【作者】 马龙；

【导师】 李敏；

【作者基本信息】 第二军医大学 ， 军事预防医学， 2008， 博士

【摘要】 铁是人体内一种必需的金属元素,它可参与组成多种含铁酶,参与各种生命活动,适量的铁对于生物体的生存、分化和繁衍都具有十分重要的意义。但过量的铁具有毒性作用。铁通过Fenton反应(Fe²⁺+H₂O₂→Fe³⁺+·OH+OH^-)产生羟自由基,加剧体内的氧化应激,导致组织、细胞损伤。脑内铁代谢是十分精细的平衡过程,从血清铁跨越血脑屏障,到脑细胞摄取铁,每个环节都受到复杂而严密的调控。脑内铁稳态（iron homeostasis）的维持主要依赖于多种脑铁代谢蛋白的正常表达和相互协作。研究发现,在一些神经变性性疾病,例如:AD、PD、亨廷顿舞蹈病中,病人脑内出现铁的重新分布和部分脑区的铁沉积。研究也表明,脑内高铁和可能存在的脑内抗氧化防御机制缺陷所导致的氧化应激与这些神经变性性疾病中的神经元死亡密切相关。但是脑内出现铁重新分布和沉积的机制尚未阐明。以往的研究表明:某些躯体应激（热应激、运动应激、晕船）状态下,动物脑内可出现铁含量的变化。本课题组前期的一些实验中也发现了这一现象。上述现象提示我们:躯体应激可能会引起脑内铁稳态失去平衡。为了验证这一想法,我们需要明确不同应激源暴露对大鼠脑铁代谢的影响,并且初步探讨躯体应激影响大鼠脑铁代谢的机制。通过这些实验,为探索临床上以脑铁代谢紊乱为主要病理改变的一些疾病（如神经变性性疾病）的治疗方法提供新的线索。为此,我们进行了如下研究。第一部分:不同躯体应激对大鼠脑铁含量的影响目的:观察不同躯体应激源暴露对大鼠全脑和各脑区铁含量的影响。方法:1.实验动物选用成年SD雄性大鼠（体重120±10g）为实验动物,动物饲养符合国际清洁级环境,温度（24±1）℃,湿度50%～60%。不锈钢笼具分笼饲养,自由饮食,自然昼夜节律变化光照。2.实验动物处理大鼠适应性饲养7天后按体重随机分为足底电击组、水浸束缚组、截肢创伤组和对照组。各模型组处理如下:足底电击应激模型:参照文献所述方法,制作足底电击应激模型,大鼠每日上午8:00至10:00接受30 min的足底电击（电压90V,电流0.80mh）,连续暴露7天。水浸束缚应激模型:将大鼠四肢和头部束缚于鼠板上以限制其活动,将鼠板垂直浸入19℃±1℃水浴中,使液面位于正立位大鼠胸骨剑突水平,6小时后从水中取出,解除束缚,连续暴露7天。截肢创伤应激模型:将大鼠用10%水合氯醛麻醉后行右后肢截肢术,酒精消毒后用4号医用缝合线于大鼠右后肢膝关节上方约0.5cm处结扎,然后用手术剪于膝关节处截去右后肢,酒精棉球消毒断端后缝合伤口。各应激组大鼠分别接受7天的实验暴露,对照组不予处理。最后一次应激暴露后,将对照组和躯体应激组大鼠用10%水合氯醛麻醉,固定于鼠板上,开胸,经左心室取血,置于离心管中。用预冷的生理盐水经心脏灌洗,分别将大鼠全脑或前额皮层、海马、纹状体、脑干和小脑迅速取出,放入液氮中,随后放入-70℃冰箱中保存。3.原子吸收分光光度法测定大鼠全脑及各脑区铁含量取各组大鼠全脑及各脑区组织适量,称重,用20mmol/l HEPES缓冲液1:20（wt/v）稀释,匀浆,取30 uL匀浆液加入等体积的超纯硝酸50℃水浴下消化48小时,然后用3.12 mmol/L的硝酸1:10稀释。用5%硝酸稀释标准铁溶液（50mg/L）,绘制标准曲线。用原子吸收分光光度计火焰法连续测定空白管和样品管三次,记录248.3 nm的吸光度,用于分析。结果:1.与对照组相比,各应激组大鼠全脑铁含量均无显著变化;各应激组间大鼠全脑铁含量无显著差异。2.与对照组相比,足底电击组、水浸束缚组和截肢创伤组大鼠大脑前额皮质内铁含量分别增加37.1%、34.5%和25.3%（P＜0.05）,三组之间无显著差异;海马内铁含量分别增加58.3%、77.4%和95.2%（P＜0.05）;纹状体内铁含量分别增加70.7%、75.2%和61.2%（P＜0.05）;小脑和脑干内铁含量无显著变化。三个应激组间大鼠相应脑区铁含量均无显著差异。第二部分:躯体应激对大鼠脑铁摄取和储存蛋白的影响目的:研究躯体应激对大鼠脑铁摄取和储存蛋白的影响,初步阐释躯体应激条件下大鼠脑铁稳态失衡的原因。方法:1.动物模型:采用大鼠足底电击模型（同前）,实验动物同前。2.ELISA法测定海马内铁蛋白和转铁蛋白受体（transferring receptor,TfR）含量:两组各取10只大鼠的适量海马组织,称重,加入含有蛋白酶抑制剂、磷酸酶抑制剂和PMSF的RIPA裂解液,4℃下匀浆。10000×g离心30min,收集上清液,分装后于-70℃下保存备用。用BCA法测定样品蛋白质含量。严格按照铁蛋白和转铁蛋白受体ELISA试剂盒说明书操作,测定各样品的铁蛋白和转铁蛋白受体含量。3.Western blot法测定海马铁蛋白、TfR、二价金属离子转运体（divalent metaltransporter 1,DMT1）和乳铁蛋白（lactoferrin,Lf）:取含50ug蛋白的海马裂解液经10%非变性SDS—PAGE电泳分离后,电转移至硝酸纤维素膜。用丽春红染色,以证实蛋白质已转移至膜上。膜用脱脂奶粉室温封闭2h后,分别与1:1000小鼠抗人铁蛋白抗体、1:3000小鼠抗人TfR抗体、1:3000兔抗大鼠DMT1（IRE）抗体、1:3000兔抗大鼠DMT1（无IRE）抗体、1:1000兔抗大鼠Lf抗体和1:1000的β-actin抗体4℃孵育过夜。TBST洗膜3次后,与1:5000辣根过氧化酶标记的二抗室温孵育1h。用ECL检测试剂检测膜上蛋白质,并用ImageJ软件进行条带分析。结果:1.ELISA实验结果显示,与对照组相比,足底电击组大鼠海马内铁蛋白含量降低57.4%,转铁蛋白受体含量升高75.0%（P＜0.05）。2.Western blot结果显示,与对照组相比,足底电击组大鼠海马内铁蛋白表达减少,TfR表达增加（P＜0.05）,DMT1（有或无IRE型）和Lf蛋白量无显著变化。第三部分:躯体应激对大鼠脑铁调节蛋白（iron regulatory proteins,IRPs）的影响目的:在转录和翻译水平测定IRPs基因表达情况,初步探讨躯体应激条件下大鼠脑铁摄取和储存机制变化的原因。方法:1.动物模型:采用大鼠足底电击模型（同前）,实验动物同前。2.Real-time PCR法测定IRP1和IRP2的mRNA含量:两组各取3只大鼠的海马组织,按照试剂说明书用Trizol试剂抽提海马组织的总RNA,用10个单位的DNase I（TAKARA）在37℃下处理20ug RNA30min。纯化后,用Oligo dT引物进行cDNA合成。将1ul的cDNA加入到含有27.5ul Real Time PCR Master Mix、15pmol引物和7.5pmol TaqMan探针的反应体系中,总体积为30ul。引物和探针由软件PrimerPremier5.0设计。3.Western blot法测定海马内IRP1:取含50ug蛋白的海马裂解液经10%非变性SDS—PAGE电泳分离后,电转移至硝酸纤维素膜。