心理应激对大鼠脑细胞铁储存和释放机制的影响
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摘要
铁是人体必需的微量元素之一,是多种含铁酶的组成部分,广泛参与机体的代谢过程。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,但是过量的铁具有毒性作用。铁通过Fenton反应(Fe~(2+)+H_2O_2→Fe~(3+)+·OH+OH~-)产生羟自由基,也可通过铁催化的Haber-Weiss反应(Fe~(3+)+O_2~-→Fe~(2+)+O_2;Fe~(2+)+H_2O_2→Fe~(3+)+·OH+OH~-(iron catalayzed))加剧体内的氧化应激,导致组织、细胞损伤。因此,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。以前认为脑内铁稳态的维持,主要是依靠对铁摄取的调控来实现的。但越来越多的研究表明,脑铁的储存和释放也对脑铁稳态的维持起着重要作用。
研究发现,在一些神经变性性疾病,例如:AD(Alzheimer's disease)、PD(Parkinson's disease)、亨廷顿(Huntington's disease)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。现已证实,过量铁是导致上述神经退行性疾病发病的重要原因之一。但是,引起部分脑区铁蓄积的原因尚未完全阐明。
国内外有研究者发现,应激机体存在铁重新分布的现象。本室前期所做的一些实验也发现诸如晕船、运动和疲劳刺激可以使得铁元素在体内出现重新分布。我们的研究还发现,心理应激大鼠皮层、海马和纹状体内总铁含量以及非蛋白结合铁含量显著升高,该脑区铁调节蛋白1和转铁蛋白受体表达增多。而应激可通过提高GR和STAT5的转录调节活性,增加大鼠海马内铁调节蛋白1的表达量,进而使转铁蛋白受体表达量增加。这些结果显示,应激反应可影响脑组织铁摄取机制。但是,应激反应是否对铁的储存和释放也有影响还不清楚。本文为阐明此问题进行了系统的研究。
目的
了解心理应激对大鼠脑铁储存和释放蛋白的影响及其机制,为阐明心理应激与中枢神经退行性疾病的关系以及深入探讨军事应激损伤的生物学机制提供实验基础,并为预防以脑铁代谢紊乱为主要病理改变的疾病治疗提供新的思路。
方法
1.心理应激对大鼠脑内非血红素三价铁(NHF III)和非血红素二价铁(NHF II)的影响
1.1实验动物分组
雄性SD大鼠(购自上海西普尔-必凯公司),体重(120士5)g。按体重随机分为空白对照组(CG)、心理应激组(PSG)和足底电击组(FSG)。动物饲养实验室环境温度24℃±1℃,湿度50%～60%;使用不锈钢笼具单笼饲养,自由饮食,自然昼夜节律变化光照。
1.2大鼠心理应激模型的制作
采用Communication Box System制作大鼠心理应激模型。CommunicationBox System由透明丙烯酸板组成,一半小室(A室)底部铺板绝缘,另一半小室(B室)通电。在B室的实验大鼠接受30min/d,足底电击(电击信号由JL-B双脉冲电刺激器发生,经放大器产生电压70V,电流0.70mA);电击组大鼠跳跃,尖叫,在A室的实验大鼠通过视觉听觉产生恐惧的心理反应,即为心理应激大鼠模型。共7天。
1.3大鼠脑组织灌注铁染色
1.3.1大鼠脑组织普鲁士蓝灌注铁染色(perfusion-Prels method)
在第七天造模完成后即刻腹腔注射10%水合氯醛进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏插管用含有肝素钠(5单位/ml)的PBS溶液150ml冲洗血液,然后800ml含有1%亚铁氰化钾4%多聚甲醛灌流液(PH1.0)固定,取脑组织采用石蜡切片制成5μm切片用于铁染色。参照包新民大鼠脑立体定位图谱,在每只大鼠耳间线嘴侧3.6-3.8mm处行冠状切片显示大鼠海马;耳间线嘴侧7.8mm-8.0mm处冠状切片显示大鼠纹状体及额叶皮层。
1.3.2大鼠脑组织滕氏蓝灌注铁染色(perfusion-Turnbull method)
在第七天造模完成后即刻腹腔注射10%水合氯醛进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏插管用含有肝素钠(5单位/ml)的PBS溶液150ml冲洗血液,然后800ml含有1%铁氰化钾4%多聚甲醛灌流液(PH1.0)固定,取脑组织采用石蜡切片制成5μm切片用于铁染色。参照包新民大鼠脑立体定位图谱,在每只大鼠耳间线嘴侧3.6-3.8mm处行冠状切片显示大鼠海马;耳间线嘴侧7.8mm-8.0mm处冠状切片显示大鼠纹状体及额叶皮层。
2.心理应激对大鼠脑细胞铁储存蛋白和铁释放蛋白的影响
2.1实验动物分组及模型制作:同前
2.2心理应激对大鼠皮层、海马以及纹状体内膜铁转运蛋白Fp1(Ferroportin)、膜铁转运辅助蛋白HP(Hephaestin)mRNA表达的影响
采用实时定量荧光PCR法测定心理应激组和对照组大鼠皮层、海马以及纹状体内膜铁转运蛋白Fp1、膜铁转运辅助蛋白HP的mRNA表达水平。两组各取5只大鼠的皮层、海马和纹状体组织,按照试剂说明书用Trizol试剂抽提组织的总RNA,用10个单位的DNase I在37℃下处20ug RNA 30min。纯化后,RevertAid~TMFirst Strand cDNA synthesis Kit逆转录为cDNA。将1ul的cDNA加入含有10×PCRbuffer 2.5ul,25mM Mg~2+3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase0.5ul,10×SYBR-green I荧光染料0.7ul,5uM上下游引物2.0ul和DNAase freewater 12.8ul.反应体系中,总体积为25ul。引物由软件Primer Premier5.0设计。
2.3心理应激对大鼠皮层、海马以及纹状体内内膜铁转运蛋白Fp1(Ferroportin)、铁蛋白Fn(Ferritin)含量的影响
Western blot法测定实验组和对照组大鼠皮层、海马以及纹状体内Fp1以及Fn的含量。取含50ug蛋白的裂解液经10%非变性SDS—PAGE电泳分离后,电转移至硝酸纤维素膜。膜用脱脂奶粉室温封闭2h后,分别与1:1000兔抗人铁蛋白抗体、1:2000兔抗小鼠Fp1抗体、和1:2000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1 h。用ECL检测试剂检测膜上蛋白质,并用Quantity One软件进行条带分析。
3.心理应激大鼠脑细胞铁储存蛋白和铁释放蛋白改变机制的探讨
3.1实验动物分组及模型制作:同前
3.2心理应激对大鼠脑皮层、海马、纹状体内白介素-6(IL-6)、铁调素Hpec(Hepeidin)mRNA的含量影响
采用实时定量荧光PCR法测定心理应激组和对照组大鼠皮层、海马以及纹状体内铁调素Hpec、IL-6的mRNA表达水平。两组各取5只大鼠的皮层、海马和纹状体组织,按照试剂说明书用Trizol试剂抽提组织的总RNA,用10个单位的DNaseI在37℃下处理20ug RNA 30min。纯化后,RevertAid~TM First Strand cDNA synthesisKit逆转录为cDNA。将1ul的cDNA加入含有10xPCR buffer 2.5ul,25mM Mg~(2+)3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10xSYBR-green I荧光染料0.7ul,5uM上下游引物2.0ul和DNAase free water 12.8ul.反应体系中,总体积为25ul。引物由软件Primer Premier5.0设计。
