Effects of Tocotrienol and Lovastatin Combination on Osteoblast and Osteoclast Activity in Estrogen-Deficient Osteoporosis

Effects of Tocotrienol and Lovastatin Combination on Osteoblast and Osteoclast Activity in Estrogen-Deficient Osteoporosis HindawiPublishingCorporation Evidence-BasedComplementaryandAlternativeMedicine Volume2012,ArticleID960742,9pages doi:10.1155/2012/960742 ResearchArticle EffectsofTocotrienolandLovastatin CombinationonOsteoblastandOsteoclastActivityin Estrogen-De,cientOsteoporosis SaifAbdul-Majeed,NorazlinaMohamed,andIma-NirwanaSoelaiman DepartmentofPharmacology,FacultyofMedicine,UniversitiKebangsaanMalaysia,JalanRajaMudaAbdulAziz, 50300KualaLumpur,Malaysia CorrespondenceshouldbeaddressedtoIma-NirwanaSoelaiman,imasoel@medic.ukm.my Received31May2012;Revised12July2012;Accepted13July2012 AcademicEditor:AhmadNazrunShuid Copyright?2012SaifAbdul-Majeedetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly cited. StatinsareHMGCoAreductaseinhibitorsandhadbeendemonstratedtostimulateboneformationinrodentsafterhighoral doses.Observationalstudiesonpatientstreatedwithoralstatinswerevaried.Delta-tocotrienolhadbeenfoundtostimulatethe cleavageofHMGCoAreductaseandinhibititsactivity.Tocotrienolswerefoundtohavebothcatabolicandanabolice,ectsonbone indi,erentanimalmodelsofosteoporosis.Thecurrentstudyaimedtoascertainthee,ectsofdelta–tocotrienolandlovastatin combinationonbiochemicalandstaticbonehistomorphometricparametersinapostmenopausalratmodelatclinicallytolerable doses.48SpragueDawleyfemaleratswererandomlydividedinto6groups:(1)baselinecontrolgroup;(2)sham-operatedcontrol group;(3)ovariectomisedcontrolgroup;(4)ovariectomisedand11mg/kglovastatin;(5)ovariectomisedand60mg/kgdelta- tocotrienol;(6)ovariectomisedand60mg/kgdelta-tocotrienol+11mg/kglovastatin.Thesetreatmentsweregivendailyviaoral gavagefor8weeks.Delta-tocotrienolpluslovastatintreatmentsigni,cantlyincreasedboneformationandreducedboneresorption comparedtotheothergroups.Therefore,thecombinedtreatmentmayhavesynergisticoradditivee,ectsandhavethepotential tobeusedasanantiosteoporoticagentinpatientswhoareatriskofbothosteoporosisandhypercholesterolemia,especiallyin postmenopausalwomen. 1.Introduction and increase bone mineral density [2]. However, the use ofparathyroidhormoneisassociatedwithsomedrawbacks Osteoporosisisknownasasilentage-relateddisorder,and suchasdailyinjection,andthepossibilityoftumorigenesis itisconsideredasamajorpublichealthproblem.Patients [3].Theidenti,cationofawell-toleratedanabolicagentthat withosteoporosishavedecreasedbonedensityandmicroar- canincreaseboneformationandrestorebonestrengthwould chitectural disruption of bone tissue, leading to skeletal representamajortherapeuticbreakthroughinthetreatment fragility and fractures. Postmenopausal osteoporosis is the ofanyformofboneloss. mostcommontypeassociatedwithhighboneturnoverand 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) isduetoestrogende,ciency[1].Currentavailabletherapies reductasecatalyzestheconversionofHMGCoAtomevalonic are e,ective in the prevention of bone loss by stabilizing acid. Statins are competitive and reversible inhibitors of the bone mass through inhibition of osteoclast activity, HMGCoA reductase. They are safely used as cholesterol- but they are not favored to treat established osteoporosis lowering agents and have pleiotropic actions in various wherethereisaneedtoincreasebonevolume.TheUnited systemssuchasthecardiovascularsystem,immunesystem, StatesFoodandDrugAdministrationapprovedparathyroid and nervous system [4]. Lovastatin is a prodrug and is hormone (Teriparatide) in 2002 as the ,rst bone anabolic converted to the active open-ring acid from its lactone by agent that can