用丽春红染色,以证实蛋白质已转移至膜上。膜用脱脂奶粉室温封闭2h后,与1:1000兔抗大鼠IRP1抗体和1:1000的β-actin抗体4℃孵育过夜。TBST洗膜3次后,与1:5000辣根过氧化酶标记的二抗室温孵育1h。用ECL检测试剂检测膜上蛋白质,并用ImageJ软件进行条带分析。结果:1.Real-time PCR结果显示,与对照组相比,足底电击组大鼠海马内IRP1 mRNA水平升高57.0%（P＜0.05）,IRP2 mRNA表达量无显著变化。2.Western blot结果显示,与对照组相比,足底电击组大鼠海马内IRP1表达增加（P＜0.05）。第四部分:躯体应激条件下大鼠海马内IRP1转录调控研究目的:通过对大鼠IRP1基因进行生物信息学分析,并检测躯体应激条件下大鼠海马内与IRP1基因有关的转录因子活性,初步探讨躯体应激条件下大鼠海马内IRP1的转录调控机制。方法:1.动物模型:采用大鼠足底电击模型（同前）,实验动物同前。2.IRP1基因启动子区潜在转录因子结合位点分析:通过PubMed检索人、小鼠和大鼠的IRP1基因序列。选取-2000到+100 bp序列为启动子区,通过TESS（Transcription Element Search System）软件（http://www.cbil.upenn.edu/tess/）和TRANSFAC数据库（http://www.gene-regulation.com/pub/databases.html#transfac）对启动子区进行潜在转录因子结合位点分析,并筛选出保守的结合位点序列。3.转录因子活性芯片测定:对照组和应激组各取两只大鼠的海马组织,严格按照芯片试剂盒说明书进行主要转录因子活性测定。简而言之,首先用Panomics核蛋白抽提试剂盒按照说明书抽提海马组织的核蛋白。经蛋白定量后,取10ug核蛋白抽提物与10ul TranSignal探针混合物混合、孵育。利用Panomics离心柱分离、回收与生物素标记的寡核苷酸结合的转录因子。将与转录因子特异结合的寡核苷酸探针洗脱后,在42℃下与TranSignal芯片膜孵育过夜。洗膜后与20μl HRP耦联链亲和素抗体孵育。去除过量溶液,将膜以X光胶片曝光。将胶片扫描为电子图片后用ScanAlyze软件进行分析。4.EMSA实验:通过EMSA实验对GR和STAT5转录因子活性测定结果进行验证。用非变性聚丙烯酰胺凝胶对核蛋白抽提物进行电泳分离,然后电转移至尼龙膜上,以254nm紫外光源激发光交联,封闭、洗膜后,加入发光反应液,以X光胶片曝光,用ImageJ软件进行分析。结果:1.IRP1基因启动子区潜在转录因子结合位点分析:通过TESS软件和TRANSFAC数据库进行分析后,选择匹配分值较高的序列,发现GR、STAT家族等保守序列。2.转录因子活性芯片测定:结果显示,与对照组相比,表达上调2倍以上的基因有GR、STAT5等32种,表达下调2倍以上的基因有GATA等124种。3.EMSA实验:转录因子活性芯片测定结果与转录因子结合位点分析结果进行比较,在表达上调2倍以上的基因中选择可能与IRP1基因启动子区结合并且与应激反应有关的转录因子GR和STAT5进行验证。结果显示,GR和STAT5与相应序列的结合活性增大。结论:本研究通过整体动物实验发现:1.足底电击、水浸束缚和截肢创伤三种躯体应激均可使大鼠部分脑区（大脑前额皮层、海马和纹状体）内铁含量增高,而小脑和脑干内铁含量不变,各躯体应激组间无差异,表明不同躯体应激均可改变大鼠脑内铁稳态,使大脑前额皮层、海马和纹状体内铁含量显著增多;2.躯体应激（足底电击应激模型）使大鼠海马内铁蛋白含量降低,TfR表达量增加,而Lf、DMT1（有和无IRE型）表达量不变,表明躯体应激使大鼠脑内铁代谢主要相关蛋白表达发生变化,TfR表达增多而铁蛋白表达减少,导致细胞铁摄入增加,而铁储存能力下降,这可能是导致大鼠海马等脑区内铁含量增多的原因之一;3.躯体应激使IRP1蛋白质和mRNA水平均升高,而IRP2表达量无显著变化,提示躯体应激条件下大鼠海马内TfR表达增加、铁蛋白表达减少可能与IRP1的转录后调控变化有关;4.躯体应激使大鼠海马内转录因子GR和STAT5的DNA结合活性增强,提示GR和STAT5的激活可能是躯体应激条件下大鼠海马内IRP1基因转录增强的原因。综合上述结果,提示躯体应激可能通过提高GR和STAT5的转录调节活性,提高大鼠海马内IRP1的表达量,进而使TfR表达量增加,铁蛋白表达量减少,最终导致大鼠脑内铁稳态失衡,相应脑区铁含量增加。但确切的调控机制以及是否存在其他通路,有待于进一步研究。更多还原

【Abstract】 Iron is one of the essential metal elements for human,which can be part of various iron-containing enzymes and participate in many physical activities.Adequate iron is of great importance for organisms to grow,differentiate and multiply.But excess iron can be deleterious.Iron can catalyze the production of hydroxyl radical through Fenton reaction(Fe²⁺+H₂O₂→Fe³⁺+·OH+OH^-),aggravating oxidative stress in the body and resulting in damage to tissues and cells.Brain iron metabolism is well controlled homeostasis.From serum iron crossing the blood-brain-barrier to neural cells uptaking iron,each process is strictly regulated. Brain iron homeostasis is mainly maintained through coordinating of expression and function of many iron metabolism related proteins.It has been demonstrated that in some neurodegenerative diseases such as Alzheimer disease（AD）,Parkinson’s disease（PD） and Huntington’s chorea,iron was redistributed and accumulated in some regions of brain.It is believed that oxidative stress resulting