心理应激对脑铁代谢的影响及其机制的探讨
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摘要
铁是一切生命体必不可缺少的微量元素,由于铁元素参与了多种酶和辅基的构成,保证足够的铁元素含量对于所有哺乳动物、植物和微生物的生存、繁衍和分化都是极为重要的。然而,近年来大量的研究发现铁在局部的沉积将对机体产生巨大的毒害作用,特别是铁过剩现象出现在中枢神经系统的组织和细胞,甚至是神经细胞的细胞器内,将导致中枢神经系统发生不可逆转的病理性改变,而导致脑铁代谢紊乱的原因却不甚清楚。研究表明脑区内铁浓度增高以及铁代谢相关蛋白发生改变直接参与神经元的损伤,并且作为中枢神经退行性病变发生前的特征性的病理性表现。
以往的研究包括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的现象,同时也有研究发现处于长期应激状态的大鼠在海马CA2和CA3区出现神经元变性死亡的现象,因此强烈提示心理应激可能是导致铁元素在脑内重新分布和铁调节因素发生紊乱的重要原因。军人作为一个特殊的职业,经常面对各种突发性事件的挑战,军事应激损伤已被确定为部队非战斗减员的主要原因,阐明应激对铁代谢的影响对防治军人应激损伤有积极的意义。
目的
了解心理应激对大鼠脑铁代谢影响及其机制,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,并为预防临床上以脑铁代谢乱为主要病理改变的疾病治疗提供新的思路。
方法
1.心理应激对大鼠脑铁含量的影响
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±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),每组10只。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。Communication BoxSystem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(电压90V,电流0.80mA):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。
ELISA法测定血清促肾上腺皮质激素(ACTH)和皮质酮(CORT)含量,使用放免试剂盒测定下丘脑内去甲肾上腺素(NE)含量。
1.3脑组织铁含量的测定
1.3.1大鼠全脑以及各脑区铁含量的测定
采用原子吸收分光光度计(日本日立公司Z-2000型)火焰法测定。100μg/ml铁标准贮备液(GBW08616)由国家标准物质中心提供。
1.3.2大鼠皮层、海马、纹状体、小脑以及脑干内非蛋白结合铁的测定
取各脑区组织50mg,采用BPS法测定。
1.3.3大鼠皮层perl's铁染色
在第七天造模完成后即刻腹腔注射2%戊巴比妥进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏冷PBS冲洗10 mins,灌流固定,取脑组织采用石蜡切片制成30μm切片用于perl's铁染色。
2.心理应激对大鼠脑铁代谢机制的影响
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TfR1、Fn 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、海马以及纹状体内转铁蛋白受体1以及铁蛋白mRNA的表达水平
2.3心理应激对大鼠皮层、海马以及纹状体内TfR1、Fn含量的影响
ELISA法测定实验组和对照组大鼠皮层、海马以及纹状体内转铁蛋白受体1以及铁蛋白的含量
2.4心理应激对大鼠各脑区IRP1以及Lf表达的影响
Western Blot检测两组大鼠皮层、海马以及纹状体内铁调节蛋白1以及乳铁蛋白的表达情况
3.心理应激对大鼠脑内抗氧化应激机制的影响
3.1实验动物分组及模型制作:同前
3.2心理应激对大鼠各脑区SOD活性的影响:
使用SOD分析试剂盒.WST(水溶性四唑盐)试剂盒测定心理应激组与对照组大鼠皮层、海马、纹状体、小脑以及脑干内SOD的活性。
3.3心理应激对大鼠各脑区GSH含量的影响:
总谷胱甘肽测定试剂盒测定心理应激组与对照组大鼠皮层、海马、纹状体、小脑以及脑干内总GSH含量。
3.4心理应激对大鼠各脑区HO-1表达的影响
WESTERNBLOT法检测两组大鼠皮层、海马以及纹状体内HO-1的表达情况。
3.5心理应激对大鼠各脑区MDA含量的影响
丙二醛(MDA)测定试剂盒测定心理应激组和对照组大鼠皮层、海马以及纹状体内MDA的含量。
4.心理应激引起大鼠脑铁代谢改变机制的探讨
4.1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±5)g,用于侧脑室注射α-CRF进行应激激素对大鼠脑铁代谢影响的研究。按体重随机分为ACF-心理应激组(P+ACF)、α-CRF-心理应激组(P+CRF)、ACF-对照组(P+ACF)和α-CRF-对照组(P+CRF),每组10只。
4.2心理应激模型的制作:同前
4.3细胞实验
选用原代培养的新生大鼠皮层神经细胞,通过给予糖皮质激素(1.0μmol/L)刺激建立应激细胞模型,同时在细胞培养基中加入一氧化氮合酶抑制剂L精氨酸和氨基胍(浓度分别为1.5μmol/L和1.2μmol/L)。
4.4大鼠脑组织NOS阳性神经元活性的测定
