2004-2005 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe

Chang, YZ

Duan, XL

Liu, JG

Liu, YQ

Qian, ZM

PolyU Institutional Repository

????:河北省自然基金项目(303158), 河北省教育厅博士科研基金(B2003124)和河北师范大学博士科研基金(L2003B15)资助。????:段相林 , Email:xlduan0311@163.com;Tel:0311-6269480;0311-6268313??????????????????????刘玉倩1　常彦忠1　钱忠明1 , 2　刘建国3　段相林11 河北师范大学生命科学学院动物细胞生物学研究室(石家庄 050016)2 香港理工大学应用生物及化学科技学系　　3河北师范大学体育学院??　目的:研究游泳训练后大鼠腓肠肌二价金属离子转运体1(divalent metal transporter 1 , DMT1)、转铁蛋白受体(transferrin receptor , TfR)及膜铁转运蛋白 1(ferroportin1 , FPN1)表达的变化 ,探究运动对腓肠肌铁代谢的影响机制。方法:40只雌性 SD大鼠随机分为对照组和游泳训练组 ,分别于游泳训练5周及10周后取材 ,测定血清铁状态 、腓肠肌非血红素铁含量 ,采用Western Blot方法检测腓肠肌铁代谢相关蛋白表达的变化。结果:(1)游泳训练 5周及10周组大鼠血清铁 、Hb及 RBC均较对照组显著升高 ,游泳运动 10周组大鼠腓肠肌非血红素铁含量显著增加(P<0.01);(2)游泳训练组大鼠腓肠肌DMT1(IRE)和TfR表达增加 ,FPN1表达减少(P<0.05),而 DMT1(non-IRE)表达无明显变化。结论:游泳训练可以增加腓肠肌非血红素铁含量 ,促进机体铁动员。这可能与腓肠肌细胞膜上向胞内转运铁的TfR和 DMT1(IRE)蛋白表达增加 ,而向胞外转运铁的FPN1表达下降有关。???　腓肠肌;非血红素铁;膜铁转运蛋白 1;二价金属离子转运体 1;大鼠Effects of Swim Training on the Expression of Gastrocnemius' Iron Transport Proteins in RatsLiu Yuqian1 , Chang Yanzhong1 , Qian Zhongming1 , 2 , et al .1 Department of Animal Cytobiology , College of Life Science , Hebei Normal University ,Shijiazhuang , China 0500162 Laboratory of Iron Metabolism , Department of Applied Biology and Chemical Technology ,The Hong Kong Polytechnic University , Kowloon , HongKong , PRCAbstract　Objective To investigate the effects of swim training on the expression of divalent metal trans-porter 1(DMT1), transferrin receptor(TfR)and ferroportin 1(FRN1)in rats' gastrocnemius.Methods 40 fe-male SD rats were randomly divided into two groups:control group(CG)and exercise group(EG), and weresampled after 5 and 10-week swimming.Determined serum iron status and non-heam iron of gastrocnemius.Observed the expression of TfR , DMT1 and FPN1 by Western Blot.Results(1)The serum iron , Hb and RBCwere significantly higher than those in control group.The gastrocnemius' non-heam iron of exercise groupwere significantly increased after 10-week training(P<0.01).(2)The expression of DMT1(IRE)and TfRof exercise group was increased , but FPN1 expression was decreased , and DMT1(non-IRE)had no change.Conclusion Suitable training could improve body iron homeostasis , increase gastrocnemius' non-heam iron.The mechanism of changes were that the expression of DMT1(IRE)and TfR were increased but that of FPN1was decreased.Key words　gastrocnemius , non-heam iron , ferroportin 1 , divalent metal transporter 1 , rats　　?????????????? ,?????O2?????????????? ATP??? ,???????? 。???????????? ,??????????????????????????????? 2005 ? 3?? 24 ?? 2? Chin J Sports Med ,March 2005 , Vol.24 , No.2 ·147　·DOI :10.16038/j.1000-6710.2005.02.004???? ,????????? 。?????????????????????????????? ,?????? (transferrin receptor , TfR),?????(iron regulatory proteins , IRP),?????(Ironresponsive element , IRE),????????? 1(di-valent metal transporter 1 , DMT1)??????? 1(ferroportin1 , FPN1)? ?? ??? ?? (hep-cidin)[ 1-4]?????????? ,?????????????????????? 。????????????????????????? ,???????????????????????????? ,?? ,????????????????????????? 、????????????????? DMT1 、TfR? FPN1??? ,?????????????????????? ,???????????????????。1　?????1.1　?????? 90 ～ 100g ? Sprague -Dawley ???? 40? ,?????????????? ,??????????????? ,???? 。???? 22℃～24℃,????? 