4.数据的统计与处理
灌注铁染色图像数据采用image-pro plus 6.0图像分析软件进行灰度分析:铁离子染色阳性颗粒呈深棕黑到黑色,镜检每例组织,在额叶脑皮层次级区,纹状体尾壳核区(CPu区)海马多形细胞层随机选取8个视野,用显微图像分析软件Image-pro plus 6.0测定阳性目标的平均灰度值、视场平均灰度值、面积密度,换算成阳性单位(positive unit,PU),以PU值大小代表阳性产物表达的多少。同时观察各部位细胞形态。Western-blot蛋白条带数据扫描为Tiff格式图像,QuantityOne一维胶分析软件进行灰度分析得到相对浓度;实时荧光定量数据通rotor-gene6.0.14 software用2~△△CT方法得到相对浓度。
实验数据采用SPSS13.0统计软件包进行。采用两独立样本t检验进行数据分析,实验数据以平均数±标准差([x]±S)表示,P<0.05表示有统计学意义。
结果
1、心理应激对大鼠脑组织非血红素铁含量的影响
1.1心理应激对大鼠大脑皮层、海马、纹状体非血红素三价铁(NHF III)影响。
心理应激大鼠脑皮层、海马内NHF III染色较对照组显著升高(P<0.05),纹状体内NHF III染色未见明显改变。海马CA1,CA2区神经元及其周围明显NHFIII染色增强,神经元细胞的数目减少。在侧脑室前角与周围额叶皮层之间大量NHF III密集。
1.2心理应激对大鼠大脑皮层、海马、纹状体非血红素二价铁(NHF II)的影响
心理应激组大鼠皮层、海马、纹状体内NHF II染色较对照组显著升高,(P<0.05)。海马CA1,CA2区可见NHF II染色增强伴有神经元细胞的数目减少和结构松散。
2.心理应激对大鼠脑铁储存和释放蛋白的影响
2.1心理应激对大鼠皮层、海马以及纹状体Fp1、HPmRNA表达的影响
实时荧光定量PCR结果显示心理应激组大鼠皮层及海马内Fp1 mRNA含量较对照组显著降低(P<0.05),心理应激组纹状体内Fp1 mRNA含量较对照组有显著增加(P<0.05);心理应激组大鼠皮层、海马内HP mRNA含量较对照组明显降低(P<0.05)。纹状体内HP mRNA含量较对照组没有明显改变。
2.2心理应激对大鼠皮层、海马以及纹状体内Fp1、Fn含量的影响
Western-blot结果显示心理应激组大鼠皮层及海马内Fp1含量在较对照组比较显著降低(P<0.05);纹状体内Fp1含量较对照组有增高趋势无统计学意义(P>0.05)。心理应激组大鼠皮层、海马内Fn含量较对照组比较显著下降(P<0.05)。纹状体内Fn含量较对照组有增高趋势,无统计学意义(P>0.05)。
3.心理应激引起大鼠脑铁储存和释放蛋白表达变化的机制探讨
3.1心理应激对大鼠皮层、海马以及纹状体内Hpec mRNA的影响
实时荧光定量PCR结果显示:(1)正常情况下(未受到任何刺激)大鼠的皮层、海马和纹状体内都有Hepcidin mRNA的表达。皮层Hepcidin mRNA的表达丰度比海马和纹状体显著高,大约20-100倍。(2)心理应激组大鼠皮层HepcidinmRNA较对照组显著升高(P<0.05),海马Hepcidin mRNA较对照组有降低趋势(P>0.05),纹状体较对照组没有变化。
3.2心理应激对大鼠皮层、海马以及纹状体IL-6表达的影响
心理应激大鼠皮层、海马、纹状体内IL-6 mRNA较对照组显著升高(P<0.05)
结论
本研究通过整体动物实验发现:
1.灌注铁染色法结果显示,心理应激组大鼠皮层、海马内NHF III含量和NHFII含量显著升高,纹状体内NHF II含量升高但NHF III没有明显改变。铁离子在皮层、海马特定部位大量沉积,海马CA1,CA2区可见神经元神经元细胞数目减少。表明,心理应激可以导致非血红素结合铁在大鼠部分脑区含量增加。而二价铁离子升高而产生的过多的羟自由基可能是海马CA1,CA2区可见神经元神经元细胞数目减少的原因之一。
2.心理应激组大鼠皮层和海马内的Fn含量和HP mRNA表达减少,神经细胞内亚铁氧化酶减少(Fn的H链和HP),进而导致二价铁离子转化为三价铁离子受到影响,细胞内低分子量铁池扩大,二价铁离子增多。另一方面,增多的二价铁离子不能及时被氧化为三价铁离子而进入铁蛋白存储或进入其他细胞器利用,只能通过自发氧化形成三价铁离子,最终异常沉积于细胞内。上述机制可能是大鼠皮层和海马内的二价铁离子增多的原因。
3.心理应激可导致皮层、海马神经元和神经胶质细胞的铁释放蛋白Fp1减少,同时伴有膜铁转运蛋白辅助蛋白HP减少。表明,脑细胞内的铁释放蛋白受到影响而引起铁排除减少,也可能是神经细胞内铁沉积的原因之一。
4.心理应激导致海马、皮层、纹状体内细胞因子IL-6 mRNA的表达增多,皮层内Hepc mRNA表达增多。结合国内外相关研究结果,提示心理应激情况下IL-6表达的增加可能是脑皮层的Hepc mRNA表达增加的原因之一。
综上所述,本研究结果提示,心理应激可能通过上调炎性细胞因子IL-6mRNA的表达,调节脑皮层Hepc表达增加,增高的Hepc同其受体-脑细胞胞膜上Fp1结合,引起内陷,降解,细胞内的铁释放减少,而亚铁氧化酶膜铁转运辅助蛋白HP的减少加剧了这一过程。Fn和HP的减少引起神经细胞内二价铁离子增多,铁储存的障碍,进而引起细胞内氧化应激的加强,可能是海马内CA1,CA2区可见神经元神经元细胞数目减少的原因。因此,我们认为,应激作为一个全身性神经—内分泌—机体免疫网络,在对抗外界不良刺激的同时,也影响了机体内环境稳定,包括铁代谢稳定。而铁代谢紊乱所导致的脑细胞功能和结构损伤,可能是过度应激后造成健康危害的原因之一。
Iron is essential trace metal for almost all living organism by participating in a wide variety of metabolic process.Adequate iron is of great importance for organism growth,cell differentiation and multiply.Iron homeostasis is critical for normal brain growth and physiologic function.But excess iron can be deleterious.Iron can catalyze the production of hydroxyl radical through Fenton -reaction(Fe~(2+)+H_2O_2→Fe~(3+)+OH +OH-),and the Haber-Weiss reaction(Fe~(3+)+O_2→Fe~(2+)+O_2;Fe~(2+)+H_2O_2→Fe~(3+) +OH+OH-(iron catalayzed)) aggravating oxidative stress in the body and resulting in damage to tissues and cells,more and more researches indicate that excess iron accumulation in the body will bring a huge damage,especially iron overload in the central nervous system.Hence,the brain have elegant systems for mobilization and of iron sequestration.From serum iron crossing the blood-brain-barrier to neural cells uptaking iron,each process is strictly regulated.
