Effect of CMP on the SOD activity in the hearts, livers and kidneys of the immunosuppressed mice.. 5.Effect of CMP on the CAT activity in the hearts, livers and kidneys of the immunosupp
Trang 1j our na l h o me p ag e : w w w e l s e v i e r c o m / l o c a t e / c a r b p o l
Cordyceps militaris polysaccharides can enhance the immunity and antioxidation activity in immunosuppressed mice
Mi Wanga,b, Xin Yu Mengb, Rui Le Yangb, Tao Qina, Xiao Yang Wangb, Ke Yu Zhangb, Chen Zhong Feib, Ying Lib, Yuan liang Hua,∗, Fei Qun Xueb,∗∗
a Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China
b Department of Pharmacy, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, PR China
a r t i c l e i n f o
Article history:
Received 18 January 2012
Received in revised form 28 February 2012
Accepted 8 March 2012
Available online 19 March 2012
Keywords:
Cordyceps militaris polysaccharides
Cyclophosphamide-induced
immunosuppression
Immunomodulation
Anti-oxidation activity in vivo
a b s t r a c t
ToevaluatetheimmuneactivationandreactiveoxygenspeciesscavengingactivityofCordyceps mili-tarispolysaccharides(CMP)invivo,90maleBALB/cmicewererandomlydividedintosixgroups.The miceinthethreeexperimentalgroupsweregivencyclophosphamideat80mg/kg/dviaintraperitoneal injectionand17.5,35,or70mg/kgbodyweightCMPviagavage.Thelymphocyteproliferation, phago-cyticindex,andbiochemicalparametersweremeasured.Theresultsshowthattheadministrationof CMPwasabletoovercometheCY-inducedimmunosuppression,significantlyincreasedthespleenand thymusindices,andenhancedthespleenlymphocyteactivityandmacrophagefunction.CMPcanalso improvetheantioxidationactivityinimmunosuppressedmice,significantlyincreasethesuperoxidase dismutase,catalase,andglutathioneperoxidaselevelsandthetotalantioxidantcapacity,anddecrease themalondialdehydelevelsinvivo
© 2012 Elsevier Ltd All rights reserved
1 Introduction
In recent years, many natural polysaccharides and
polysaccharide-protein complexes were isolated from fungi
and usedas a source oftherapeutic agents (Novak &Vetvicka,
2008) Among them, Cordyceps militaris, an entomopathogenic
fungusbelongingtotheclassAscomycetes,hasbeenextensively
usedasacrudedrugandafolktonicfoodinEastAsia.C.militaris
isknownastheChineserarecaterpillarfungusand hassimilar
pharmacologicalactivitiestothewell-knownChinesetraditional
medicineCordycepssinensis(Gai,Jin,Wang,Li,&Li,2004;Zheng&
Cai,2004).ThebeneficialeffectsofCordycepsonrenalandhepatic
functionsand immunomodulation-related antitumouractivities
aremostpromisinganddeservefurtherattention(Paterson,2008)
VariousbioactiveconstituentsfromtheCordycepsspecieshave
beenreported,suchascordycepin,polysaccharides,antibacterial
andantitumouradenosinederivatives,ophicordin,anantifungal
agent,and L-tryptophan.Polysaccharides areconsideredone of
themajoractivecomponentsofCordyceps.Purified
polysaccha-ridesfromC.militarishavenumerousbiologicalactivities,suchas
antioxidant(Li,Li,Dong,&Tsim,2001;Lietal.,2003),
immunomod-ulatory(Cheungetal.,2009;Kimetal.,2008),antitumour(Park,
∗ Corresponding author Tel.: +86 25 84395203; fax: +86 25 84398669.
∗∗ Corresponding author Tel.: +86 21 34293460; fax: +86 21 34293396.
E-mail addresses: ylhu@njau.edu.cn (Y.l Hu), feiqun@gmail.com (F.Q Xue).