在第七天造模完成后即刻腹腔注射2%戊巴比妥进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏冷PBS冲洗,分离固定脑组织,冰冻切片,片厚40 um,收集于0.1 mol/L Tris-HC1 buffer(pH 7.6)中备用。染色方法为NADPH-d酶组织化学法:经Tris HC1 buffer漂洗5 min×3次,入孵育液37℃水浴1-2 h。对照液:无NADPH的孵育液。阳性神经元可分为强阳性、中等和弱阳性三种类型。强阳性NOS神经元染成深蓝色,弱阳性NOS神经元着色较淡,中等强度介于二者之间。统计NOS阳性细胞数,算出每个视野的均数。
4.5 CCK-8法测定神经元细胞的活性
4.6 Griess法测定神经元细胞内NO代谢物的含量
4.7电感耦合等离子体质谱(ICP-MS)分析法测定细胞内铁含量
4.8采用凝胶阻滞电泳法测定神经元内IRE反应元件结合活性
5.数据的统计与处理
实验数据采用SPSS10.0统计软件包采用one—way ANOVA检验进行数据分析,实验数据以平均数±标准差((?)±S)表示,显著性水平为P＜0.05,非常显著性水平为P＜0.01。
结果
1.心理应激对大鼠脑铁含量的影响
1.1大鼠血清CORT、ACTH含量以及下丘脑NE含量的测定结果
心理应激组大鼠血清CORT、ACTH含量与下丘脑NE含量较对照组明显升高(P＜0.05)。
1.2心理应激对大鼠全脑、皮层、海马、纹状体、小脑和脑干铁含量的影响
心理应激组大鼠全脑铁含量与对照组无差别,皮层、海马内铁含量较对照组比较有显著上升(P＜0.05),升高幅度分别达到50.88%和24.35%;心理应激组大鼠脑干铁含量显著低于对照组(P＜0.05),下降幅度达23.66%;心理应激组大鼠小脑铁含量与对照组无显著差异(P＞0.05)。
1.3心理应激对大鼠皮层、海马、纹状体、小脑和脑干NPBI含量的影响
心理应激组大鼠大脑皮层,海马以及纹状体内NPBI含量均显著高于对照组(P＜0.05);而脑干及小脑较对照组比较有下降的趋势,但没有统计学差异(P＞0.05)。
1.4心理应激对大鼠皮层中铁分布情况的影响
心理应激组大鼠大脑皮层运动区的神经元周围有大量铁颗粒的沉积,胶质细胞铁染色呈阳性。
2.心理应激对大鼠脑铁代谢机制的影响
2.1心理应激对大鼠皮层、海马以及纹状体内TfR1、Fn mRNA表达的影响
心理应激组大鼠海马及纹状体内TfR1 mRNA含量较对照组显著升高(P＜0.05),心理应激组皮层TfR1 mRNA含量有增加的趋势但没有显著性(P＞0.05);心理应激组大鼠皮层及纹状体内Fn mRNA含量较对照组明显降低(P＜0.05)。
2.2心理应激对大鼠皮层、海马以及纹状体内TfR1、Fn含量的影响
心理应激大鼠TfR1含量在皮层及海马内较对照组比较显著升高(P＜0.05);而心理应激组Fn含量在大脑、海马及纹状体较对照组比较显著下降(P＜0.05)。
2.3心理应激对大鼠皮层、海马以及纹状体内IRP1以及Lf表达的影响
心理应激导致大鼠皮层、海马以及纹状体内IRP1表达显著增加,而Lf在心理应激大鼠海马内表达显著增加。
3.心理应激对大鼠脑内抗氧化应激机制的影响
3.1心理应激对大鼠皮层、海马、纹状体、脑干和小脑内SOD活性的影响
心理应激组大鼠皮层、海马以及脑干内SOD活性均显著下降(P＜0.05);而SOD活性心理应激组大鼠纹状体内则显著上升(P＜0.05);心理应激组与对照组大鼠小脑内SOD活性没有明显的统计学差异(P＞0.05)。
3.2心理应激对大鼠皮层、海马、纹状体、脑干和小脑内总GSH含量的影响
心理应激组与对照组大鼠各脑区的总GSH含量没有明显的统计学差异(P＞0.05)。
3.3心理应激对大鼠皮层、海马以及纹状体内MDA含量的影响
心理应激组大鼠大脑皮层、海马以及纹状体内MDA含量较对照组比较均显著升高(P＜0.05)。
3.4心理应激对大鼠皮层、海马以及纹状体内HO-1的表达情况:
HO-1在心理应激大鼠皮层、海马以及纹状体内表达显著增加。
4.心理应激引起大鼠脑铁代谢改变的机制探讨
4.1应激激素对大鼠皮层、海马以及纹状体内铁含量的影响
侧脑室注射α-CRF的心理应激大鼠与注射ACF的心理应激大鼠、注射α-CRF和ACF的对照组大鼠在海马以及纹状体内铁含量均显著下降(P＜0.05);而侧脑室注射α-CRF的心理应激大鼠与注射ACF的心理应激大鼠、注射α-CRF和ACF的对照组大鼠皮层铁含量有降低的趋势:而侧脑室注射α-CRF的心理应激大鼠海马铁含量对照组比较铁含量显著升高(P＜0.05)。
4.2心理应激对大鼠脑组织NOS阳性神经元活性和数量的影响
心理应激组大鼠皮层、海马CA3区以及纹状体尾状核内NOS阳性神经元活性和数目较对照组显著升高(P＜0.05)。
4.3糖皮质激素和NOS抑制剂对大鼠皮层神经元NO含量的影响
糖皮质激素显著提高皮层神经元内NO代谢物的含量(P＜0.05);而CORT/L-NAME组和CORT/AG组皮层神经元之间NO代谢物的含量并没有明显的差别(P＞0.05),但较对照组比较含量则显著下降(P＜0.05)。
4.4糖皮质激素和NOS抑制剂对大鼠皮层神经元铁含量的影响
糖皮质激素具有显著提高皮层神经元内铁含量的作用(P＜0.05);而CORT/L-NAME组和CORT/AG组之间神经元铁含量并没有明显的差别,但较对照组比较含量显著下降(P＜0.05)。
4.5糖皮质激素和NOS抑制剂对大鼠皮层神经元内IRE反应元件结合活性的影响
糖皮质激素的刺激可显著提高神经元内IRE的结合活性,而两种NOS抑制剂则显著降低了神经元内IRE的结合活性。
结论
1.本研究所采用Communication Box System成功复制了大鼠的心理应激模型。心理应激在未改变全脑铁含量的情况下,大鼠皮层、纹状体以及海马内总铁含量以及毒性游离铁离子含量显著升高,铁染色的结果也发现铁在皮层组织间隙大量沉积,铁染色阳性神经元增多,表明心理应激可以导致铁元素在大鼠部分脑区含量增加。