from excessly high iron content and defects of anti-oxidation defense in the brain is associated with death of neurons.But how iron is redistributed and accumulates in the brain is still not elucidated.Previous studies showed that brain iron content of animals was altered after some physical stresses such as exposure to high temperature,exercise and motion sickness. This is supported by experiments of our research group.All these results suggested that various physical stresses can alter brain iron homeostasis.To test this idea,we should determine the effects of various physical stresses on brain iron metabolism and explore the way in which physical stress affects brain iron metabolism.Results from these experiments will provide theoretical and experimental support for treatment of some neurodegenerative diseases characterized by misregulation of brain iron metabolism.The procedure of this study was as followed.Part 1:Effects of various physical stresses on iron metabolism in rat brainObjective:To determine the effects of various physical stresses on iron contents of whole rat brain and various regions of rat brain.Methods: 1.Establishing rat physical stress modelThree physical stress model:foot shock（FS）,Water-immersion restraint（WRS） and trauma（TR） stress model.Foot shock stress model:the rats were administered 30 min’s foot shock（90 V, 0.80mA） between 8:00AM and 10:00AM in the morning for successive 7 days（referring to Communication Box Stress Model）.Water-immersion restraint stress model:the rats were bound to restrict motion of extremities and head.The rats were immerged into water bath（19℃±1℃） vertically with sternoxiphoid just above the water for 6 hours for successive 7 days.Trauma stress model:the rats were amputated of right hind limb after anesthesia by 10%Chloral Hydrate solution.2.GroupingHealthy adult SD rats（body weight:120±10g） were used as experimental animals and randomly assigned to control（CTL）,foot shock（FS）,water-immersion restraint（WRS） and trauma（TR） groups.Rats were raised in SPF-compliant breeding room with a temperature of（24±1）℃and humidity of 50%～60%.Rats were raised individually in stainless steel cages with access to feeds and water ad libitum and in an 12/12 hours circadian rhythm.Rats of three different physical stress group were treated for successive 7 days while rats from control group received no treatment.After the last stress treatment, rats of all four groups were anesthetized by 10%Chloral Hydrate solution.Blood were collected from the hearts and centrifuged.Rats were perfused with cold saline through the heart and brains were removed.Some of the brains were dissected into such regions as prefrontal cortex,hippocampus,striatum,brain stem and cerebellum.Tissue samples were immediately put into liquid nitrogen.3.Atomic absorption spectrophotometry determination of iron content of whole rat brain and various regions of brainWhole and various regions of rat brain were weighted,1:20（wt/v） diluted with 