reduce the risk of osteoporotic fractures esterases. Lovastatin was the ,rst compound identi,ed as 2 Evidence-BasedComplementaryandAlternativeMedicine a promising bone anabolic agent after examining about foundthatthetocopherolisomersdonotpreventboneloss 30,000compounds[5].Statinsactasananabolicagentby inorchidectomisedrats[45,46].Thus,itisimportanttouse promotingboneformationinvitroandalsoinvivoinrodents atocopherolfreeextractinthisstudy. after high oral doses [5–11]. Several observational clinical Ovariectomisedratsareawidelyacceptedmodelofpost- studiesonpatientstreatedwithoralstatinsshowedvarying menopausalosteoporosisduetotheirappropriateness,con- results. Some hadsuggestedthat oral statins minimize the venience, and relevance. Furthermore, the ovariectomised risk of fractures and increase bone mineral density [12– rats exhibit skeletal response similar to postmenopausal 17], while others reported that they had no e,ects on women[47]. bone [18–23]. Several clinical studies that compared bone Biochemicalmarkersofboneresorptionandformation biochemical markers between statin-treated patients and are sensitive markers that re,ect the di,erent processes control populations have had varying outcomes [24–26]. involved in bone metabolism by detecting the activity of However,these,ndingsasawholesuggestedthattheoral osteoclastsandosteoblasts.However,theydonotshowthe statinsdonothavesu,cientanabolice,ectsinvivo when changesinbonemassandstructure[48,49].Osteocalcinis given in cholesterol lowering doses. Therefore, high doses anosteoblast-speci,cnoncollagenousprotein.Itformsabout ofstatinsareneededtoprotecttheboneandinducebone 10% of noncollagenous proteins of the bone matrix and formationinvivo.However,highdosesofstatinshadbeen generally serves as a speci,c marker for osteoblast activity associatedwithmyotoxicityandhepatotoxicity[27–29]. and bone formation [50]. Cross-linked C-terminal (CTX) Tocotrienolsandtocopherolsaremembersofthevitamin telopeptides are proteolytic fragments of type 1 collagen E family. They are further subdivided into alpha, beta, formedduringboneresorption.CTXisknownasaspeci,c gamma, and delta isomers. All the vitamin E isomers marker for osteoclast activity and bone resorption [51]. have antioxidant properties. In addition, tocotrienols have Staticbonehistomorphometricindicesareusedtoexamine anticancer, neuroprotective, antiplatelet, and cholesterol- bone histology and quantitatively evaluate the activity of lowering activities [30]. Studies have shown that vitamin thebonecellsataspeci,ctime.Therefore,astrongtoolto E, speci,cally the tocotrienols was able to maintain bone study bone metabolism and bone morphology is through density and prevent further bone loss in di,erent animal a combination of bone biochemical analysis and static modelsofosteoporosis[31].Recentstudieso,eredevidence histomorphometricindices. for tocotrienols as a bone anabolic agent in normal male, The current study was designed to evaluate the com- ovariectomised female and nicotine-treated male rats [32– bined e,ects of delta-tocotrienol and lovastatin and to 35].Tocotrienols,similartostatins,suppresstheactivityof compare it with delta-tocotrienol and lovastatin given HMGCoAreductase(Figure1),althoughthroughdi,erent individually on bone biomarkers and static bone histo- mechanisms [36, 37]. Statins inhibit the enzyme activity morphometric parameters in the ovariectomised estrogen- throughcompetitiveinhibition,whiletocotrienolsmodulate de,cient female rat. The ,ndings from this study may the intracellular mechanism of controlled degradation of provideanalternativemedicationtotreatpostmenopausal the reductaseprotein [38, 39]. A prior study revealed