<0.
05),eventheywerelowerthanthevaluesofcontrolgroup(p<0.
05controlvsbothASEadministrationgroups).
Onthecontrary,geneexpressionofLdlrinliverofhyperlipidemicratswasdown-regulatedascomparedtothecontrolgroup(P<0.
05),andsignificantlyincreasedinhyperlipidemicratswithbothASEadministration(P<0.
05)althoughtheydidnotreachthevaluesofcontrolgroup(p<0.
05controlvsbothASEadministrationgroups).
ComparedwithASEtreatmentgroup,geneexpressionofCyp7a1andLdlrofratsinASEpreventiongroupweresignificantlyincreased(P<0.
05).
ELISAdatapresentedinFigure6showedthattherewasthesametrendonactivitiesoftheseenzymesintheliverasgeneexpression.
Figure5.
EffectsofalfalfasaponinextractonmRNAexpressionofgenesinratliver.
A.
HmgcrmRNA.
B.
Acat2mRNA.
C.
Cyp7a1mRNA.
D.
LdlrmRNA.
n=10.
Hmgcr,3-Hydroxy-3-methylglutarylCoAreductase;Acat2,acyl-CoA:cholesterolO-acyltransferase2;Cyp7a1,cytochromeP450,family7,subfamilya,polypeptide1;Ldlr,low-densitylipoproteinreceptor.
*P<0.
05,HyperlipidemicgroupVS.
controlgroup;#P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
hyperlipidemicgroup;$P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
controlgroup;+P<0.
05,ASEpreventiongroupVS.
ASEtreatmentgroup.
doi:10.
1371/journal.
pone.
0088282.
g005Figure6.
EffectsofalfalfasaponinextractonenzymaticactivityandconcentrationofLDLRinratliver.
A.
HMGCRactivity.
B.
ACAT2activity.
C.
CYP7A1activity.
D.
LDLRconcentration.
n=10.
HMGCR,3-Hydroxy-3-methylglutarylCoAreductase;ACAT2,acyl-CoA:cholesterolO-acyltransferase2;CYP7A1,cytochromeP450,family7,subfamilya,polypeptide1;LDLR,low-densitylipoproteinreceptor.
*P<0.
05,页码,4/8(W)w2014/2/27http://www.
plosone.
org/article/info%3Adoi%2F10.
1371%2Fjournal.
pone.
0088282CreatePDFfileswithoutthismessagebypurchasingnovaPDFprinter(http://www.
novapdf.
com)HyperlipidemicgroupVS.
controlgroup;#P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
hyperlipidemicgroup;$P<0.
05,ASEgroup(bothASEtreatmentandpreventiongroup)VS.
controlgroup;+P<0.
05,ASEpreventiongroupVS.
ASEtreatmentgroup.
doi:10.
1371/journal.
pone.
0088282.
g006DiscussionAnti-hyperlipidemiceffectsofASEinhyperlipidemicratsDyslipidemia(usuallyelevatedserumlevelsofTG,TCandLDL-C,accompaniedbyreducedHDL-Clevel)isametabolicdisorderthatconstitutesacrucialriskfactorofatherosclerosisandcardiovasculardisease[29].
LDL-ChasbeenfoundtobethemostdangerousfactoramongserumlipidsowingtoincreasedpenetrationofoxidatedLDL-Cintoarterialwalls[30]andthentheexcessofLDLiseasilydepositedintothebloodvesselwalls,whichisinvolvedintheinitiationandpromotionofatherosclerosisandbecomesamajorcomponenttocauseatheroscleroticplaquelesions[31].
HDLcarriescholesterolandcholesterolestersfromtheperipheraltissuesandcellstotheliver,wherecholesterolismetabolizedintobileacids.
SoHDLplaysaveryimportantroletoreducecholesterollevelsinthebloodandperipheraltissues,andtoinhibitatheroscleroticplaqueformationintheaorta[32].
Therefore,decreasingserumTCandLDL-ClevelsandincreasingserumHDLlevelarepivotalforreducingtheriskofatherosclerosis[33].
Dietplaysacrucialroleinthecontrolofcholesterolhomeostasis.