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 the key point to death of neurons.But how iron is redistributed and accumulates in the brain is still not elucidated.With the progress of related research, more and more evidence to support the idea that iron efflux was also played a vital role in iron homeostasis regulation.
Previous researches including some experiments results our office did showed that motion sickness,sports and fatigue can make iron redistribution in the rat body.The facts indicate that mental stress may be one of reasons for the brain iron metabolism disorders.Our previous study on psychological stress rat reveal that some brain region with iron overload was up-regulated in IRP1 by enhancing the transcriptional regulation of GR and STAT5 on IRP1 gene,resulting in higher expression of IRP1 and consequently higher expression of TfR.We presume that psychological stress not only changes the iron uptaking but also changes the iron storage and iron efflux.The procedure of this study was as followed.
Objective
To study the characteristic effects of psychological stress on brain iron storage proteins and efflux proteins and establish a useful experimental basis for further study involving how stress changes cell normal iron homeostasis and the consequent effects on physiological function of the human body.
Method
1.Effects of psychological stress on NHFⅡ(nonheme ferrous iron )and NHFⅢ(nonheme ferric iron) concentrations in the rat brain
1.1 To divide experimental animals into groups
All experimental procedures involving animals received the approval from the Animal Care and Use Committee of the Second Military Medicine University. Guidelines and Policy on using and caring of the laboratory animals were followed at all time.Male SD rats(120±5g body weight) fed with a standard diet were purchased from the Shanghai-BK Ltd.Co,and were housed individually in a cage in a temperature-controlled room(24±1℃,55±5%humidity) with a 12-hour light and 12-hour dark cycle.After adaptation for 3 days,the rats were divided into the foot-shock group(FSG),psychological stress group(PSG) and the control group(CG). Each rat was exposed to stress for 30 minutes every day,successive 7 days.
1.2 To build psychological stress model of SD rats
Using a communication box system,footshock stress(FS) and psychological stress(PS) were administered to the rats.The communication box was divided into two parts with a transparent acrylic board,i.e.,Part A including ten rooms with a plastic board-covered floor for electric insulation and part B including ten rooms with a metal grid-exposed floor.Rats in part B were administered an electrical shock through the floor(90 V,0.8 mA for 1 second) randomly for 30 min,90 times in total, and then exhibited a nociceptive stimulation-evoked response such as jumping up, defecation and crying.Thus they were exposed to systemic(physical) stress.Rats in part A were not directly administered the electrical shock,but were exposed to psychological stress in response to the actions of the rats in Room B.
1.3 Perfusion-Perls and -Turnbull methods supplemented by DAB intensification for nonheme iron histochemistry in rats brian
1.3.1 Perfusion-Perls methods supplemented by DAB intensification for NHFⅢhistochemistry in rats brian
After successive 7 days ps treatment,The animals were anesthetized with intraperitoneal pentobarbital sodium(50 mg/kg) and transcardially perfused with the 130 ml of phosphate buffered saline(PBS,pH7.4) containing heparin(5 units/ml)for 5 min,to flush out the blood,then 800 ml of the solution(pH 1.0) containing 1% potassium ferrocyanide and 4%PFA in distilled water was perfused for tissue fixation and insute stain.After the perfusion,the brain was excised and cut off.To prepare 5 um thick sections,paraffin embedding and tissue cutting were done by the conventional methods.The sections were mounted on silan-coated slides,dewaxed and hydrated for further treatments.
1.3.2 Perfusion- Turnbull methods supplemented by DAB intensification for NHFⅡhistochemistry in rats brian
After successive 7 days ps treatment,The animals were anesthetized with intraperitoneal pentobarbital sodium(50 mg/kg) and transcardially perfused with the 130 ml of phosphate buffered saline(PBS,pH 7.4 ) containing heparin(5 units/ml)for 5 min,to flush out the blood,then 800 ml of the solution(pH 1.0) containing 1% potassium ferricyanide and 4%PFA in distilled water was perfused for tissue fixation and insute stain.After the perfusion,the brain was excised and cut off.To prepare 5 um thick sections,paraffin embedding and tissue cutting were done by the conventional methods.The sections were mounted on silan-coated slides,dewaxed and hydrated for further treatments.
2.Effects of psychological stress on brain iron storage protein and iron release protein
2.1 Determination of Ferroportin and Hephaestin mRNA levels
Real-time PCR determination of Ferroportin and Hephaestin mRNA:
RNA was extracted from rat cortex,hippocampus and striatum of both control and PS groups(n=5) with Trizol reagents according to the instructions of manufacturer. 20ug RNA was treated with 10 units of DNase 1 for 30min at 37℃.First strand cDNA was synthesized with M-MuLV Revers transcriptase after the purification of total RNA being incubated with Oligo dT primers.1 ul cDNA was added to a PCR system of 25ul containing 10×PCR buffer 2.5ul,25mM Mg~(2+) 3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10×SYBR-green I fluorescence dye 0.7ul,5uM forword primer and reverse primer 2.0ul and DNAase free water 12.8ul.(primers were designed with Primer Premier5.0 software).
2.2 Western blotting analysis of Ferroportin and Ferritin expression
Dissected tissues from the cortex,hippocampus and striatum from both control and PS groups(n=3) were homogenized separately by a dounce homogenizer in lysis buffer.Each group extractions 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,1:2000 diluted rabbit anti-rat Ferroportin antibody and 1:2000 mouse anti-humanβ-actin antibody for 1 hours at room temperature.Blots were washed 3 times with PBST 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 quantity one software.
3.Effects of molecular mechanisms of psychological stress on brain iron storage protein and iron efflux protein
Determination of Hepc mRNA and IL-6 mRNA levels
RNA was extracted from rat cortex,hippocampus and striatum of both control and PS groups(n=5) with Trizol reagents according to the instructions of manufacturer. 20ug RNA was treated with 10 units of DNase I for 30min at 37℃.First strand cDNA was synthesized with M-MuLV Revers transcriptase after the purification of total RNA being incubated with Oligo dT primers.1ul cDNA was added to a PCR system of 25ul containing 10×PCR buffer 2.5ul,25mM Mg~(2+) 3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10×SYBR-green I fluorescence dye 0.7ul,5uM forword primer and reverse primer 2.0ul and DNAase free water 12.8ul.(primers were designed with Primer Premier5.0 software).
4.Statistical analysis
Perfusion-Perls and -Turnbull methods for nonheme iron histochemistry image were analyzed by image-pro plus 6.0 software.Microscopic examination in ervry section and choose site as follows:Brain sub-district for cortex,Polymorphic cell layer for hippocampus and CPu region for striatum.Eight visual fields were selected by random.Accouding to the mean object gray scale and object area density,PU(positive units )were defined.The quantity of PU represent stained iron ion.Western-blot data were analyzed by Quantity One software.Real-time PCR data were analyzed by rotor-gene 6.0.14 software.
All results were expressed as mean±S.D.Statistical analysis was carried out by using SPSS 13.0 in independent sample T test method.All values below the detection limits were set to zero and absolute values without correction for recovery rate were used in analyses.A P value less than 0.05 was considered statistically significant.