45%～ 55%。???????????(??)??????(????????????????????? 90min[ 5 ,6] ),?? 20? ,????? 5???? 10???? 。?????????130cm×90cm×70cm ,?? 32℃±1℃。1.2　??????? 36h?? ,?????????? 。0.4%?????(1ml/100g)???????? , EP?????? EDTA-K2 ????;??? EP ???? 1.5ml ,845g/min ,4℃?? 20min??? , -70℃???????? 。????? ,?????(Mill-Q ,France)?? 4℃???????? ,?????? 80 ～ 100mg ,?????? ,???????????? ,???????????。?????? ,??????? ,110℃? 4h ,?-70℃????????????。1.3　?????:?? Beckman Coulter AC Tdiff 2??????????。1.4　????????:?? Iron and Iron-bindingCapacity ???(SIGMA ,USA),???????? 。1.5　??????????:?110℃????????? ,???? 12.5?????? HCl 、????????????????? , 65℃?? 20h。12000g/min ,?? 10min ,? 0.025ml???????(1μg/ml),?????1.25ml ,10mim ? 540nm??。?????(μg/g)=[(??????/??????)×????×??????] / ???(g)1.6　Western Blot[ 7]??:????????:?????????(1ml/100mg):1mlTBS(pH8.0)??? 10μl NP -40 , 1μlPepstatin(1μg/ml),1μl Leupeptin(1μg/ml),1μl Apro-tinin(1μg/ml), 5μl PMSF(phenyl methylsulfonyl fluo-ride)(100μg/ml)。?????? ,??(4℃,12000g/min ,20min),????-70℃??。Bradford??????(Eppendorf Biophotometer)。SDS-PAGE(sodi-um dodecly sulphate -polyacrylamide gel electrophore-sis):Tris base ,glycine , acrylamide ,N , N' -methylene-bisacrylamide , 2-mercaptoethanol ? ammonium persul-fate ??????SIGMA。10%??? ,4%??? ,???? 50μg ,???? Marker (Molecular weightstandards , Amersham Pharmacia Biotech), ????(Bio -Rad)100V , 20min ,? 200V , 45min;????NC?(Trans-Blot★R Transter Medium Pure Nitrocellu-lose Membrane , Bio -RAD , 0.45μm)300mA , 45min。5%????????? 2h ,???? 1∶5000???? FPN1 、DMT1(IRE)、DMT1(non -IRE)? TfR(ADI , San Antonio , TX)??? , 4℃??;TBST (20mM Tris-Cl , pH 7.6 , 137 mM NaCl , 0.1%Tween-20)????? 1∶5000???????(HRP)?????(???? IgG(H+L)),?????? 1h;????????(Super Signal★RWest Pico Trial Kit ,PIERCE , USA)??;???????(Chemilumines-cence imaging system , ΨMEGA12ic , USA)???? ,Gel-Pro Analyzer★R(MEDIA CYBERNETICS , USA)????????。TBST????????? ,??? β -actin??(anti-actin ,Sigma-Aldrich , MO)????? 。???????????? β-actin?OD?? 。1.7 　????:??????  x ± s ?? , ?SPSS11.0 ?????????(One -Way ANO-VA),P <0.05???????? , P <0.01??????????。2　??2.1　???????????????(? 1)?????? ,5???????? Hb 、Hct????????????(P <0.05 , P <0.01),???????????(P<0.05)。10???????? Hct? Hb ??????　·148　· ???????? 2005 ? 3 ??24?? 2 ? Chin J Sports Med ,March 2005 , Vol.24 , No.2??(P<0.05),??? 、???????????????(P <0.01),???????????????。????5??????????????????(P >0.05)。????10???????????????????????(P <0.01)。? 1　?????????? 、???????????????????(n=10)( x±s)Tab.1　Effects of swim training on rats' blood iron status and non-heam iron in gastrocnemius5 周对照组control游泳训练组exercise group10 周对照组control游泳训练组exercise group红细胞压积 Hct(%) 30.17±1.56 34.73±2.84＊＊ 32.58±3.23 35.04±3.55△血红蛋白 Hb (g/ dl) 13.33±0.63 14.23±0.37＊ 13.55±0.85 14.46±0.52△血清铁 SI(μg/ dl) 207.41±25.61 225.56±17.54 193.95±8.58 219.14±6.31△△未饱和铁结合力 UIBC(μg/ dl) 256.47±21.36 223.89±30.91＊ 295.99±11.74 260.72±23.03△总铁结合力 TIBC(μg/dl) 463.95±26.35 449.45±46.10 489.89±17.34 479.86±33.43转铁蛋白饱和度 TS(%) 44.61±4.38 50.31±2.24＊＊ 38.86±2.59 45.67±3.67△△腓肠肌非血红素铁gastrocnemius' non-heam iron(μg/g) 103.2±18.07 125.95±38.09 66.92±15.39 125.16±20.91△△注:＊ P <0.05 , ＊＊ P<0.01 ,与 5 周对照组比较 , compared with control group after 5-week swim training.△P<0.05 , △△P<0.01 ,与 10 周对照组比较 , compared with control group after 10-week swim training.2.2　??????????????????? ???　　