Theconsumptionofcholesterol-enricheddietisregardedasakeyriskfactorinthedevelopmentofcardiovasculardiseasesasitleadstothedevelopmentofhyperlipidemiaandatherosclerosis.
OurresultsshowedthatthehyperlipidemicratsdevelopedhigherserumlevelsofTG,TCandLDL-C,aswellasadecreasedconcentrationofHDL-C.
Theresultsobtainedwereconsistentwiththepreviousstudies[20],[21],[34],[35].
However,theelevatedserumlevelsofTG,TCandLDL-CinhyperlipidemicratsweresignificantlyreducedbyASEadministration.
Conversely,thedeclinedserumHDL-ClevelwassignificantlyincreasedbyASEadministration.
TheseresultssuggestedthatASEwasaneffectivelipid-loweringagent.
Thefindingsalsoagreedwiththepreviousstudiesonthecholesterol-loweringeffectsofASinmonkeysandratsreportedbyMalinowetal[10]–[12]andStoryetal[14],theyconcludedthatthehypocholesterolemiceffectsofAScouldbeascribedtoitsinhibitionofcholesterolabsorption.
However,whetherthecholesterol-loweringeffectsofASEaremediatedbysomekeygenesinvolvedincholesterolmetabolismisnotknown.
Therefore,inthepresentstudy,weinvestigatedthehepaticmetabolicpathwayofcholesterolanddetectedtheexpressionandactivityofHMGCR,ACAT2,CYP7A1andLDLRintheliver.
InhibitoryeffectsofASEonHMGCRandACAT2inhyperlipidemicrats3-Hydroxy-3-methylglutarylCoAreductase(HMGCR)istherate-limitingenzymeincholesterolbiosynthesis.
TheinhibitionofHMGCRexpressionoractivitywillleadtoinhibitcholesteroldenovosynthesisintheliverandthusreduceserumcholesterollevel[16],[36].
Acyl-CoA:cholesterolO-acyltransferase2(ACAT2),isthemajortissuecholesterol-esterifyingenzyme,whichisfoundwithinlipoprotein-producingcellssuchasenterocytesandhepatocytes[18].
IthasbeenpreviouslydocumentedthatACAT2convertsfreecholesterolintocholesterylestersinresponsetoexcessintracellularcholesterol[37].
ACAT2-derivedcholesterylestersmayalsobeincorporatedintohepaticapoB-containinglipoproteinsandsecretedintoplasma.
SoACAT2playsacriticalroleintheproductionofatherogenicapoB-containinglipoproteinsandthatACAT2-specificinhibitorsareextremelyeffectiveinpreventingmurineatherosclerosis[38].
Inourstudy,geneexpressionofHmgcrwassuppressedinhyperlipidemicrats,andfurthersignificantlyinhibitedbyASEadministration,andmarkedlyreducedTClevelintheliverandserumofhyperlipidemicrats.
SothedecreasingTClevelintheliverandserumobservedinthecurrentstudycouldbeexplainedbythedown-regulationofHmgcr,whichreducedtheconversionofHMG-CoAintomevalonate,andinhibitedthesynthesisofcholesterol[39].
AlthoughtherewasnoreportontheeffectofASEontheexpressionofcholesterolmetabolismrelatedgenes,itwasfoundthatFTZ(FufangZhenshuTiaoZhi)extractedfromChineseherbscouldregulatethegeneexpressionofHmgcr[28].
Cynomolgusmonkeysfedahigh-cholesteroldietexpressincreasedhepaticAcat2mRNAlevels,andpatientstreatedwithstatinshaveadose-dependentdecreaseinAcat2expression[40].
Theintakeofhigh-lipiddietledtoanincreaseinAcat2mRNAlevelsinthepresentstudy,however,theadministrationofASEremarkablydecreasedgeneexpressionofAcat2.
StudiesofAlgeretal[41]demonstratedthatliver-specificdepletionofAcat2withantisenseoligonucleotidespreventeddietarycholesterol-associatedhepaticsteatosisbothinaninbredmousemodelofnon-alcoholicfattyliverdisease(SJL/J)andinahumanizedhyperlipidemicmousemodel(LDLr,apoB),andACAT2-specificinhibitorsmightholdunexpectedtherapeuticpotentialtotreatbothathero-sclerosisandnon-alcoholicfattyliverdisease.