Results
1.Psychological stress exposure increased both NHFⅢand NHFⅡstained positive units levels in some brain regions
1.1 Effects of psychological stress on NHFⅢconcentrations
We found that the NHFⅢstained positive units levels in the frontal cortex, hippocampus and were significantly higher in the PS exposure group than in the control group(P＜0.05);however,no significant higher was observed in NHFⅢstained positive units levels striatum between the two groups.(P＞0.05) The neurons of CA1,CA2 region in PS exposure group hippocampus were significantly stained stronger than in the control group,a great quantity of NHFⅢstained positive units were deposit in the area between anterior horn of lateral ventricle and frontal lobe,where in exposure group is significant higher than in the control group.
1.2 Effects of psychological stress on NHFⅡconcentrations
We found that the NHFⅡstained positive units levels in the frontal cortex, hippocampus and striatum were significantly higher in the PS exposure group than in the control group(P＜0.05).The CA1,CA2 region in PS exposure group hippocampus were stained stronger than that in the control group,the structure of pyramidal neuron cell boby were demolished can be observed,as well as the quantity of pyramidal neuron were diminished.
2.Effects of Psychological stress on brain iron storage protein and iron efflux protein
2.1 Psychological stress exposure caused changes in Ferroportin、Hephaestin mRNA
Real time-PCR analysis showed that PS exposure decreased Ferroportin mRNA levels in the cortex,hippocampus(P＜0.05) but increase Ferroportin mRNA in the striatum significantly(P＜0.05).The Hephaestin mRNA level of PS exposure group was significantly lower than that in the control group(P＜0.05),though the decrease was not significant in the striatum.
2.2 Psychological stress exposure caused changes in Ferroportin and Ferritin levels
Western blot analysis showed that Ferroportin concentrations levels in the cortex and hippocampus were significantly lower than those in the control group (P＜0.05),but a little higher in striatum(P＞0.05);Ferritin concentrations levels in the cortex and hippocampus were significantly lower than those in the control group (P＜0.05),but little higher in striatum(P＞0.05)
3.Effects of molecular mechanisms of psychological stress on brain iron storage protein and iron efflux protein
3.1 Effects of psychological stress on brain Hepcidin mRNA expression
Real time-PCR analysis showed that the level of cortex Hepcidin mRNA expression both in PS exposure group and control group were extremely higher than that in the hippocampus and striatum.PS exposure increased Hepcidin mRNA levels in the cortex significantly(P＜0.05),decreased in hippocampus(P＞0.05 )and no obvious changed in striatum.
3.2 Effects of psychological stress on brain IL-6 mRNA expression
Real time-PCR analysis manifested that Psychological stress exposure increased IL-6 mRNA levels in the cortex,hippocampus and striatum.significantly(P＜0.05)
Conclusions
1.Our study was built the psychological stress rats model by means of the Communication Box System.Take the advantage of the perfusion-Perls and -Turnbull methods supplemented by DAB intensification(+ DAB),we demonstrated visualization of nonheme ferric and ferrous iron in situ sensitively.We found that the NHFⅢstained positive units levels in the frontal cortex,hippocampus and were significantly higher in the PS exposure group than that in the control group,as well as the NHFⅡstained positive units levels.Furthermore,the structure of pyramidal neuron cell body were demolished can be observed in CA1 and CA2 region.All of these suggested psychological stress on rat brain lead to iron overload in specific encephalic region including cerebral cortex and hippocampus.The increasing of oxidative stress by NHFⅡmay be the reason to resulting the changes of hippocampus CA1,CA2 region.
2.We found that Ferritin concentrations levels in the cortex and hippocampus of psychological stress group were significantly lower while the Hephaestin mRNA levels and the Ferroportin concentrations levels and mRNA likewise.According to our previous research,psychological stress can evaluate iron intaking in cortex and hippocampus.Considerable iron ion was imported via DMT1,keeping themselves in divalence state.In cytosol,ferrous ion is considered to loosely complex with low-molecular weight organic bases forming the so-called low-molecular weight Iron pool(LIP),which contains some ferric iron as due to cytosolic oxidation.Because of its reactivity and cytotoxicity,the LIP size is maintained in very small amount.We speculate that LIP was expanded,meanwhile,ferritin(which has a ferroxidase core in H chain) and hephaestin(ferroxidase)were reduced,ferrous iron could not be oxidized into ferric iron immediately for storage in ferritin or final use in mitochondria and other organelles.Finally,ferrous iron was auto-oxidized to trivalence state and in a abnormal deposit form in cell.
3.Ferroportin is the only iron efflux protein can export the iron out of cell,which process need hepaestin facilitating.We found both of them were decreased in cortex and hippocampus in the psychological stress rat brain.The iron export was blocked.As a result,iron was sequestered in neuron and glia cell.
4.Our data showed that the psychological stress induced interleukin-6 in rat brian, interleukin-6(IL-6) appears to stimulate the Hepcidin,which is the important upstream regulator of ferroportin.Hepcidin binds to ferroportin,inducing its internalization and degradation,iron export is blocked and iron is trapped within.We believe that interleukin-6 was the initial factor contribute to brain iron overloaded.
In conclusion,we found in the present study that the contents of iron and ferrous iron were increased in the cerebral cortex,hippocampus,and partial in striatum of rats exposed to psychological stress,accompanied by intense oxidative stress response and disorder of brain iron storage and efflux.We believe that as a systemic nerves -internal hormone—immunological network,psychological stress was against to stimulant,also influence the organism homeostasis,including iron metabolism.It may be the important reason resulting in brian dysfunction after excessive psychological stress.