Kim,Lee,Yoo,&Cho,2009;Rao,Fang,Wu,&Tzeng,2010),and anti-inflammatory(Raoetal.,2010)
Previous studiesontheimmunomodulatory andantioxidant effects ofC.militarispolysaccharides(CMPs)ininvitrosystems havebeenconducted.CMPscaninducethefunctionalactivationof macrophagesthroughtheupregulationofcytokineexpressionand nitricoxide(NO)release(Leeetal.,2010),induceT-lymphocyte proliferationandsecretionofinterleukin(IL)-2,IL-6,andIL-8(Chen, Zhang,Shen, &Wang,2010), andstimulate thephagocytosisof macrophagesinvitro.Theseresultsconfirmtheimportantroleof CMPsintriggeringimmuneresponses.TheCMPsfractions
P70-1and CBP-1werefoundtoexhibit hydroxylradical-scavenging activityinvitro(Yuetal.,2007,2009)
In the present study, the fruiting body of C militaris came fromShanghai,whichhasbeenscarcelyinvestigated.Successive testswereconductedtoevaluatetheimmuneactivationand reac-tiveoxygenspecies(ROS)-scavengingactivityofCMPinvivo.The detailsarereportedinthecurrentstudy
2 Materials and methods
2.1 Material DryculturedC.militariswasobtainedfromShanghaiDianzhi Bioengineering Corp (Shanghai, China) The material (No 06-01-0727) was identified by Associate Researcher X.H Gao
of the Research Group of Dong Chong Xia Cao, Shanghai
0144-8617/$ – see front matter © 2012 Elsevier Ltd All rights reserved.
Trang 2Cyclophosphamide (CY) was purchased from Jiangsu Hengrui
Medicine Co., Ltd (Lianyungang, Jiangsu, China) Bovine serum
albumin,CoomassieBrilliantBlueG-250,andcellulosesackswere
purchasedfromSigmaChemicalCo.(St.Louis,MO,USA).Thefilter
membranewaspurchasedfromMilliporeCorp.(Billerica,MA,USA)
Allchemicalsusedintheexperimentswereofanalyticalgrade
2.2 Polysaccharideextraction
PolysaccharidesfromC.militariswerepreparedaspreviously
described(Li,Yang,&Tsim,2006;Yuetal.,2007).Thedried
pow-derofculturedC.militariswasdefattedwithethanolfor10hand
subsequentlyextracted three timeswithhot water (100◦C) for
2h.Theresultingsuspensionwascentrifuged(8000× gfor20min)
andfilteredthrougha0.45mmembrane(Millipore).Thefiltered
aqueoussolution wasconcentrated to a specific volume under
reducedpressure.The darkbrownprecipitate wascollectedvia
centrifugationandwashedtwicewithethanol.Theprecipitatewas
thensuspendedinwaterandlyophilizedtoyieldCMPwith41.2%
(w/w)polysaccharidecontent,whichwasmeasuredusing
vitriol-anthronewithanhydrousglucoseasthestandardcontrol
2.3 Animalandexperimentaldesign
MaleBALB/cmice(8weeksold,18hto20g)werepurchased
from Shanghai Slac Laboratory Animal Center of the Chinese
Academyof Sciences (Shanghai, China) Theanimals were
pro-videdwithwater andmousechowadlibitumandwerehoused
inarodentfacilityat(22±1)◦Cwitha12hlight-darkcyclefor
acclimatization.Allproceduresinvolvinganimalsandtheircare
wereapprovedbytheEthicsCommitteeoftheChineseAcademy
ofAgriculturalSciences.Themicewererandomlydividedinto6
groupsconsistingof15miceeach.Threemicefromeachgroupwere
usedforphagocyticindexdeterminationinthecarbonclearance
test,3wereusedforlymphocyteproliferation,and9wereusedfor
theotherexperiments.Allanimalswereallowedoneweektoadapt
totheirenvironmentbeforethetreatment.Twogroupsofhealthy
micewereusedasnormalcontrol(NSgroup)andpositivecontrol
groupsandtreatedoncedailywithphysiologicalsalinesolutionand