Sothedown-regulationofAcat2inthecurrentstudymightpreventdietarycholesterol-associatedhepaticsteatosis.
FurtherstudyontheeffectofASEonthegeneexpressionofAcat2couldgivemoreinsightsonthepharmacologicaleffectsofAS.
Up-regulatingeffectsofASEonCYP7A1andLDLRinhyperlipidemicratsCholesterolconversionintobileacidsintheliverisapivotalpathwayinreducingtheserumcholesterollevel.
Bileacidsynthesisandexcretioncontributetomostofthecholesterolremovedfromthebody[42].
CytochromeP450,family7,subfamilya,polypeptide1,alsoknownascholesterol7-alpha-hydroxylase(CYP7A1)istherate-limitingenzymeintheclassicalbileacidbiosyntheticpathway,whichaccountsforatleast75%ofthetotalbileacidpool[43].
TheincreaseofCYP7A1expressionoractivitywillenhancethecatabolicpathwayofcholesterolandledtothereductionofserumandlivercholesterollevel[44].
Low-densitylipoproteinreceptor(LDLR)isacellsurfaceglycoprotein,whichbindstwoproteins:apoB-100,whichisthesoleproteinofLDL,andapoE,whichisfoundinmultiplycopiesinIDLandasubclassofHDL[19].
LDLRhasdualroleinLDLmetabolism.
First,itlimitsLDLproductionbyenhancingtheremoveoftheprecursor,IDL,fromthecirculation.
Second,itenhancesLDLdegradationbymediatingcellularuptakeofLDL.
AdeficiencyofLDLreceptorscausesLDLtoaccumulateasaresultbothofoverproductionandofdelayedremoval[45].
SoLDLRisacruciallyimportantmodulatorofplasmaLDLlevelsinhumansandanimals.
TheincreaseofLDLRexpressionoractivitywillresultinthereductionofserumLDLcholesterollevelbyenhancingtheuptakeandclearanceofLDLcholesterol[46].
/100/100页码,5/8(W)w2014/2/27http://www.
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0088282CreatePDFfileswithoutthismessagebypurchasingnovaPDFprinter(http://www.
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ViewArticlePubMed/NCBIGoogleScholarOurstudyshowedthatgeneexpressionofCyp7a1wasenhancedinliverofhyperlipidemicratsandfurthersignificantlyincreasedbyASEadministration,whichresultedinanincreaseofthecholesterolconversionintobileacids.
GeneexpressionofLdlrwasinhibitedinhyperlipidemicrats,however,ASEpromotedhepaticuptakeandclearanceofplasmacholesterolbyup-regulatinggeneexpressionofLdlr.
Theliverplaysanimportantroleinmaintainingwhole-bodycholesterolhomeostasis.
Itisthemajorsiteforeliminationofcholesterolfromthebodyviabilethroughconvertingcholesterolintobileacids,andalsoamajorcatabolicsitefortheLDLreceptor-mediatedpathway[47],[48].
Ingeneral,theriseinexpressionofCyp7a1andLdlrwouldincreaseuptakeofLDLcholesterolandenhancethecatabolicpathwaywhichconvertscholesteroltobileacid.
Asaresult,hepaticTBAlevelwouldincrease,hepaticandserumTClevelwouldreduce.
Thiscorrespondedtothechangesintheseparametersobservedinthepresentstudy.
Reenaetal.
[46]reportedthehypocholesterolemiceffectsofinteresterifiedoilsweremediatedbyup-regulatingCyp7a1andLdlrmRNAexpressioninrats.
Wuetal.
[49]alsoreportedPNS(Panaxnotoginsengsaponins)supplementationcouldup-regulatethemRNAexpressionofCyp7a1andsupressthediet-inducedhypercholesterolaemia.
SimilarmechanismwasalsoobservedwithsoyisoflavoneandpuerarinwhichdecreasedserumTClevelbymainlyenhancingtheexpressionofCyp7a1[50],[51].
CholesterolhomeostasisistightlycontrolledbycoordinatedchangesintheconcentrationsofmRNAencodingmultipleenzymes[15].