研究发现,在一些神经变性性疾病,例如:AD(Alzheimer's disease)、PD(Parkinson's disease)、亨廷顿(Huntington's disease)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。现已证实,过量铁是导致上述神经退行性疾病发病的重要原因之一。但是,引起部分脑区铁蓄积的原因尚未完全阐明。
国内外有研究者发现,应激机体存在铁重新分布的现象。本室前期所做的一些实验也发现诸如晕船、运动和疲劳刺激可以使得铁元素在体内出现重新分布。我们的研究还发现,心理应激大鼠皮层、海马和纹状体内总铁含量以及非蛋白结合铁含量显著升高,该脑区铁调节蛋白1和转铁蛋白受体表达增多。而应激可通过提高GR和STAT5的转录调节活性,增加大鼠海马内铁调节蛋白1的表达量,进而使转铁蛋白受体表达量增加。这些结果显示,应激反应可影响脑组织铁摄取机制。但是,应激反应是否对铁的储存和释放也有影响还不清楚。本文为阐明此问题进行了系统的研究。
目的
了解心理应激对大鼠脑铁储存和释放蛋白的影响及其机制,为阐明心理应激与中枢神经退行性疾病的关系以及深入探讨军事应激损伤的生物学机制提供实验基础,并为预防以脑铁代谢紊乱为主要病理改变的疾病治疗提供新的思路。
方法
1.心理应激对大鼠脑内非血红素三价铁(NHF III)和非血红素二价铁(NHF II)的影响
1.1实验动物分组
雄性SD大鼠(购自上海西普尔-必凯公司),体重(120士5)g。按体重随机分为空白对照组(CG)、心理应激组(PSG)和足底电击组(FSG)。动物饲养实验室环境温度24℃±1℃,湿度50%～60%;使用不锈钢笼具单笼饲养,自由饮食,自然昼夜节律变化光照。
1.2大鼠心理应激模型的制作
采用Communication Box System制作大鼠心理应激模型。CommunicationBox System由透明丙烯酸板组成,一半小室(A室)底部铺板绝缘,另一半小室(B室)通电。在B室的实验大鼠接受30min/d,足底电击(电击信号由JL-B双脉冲电刺激器发生,经放大器产生电压70V,电流0.70mA);电击组大鼠跳跃,尖叫,在A室的实验大鼠通过视觉听觉产生恐惧的心理反应,即为心理应激大鼠模型。共7天。
1.3大鼠脑组织灌注铁染色
1.3.1大鼠脑组织普鲁士蓝灌注铁染色(perfusion-Prels method)
在第七天造模完成后即刻腹腔注射10%水合氯醛进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏插管用含有肝素钠(5单位/ml)的PBS溶液150ml冲洗血液,然后800ml含有1%亚铁氰化钾4%多聚甲醛灌流液(PH1.0)固定,取脑组织采用石蜡切片制成5μm切片用于铁染色。参照包新民大鼠脑立体定位图谱,在每只大鼠耳间线嘴侧3.6-3.8mm处行冠状切片显示大鼠海马;耳间线嘴侧7.8mm-8.0mm处冠状切片显示大鼠纹状体及额叶皮层。
1.3.2大鼠脑组织滕氏蓝灌注铁染色(perfusion-Turnbull method)
在第七天造模完成后即刻腹腔注射10%水合氯醛进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏插管用含有肝素钠(5单位/ml)的PBS溶液150ml冲洗血液,然后800ml含有1%铁氰化钾4%多聚甲醛灌流液(PH1.0)固定,取脑组织采用石蜡切片制成5μm切片用于铁染色。参照包新民大鼠脑立体定位图谱,在每只大鼠耳间线嘴侧3.6-3.8mm处行冠状切片显示大鼠海马;耳间线嘴侧7.8mm-8.0mm处冠状切片显示大鼠纹状体及额叶皮层。
2.心理应激对大鼠脑细胞铁储存蛋白和铁释放蛋白的影响
2.1实验动物分组及模型制作:同前
2.2心理应激对大鼠皮层、海马以及纹状体内膜铁转运蛋白Fp1(Ferroportin)、膜铁转运辅助蛋白HP(Hephaestin)mRNA表达的影响
采用实时定量荧光PCR法测定心理应激组和对照组大鼠皮层、海马以及纹状体内膜铁转运蛋白Fp1、膜铁转运辅助蛋白HP的mRNA表达水平。两组各取5只大鼠的皮层、海马和纹状体组织,按照试剂说明书用Trizol试剂抽提组织的总RNA,用10个单位的DNase I在37℃下处20ug RNA 30min。纯化后,RevertAid~TMFirst Strand cDNA synthesis Kit逆转录为cDNA。将1ul的cDNA加入含有10×PCRbuffer 2.5ul,25mM Mg~2+3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase0.5ul,10×SYBR-green I荧光染料0.7ul,5uM上下游引物2.0ul和DNAase freewater 12.8ul.反应体系中,总体积为25ul。引物由软件Primer Premier5.0设计。
2.3心理应激对大鼠皮层、海马以及纹状体内内膜铁转运蛋白Fp1(Ferroportin)、铁蛋白Fn(Ferritin)含量的影响
Western blot法测定实验组和对照组大鼠皮层、海马以及纹状体内Fp1以及Fn的含量。取含50ug蛋白的裂解液经10%非变性SDS—PAGE电泳分离后,电转移至硝酸纤维素膜。膜用脱脂奶粉室温封闭2h后,分别与1:1000兔抗人铁蛋白抗体、1:2000兔抗小鼠Fp1抗体、和1:2000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1 h。用ECL检测试剂检测膜上蛋白质,并用Quantity One软件进行条带分析。
3.心理应激大鼠脑细胞铁储存蛋白和铁释放蛋白改变机制的探讨
3.1实验动物分组及模型制作:同前
3.2心理应激对大鼠脑皮层、海马、纹状体内白介素-6(IL-6)、铁调素Hpec(Hepeidin)mRNA的含量影响
采用实时定量荧光PCR法测定心理应激组和对照组大鼠皮层、海马以及纹状体内铁调素Hpec、IL-6的mRNA表达水平。两组各取5只大鼠的皮层、海马和纹状体组织,按照试剂说明书用Trizol试剂抽提组织的总RNA,用10个单位的DNaseI在37℃下处理20ug RNA 30min。纯化后,RevertAid~TM First Strand cDNA synthesisKit逆转录为cDNA。将1ul的cDNA加入含有10xPCR buffer 2.5ul,25mM Mg~(2+)3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10xSYBR-green I荧光染料0.7ul,5uM上下游引物2.0ul和DNAase free water 12.8ul.反应体系中,总体积为25ul。引物由软件Primer Premier5.0设计。
4.数据的统计与处理
灌注铁染色图像数据采用image-pro plus 6.0图像分析软件进行灰度分析:铁离子染色阳性颗粒呈深棕黑到黑色,镜检每例组织,在额叶脑皮层次级区,纹状体尾壳核区(CPu区)海马多形细胞层随机选取8个视野,用显微图像分析软件Image-pro plus 6.0测定阳性目标的平均灰度值、视场平均灰度值、面积密度,换算成阳性单位(positive unit,PU),以PU值大小代表阳性产物表达的多少。同时观察各部位细胞形态。Western-blot蛋白条带数据扫描为Tiff格式图像,QuantityOne一维胶分析软件进行灰度分析得到相对浓度;实时荧光定量数据通rotor-gene6.0.14 software用2~△△CT方法得到相对浓度。
实验数据采用SPSS13.0统计软件包进行。采用两独立样本t检验进行数据分析,实验数据以平均数±标准差([x]±S)表示,P<0.05表示有统计学意义。
结果
1、心理应激对大鼠脑组织非血红素铁含量的影响
1.1心理应激对大鼠大脑皮层、海马、纹状体非血红素三价铁(NHF III)影响。
心理应激大鼠脑皮层、海马内NHF III染色较对照组显著升高(P<0.05),纹状体内NHF III染色未见明显改变。海马CA1,CA2区神经元及其周围明显NHFIII染色增强,神经元细胞的数目减少。在侧脑室前角与周围额叶皮层之间大量NHF III密集。
1.2心理应激对大鼠大脑皮层、海马、纹状体非血红素二价铁(NHF II)的影响
心理应激组大鼠皮层、海马、纹状体内NHF II染色较对照组显著升高,(P<0.05)。海马CA1,CA2区可见NHF II染色增强伴有神经元细胞的数目减少和结构松散。
2.心理应激对大鼠脑铁储存和释放蛋白的影响
2.1心理应激对大鼠皮层、海马以及纹状体Fp1、HPmRNA表达的影响
实时荧光定量PCR结果显示心理应激组大鼠皮层及海马内Fp1 mRNA含量较对照组显著降低(P<0.05),心理应激组纹状体内Fp1 mRNA含量较对照组有显著增加(P<0.05);心理应激组大鼠皮层、海马内HP mRNA含量较对照组明显降低(P<0.05)。纹状体内HP mRNA含量较对照组没有明显改变。
2.2心理应激对大鼠皮层、海马以及纹状体内Fp1、Fn含量的影响
Western-blot结果显示心理应激组大鼠皮层及海马内Fp1含量在较对照组比较显著降低(P<0.05);纹状体内Fp1含量较对照组有增高趋势无统计学意义(P>0.05)。心理应激组大鼠皮层、海马内Fn含量较对照组比较显著下降(P<0.05)。纹状体内Fn含量较对照组有增高趋势,无统计学意义(P>0.05)。
3.心理应激引起大鼠脑铁储存和释放蛋白表达变化的机制探讨
3.1心理应激对大鼠皮层、海马以及纹状体内Hpec mRNA的影响
实时荧光定量PCR结果显示:(1)正常情况下(未受到任何刺激)大鼠的皮层、海马和纹状体内都有Hepcidin mRNA的表达。皮层Hepcidin mRNA的表达丰度比海马和纹状体显著高,大约20-100倍。(2)心理应激组大鼠皮层HepcidinmRNA较对照组显著升高(P<0.05),海马Hepcidin mRNA较对照组有降低趋势(P>0.05),纹状体较对照组没有变化。
3.2心理应激对大鼠皮层、海马以及纹状体IL-6表达的影响
心理应激大鼠皮层、海马、纹状体内IL-6 mRNA较对照组显著升高(P<0.05)
结论
本研究通过整体动物实验发现:
1.灌注铁染色法结果显示,心理应激组大鼠皮层、海马内NHF III含量和NHFII含量显著升高,纹状体内NHF II含量升高但NHF III没有明显改变。铁离子在皮层、海马特定部位大量沉积,海马CA1,CA2区可见神经元神经元细胞数目减少。表明,心理应激可以导致非血红素结合铁在大鼠部分脑区含量增加。而二价铁离子升高而产生的过多的羟自由基可能是海马CA1,CA2区可见神经元神经元细胞数目减少的原因之一。