? 1-4 ?????????????????Western Blot??。??????????????? ,Gel-Pro Analyzer★R???????? 。CG???? , EG?????? 。???????????TfR ～ 90kDa ,DMT1 ～ 56kDa ,FPN1 ～ 60kDa , β -actin ～ 42kDa 。???? β -actin ???????( x ±s , n=10)(? 5)。Western Blot????:???? 10????????????? 2005 ? 3?? 24 ?? 2? Chin J Sports Med ,March 2005 , Vol.24 , No.2 ·149　·TfR? DMT1(IRE)?????????(TfR:CG ?0.743±0.071 ,EG? 0.854±0.033;DMT1(IRE):CG? 1.069±0.102 , EG ? 1.258±0.048 , P <0.05);DMT1(non-IRE)?????????(CG? 1.157±0.111 ,EG? 1.276±0.106),????? 10??FPN1??????????(CG? 1.174±0.117 ,EG? 0.953±0.133 , P<0.05)(?5)。? 5　10 ???????????????????????Fig.5　Effects of 10- week swim training on the expres-sion of gastrocnemius' iron transport proteins inrats＊P <0.05 ,与对照组相比 , compared with controls3　??3.1　????????????????????? 5?? 10??Hct? Hb??????。Hct?Hb???????????????????O2 ????? ,????????????? ,??????? ATP ?????? 。??????????????????? ,?????????????? ,??????????? Tf?????? ,?????????????????? 。??????????? ,??????????????。???? 5??????????????? ,????? 10???????????? ,?????????????????????? ,???Mb ??????????? ,????????????? 。3.2　???????????????????????? 5????????????????????????? ,?????10????????????????????? , ?? Kap-tanoglu?Navas ???????[ 5 , 6] 。?????????????????? ,????????????? ,????????????????? ,????????????????? 。???????????? Mb??????????? ,?????ATP ,??????????。3.3　????????????????????TfR??????????? ,????????????????????? ,?????????? 95kDa ????????????? ,????????????????????????[ 8 ,9] 。DMT1??????????????????????。DMT1????? 12???????? ,??????????。?? DMT1 mRNA?? ,DMT1?????? ,3' ????????????(iron regular element , IRE)??? DMT1(IRE),? IRE??? DMT1(non-IRE),???????? 、????????? 。???? DMT1?????????????????? ,???????????? DMT1???????????????? , ?????[ 10 , 11] 。FPN1 ? 2000 ?Donvoan?[ 1]????????????????????????????????????? ,???? ferroportin 1。FPN1??????????????? ,?????? (hephaestin)?/????????? ,?????????????。???? , ???? 10 ??????? TfR ?DMT1(IRE)???? ,???????????????? 。? FPN1???????? ,?????????????????。???????????Mb? ATP ?????? ,?????????。3.4　???????????????????????????????? mRNA ?????(untranslated region , UTR)??? IRE ,?????????????????(iron regulation protein ,IRP)? IRE ????????????????? 。DMT1(IRE)? TfR? IRE??? mRNA 3′????? ,?????????? , IRP ? mRNA 3′?? IRE?? ,mRNA ???? ,DMT1(IRE)? TfR?????? 。??????? , IRP ? IRE?????? ,mRNA ????? ,?????? ,?????????[ 13] 。? FPN1? IRE?? mRNA 5′????? ,???????? , IRE/ IRP ?????mRNA??? ,?????? ,?????????　·150　· ???????? 2005 ? 3 ??24?? 2 ? Chin J Sports Med ,March 2005 , Vol.24 , No.2????? 。?? , IRE?? mRNA? 5' ?? ,??? IRE/ IRP??? ,???? mRNA ?????????????????;?? IRE?? mRNA ?3' ?? ,? IRE?????? ,????mRNA???? ,???????? ,????????[ 12] 。???????? 10????????? DMT1(IRE)??????????? ,? DMT1(non -IRE)?????。???????????????????????? IRE? IRP ???????。???? 10????? TfR? DMT1(IRE)?????????? , FPN1 ????????(P<0.05),?????????????? ,??????????? ,???????????????????。IRE? IRP ??????? ,?????????????:(1)??? NO ?????? IRE ?IRP????[ 14-17] 。(2)Hepcidin ???????[ 18] :?????? Hepcidin??????????????? ,???????? ,???? IRE?IRP????? ,?? DMT1 、????? B 、????????(hephaesin , HP)? FPN1???? ,??????????? 。???? Hepcidin??????? IRE?? ,?? Hepcidin mRNA?????????????? 。?????Hepcidin???????NO ?Hepcidin??????????????? 。4　??(1)?????????? Hb?? ,????????? ,???????? ,???????? ,??????????????? 