2.导致心理应激大鼠某些脑区内铁沉积的原因是铁调控因素发生变化:虽然铁在部分脑区含量增加,但主要功能为储存铁的铁蛋白的含量并没有预期的增加反而是下降的,转铁蛋白受体1含量却是增加的,而铁调节蛋白1在心理应激组大鼠皮层、海马以及纹状体内表达升高,基于文献报道IRP1在脑区内的高表达将引起TfR1 mRNA和Fn mRNA含量升高。我们还发现心理应激导致海马内另一种重要的金属转运体乳铁蛋白的表达升高,而心理应激后海马铁含量的升高要比其他区域程度更高,同时也有研究发现长期的抑郁将导致海马体积的减少同时伴有大量神经元的空泡变性,因此海马极有可能是由心理应激所引起的铁代谢紊乱后引起氧化应激损伤的易受到攻击的部位。
3.本研究发现抵御超氧自由基作为脑内抗氧化应激的第一道防御屏障的SOD活性在铁升高的那些脑区内的活性也是变化的,同时HO-1的表达以及脂质过氧化产物MDA含量也随之升高,提示心理应激导致部分脑区铁元素含量增加后引起了氧化应激损伤。
4.心理应激时HPA轴的激活是导致大鼠部分脑区铁含量升高的重要原因,因为我们在细胞实验中发现糖皮质激素对神经元内铁含量的增高有着重要的作用,糖皮质激素影响细胞铁含量则主要是通过促进细胞内NO的含量,导致铁反应元件活性增强,从而细胞内铁的吸收增强导致铁含量增加。而一氧化氮是导致心理应激大鼠局部脑区需铁量增加的一个重要因素:心理应激大鼠皮层、海马以及纹状体一氧化氮合酶阳性神经元活性和含量显著增加,基于我们的研究结果以及文献报道,心理应激的持续作用将导致NO从细胞内动员铁,造成铁的剥夺而激活IRP1结合活性,其结果导致Fn mRNA合成减少,TfR mRNA稳定性增加,从而引起心理应激大鼠部分脑区铁含量增加。
综上所述,本研究发现心理应激导致大鼠皮层、海马以及纹状体内总铁含量以及游离铁含量均增加,同时伴随着氧化应激损伤,由于心理应激导致局部脑区需铁量增加后激活铁调节因素的改变是导致心理应激大鼠脑铁含量增加的原因。因此我们认为应激作为一个全身性神经一内分泌反应,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 for vital cellular activities and as such is required to be present in a readily available form.However,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:oxidative damage is implied in the neurodegenerative diseases.Iron, especially non-protein-bound-iron(NPBI) catalyzes the formation of toxic hydroxyl radicals.Thus,iron toxicity can lead to the CNS irreversible pathological results.Previous researches including some experiments results our office did showed that motion sickness, sports and fatigue can make iron redistribution in the rat body,and we concluded previous researches and associated with modern social work tension,life pressure or study competition made people always be a more or less compressive stress.The exposure of animals to long-term stress produces deleterious effects on the brain,morphological neuronal damage in the CA2 and CA3 subfields of the hippocampus,an extensive literature demonstrate that glucocorticoids secreted during stress have a broad range of deleterious effects in the brain.Hence we guess:mental stress may be one of reasons for the brain iron metabolism disorders.
Objective
To study the characteristic effects of psychological stress on brain iron metabolism and to 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 iron concentrations in the rat brain 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±5 g 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.
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.