2.心理应激组大鼠皮层和海马内的Fn含量和HP mRNA表达减少,神经细胞内亚铁氧化酶减少(Fn的H链和HP),进而导致二价铁离子转化为三价铁离子受到影响,细胞内低分子量铁池扩大,二价铁离子增多。另一方面,增多的二价铁离子不能及时被氧化为三价铁离子而进入铁蛋白存储或进入其他细胞器利用,只能通过自发氧化形成三价铁离子,最终异常沉积于细胞内。上述机制可能是大鼠皮层和海马内的二价铁离子增多的原因。
3.心理应激可导致皮层、海马神经元和神经胶质细胞的铁释放蛋白Fp1减少,同时伴有膜铁转运蛋白辅助蛋白HP减少。表明,脑细胞内的铁释放蛋白受到影响而引起铁排除减少,也可能是神经细胞内铁沉积的原因之一。
4.心理应激导致海马、皮层、纹状体内细胞因子IL-6 mRNA的表达增多,皮层内Hepc mRNA表达增多。结合国内外相关研究结果,提示心理应激情况下IL-6表达的增加可能是脑皮层的Hepc mRNA表达增加的原因之一。
综上所述,本研究结果提示,心理应激可能通过上调炎性细胞因子IL-6mRNA的表达,调节脑皮层Hepc表达增加,增高的Hepc同其受体-脑细胞胞膜上Fp1结合,引起内陷,降解,细胞内的铁释放减少,而亚铁氧化酶膜铁转运辅助蛋白HP的减少加剧了这一过程。Fn和HP的减少引起神经细胞内二价铁离子增多,铁储存的障碍,进而引起细胞内氧化应激的加强,可能是海马内CA1,CA2区可见神经元神经元细胞数目减少的原因。因此,我们认为,应激作为一个全身性神经—内分泌—机体免疫网络,在对抗外界不良刺激的同时,也影响了机体内环境稳定,包括铁代谢稳定。而铁代谢紊乱所导致的脑细胞功能和结构损伤,可能是过度应激后造成健康危害的原因之一。
Iron is essential trace metal for almost all living organism by participating in a wide variety of metabolic process.Adequate iron is of great importance for organism growth,cell differentiation and multiply.Iron homeostasis is critical for normal brain growth and physiologic function.But excess iron can be deleterious.Iron can catalyze the production of hydroxyl radical through Fenton -reaction(Fe~(2+)+H_2O_2→Fe~(3+)+OH +OH-),and the Haber-Weiss reaction(Fe~(3+)+O_2→Fe~(2+)+O_2;Fe~(2+)+H_2O_2→Fe~(3+) +OH+OH-(iron catalayzed)) aggravating oxidative stress in the body and resulting in damage to tissues and cells,more and more researches indicate that excess iron accumulation in the body will bring a huge damage,especially iron overload in the central nervous system.Hence,the brain have elegant systems for mobilization and of iron sequestration.From serum iron crossing the blood-brain-barrier to neural cells uptaking iron,each process is strictly regulated.
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 the key point to death of neurons.But how iron is redistributed and accumulates in the brain is still not elucidated.With the progress of related research, more and more evidence to support the idea that iron efflux was also played a vital role in iron homeostasis regulation.
Previous researches including some experiments results our office did showed that motion sickness,sports and fatigue can make iron redistribution in the rat body.The facts indicate that mental stress may be one of reasons for the brain iron metabolism disorders.Our previous study on psychological stress rat reveal that some brain region with iron overload was up-regulated in IRP1 by enhancing the transcriptional regulation of GR and STAT5 on IRP1 gene,resulting in higher expression of IRP1 and consequently higher expression of TfR.We presume that psychological stress not only changes the iron uptaking but also changes the iron storage and iron efflux.The procedure of this study was as followed.
Objective
To study the characteristic effects of psychological stress on brain iron storage proteins and efflux proteins and establish a useful experimental basis for further study involving how stress changes cell normal iron homeostasis and the consequent effects on physiological function of the human body.
Method
1.Effects of psychological stress on NHFⅡ(nonheme ferrous iron )and NHFⅢ(nonheme ferric iron) concentrations in the rat brain
1.1 To divide experimental animals into groups
All experimental procedures involving animals received the approval from the Animal Care and Use Committee of the Second Military Medicine University. Guidelines and Policy on using and caring of the laboratory animals were followed at all time.Male SD rats(120±5g body weight) fed with a standard diet were purchased from the Shanghai-BK Ltd.Co,and were housed individually in a cage in a temperature-controlled room(24±1℃,55±5%humidity) with a 12-hour light and 12-hour dark cycle.After adaptation for 3 days,the rats were divided into the foot-shock group(FSG),psychological stress group(PSG) and the control group(CG). Each rat was exposed to stress for 30 minutes every day,successive 7 days.
1.2 To build psychological stress model of SD rats
Using a communication box system,footshock stress(FS) and psychological stress(PS) were administered to the rats.The communication box was divided into two parts with a transparent acrylic board,i.e.,Part A including ten rooms with a plastic board-covered floor for electric insulation and part B including ten rooms with a metal grid-exposed floor.Rats in part B were administered an electrical shock through the floor(90 V,0.8 mA for 1 second) randomly for 30 min,90 times in total, and then exhibited a nociceptive stimulation-evoked response such as jumping up, defecation and crying.Thus they were exposed to systemic(physical) stress.Rats in part A were not directly administered the electrical shock,but were exposed to psychological stress in response to the actions of the rats in Room B.