70mg/kgbodyweightCMP,respectively,for18days.Fromdays1
to3,theotherfourgroupsofmiceweregiven80mg/kg/dCYvia
intraperitonealinjection.Fromdays4to18,themicewere
admin-isteredasfollows:modelgroup,physiologicalsalinesolution;three
CMPgroups,17.5,35,or70mg/kgbodyweightCMP.CY(0.2ml)was
administeredviaintraperitonealinjection.Theotherswere
admin-isteredviagavagein0.2mlsolutions.Twenty-fourhoursafterthe
lastdrugadministration,theanimalswereweighedandthen
sacri-ficedviadecapitation.Theheart,liver,kidney,spleen,andthymus
wereexcised;thespleenandthymuswereimmediatelyweighed
Thethymusandspleenindiceswerecalculatedaccordingtothe
formula,index(mg/g)=(weightofthymusorspleen)/bodyweight
2.4 Lymphocyteproliferationassay
Themousespleenswereasepticallyremovedfromthe
sacri-ficedmice usingscissors and forceps in 0.1Mcold PBS, gently
incubatedfor4.5hwith10lMTT(5mg/ml)perwell.Theplate wascentrifugedat200×gfor15min,andthesupernatewas dis-carded.DMSO(100l)wasaddedtoeachwell,whichwasthen shakenuntilallcrystalsdissolved.Theabsorbanceat570nmwas measuredonamicroplatereader(ThermoMultiskanMK3,USA) 2.5 Phagocyticindex
Thefunctionofthemacrophagecellswasassessedviaacarbon clearancetestperformedonthreemicefromeachgroupaccording
totheprocedureofWanget al.(2011).Eachmouse was intra-venouslyinjectedwithdilutedIndiainkat100l/10gbodyweight Bloodspecimenswerecollectedat2min(t1)and10min(t2)from theretinalvenousplexuses,and20lbloodwasthenmixedwith
2ml0.1%Na2CO3.Theabsorbanceat600nmwasmeasuredona UV-visiblespectrophotometerwith0.1%Na2CO3astheblank.The liverandthespleenwereweighed,andthephagocyticindexwas calculatedasfollows:
K=lgOD1−lgOD2
t2−t1
whereOD1wasfort1andOD2wasfort2 Phagocytic index ˛=√3
K×A/(B+C), where A is the body weight,Bistheliverweight,andCisthespleenweight
2.6 Biochemicalassay Theorganhomogenates(includingtheliver,heart,andkidney) werepreparedina0.1g/mlwetweightofice-coldisotonic physio-logicalsaline.Thesampleswerecentrifugedat2000×gat4◦Cfor
10min,andthesupernateswereusedforthemeasurementofthe protein,T-AOC,MAD,CAT,SOD,andGSH-Pxlevels.TheSODactivity andtheMDAandTAOClevelsweremeasuredvia spectrophotomet-ricmethods.TheMDAlevelwasdetectedusing2-thiobarbituric acid(Uchiyama&Mihara,1978).TheSODactivitywasanalysedvia autooxidationofpyrogallol(Marland&Marklund,1974).TheTAOC levelwasmeasuredusingtheferricreducing/antioxidantpower assay(Benzie&Strain,1996).Theenzymeactivitywasexpressed
innanomolespermilligramofprotein
2.7 Statisticalanalysis Alldataarepresentedasthemean±SD,analysedusingSPSS forWindowsversion15.0 (SPSSInc.,Chicago,IL,USA).The sta-tisticalanalysiswasevaluatedviaone-wayANOVAfollowedby Scheffe’stesttodetecttheintergroupdifferences.AP<0.05values wasconsideredstatisticallysignificant
3 Results
3.1 EffectofCMPonmousespleenandthymusindices Thespleenandthymusindicescanreflecttheimmunefunction andprognosisofanorganism.AsshowninFig.1,thespleenand thy-musindicesofthemodelgroupremarkablydecreasedcompared withthoseofthenormalgroup(P<0.05).CMPincreasedthespleen
Trang 3Fig 1. Effects of CMP on the internal organ indices of the CY-induced mice *P < 0.05, **P < 0.01 compared with the NS group; # P < 0.05, ## P < 0.01 compared with the model group Values are means ± SD.