Measurement of serum CORT and ACTH,hypothalamus NE levels in rats under stress
The concentrations of serum CORT and ACTH were measured by the enzyme-linked immunoassays(ELISAs) kit;the hypothalamus NE levels were measured by the radioimmunofocus assays(RI) kit.
Measurement of brain iron concentrations in rats 1.4.1 Determination of total iron in rats
Iron concentrations were determined using a Varian SpectrAA-220G graphite furnace atomic absorption spectrometer equipped with a GTA 110 atomizer,programmable sample dispenser,and deuterium background correction.Standard addition method was used for calibration.Standards and control samples were prepared in an identical manner to the experimental samples.
1.4.2 Determination of NPBI levels in rats
NPBI levels were analyzed by a method using bathophcnanthroline disulfonate (BPS) to chelate ferrous iron,thus forming a complex that could be analyzed with spectrophotometry.Dissected brain tissues were homogenized in a glass homogenizer in 10 vol of 50 mmol/L phosphate buffer,pH 7.4.
1.4.3 Perl's iron staining
For Perl's staining,sections were processed through a series of graded alcohols,into xylene,and rehydrated back to water.Sections were counterstained with Neutral Red, dehydrated in increasing concentrations of ethanol,cleared in xylene,and mounted on slides.
2.Effects of Psychological Stress on Brain Iron Metabolism
2.1 Determination of TfR1 and Fn mRNA levels
Real time Q-RT-PCR was performed using IQ5 Real-Time PCR Detection System. Two step RT-PCR method was performed using Real Time PCR Master Mix.Primers used to analyze all the transcripts have been reported else where.The Q-RT-PCR data were analyzed by 2-~(ΔΔ)AACT method as described
2.2 Determination of TfR1 and Fn levels
The concentrations of TfR1 and Fn in the cortex,hippocampus and striatum samples were assessed using a commercially available ELISA kitswith the absorbance read on a microplate reader at a wavelength of 450 nm.
2.3 Western blotting analysis of IRP-1 and Lf expression
Dissected tissues from the cortex,hippocampus and striatum were homogenized separately by a dounce homogenizer in lysis buffer.Proteins were incubated overnight at 4℃with a primary antibody against IRP-1(monoclonal,1:1000,Santa),Lf(rabbit polyclonal,1:500,Santa),orβ-actin(rabbit polyclonal,1:10000,Sigma).The blots were developed by incubation in ECL chemiluminescence reagent and subsequently exposed to BioMax Light Film.