20mmol/l HEPES buffer and homogenated.30 uL homogenate was mixed with equal volume of ultra pure nitric acid and digested in a 50℃warm water bath for 48 hours. Iron standard solution（50mg/L） was diluted with 5%nitric acid and used in plotting of standard curve.Blank and sample tubes were successively measured for 3 times with atomic absorption flame spectrophotometry and absorbance at 248.3 nm was recorded for further analysis. Results:1.Compared to control group,all three physical stress groups showed no significant change in iron content of whole rat brains;there is on significant difference in iron content of whole rat brains between either two of these three physical stress groups.2.Compared to control group,FS,WRS and TR groups showed an increase of 37.1%,34.5%and 25.3%in iron content of prefrontal cortex,respectively（P＜0.05） with no significant difference in iron content of prefrontal cortex between either two of these three physical stress groups;Compared to control group,FS,WRS and TR groups showed an increase of 58.3%,77.4%and 95.2%in iron content of hippocampus,respectively（P＜0.05） with no significant difference in iron content of hippocampus between either two of these three physical stress groups;Compared to control group,FS,WRS and TR groups showed an increase of 70.7%,75.2%and 61.2%in iron content of striatum,respectively（P＜0.05） with no significant difference in iron content of striatum between either two of these three physical stress groups;there was no significant difference in iron content of cerebellum or brain stem between either two of all four groups.Part 2.Effects of physical stress on important iron metabolism related proteins in rat brainObjective:To determine the effects of physical stress on important iron metabolism related proteins in rat brain and give a primary explanation of how brain iron homeostasis was disrupted.Methods:1.Animal model:Foot shock stress model（see Part 1）.2.ELISA determination of ferritin and transferring receptor in rat hippocampus:Hippocampus from both control and FS groups（n=10） was weighted,added to RIPA lysis buffer containing protease inhibitors,phosphotase inhibitors and PMSF and homogenated at 4℃.Homogenates was centrifuged at 10000 g for 30 min.and supernatant was collected and stored at -70℃.Protein concentration was determined with BCA protein quantification kit.ELISA tests were carried out according to instruction of test kits to determine the content of ferritin and transferring receptor.3.Western blot analysis of ferritin,transferring receptor,DMT1 and Lf in rat hippocampus:Hippocampus extracts containing 50ug protein was separated with 10% nondenaturing polyacrylamide gel electrophoresis（SDS-PAGE） and electrically transferred to nitrocellulose membrane.Blots were dyed with ponceau red solution to show whether protein had been transferred to the membrane.After blocking with skimmed milk for 2 hours,blots were incubated with 1:1000 diluted mouse anti-human ferritin antibody,1: 