that osteoporosis. onlygammaanddeltatocotrienolsstimulatethedegradation of HMGCoA reductase, and only the delta isomer was able to block the cleavage of sterol regulatory element- 2.MethodandMaterials bindingproteins(SREBP)[39].Therefore,administrationof statinsanddelta-tocotrienoltogethermayhavesynergisticor 2.1. Animals. Forty eight female Sprague-Dawley rats that additivee,ects.Additionally,withcoadministrationofdelta- were approximately 3 months old and weighed 200–250g, tocotrienol,wemaybeabletoavoidtheoccurrenceofthe werepurchasedfromtheLaboratoryAnimalResearchUnit, adversee,ectsofhighdosesoflovastatininhumans. UniversitiKebangsaanMalaysia.Theratswerekepttwoper The annatto bean is one of the major sources of cage under 12 hour light-dark cycles. The rats were fed tocotrienols, containing 90% delta and 10% gamma commercial rat chow (Gold Coin, Selangor, Malaysia) and tocotrienols.TheannattotreeisatropicalSouthAmerican tap water ad libitum. After one week of acclimatization, tree (Bixa Orellana), having spinose capsules with seeds the rats were randomly divided into 6 groups with 8 and cordate leaves that yield annatto beans. A previous rats in each group. The ,rst group, served as a baseline study reported that coadministration of a pure extract of control (BC), was not ovariectomised and was sacri,ced annatto tocotrienols lowered the e,ective dose of lovas- upon receipt. The second group was not ovariectomised tatin and o,ered a novel approach to cancer prevention butwassham-operated(SHAM)forsimulationofsurgical and therapy [40]. Small daily doses of delta and gamma stress. The third group was the ovariectomised control tocotrienols isolated from annatto bean reduced serum group(OVXC).Thefourthgroupwasovariectomisedand levelsofcholesterol,triglycerides,andLDLby15–20%[41]. treatedwith11mg/kgoflovastatin(OVX+LOV).The,fth Annatto-derivedtocotrienolwaschosenforthisstudydue was ovariectomised and treated with 60mg/kg of delta- to the reported e,cacy above, as well as the total absence tocotrienol(OVX+TT).Andthesixthwasovariectomised of any tocopherol isomers in the extract. Tocopherol may and treated with 11mg/kg of lovastatin and 60mg/kg of interfere with tocotrienol absorption and distribution and delta-tocotrienol (OVX+TT+LOV). The treatment had may attenuate the inhibitory e,ect of delta-tocotrienol on been administrated to the rats daily via oral gavage for 8 liver HMGCoA reductase [42–44]. Previous studies have weeks. Evidence-BasedComplementaryandAlternativeMedicine 3 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) Statins and delta-tocotrienol inhibit HMGCoA reductase. Mevalonate Isopentenyl pyrophosphate Geranyl diphosphate Cholesterol Squalene Farnesyl diphosphate Geranylgeranyl diphosphate Proteins prenylation Geranylgeranylated proteins Fransylated protein Figure1:Mechanismofactionoflovastatinanddelta-tocotrienolonmevalonatepathway. Priorapprovalforthestudyprotocolhadbeenobtained For bone histomorphometric analysis, the rats were from the UKM Animal Ethics Committee, (PP/FAR/2011/ sacri,ced by high dose diethyl ether after completing the IMA/27-JANUARY/352-JANUARY-2011–DECEMBER- treatment period. The left femurs were removed and the 2012). distalportionkeptin70%alcohol. 