。(2)??????? TfR?DMT1(IRE)????? ,FPN1???? ,????????? 10?????????????????。5　????[ 1] Donovan A , Brownline A , Zhuo Y , et al.Positional cloning ofZebrafish ferroportin 1 identifies a conserved vertebrate iron ex-porter.Nature , 2000 , 403(6771):776～ 781.[ 2] ??? ,??? , ??? ,?.???????????.???? , 2004 , 24(1):27 ～ 30 , 34.[ 3] Portal S , Epstein M , Dubnov G.Iron deficiency and anemia infemale athletes-causes and risks.Harefuah , 2003 , 142(10):698～ 703 , 717.[ 4] Schumacher RO , Schmid A , Grathwohl D, et al.Hematologi-cal indices and iron status in athletes of various sports and per-formances.Med Sci Sports Exerc , 2002 , 34(5):869～ 875.[ 5] Kaptanoglu B , Turgut G , Genc O , et al.Effects of acute exer-cise on the levels of iron , magnesium , and uric acid in liverand spleen tissues.Biol Trace Elem Res , 2003 , 91(2):173 ～178.[ 6] Navas FJ , Cordova A.Iron distribution in different tissues inrats following exercise.Biol Trace Elem Res , 2000 , 73(3):259～ 268.[ 7] J.????? , D.W.???.????????.??:????? , 2003.1044～ 1428.[ 8] Zhongming Qian , Desheng Xiao , Paklai Tang , et al.Increasedexpression of transferrin receptor on membrane of erythroblastsin strenuously exercised rats.Appl Physiol , 1999 , 87(2):523～ 529.[ 9] Schumacher YO , Schmid A , Konig D , et al.Effects of exer-cise on soluble transferring receptor and other variables of ironstatus.Br J Sports Med , 2002 , 36(2):195～ 200.[ 10] Zoller H , Koch RO , Theurl I , et al.Expression of the duode-nal iron transporters divalent-metal transporter1 and ferro-portin1 in iron deficiency and iron overload.Gastroenterology ,2001 , 120 (6):1412 ～ 1419.[ 11] Gunshin H , Mackenzie B , Berger UV , et al.Cloning andcharacterization of a mammalian proton-coupled metal-iontransporter.Nature , 1997 , 388(6641):482 ～ 488.[ 12] Dupic F , Fruchon S , Bensaid M , et al.Duodenal mRNA ex-pression of iron related genes in response to iron loading andiron deficiency in four strains of mice.Gut , 2002 , 51 (5):648～ 653.[ 13] Ya Ke , Yin-yin CHEN , Yan-zhong Chang , et al.Post-transcriptional expression of DMT1 in the heart of rat.Journalof Cellular Physiology , 2003 , 196(1):124～ 130.[ 14] Tatchum-Talom R, Schulz R, McNeill JR, et al.Upregula-tion of neuronal nitric oxide synthase in skeletal muscle byswim training.Am J Physiol Heart Circ Physiol , 2000 , 279(4):H1757～ 1766.[ 15] Sangwon Kim , Prem Ponka.Nitric oxide-mediated modula-tion of iron regulatory proteins:Implication for cellular ironhomeostasis.Blood Cells , Molecules and Diseases , 2002 , 29(3):400～ 410.[ 16] Qian ZM.Nitric oxide and changes of iron metabolism in exer-cise.Biol Rev Camb Philos Soc , 2002 , 77(4):529～ 536.[ 17] Galen M , Pieper Nadine L N , Halligan , et al.Non-hemeiron protein:A potential target of nitric oxide in acute cardiacallograft rejection.PNAS , 2003 , 100(6):3125～ 3130.[ 18] Gael Nicolas , Lydie Viatte , Myriam Bennoun , et al.Hep-cidin , a new iron regulatory peptide.Blood Cells , Molecules ,and Diseases , 2002 , 29(3):327 ～ 335.(2004.07.26 收稿;2004.08.24修回)???????? 2005 ? 3?? 24 ?? 2? Chin J Sports Med ,March 2005 , Vol.24 , No.2 ·151　·

Effects of swim training on the expression of gastrocnemius’ iron transport proteins in rats

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