andthymusindicesintheCY-treatedmiceinadose-dependent
mannerat17.5,35,and70mg/kg,indicatingthatCMPcanreverse
theCY-inducedatrophyofimmuneorgans
3.2 EffectofCMPoncellularimmunityinmice
Spleenlymphocyteproliferationwasexaminedtounderstand
themechanismoftheimmunoregulatoryactivityofCMP.Asshown
inFig.2,thespleenlymphocyteproliferationofthemodelgroup
remarkablydecreased comparedwiththatof thenormalgroup
(P<0.05).CMPsignificantlyincreasedthespleenlymphocyte
pro-liferationinCY-treatedmiceinadose-dependentmannerat17.5,
35,and70mg/kgcomparedwiththemodelgroup,suggestingthat
CMPisdirectlymitogenicformousesplenocytes
3.3 EffectofCMPonthephagocyticactivityofthemacrophage
system
Carbonclearancetestswereperformedtodeterminetheeffect
ofCMPonmacrophageactivation.Thephagocyticindex˛ofthe
modelgroupwaslowercomparedwiththatoftheNSgroup(Fig.3)
CMPeffectivelyincreasedthe˛valueoftheCY-treatedmiceina
dose-dependentmanner.AtthehighCMPdose(70mg/kg/d),the
phagocyticactivitywasrestoredtoabovethenormallevel(from
4.51to4.74),demonstratingthatCMPcanenhancethemacrophage
functioninCY-treatedmice
3.4 AntioxidantactivityofCMPinvivo
3.4.1 EffectofCMPontheactivityofSODinthedifferentorgans
oftheimmunosuppressedmice
Fig 4 shows that CY significantly reduced the SOD activity
(P<0.01)inthehearts,liversandkidneyscomparedtotheNS
con-trolgroup.AllCMPdosessignificantlyincreasedtheSODactivity
relativetothemodelgroup(P<0.01)
3.4.2 EffectofCMPontheactivityofCATinthedifferentorgans
oftheimmunosuppressedmice
Fig.5showsthemarkedreductionsCATactivity(P<0.01)inthe
hearts,liversandkidneysofmiceintheCY-treatedandNScontrol
groups.CMP(17.5,35,and70mg/kg)significantlyincreasedCAT
activitycomparedtothemodelgroup(P<0.01)
3.4.3 EffectofCMPontheactivityofGSH-Pxinthedifferent
organsoftheimmunosuppressedmice
Fig 6 shows the significant reductions in GSH-Px activity
(P<0.01)inthehearts,liversandkidneysoftheCY-treatedandNS
controlgroups.AllCMPdosessignificantlyincreasedtheGSH-Px
activitycomparedtothemodelgroup(P<0.01)
3.4.4 EffectofCMPontheactivityofTAOCinthedifferentorgans
oftheimmunosuppressedmice Fig 7 shows the remarkable reductions in TAOC activity (P<0.01)inthehearts,liversandkidneysoftheCY-treatedandNS controlgroups.CMP(17.5,35,and70mg/kg)significantlyincreased theTAOCactivitycomparedtothemodelgroup(P<0.01) 3.4.5 EffectofCMPontheactivityofMDAinthedifferentorgans
oftheimmunosuppressedmice Fig.8showsthesignificantincreasesinMDAlevels(P<0.01)
in thehearts,livers and kidneysof theCY-treatedand NS con-trolgroups.AllCMPdosessignificantlydecreasedtheMDAlevels comparedtothemodelgroup(P<0.01)
4 Discussions
CYisacytotoxicchemotherapeuticdrugthatactsasan impor-tantagentintumourtreatment.However,itsadministrationleads
toimmunosuppression,whichmaybelife-threatening(Hong,Yan,
& Baran, 2004) Traditional Chinese medications for immuno-suppression treatment are available In the present study, the protective effects of CMP in reversing the immunosuppression causedbyCYtreatmentwereinvestigated.Theresultsindicatethat CMPcanreversetheCY-inducedatrophyofimmuneorgans
InlinewiththeusageofCordycepsinChina,Chinesemedicines arestronglyrecommendedfortheageingpopulationtoenhance theirimmune system and preventpossible infection Immuno-stimulationitselfisregardedasoneoftheimportantstrategiesto improvethebodysdefensemechanisminelderlypeopleaswellas
incancerpatients.Asignificantamountofexperimentalevidence suggeststhatpolysaccharidesfrommushroomsenhancethehost immunesystembystimulatingnaturalkillercells,T-cells,B-cells, andmacrophage-dependentimmunesystemresponses(Dalmo& Boqwald,2008;Dennert&Tucker,1973).Polysaccharidesobtained fromdifferentnaturalsourcesrepresentastructurallydiverseclass
ofmacromolecules,whichexerttheirantitumouractionmostlyby activatingvariousimmunesystemresponses(Schepetkin&Quinn,
2006).Inpreviousstudies,Cordycepspolysaccharideswerefound
toinducethefunctional activationofmacrophages throughthe upregulationofcytokineexpression(tumournecrosisfactoralpha andIL-1)andnitricoxide(NO)release(Leeetal.,2010),aswell
as theproduction of IL-6 and IL-10 in a dose-dependent man-ner.TheypromotethemRNAandproteinexpressionsofinducible nitricoxidesynthase,induceT-lymphocyteproliferationandthe secretionofIL-2,IL-6,andIL-8,andincreasethephagocyticand enzymaticactivitiesoftheacidphosphataseofmacrophages.In thecurrentstudy,theadministrationofCMPsignificantlyenhanced thespleenlymphocyteproliferationandincreasedthephagocytic index˛inadose-dependentmanner,therebyimplyingthatCMP canalsoenhancethespleenlymphocyteactivityandmacrophage functioninCY-treatedmice
Trang 4Fig 2.Effect of CMP on the spleen lymphocyte proliferation in CY-treated mice *P < 0.05, **P < 0.01 compared with the NS group; # P < 0.05, ## P < 0.01 compared with the model group Values are means ± SD.