2.2.PreparationofTreatment. TheDeltaGold70viscousoil (American River Nutrition, Hadely, USA) is a rich delta- 2.4.BiochemicalAnalysis. Levelsofbonebiochemicalmark- tocotrienol extract from the annatto bean consisting of ers,osteocalcinandCTXinserumweremeasuredusingan 90% delta-tocotrienol and 10% gamma-tocotrienol. The ELISA microplate reader (VERSA max, Sunnyvale, USA). orange-red oil was diluted in olive oil (Bertolli Classico, ThekitsusedwereRat-MidOsteocalcinELISAkit(IDS,UK) Italy) and administrated daily via oral gavage at a dose andRatLapsCTX-1ELISAkit(IDS,UK). of 60mg/kg delta-tocotrienol for 8 weeks. This dose was roughlyequivalentto420mg/dayforanadulthuman. Mevacor tablet, containing 40mg of lovastatin, was 2.5.BoneHistomorphometry. Theleftfemurwasdecalci,ed crushed and suspended in 0.5% carboxymethylcellulose withEDTA(SigmaAldrich,St.Louis,USA)for2monthsand (Sigma-Aldrich,St.Louis,USA)solutionandgivendailyto thenembeddedinhistologicalpara,nwax.Thedecalci,ed ratsviaoralgavageatadoseof11mg/kgfor8weeks.This para,n blocks were sectioned at 6μm with a microtome dosewasequivalentto80mg/dayforanadulthuman.Oral (Leica,Wetzlar,Germany)andstainedwithHematoxylinand gavagesofthevehiclesweregiventoSHAMandOVXgroups Eosin. forasimilardurationoftreatment.Thedurationofthestudy The static parameters, namely, osteoblast surface/bone wasbasedonapreviousstudy,inwhich8weekswasshown surface(ObS/BS),osteoclastsurface/bonesurface(OcS/BS), tobeadequateforsigni,cantchangesinboneparametersto eroded surface/bone surface (ES/BS), osteoid surface/bone beobserved[52]. surface(OS/BS),andosteoidvolume/bonevolume(OV/BV) wereanalysedusingaquantitativestereologicalmethodfor histologyknownastheWeibeltechnique. 2.3. Sample Collection. For the biochemical study, blood Thestatichistomorphometricindiceswereperformedat sampleswerecollectedatthestart(pretreatment)andafter the secondary spongiosa area, which is rich in trabecular 8 weeks of treatment (posttreatment) from all the groups exceptBCbecausetheyweresacri,ceduponreceipt.Blood bone. The selected metaphyseal region was located 1mm sampleswereobtainedfromtheretroorbitalvesselafterthe fromthelateralcortexand3–7mmfromthelowestpoint ratwasanesthetizedwithdiethylether.After3hours,blood ofthegrowthplate. wascentrifugedfor10minat3000rpm,andtheserumstored Bone cellular average changes were analyzed and ex- pressed using bone histomorphometric measurements as at?70 Cforfurtheruse. ? 4 Evidence-BasedComplementaryandAlternativeMedicine 450 ? 70 400 ? acd df 60 350 bef ce ab 300 50 250 ? ? 40 ce 200 bef df acd 30 ab 150 20 100 50 10 0 0 Pre-treatment Pre-treatment Post-treatment Post-treatment Figure 2: Serum osteocalcin levels in treatment groups. Data Figure3:SerumCTXlevelsintreatmentgroups.Datalabeledwith labeledwiththesameletterindicatessigni,cantdi,erencebetween ,erence between treatment the same letter indicates signi,cant di? groups. ?Indicatessigni,cantdi,erencebetweenpretreatmentand treatment groups. Indicates signi,cant di,erence between pre- posttreatment values for the same group. Data was presented as treatmentandposttreatmentvaluesforthesamegroup.Datawas mean? SEM.Signi,cantlevelwastakenatP<0.05. presentedasmean? SEM.Signi,cantlevelwastakenatP<0.05. recommended by The American Society of Bone Min- from the pretreatment level for the remaining groups No eralResearchHistomorphometryNomenclatureCommittee signi,cant di,erences were observed between the groups [53]. beforetreatment.AftertreatmenttheserumCTXlevelfor the OVXC group was signi,cantly higher than the SHAM 2.6.StatisticalAnalysis. Dataanalysiswasperformedusing group. The OVX+TT and OVX+TT+LOV groups had theStatisticalPackageforSocialSciencessoftware(19,SPSS, signi,cantlylowerserumCTXlevelscomparedtotheOVXC Chicago,IL,USA).TheKolmgorov-Smirnovtestwasused and OVX+LOV groups, but they did not di,er from the asanormalitytest.Thepaired-samplettestwasutilizedto SHAM group. While the OVX+LOV group did not di,er compare the same group before and after treatment. The signi,cantly from the OVXC group but was signi,cantly ANOVA followed by post hoc Tukey’s tests were used to higherthantheSHAMgroup(Figure3). determine