3000 diluted mouse anti-human TfR antibody,1:3000 diluted rabbit anti-rat DMT1（with IRE） antibody,1:3000 diluted rabbit anti-rat DMT1（without IRE） antibody,1:1000 rabbit anti-rat Lf antibody and 1:1000 mouse anti-humanβ-actin antibody for 12 hours at 4℃. Blots were washed 3 times with TBST before incubated with HRP-labeled secondary antibody for 1 hour at room temperature.Blots were detected with ECL reagents and gel images were analyzed with Image J software.Results:1.Results of ELISA tests showed a decrease of 57.4%in content of ferritin and an increase of 75.0%in content of TfR respectively in rat hippocampus of FS group compared with control group（P＜0.05）.2.Results of Western blot analysis showed decrease in ferritin expression and increase in TfR expression and no change in expression of DMT1（with or without IRE） and Lf in rat hippocampus of FS group compared with control group.Part 3:Effects of physical stress on iron regulatory mechanism in rat hippocampusObjective:To determine the expression of iron regulatory proteins at transcriptional and translational levels and give a primary explanation of how iron metabolism was affected by physical stress in rat hippocampus.Methods:1.Animal model:Foot shock stress model（see Part 1）.2.Real-time PCR determination of IRP1和IRP2 mRNA:RNA was extracted from rat hippocampus of both control and FS groups（n=3） with Trizol reagents according to the instructions of manufacturer.20ug RNA was treated with 10 units of DNase I（TAKARA） for 30min at 37℃.cDNA was synthesized with Oligo dT primers after purification of total RNA.1 ul cDNA was added to a PCR system of 30ul containing 27.5ul Real Time PCR Master Mix,15pmol primers and 7.5pmol TaqMan probe（primers and probes were designed with Primer Premier5.0 software）.3.Western blot analysis of IRP1 in rat hippocampus:Hippocampus extracts containing 50ug protein was separated with 10%nondenaturing polyacrylamide gel electrophoresis（SDS-PAGE） and electrically transferred to nitrocellulose membrane.Blots were dyed with ponceau red solution to show whether protein had been transferred to the membrane.After blocking with skimmed milk for 2 hours,blots were incubated with 1: 1000 diluted rabbit anti-rat IRP1 antibody and 1:1000 mouse anti-humanβ-actin antibody for 12 hours at 4℃.Blots were washed 3 times with TBST before incubated with HRP-labeled secondary antibody for 1 hour at room temperature.Blots were detected with ECL reagents and gel images were analyzed with Image J software.Results:1.Results of Real-time PCR analysis showed an increase of 57.0%（P＜0.05） in IRP1 mRNA level and no change in IRP2 mRNA level in rat hippocampus of FS group compared with control group.2.Results of Western blot analysis showed increase in IRP1 expression in rat hippocampus of FS group compared with control group（P＜0.05）.Part 4:Study on transcriptional regulation of IRP1 gene in rat hippocampus after physical stressObjective:To determine the transcriptional regulation of IRP1 gene