TheactivityoftheseenzymesinthepresentstudyalsoparalleledtheobservedchangesinmRNAlevels.
Down-regulatinggeneexpressionofHmgcrandAcat2thusdecreasingtheiractivitybyASEadministrationmayhaveresultedinthereductionofliverandserumTClevelinhyperlipidemicrats.
Conversely,up-regulatinggeneexpressionofCyp7a1andLdlrthusincreasingtheiractivitybyASEadministrationmayhaveledtotheriseofTBAlevelandthereductionofTClevelintheliverandserumofhyperlipidemicrats.
ThedatainourstudysuggestedASEcouldregulatecholesterolmetabolismmainlyfromthreepathways,i.
e.
enhancingthecatabolicpathwayanduptakeofLDL-Candinhibitingthesynthesispathwayofcholesterol.
Thehypocholesterolemiceffectsofsomeplantextractswasalsofoundtoberelatedtotheexpressionofgenesimplicatedincholesterolmetabolism,suchasHmgcrandCyp7a1inFTZ[28],Cyp7a1inPNS[49],soyisoflavone[50]andpuerarin[51].
Althoughtheexactmechanismofactionfortheseplantextractsoncholesterolmetabolismremainstobeelucidated,theresultsobtainedprovidepowerfulsupportforseekingnewnaturalcholesterol-loweringagents.
OurstudyalsoshowedthatlevelsofTBAandTCinfecesofhyperlipidemicratsweresignificantlyincreasedbyASEadministration,whichalsosuggestedthattheincreaseinexcretionofcholesterolanditsmetabolitewasanotherimportantpathwayofASEtoreduceserumcholesterollevelinhyperlipidemicrats.
Previously,ithasbeendemonstratedthatASreducedserumand/orlivercholesterolaccumulation,ButthesestudiesmainlyfocusedontheeffectsofASonintestinalcholesteroladsorption,whichindicatedASbindtocholesterolpreventingitsreabsorption,increasingthecholesterolcontentinthefecesanddecreasingcholesterollevelsintheblood[10]–[14].
Inthepresentstudy,besidestheadsorptionofASEoncholesterol,itwasnotablethatourfindingsdemonstratedthattheregulationofASEonsomekeygenesimplicatedincholesterolmetabolismmightberesponsibleforthehypocholesterolemiceffectsofASE.
However,whetherASEhasadirecteffectinmRNAexpressionandactivityofthesegenes,furtherstudyisneededtoclarifythedetailedmechanism.
Althoughmostparametersevaluateddidn'treturntonormalvaluesinbothASEadministrationgroupscomparedwiththecontrolgroup,buttheseparametersweresignificantlyimprovedinbothASEadministrationgroupscomparedwithhyperlipidemicgroup,whichsuggestedthatoraladministrationofASEwasabletoameliorateplasmaandlivercholesterol/lipidparameters.
ThesechangeswerecorrelatedwithalterationsinthemRNAexpressionandactivityofkeygenesimplicatedincholesterolmetabolism.
TherewasnosignificantdifferencebetweenASEtreatmentandASEpreventiongroupformostparametersevaluated,whichindicatedthatASEpreventiongrouphadnoextraeffectsincomparisonwithASEtreatmentgroup,sofurtherexperimentsshouldbeconductedtoinvestigatethepreventiveeffectsofASEonhyperlipidemia.
ConclusionsOurpresentstudyindicatedthatASEhadcholesterol-loweringeffects.
Thepossiblemechanismcouldbeattributedto:(1)thedown-regulationofHmgcrandAcat2,aswellasup-regulationofCyp7a1andLdlrinliverofhyperlipidemicrats,whichinvolvedincholesterolbiosynthesis,uptake,andeffluxpathway;(2)theincreaseinexcretionofcholesterol.
ThefindingsinourstudysuggestedASEhadgreatpotentialusefulnessasanaturalagentfortreatinghyperlipidemia.
AuthorContributionsConceivedanddesignedtheexperiments:CZWYHSXKW.
Performedtheexperiments:RGXKWDDY.
Analyzedthedata:YHSRGXKWDDY.
Contributedreagents/materials/analysistools:SHZJWXBY.
Wrotethepaper:YHSCZW.
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