Fig 3. Effect of CMP on the phagocytic index in the CY-treated mice *P < 0.05,**P < 0.01 compared with the NS group; # P < 0.05, ## P < 0.01 compared with the model group Values are means ± SD.
Fig 4. Effect of CMP on the SOD activity in the hearts, livers and kidneys of the immunosuppressed mice *P < 0.05, **P < 0.01 compared with the NS group; # P < 0.05, ## P < 0.01 compared with the model group Values are means ± SD.
Fig 5.Effect of CMP on the CAT activity in the hearts, livers and kidneys of the immunosuppressed mice *P < 0.05, **P < 0.01 compared with the NS group; # P < 0.05, ## P < 0.01
±
Trang 5Fig 6.Effect of CMP on the GSH-Px activity in the hearts, livers and kidneys of the immunosuppressed mice *P < 0.05, **P < 0.01 compared with the NS group; # P < 0.05,
## P < 0.01 compared with the model group Values are means ± SD.
Fig 7. Effect of CMP on the TAOC activity in the hearts, livers and kidneys of the immunosuppressed mice *P < 0.05, **P < 0.01 compared with the NS group; # P < 0.05,
## P < 0.01 compared with the model group Values are means ± SD.
Free-radical-induced lipid peroxidation has been associated
withanumberofdiseases.Theexcessiveproductionoffreeradicals
suchassuperoxide,hydroxylradicals,hydrogenperoxide,andNO
(collectivelyreferredtoasROS)playsmultipleimportantrolesin
tissuedamageandlossoffunctioninanumberoftissuesandorgans
(Simic,Bergtold,&Karam,1989;Zheng&Huang,2001).An
increas-ingamountofevidenceindicatesthatmanykindsof
polysaccha-rideshavepotentialandpotentcapabilitiesofpreventingoxidative
damageinlivingorganismsfromfreeradicalscavenging(Liu,Ooi,
&Chang,1997;Peterszegi,Robert,&Robert,2003;Zhangetal.,
2003).Cordycepspolysaccharidescanscavengefreeradicals,and
theantioxidantactivityofC.militariswasevenstrongerthanthat
ofthe C.sinensisand Cordycepskyushuensis(Chen,Luo,Li, Sun,
& Zhang, 2004) The polysaccharide fractions P70-1and CBP-1
fromC.militarisshowedhydroxylradical-scavengingactivitiesin
aconcentration-dependentmanner,withIC50valuesof0.548and
0.638mg/mlinvitro(Yuetal.,2007,2009).Theresultofthepresent studyisconsistentwiththatofP70-1andCBP-1invitro.When themiceweretreatedwithCY,theT-AOC,CAT,SOD,and
GSH-Px levels in the heart, liver,and kidney remarkably decreased andtheMDAlevelsclearlyincreased.However,the administra-tionofCMP(17.5,35,and70mg/kg)cancausesignificantincreases
intheSOD,CAT,GSH-Px,andTAOClevelsaswellasadecrease
in the MDA levels, thereby indicating that CMP can be effec-tiveinscavengingvarioustypesofoxygenfreeradicalsandtheir products
Inconclusion,thecurrentstudydemonstratesthatCMPalone improvedtheimmunefunctionsandexhibitedeffective antioxi-dantactivitiesintheCY-treatedmice.CMPshouldbeexploredasa goodimmunomodulatoryagentwithantioxidantactivityandmay
beappliedtoantineoplasticimmunotherapyincombinationwith chemotherapeuticagents.However,furtherinvestigationonthe
Fig 8.Effect of CMP on the MDA level in the hearts, livers and kidneys of the immunosuppressed mice *P < 0.05, **P < 0.01 compared with the NS group; # P < 0.05, ## P < 0.01 compared with the model group Values are means ± SD.
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