in rat hippocampus after physical stress through bioinformatic analysis of rat IRP1 gene promoter and determination of activities of some transcription factor related to IRP1 gene expression.Methods:1.Animal model:Foot shock stress model（see Part 1）.2.Prediction of potential transcription factors binding sites in IRP1 gene promoter:Human,mouse and rat IRP1 genes were retrieved in PubMed website. Promoter region of IRP1 gene（-2000 to +100 bp） was analyzed with TESS（Transcription Element Search System） software（http://www.cbil.upenn.edu/tess/） and TRANSFAC database（http://www.gene-regulation.com/pub/databases.html#transfac） for potential transcription factors binding sites and conserved sites were picked out.3.Array test of activity of transcription factors:Tests were carried out with Panomics protein/DNA array kit according to the manufacturer’s instruction.Briefly, nuclear proteins were extracted from rat hippocampus with Panomics Nuclear Extraction Kit according to manufacture’s instruction.After quantification of protein concentration, 10ug nuclear extract was incubated with 10ul TranSignal probe mix.Transcription factors attached to biotin-labeled oligonucleotide were separated and collected with Panomics spin columns.Oligonucletides attached specifically to transcription factors were eluted and incubated with TranSignal array membranes at 42℃.The membranes were washed and incubated with 20μl HRP-labeled streptavidin.Throw off excess water and image with X-ray films.Scan the films,save the images as pictures and analyze them with ScanAlyze software.4.EMSA tests:EMSA tests of GR and STAT5 were carried out to verify the results of Protein/DNA array tests.Nuclear extracts were separated with non-denaturing polyacrylamide gel electrophoresis（SDS-PAGE） and electrically transferred to nylon membranes.The membranes were incubated with specific GR or STAT5 DNA probes. After cross-linking with 254nm ultraviolet light,blocking and washing,the membranes were incubated with chemiluminescent reagents.After exposure,development and fixation, X-ray films were scanned and analyzed with Image J software.Results:1.Prediction of potential transcription factors binding sites in IRP1 gene promoter:After analysis of the promoter with TESS software and TRANSFAC database, we found some well-matched conserved binding sites including ones responding to GR and STAT5.2.Panomics Protein/DNA array tests:results of array tests showed 32 transcription factors including GR and STAT5 with binding activities 2 times higher in FS group than that in control group and 124 transcription factors with binding activities 2 times lower in FS group than that in control group.3.EMSA tests:Comparing the results of Protein/DNA array tests with the results of analysis of potential transcription factors binding sites in IRP1 gene promoter,we found two transcription factors（GR and STAT5 ） with 2 times higher binding activities in FS group than that in control group responding to