the statistical signi,cance between groups. The TheOVXCgrouphadsigni,cantlylowerObS/BS,OS/BS results were expressed as mean values ? standard error of andOV/BVvaluesthantheBCandSHAMgroups(Figures4, themean(SEM).Thestatisticaldi,erenceswereconsidered 5,6,7,and8).Therewerenosigni,cantchangesinallstatic signi,cantatP<0.05. bone parameters between the BC and SHAM groups. The OVX+TT+LOV group had signi,cantly higher ObS/BS and OV/BV values compared to the OVX+TT group; 3.Results signi,cantlyhigherObS/BS,OS/BS,andOV/BVvaluescom- pared to OVX+LOV and OVXC groups; and signi,cantly Serum osteocalcin level was signi,cantly lower post- treatment compared to pretreatment for the OVXC and higher ObS/BS, OS/BS, and OV/BV values than the BC OVX+LOVgroups.Theposttreatmentlevelofserumosteo- andSHAMgroups.TheOVX+TTgrouphadsigni,cantly calcin did not di,er signi,cantly from the pre-treatment higher ObS/BS, OS/BS, and OV/BV values compared to theOVX+LOVandOVXCgroups,andsigni,cantlyhigher level for the remaining groups. No signi,cant di,erences ObS/BS,OS/BS,andOV/BVvaluesthantheBCandSHAM wereseenbetweenthegroupsbeforetreatment.Aftertreat- groups.TheOVX+LOVdidnotdi,erfromtheOVXCinall ment,theserumosteoclacinlevelintheOVXCgroupwas signi,cantly lower than the SHAM group. The OVX+TT staticboneparametersbuthadsigni,cantlylowerObS/BS, andOVX+TT+LOVgroupshadsigni,cantlyhigherserum OS/BS,andOV/BVvaluesthantheBCandSHAMgroups osteocalcinlevelscomparedtotheOVXCandOVX+LOV (Figures4,5,6,7,and8). groups,buttheydidnotdi,erfromtheSHAMgroup.While The OVXC group had signi,cantly higher OcS/BS theOVX+LOVgroupdidnotdi,ersigni,cantlyfromthe and ES/BS values than the BC and SHAM groups. The OVXC group but was signi,cantly lower than the SHAMOVX+TT+LOV group had signi ,cantly lower OcS/BS group(Figure2). valuecomparedtotheOVX+TTgroup;signi,cantlylower OcS/BSandES/BSvaluescomparedtotheOVX+LOVand SerumCTXlevelwassigni,cantlyhigherposttreatment comparedtopretreatmentfortheOVXCgroup.Thepost- OVXC groups; signi,cantly lower OcS/BS value than the treatment level of serum CTX did not di,er signi,cantly BCandSHAMgroups;signi,cantlylowerES/BSvaluethan Evidence-BasedComplementaryandAlternativeMedicine 5 90 50 bdhi dhjmn 45 80 acfg 40 cgikn 70 35 abcd cde 60 efgh 30 fh ab egi 50 25 bfkm aeij 20 40 15 30 10 20 5 10 0 0 Figure6:ErodedSurface/BoneSurface%(ES/BS%)intreatment Figure4:OsteoblastSurface/BoneSurface%(ObS/BS%)intreat- groups. Data labeled with the same letter indicates signi,cant mentgroups.Datalabeledwiththesameletterindicatessigni,cant di,erencebetweentreatmentgroups.Datawaspresentedasmean ,erencebetweentreatmentgroups.Datawaspresentedasmean di? SEM.Signi,cantlevelwastakenatP<0.05. ? SEM.Signi,cantlevelwastakenatP<0.05. 40 dhjm 35 cgik aeij 35 bfkm 30 abcd efgh 30 efgh 25 abcd 25 bfkm aeij 20 cgikn 20 15 15 dhjmn 10 10 5 5 0 0 Figure7:OsteoidSurface/BoneSurface%(OS/BS%)intreatment Figure5:OsteoclastSurface/BoneSurface%(OcS/BS%)intreat- groups. Data labeled with the same letter indicates signi,cant mentgroups.Datalabeledwiththesameletterindicatessigni,cant di,erencebetweentreatmentgroups.Datawaspresentedasmean ,erencebetweentreatmentgroups.Datawaspresentedasmean di? SEM.Signi,cantlevelwastakenatP<0.05. ? SEM.Signi,cantlevelwastakenatP<0.05. the bone matrix resulting in the formation of the eroded the SHAM group. The OVX+TT group had signi,cantly surfacesandthereleaseofCTX[54]. lowerOcS/BSandES/BSvaluescomparedtotheOVX+LOV The results of the current study showed that daily and OVXC groups; and signi,cantly lower OcS/BS value supplementation of delta-tocotrienol in combination with thantheBCandSHAMgroups.TheOVX+LOVgrouphad lovastatin increased the osteoblastic bone