1.3 Perfusion-Perls and -Turnbull methods supplemented by DAB intensification for nonheme iron histochemistry in rats brian
1.3.1 Perfusion-Perls methods supplemented by DAB intensification for NHFⅢhistochemistry in rats brian
After successive 7 days ps treatment,The animals were anesthetized with intraperitoneal pentobarbital sodium(50 mg/kg) and transcardially perfused with the 130 ml of phosphate buffered saline(PBS,pH7.4) containing heparin(5 units/ml)for 5 min,to flush out the blood,then 800 ml of the solution(pH 1.0) containing 1% potassium ferrocyanide and 4%PFA in distilled water was perfused for tissue fixation and insute stain.After the perfusion,the brain was excised and cut off.To prepare 5 um thick sections,paraffin embedding and tissue cutting were done by the conventional methods.The sections were mounted on silan-coated slides,dewaxed and hydrated for further treatments.
1.3.2 Perfusion- Turnbull methods supplemented by DAB intensification for NHFⅡhistochemistry in rats brian
After successive 7 days ps treatment,The animals were anesthetized with intraperitoneal pentobarbital sodium(50 mg/kg) and transcardially perfused with the 130 ml of phosphate buffered saline(PBS,pH 7.4 ) containing heparin(5 units/ml)for 5 min,to flush out the blood,then 800 ml of the solution(pH 1.0) containing 1% potassium ferricyanide and 4%PFA in distilled water was perfused for tissue fixation and insute stain.After the perfusion,the brain was excised and cut off.To prepare 5 um thick sections,paraffin embedding and tissue cutting were done by the conventional methods.The sections were mounted on silan-coated slides,dewaxed and hydrated for further treatments.
2.Effects of psychological stress on brain iron storage protein and iron release protein
2.1 Determination of Ferroportin and Hephaestin mRNA levels
Real-time PCR determination of Ferroportin and Hephaestin mRNA:
RNA was extracted from rat cortex,hippocampus and striatum of both control and PS groups(n=5) with Trizol reagents according to the instructions of manufacturer. 20ug RNA was treated with 10 units of DNase 1 for 30min at 37℃.First strand cDNA was synthesized with M-MuLV Revers transcriptase after the purification of total RNA being incubated with Oligo dT primers.1 ul cDNA was added to a PCR system of 25ul containing 10×PCR buffer 2.5ul,25mM Mg~(2+) 3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10×SYBR-green I fluorescence dye 0.7ul,5uM forword primer and reverse primer 2.0ul and DNAase free water 12.8ul.(primers were designed with Primer Premier5.0 software).
2.2 Western blotting analysis of Ferroportin and Ferritin expression
Dissected tissues from the cortex,hippocampus and striatum from both control and PS groups(n=3) were homogenized separately by a dounce homogenizer in lysis buffer.Each group extractions 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,1:2000 diluted rabbit anti-rat Ferroportin antibody and 1:2000 mouse anti-humanβ-actin antibody for 1 hours at room temperature.Blots were washed 3 times with PBST 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 quantity one software.
3.Effects of molecular mechanisms of psychological stress on brain iron storage protein and iron efflux protein
Determination of Hepc mRNA and IL-6 mRNA levels
RNA was extracted from rat cortex,hippocampus and striatum of both control and PS groups(n=5) with Trizol reagents according to the instructions of manufacturer. 20ug RNA was treated with 10 units of DNase I for 30min at 37℃.First strand cDNA was synthesized with M-MuLV Revers transcriptase after the purification of total RNA being incubated with Oligo dT primers.1ul cDNA was added to a PCR system of 25ul containing 10×PCR buffer 2.5ul,25mM Mg~(2+) 3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10×SYBR-green I fluorescence dye 0.7ul,5uM forword primer and reverse primer 2.0ul and DNAase free water 12.8ul.(primers were designed with Primer Premier5.0 software).
4.Statistical analysis
Perfusion-Perls and -Turnbull methods for nonheme iron histochemistry image were analyzed by image-pro plus 6.0 software.Microscopic examination in ervry section and choose site as follows:Brain sub-district for cortex,Polymorphic cell layer for hippocampus and CPu region for striatum.Eight visual fields were selected by random.Accouding to the mean object gray scale and object area density,PU(positive units )were defined.The quantity of PU represent stained iron ion.Western-blot data were analyzed by Quantity One software.Real-time PCR data were analyzed by rotor-gene 6.0.14 software.
All results were expressed as mean±S.D.Statistical analysis was carried out by using SPSS 13.0 in independent sample T test method.All values below the detection limits were set to zero and absolute values without correction for recovery rate were used in analyses.A P value less than 0.05 was considered statistically significant.
Results
1.Psychological stress exposure increased both NHFⅢand NHFⅡstained positive units levels in some brain regions
1.1 Effects of psychological stress on NHFⅢconcentrations
We found that the NHFⅢstained positive units levels in the frontal cortex, hippocampus and were significantly higher in the PS exposure group than in the control group(P＜0.05);however,no significant higher was observed in NHFⅢstained positive units levels striatum between the two groups.(P＞0.05) The neurons of CA1,CA2 region in PS exposure group hippocampus were significantly stained stronger than in the control group,a great quantity of NHFⅢstained positive units were deposit in the area between anterior horn of lateral ventricle and frontal lobe,where in exposure group is significant higher than in the control group.
1.2 Effects of psychological stress on NHFⅡconcentrations
We found that the NHFⅡstained positive units levels in the frontal cortex, hippocampus and striatum were significantly higher in the PS exposure group than in the control group(P＜0.05).The CA1,CA2 region in PS exposure group hippocampus were stained stronger than that in the control group,the structure of pyramidal neuron cell boby were demolished can be observed,as well as the quantity of pyramidal neuron were diminished.
2.Effects of Psychological stress on brain iron storage protein and iron efflux protein
2.1 Psychological stress exposure caused changes in Ferroportin、Hephaestin mRNA
Real time-PCR analysis showed that PS exposure decreased Ferroportin mRNA levels in the cortex,hippocampus(P＜0.05) but increase Ferroportin mRNA in the striatum significantly(P＜0.05).The Hephaestin mRNA level of PS exposure group was significantly lower than that in the control group(P＜0.05),though the decrease was not significant in the striatum.
2.2 Psychological stress exposure caused changes in Ferroportin and Ferritin levels
Western blot analysis showed that Ferroportin concentrations levels in the cortex and hippocampus were significantly lower than those in the control group (P＜0.05),but a little higher in striatum(P＞0.05);Ferritin concentrations levels in the cortex and hippocampus were significantly lower than those in the control group (P＜0.05),but little higher in striatum(P＞0.05)
3.Effects of molecular mechanisms of psychological stress on brain iron storage protein and iron efflux protein
3.1 Effects of psychological stress on brain Hepcidin mRNA expression
Real time-PCR analysis showed that the level of cortex Hepcidin mRNA expression both in PS exposure group and control group were extremely higher than that in the hippocampus and striatum.PS exposure increased Hepcidin mRNA levels in the cortex significantly(P＜0.05),decreased in hippocampus(P＞0.05 )and no obvious changed in striatum.