3.Effects of Psychological Stress on Brain oxidative status
3.1 Measurement of SOD activity in rat brain
Tissue samples were preserved in 50μl of 5 mM butylated hydroxytoluene to prevent further lipid peroxidation.SOD activity was measured using WST-1 kit with the absorbance read on a microplate reader at a wavelength of 450 nm.SOD activity of each region were normalized to wet tissue weight(mg) and expressed asμmol/mg.
3.2 Measurement of GSH levels in rat brain
GSH level was measured using kit with the absorbance read on a microplate reader at a wavelength of 490 nm.GSH concentration of each region were normalized to wet tissue weight(mg) and expressed asμmol/mg.
3.3 Measurement of MDA concentrations in rat cerebral cortex,hippocampus and striatum
MDA concentration was assessed using a MDA assay kit with the absorbance read on a microplate reader at a wavelength of 586 nm.MDA concentration of each region were normalized to wet tissue weight(mg) and expressed asμg/mg.
3.4 Western blotting analysis of HO-1 expression
Dissected tissues from the cortex,hippocampus and striatum were homogenized separately by a dounce homogenizer in lysis buffer.Proteins were incubated overnight at 4℃with a primary antibody against HO-1(monoclonal,1:1000,Santa) orβ-actin(rabbit polyclonal,1:10000,Sigma).
4.Effects of molecular mechanisms of psychological stress on brain iron metabolism
4.1 Effects stress hormones on brain iron contents
Male SD rats(120±5g body weight) were divided into the ACF-psychological stress group(P+ACF),α-CRF-psychological stress group(P+CRF),ACF-control group (P+ACF) andα-CRF-control group(P+CRF).Each rat was exposed to stress for 30 minutes every day.Iron concentrations were also determined using a Varian SpectrAA-220G graphite furnace atomic absorption spectrometer.
4.2 Measurement of the number and activity of nitric oxide synthase(NOS) positive neurons
The sections were processed using the NADPH-diaphorase(NADPH-d) histochemical method.NOS positive neurons were counted in 4 fields of the cerebral cortex,hippocampal CA3 and caudate putamen.Data were analyzed by One-way ANOVA.
4.3 Cell preparation for intraceUular NO and iron analysis
The primarily cultured cortical neurons were plated into in a poly 1-lysine-coated 6-well plates at a density of about 1×106 cells/ml.The cells were treated with corticosterone(CORT,1μmol/L) or corticosterone/ L-NAME or corticosterone/ AG (CORT,1μmol/l;L-NAME 1.5μmol/L)(CORT,1μmol/l;AG 1.2μmol/L) for 24 h. Untreated neurons served as control.Counting Kit-8 was used to count the cells in three groups.
4.4 NO measurement
NO production was assayed by measuring the nitrite concentrations with the Griess assays.Plates wcrc incubated at 25℃for 10 min,and the absorbance at 550 nm was measured with a microplatc reader.Nitrite concentrations wcrc calculated with sodium nitrite standard curve as a reference.
4.5 Analysis of intracellular iron
The experiments were carried out with a quadrupole ICP-MS X7 equipped with collision cell technology.A blank sample was included for baseline check for every run. Standards and control samples were prepared in an identical manner to the samples.
a) Measurement of IRE binding activity
We used gel retardation to detect the IRE binding activity in the neuron,and the autoradiographic images were analyzed by immunosorbent assay method. Statistical analysis
All results were expressed as mean±SE.Statistical analysis was carried out by using SPSS 11.0.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.PS exposure increased the iron concentrations in some brain regions
1.1 Determination of the PS model estabolishment
We found that the levels of serum ACTH,corticosterone and hypothalamus noradrenalin in the model animals were significantly higher than those in the control animals(P＜0.05),indicating the successful of model establishment.
1.2 Effects of psychological stress on iron concentrations
We found that the iron levels in the right frontal cortex,hippocampus and striatum were significantly higher in the PS exposure group than in the control group(P＜0.05); however,no significant difference was observed in iron levels in the whole brain and the cerebellum between the two groups.Moreover,the iron level in the brain stem of the PS exposure group was significantly lower than that of the control group(P＜0.05)
1.3 Effects of psychological stress on NPBI levels
We also found that the concentrations of NPBI in the right frontal cortex, hippocampus and striatum were significantly higher in the PS exposure group than in the control group(P＜0.05,P＜0.01 for hippocampus),and no significant difference was observed in NPBI concentration in the brain stem and cerebellum between the two groups.