formation and signi,cantly higher OcS/BS and ES/BS values than the BC decreased osteoclastic bone resorption in ovariectomised andSHAMgroups(Figures4,5,6,7,and8). ratsasindicatedbytheOVX+TT+LOVgroupwhichhad signi,cantlyhigherserumosteocalcin,ObS/BS,OS/BS,and OV/BVvaluesandsigni,cantlylowerserumCTX,OcS/BS, 4.Discussion and ES/BS values compared to the OVXC group. The role of the mevalonate pathway in the pathophysiology of Bothosteoblastandosteoclastcellsarerequiredforcontinu- ousboneremodeling.Duringboneformation,theosteoblast osteoporosissuggeststhatcriticalregulatorymechanismsare cellsstarttosecreteosteoidandsynthesizeosteocalcin,while neededtomaintainosteoblastandosteoclastfunction.Inhi- duringboneresorption,theactivatedosteoclastcellsdissolve bitionofthemevalonatepathwaybystatinsandtocotrienols 6 Evidence-BasedComplementaryandAlternativeMedicine 18 dhjmn of tocotrienols had antiosteoporotic e,ects in thyroidec- 16 cgikn tomised, orchidectomised, oxidative stressed, adrenalec- abcd efgh tomized, nicotine treated, and ovariectomised rat models 14 [62–69].Thedoseof60mg/kg/dayforratsisroughlyequiv- 12 alentto420mg/dayforhumans,takingintotheaccountthe 10 bfkm metabolicrateofrodentsisaroundtentimesfasterthanthat aeij 8 ofhumans.Thisdoseisrelativelylowhasnotoxice,ects.It 6 hadbeenreportedthatdailysupplementationof200mg/kg 4 palmvitaminEextractcontaining18.43%alpha-tocopherol, 2 14.62%alpha-tocotrienol,32.45%gamma-tocotrienol,and 0 23.93%delta-tocotrienolhasnotoxice,ectsinfemalemice [70]. Thecurrentstudyshowedthatthecombinationofdelta- tocotrienol plus lovastatin increased bone formation and reducedbonelosscomparedtolovastatinaloneasindicated by the OVX+TT+LOV group which had signi,cantly higherserumosteocalcinlevel,ObS/BS,OS/BS,andOV/BV Figure8:OsteoidVolume/BoneVolume%(OV/BV%)intreatment values and signi,cantly lower serum CTX, OcS/BS, and groups. Data labeled with the same letter indicates signi,cant ES/BSvaluescomparedtotheOVX+LOVgroup.Moreover, di,erencebetweentreatmentgroups.Datawaspresentedasmean therewerenosigni,cantchangesinallbiochemicalmarkers ? SEM.Signi,cantlevelwastakenatP<0.05. and static bone histomorphometric indices between the OVXCandOVX+LOVgroups.Therefore,lovastatinalone failed to enhance bone formation and to prevent bone resorption in ovariectomised rats at clinically tolerable hypocholesterolemicdoses.Statinshavelimiteddistribution to the peripheral tissues after oral administration [71]. (Figure1) suppresses the prenylation of GTPase binding proteins and disrupts their function. Therefore, inhibition Therefore, they yield uncertain results as bone anabolic agents when used in vivo at cholesterol lowering doses. of GTPase function reduces the activity of osteoclasts and Bjarnasonetal.[26]reportedthat,uvastatindidnota,ect induces their apoptosis [5, 55–57]. Inhibition of GTPase serumosteocalcinandserumandurinaryCTXlevelsinpost- functionalsoincreasesosteoblastactivitythroughenhance- menoposalwomenwithosteoporosisandmildhypercholes- mentofBMP-2expression[5,6,57–60].Ultimately,thiswill leadtostimulationofboneformationanddecreaseinbone terolemiawhengiveninclinicallyrelevantdoses.Acrossover resorption. clinicalstudyshowedthat40mg/dayofatorvastatinhadno e,ectonserumosteocalcinandCTXintype2diabeticmen CompetitiveinhibitionofHMGCoAreductasebystatins with baseline hypercholesterolemia compared to placebo reduces the cholesterol level. This reduction subsequently [72].Similarresultswereseen,whenarandomizedclinical stimulatesSREBPcleavageandinhibitsHMGCoAreductase trialmeasuredtheserumCTXconcentrationinhypercholes- degradation,resultinginanincreaseinmRNAandHMG- terolemicpatientstreatedwith20–80mg/dayofsimvastatin CoA reductase