3.2 Effects of psychological stress on brain IL-6 mRNA expression
Real time-PCR analysis manifested that Psychological stress exposure increased IL-6 mRNA levels in the cortex,hippocampus and striatum.significantly(P＜0.05)
Conclusions
1.Our study was built the psychological stress rats model by means of the Communication Box System.Take the advantage of the perfusion-Perls and -Turnbull methods supplemented by DAB intensification(+ DAB),we demonstrated visualization of nonheme ferric and ferrous iron in situ sensitively.We found that the NHFⅢstained positive units levels in the frontal cortex,hippocampus and were significantly higher in the PS exposure group than that in the control group,as well as the NHFⅡstained positive units levels.Furthermore,the structure of pyramidal neuron cell body were demolished can be observed in CA1 and CA2 region.All of these suggested psychological stress on rat brain lead to iron overload in specific encephalic region including cerebral cortex and hippocampus.The increasing of oxidative stress by NHFⅡmay be the reason to resulting the changes of hippocampus CA1,CA2 region.
2.We found that Ferritin concentrations levels in the cortex and hippocampus of psychological stress group were significantly lower while the Hephaestin mRNA levels and the Ferroportin concentrations levels and mRNA likewise.According to our previous research,psychological stress can evaluate iron intaking in cortex and hippocampus.Considerable iron ion was imported via DMT1,keeping themselves in divalence state.In cytosol,ferrous ion is considered to loosely complex with low-molecular weight organic bases forming the so-called low-molecular weight Iron pool(LIP),which contains some ferric iron as due to cytosolic oxidation.Because of its reactivity and cytotoxicity,the LIP size is maintained in very small amount.We speculate that LIP was expanded,meanwhile,ferritin(which has a ferroxidase core in H chain) and hephaestin(ferroxidase)were reduced,ferrous iron could not be oxidized into ferric iron immediately for storage in ferritin or final use in mitochondria and other organelles.Finally,ferrous iron was auto-oxidized to trivalence state and in a abnormal deposit form in cell.
3.Ferroportin is the only iron efflux protein can export the iron out of cell,which process need hepaestin facilitating.We found both of them were decreased in cortex and hippocampus in the psychological stress rat brain.The iron export was blocked.As a result,iron was sequestered in neuron and glia cell.
4.Our data showed that the psychological stress induced interleukin-6 in rat brian, interleukin-6(IL-6) appears to stimulate the Hepcidin,which is the important upstream regulator of ferroportin.Hepcidin binds to ferroportin,inducing its internalization and degradation,iron export is blocked and iron is trapped within.We believe that interleukin-6 was the initial factor contribute to brain iron overloaded.
In conclusion,we found in the present study that the contents of iron and ferrous iron were increased in the cerebral cortex,hippocampus,and partial in striatum of rats exposed to psychological stress,accompanied by intense oxidative stress response and disorder of brain iron storage and efflux.We believe that as a systemic nerves -internal hormone—immunological network,psychological stress was against to stimulant,also influence the organism homeostasis,including iron metabolism.It may be the important reason resulting in brian dysfunction after excessive psychological stress.
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78.Weinstein DA,Roy CN,Fleming MD,et al.Inappropriate expression of hepeidin is associated with iron refractory anemia:implications for the anemia of chronic disease.Blood,2002,100:3776
79.NicolasG,ChauvetC,Viatte L'et al.The gene encoding the iron regulatory peptide hepeidin is regulated by anemia,hypoxia,an d inflammation.J Clin Invest.2002.110:1037
80.Kulaksiz H,Theilig F,Bachmann S et al.The iron-regulatory peptide hormone hepcidin:expression and cellular localization in mammalian kidney.Journal of Endocrinolog,2005,184:361—370.
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70．蒋达和,钱忠明．小肠铁吸收机制及相关疾病研究进展．中华医学杂志,2001,811：1353—1355
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74.Hunter HN,Fulton DB,Ganz T.The solution structure of human hepcidin,a peptide-hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis[J].J Biol Chem.2002,277:37597—37603
75.Courselaud B,Pigeon C,InoueY,etal;C/EBP alpha regulates hepatic transcription of hepcidin,an antimicrobial peptide and regulator of iron metabolism crosstalk between C / EBP pathway and iron metabolism[J].J Biol Chem,2002,277:41163—41170
76.NicolasG,BennounM,Devauxl,etal.Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2(USF2)knockout mice[J].Proc Natl Aead Sci USA,2001,98(15):8780—8785.
77.Abboud S,Halle DJ.A novel mammalian iron regulated protein involved in intracellular iron metabolism[J].J Biol Chem,2000,275(26):19906—19912.
78.Weinstein DA,Roy CN,Fleming MD,et al.Inappropriate expression of hepeidin is associated with iron refractory anemia:implications for the anemia of chronic disease.Blood,2002,100:3776
79.NicolasG,ChauvetC,Viatte L'et al.The gene encoding the iron regulatory peptide hepeidin is regulated by anemia,hypoxia,an d inflammation.J Clin Invest.2002.110:1037
80.Kulaksiz H,Theilig F,Bachmann S et al.The iron-regulatory peptide hormone hepcidin:expression and cellular localization in mammalian kidney.Journal of Endocrinolog,2005,184:361—370.
81．常彦忠,段相林,钱忠明．Hepcidin和铁稳态,中华内分泌代谢杂志,2003,19(6)：502～503
82.Lee P,PengH,Gelbart T et al The IL-6 and lipopolysaccharide-induced transcription of hepcidin in HFE-,transferrin receptor2-,and beta 2-microglobulin-deficient hepatocytes[J].PNAS,2004;101:9263-9265.
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84.Dzikaite V,Holmstrom P,Stal Petal.Regulatory effects of tumor necrosis factor-alpha and interleukin-6 on HAMP expression in iron loaded rat hepatocytes[J].J Hepatol,2006;44(3):54-551.
85.Nemeth E,Rivera S,Gabayan V et al IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin[J].J Clin Invest,2004;113:1271-1276
86.Carbia-Nagashima A,Arzt E.Intracellular proteins and mechanisms involved in the control of gp130/JAK/STAT cytokine signaIing[J].IUBMB Life,2004;56:83-88.
87.WangR H,Li C,Xu X et al A role of SMAD4 in iron metabolism throngh the positive regulation of hepcidin expression[J].CellMetab,20O5;2:399-409.
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89.Weinstein DA,Roy CN,Fleming M D,etal.Inappropriate expression of hepeidin is associated with iron refractory anemia:implication for the anemia of chronic disease.Blood。2002,100(10):3776—3781.
90.SraiSK.HFE,a putative hepatocyte iron sensor?Blood,2004,103(4):1177.
91.ClarkeAT,Cadet E,BomfordA,etal.Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis.Hum Mol Genet.2003,12(17):2241—2247.
92.Nicolas G,Viatte L,Lou DQ,et al.Constitutive hepcidin expression prevents iron overload in a mouse model ofhemocromatosis.Nat Genet.2003.34(1):97—101.
93.Jacolot S,Gac GL,Scotet V.HAMP as a modifier gene that increases the phenotypie expression of the HFE pC282Y homozygous genotype.Blood,2004,103(7):2835—2840.