respective conserved binding sites in IRP1 gene promoter.EMSA tests showed binding activities of GR and STAT5 with specific DNA sequences were enhanced in FS group than that in control group,consistent with results of array tests.Conclusion:This study was designed to（1） determine effects of various physical stresses on iron metabolism in rat brain,（2）find out effects of physical stress on important iron metabolism related proteins in rat brain,（3）understand effects of physical stress on iron regulatory mechanism in rat hippocampus and（4）study the transcriptional regulation of IRP1 gene in rat hippocampus after physical stress.The results showed:1.Compared to control group,FS,WRS and TR groups showed a significant increase in iron content of prefrontal cortex,hippocampus and striatum（P＜0.05） with no significant difference in iron content of prefrontal cortex,hippocampus and striatum between either two of these three physical stress groups;there was no significant difference in iron content of cerebellum or brain stem between either two of all four groups, indicating that various physical stresses can alter iron homeostasis in rat brain,resulting in accumulation of excess iron in such regions as prefrontal cortex,hippocampus and striatum;2.Physical stress resulted in a significant decrease in ferritin expression and a significant increase in TfR expression while no significant change in Lf and DMT1（with or without IRE） expression in rat hippocampus,indicating that physical stress induced up-regulation of TfR and down-regulation of ferritin expression resulting in increased iron uptake and decreased capacity of iron storage was the reason why iron accumulated in some regions including hippocampus in rat brain;3.Physical stress resulted in a significant increase of IRP1 gene expression at both transcriptional and translational levels while no significant change in IRP2 gene expression, indicating that post-transcriptional regulation of IRP1 maybe was reason of increased expression of TfR and decreased expression of ferritin in rat hippocampus after physical stress;4.Physical stress resulted in enhanced DNA binding activities of two transcription factors（GR and STAT5） which can specifically bind to conserved sequences in IRP1 promoter,indicating that activation of binding activity of GR and STAT5 to specific DNA sequences may be the reason of enhanced gene transcription of IRP1 in rat hippocampus after physical stress.The results of this study suggested that physical stress may enhance the transcriptional regulation of GR and STAT5 on IRP1 gene,resulting in higher expression of IRP1 and consequently higher expression of TfR and lower expression of ferritin.Finally,this may lead to disturbed iron homeostasis featuring iron accumulating in some regions of rat brain including prefrontal cortex,hippocampus and striatum.However,further study is needed to explore the exact signaling pathways leading to disrupted iron homeostasis after physical stress.更多还原