protein expression [61]. In contrast, delta- tocotrienolinhibitsthecleavageofSREBPandinducesthe [24].Twentymg/dayofpravastatindidnota,ecttheserum degradation of HMGCoA reductase, thereby inducing the CTXlevelinhypercholesterolemicpostmenopausalwomen [25].Metaanalysisofbothobservationalstudiesandclinical reductioninmRNAandproteinHMGCoAreductaselevels trials of around 300,000 patients found that there was [61]. Therefore, the combination of lovastatin and delta- clinicalbene,tfromtheuseoforalstatinsbuttherewasno tocotrienolmayhavesynergisticoradditivee,ectsonbone signi,cantreductioninfractureincidenceinolderwomen metabolism,whileatthesametimeavoidingtheunwanted [73].Yaoandhiscoworkersascertainedthatthe0.3,0.6,3,6, e,ectsofhighdosesandlowbioavailabilityoflovastatin. and10mg/kgofsimvastatinfor60dayscouldnotprevent The current study found that delta-tocotrienol com- bined with lovastatin provided better bone formation and or restore ovariectomy-induced osteoporosis [74]. On the other hand, previous studies showed that lovastatin and bone protection against ovariectomy-induced bone loss other statins enhanced bone formation and reduced bone compared to delta-tocotrienol alone as indicated by the resorption after high oral doses in rodents [5, 7–10]. This OVX+TT+LOV group which had signi,cantly higher indicatesthatclinicallynontolerabledosesoforalstatinsare ObS/BS,andOV/BVvaluesandsigni,cantlylowerOcS/BS requiredtoachievesuccessfulpreventionandtreatmentof value compared to the OVX+TT group. The improve- osteoporosis.Myotoxicityandhepatotoxicitywereassociated ment in bone metabolism by the combined treatment may be due to synergistic or additive inhibition of the with the high doses of oral statins [27–29]. In this study, mevalonate pathway. Moreover, the OVX+TT group had 11mg/kgoflovastatinwaschosen,whichifextrapolatedto humanisroughlyequivalentto80mg/day,thehighestdose signi,cantlyhigherserumosteocalcin,ObS/BS,OS/BS,and oflovastatinusedasanantihyperlipidemicagent. OV/BVvaluesandsigni,cantlylowerserumCTX,OcS/BS and ES/BS values compared to the OVXC group. These The results of the current study found that the resultswereconsistentwiththosewhofoundthat60mg/kg OVX+TT+LOV group had signi,cantly higher ObS/BS, Evidence-BasedComplementaryandAlternativeMedicine 7 cortical bone formation,” Bone, vol. 34, no. 4, pp. 609–618, OV/BV and OS/BS values and signi,cantly lower OcS/BS 2004. and ES/BS values than the SHAM group. These current [8] H. Oxlund, M. Dalstra, and T. T. Andreassen, “Statin given ,ndingsindicatethatdelta-tocotrienolincombinationwith perorally to adult rats increases cancellous bone mass and lovastatinpromotedbettercellularbonehistomorphometric compressivestrength,”Calci,edTissueInternational,vol.69, parameters than the SHAM group, thus exhibiting bone no.5,pp.299–304,2001. anabolice,ects.Therefore,thecombinedtreatmenthasthe [9] M.L.Ho,Y.H.Chen,H.J.Liaoetal.,“Simvastatinincreases potential to increase bone strength. Recently, tocotrienols osteoblasts and osteogenic proteins in ovariectomized rats,” wereshowntohaveboneanabolicactivityinovariectomised EuropeanJournalofClinicalInvestigation,vol.39,no.4,pp. female,intactmaleandnicotine-treatedmalerats[32–35], 296–303,2009. andthese,ndingshadbeencon,rmedbytheresultsofthe [10] F.J.Maritz,M.M.Conradie,P.A.Hulley,R.Gopal,andS. currentstudy(Figures4,5,7,and8).Therefore,combination Hough,“E,ectofstatinsonbonemineraldensityandbone of delta-tocotrienol plus lovastatin may have the ability to histomorphometry in rodents,” Arteriosclerosis, Thrombosis, furtherimprovethebonedensityinnormalbone. andVascularBiology,vol.21,no.10,pp.1636–1641,2001. 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