1.4 Iron staining results in the cortex
Perl's iron staining revealed weak staining for iron in the cortex of control group, and very strong staining in the cortex of PS rats.
2.Effects of Psychological Stress on Brain Iron Metabolism
2.1 PS exposure caused changes in TfR1 and Fn mRNA
Real time-PCR analysis showed that PS exposure increased TfR1 mRNA levels in the cortex,hippocampus and striatum(P＜0.05 for hippocampus and striatum),though the increase in the cortex was not significant;the Fn mRNA level of PS exposure group was significantly lower than that in the control group(P＜0.01 for cortex,P＜0.05 for striatum), though the decrease was not significant in the hippocampus.
2.2 PS exposure caused changes in TfR1 and Fn levels
TfR1 levels in the cortex,stfiatum and hippocampus were significantly higher than those in the control group(P＜0.05);Fn concentrations in the cortex and hippocampus were significantly lower in PS exposure group than in the control group(P＜0.05);and the Fn concentration in the striatum was also lower than that of the control group,but the difference was not significant.
2.3 PS exposure increased IRP1 and Lf expression in different parts of rat brain
Western blot analysis showed that PS exposure increased IRP-1 immunoreactivity in the cortex,hippocampus and striatum in the rat brain;and the expression of Lf was also increased in the hippocampus of the PS-exposed rats.
3.PS exposure intensified the oxidative reaction in rat brain
3.1 SOD activities in rat brain of two groups
SOD activities in the cortex and hippocampus in the PS group were significantly lower than those in the control group(P＜0.01 for cortex,P＜0.05 for hippocampus);SOD activity in the striatum was significantly higher in the PS exposure group than in the control group(P＜0.01).No significant difference in SOD activities in the cerebellums and brain stem was observed between the two groups.
3.2 MDA levels in rat brain of two groups
We also found that MDA levels were significantly increased in the cortex, hippocampus and striatum of the PS exposure group compared to the control group (P＜0.01).
4.Effects of molecular mechanisms of psychological stress on brain iron metabolism
4.1 PS exposure increased NOS positive neurons in different brain regions
The number of NOS positive neurons in the cerebral cortex,hippocampal CA3 and caudate putamen were significantly higher in PS-exposed rats than in the control animals(P＜0.05).
4.2 Effects of CORT and NOS inhibitors on NO concentrations in primarily cultured nerve cells
The NO production of the CORT cultured nerve cells was increased significantly compared to the control group and L-NAME cultured cells(P＜0.05);and the NO production of the NOS inhibitor cultured nerve cells was decreased significantly compared to the control group(P＜0.05).
4.3 Effects of CORT and NOS inhibitors on iron concentrations in primarily cultured nerve cells
Iron concentrations of the CORT cultured nerve cells were increased significantly compared to the control group and the NOS inhibitor group(P＜0.05);and the iron concentrations of the NOS inhibitor cultured nerve cells were decreased significantly compared to the control group(P＜0.05).
Conclusions
1.we speculate that stress might have caused change in normal iron metabolism,and our speculation was confirmed in this study:We found that,although PS did not alter the total content of brain iron under normal dietary iron levels,the iron contents were increased in the cerebral cortex,striatum,and hippocampus,which happen to be the regions involved in degeneration diseases.The staining result of iron also revealed iron deposition in the cerebral cortex.Therefore,it can be concluded that the iron concentrations are increased in some specific regions of the brain after PS exposure.
2.We believe that PS induces iron deposition in certain cerebral regions by changing the iron regulation factors.Although we found that there was iron accumulation in some areas of the PS rats brain,there was no compensatory increase in Fn protein.More interestingly,there was an increase in TfR1 levels as would not be expected under these conditions.When TfR1 levels were increased,the cell can increase uptake of iron from extra cellular transferrin.Increased IRP1 expression in the cortex,hippocampus and striatum of PS-exposed rats might have caused the changes in TfR1 mRNA and Fn mRNA.In addition,we noticed that PS exposure also caused higher expression of Lf, another important metal transporter,in the hippocampus;and the iron deposition in the hippocampus after PS exposure was significantly higher than those of other regions. Studies have found that long-term depression could lead to decreased volume and neuron death in the hippocampus.Hence we speculate that the hippocampus is most vulnerable brain parts to the oxidative stress induced by PS-associated disorder of iron metabolism, which deserves further study in the future.
3.Our data showed that PS did induced oxidative damages in some regions of rat brain:SOD activity,which catalyzes the breakdown of superoxide radicals and provides the first line defense against oxygen toxicity,had undergone changes,and the MDA level, which is a by-product of the lipid peroxidation process.
4.We believe that NO is an important factor for the increased iron demand in local cerebral regions of PS-exposed rats.The increased number of NOS positive neurons will unavoidably lead to the increase of NO secretion.Glucocorticoids can greatly influence NO diffusion to different brain areas and NO is very important to protect the neurons under stress.Our results showed that corticosterone increased the NO production in cortex nerve cells,and NOS inhibitor decreased the NO production in the corticosterone cultured cells.Meanwhile,we also found that iron concentrations were significantly increased in the corticosterone treated cells and significantly decreased in NOS inhibitor treated ceils.Therefore,we believe that the activation NO induced by glucocorticoids under background of stress is an important reason for the upregulation of iron in some brain areas.
In conclusion,we found in the present study that the contents of iron and NPBI were both increased in the cerebral cortex,hippocampus,and striatum of rats exposed to PS,accompanied by intense oxidative stress response,which is caused by PS-induced increase of local iron demand and the subsequent activation of iron regulation system. We believe that PS-induced location iron deposition and subsequent intensification of oxidative stress response is one of the important reasons for neurodegenerative disease.
以往的研究包括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的现象,同时也有研究发现处于长期应激状态的大鼠在海马CA2和CA3区出现神经元变性死亡的现象,因此强烈提示心理应激可能是导致铁元素在脑内重新分布和铁调节因素发生紊乱的重要原因。军人作为一个特殊的职业,经常面对各种突发性事件的挑战,军事应激损伤已被确定为部队非战斗减员的主要原因,阐明应激对铁代谢的影响对防治军人应激损伤有积极的意义。
目的
了解心理应激对大鼠脑铁代谢影响及其机制,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,并为预防临床上以脑铁代谢乱为主要病理改变的疾病治疗提供新的思路。
方法
1.心理应激对大鼠脑铁含量的影响
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±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),每组10只。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。Communication BoxSystem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(电压90V,电流0.80mA):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。
ELISA法测定血清促肾上腺皮质激素(ACTH)和皮质酮(CORT)含量,使用放免试剂盒测定下丘脑内去甲肾上腺素(NE)含量。
1.3脑组织铁含量的测定
1.3.1大鼠全脑以及各脑区铁含量的测定
采用原子吸收分光光度计(日本日立公司Z-2000型)火焰法测定。100μg/ml铁标准贮备液(GBW08616)由国家标准物质中心提供。
1.3.2大鼠皮层、海马、纹状体、小脑以及脑干内非蛋白结合铁的测定
取各脑区组织50mg,采用BPS法测定。
1.3.3大鼠皮层perl's铁染色
在第七天造模完成后即刻腹腔注射2%戊巴比妥进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏冷PBS冲洗10 mins,灌流固定,取脑组织采用石蜡切片制成30μm切片用于perl's铁染色。
2.心理应激对大鼠脑铁代谢机制的影响
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TfR1、Fn 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、海马以及纹状体内转铁蛋白受体1以及铁蛋白mRNA的表达水平
2.3心理应激对大鼠皮层、海马以及纹状体内TfR1、Fn含量的影响
ELISA法测定实验组和对照组大鼠皮层、海马以及纹状体内转铁蛋白受体1以及铁蛋白的含量
2.4心理应激对大鼠各脑区IRP1以及Lf表达的影响
Western Blot检测两组大鼠皮层、海马以及纹状体内铁调节蛋白1以及乳铁蛋白的表达情况
3.心理应激对大鼠脑内抗氧化应激机制的影响
3.1实验动物分组及模型制作:同前
3.2心理应激对大鼠各脑区SOD活性的影响:
使用SOD分析试剂盒.WST(水溶性四唑盐)试剂盒测定心理应激组与对照组大鼠皮层、海马、纹状体、小脑以及脑干内SOD的活性。
3.3心理应激对大鼠各脑区GSH含量的影响:
总谷胱甘肽测定试剂盒测定心理应激组与对照组大鼠皮层、海马、纹状体、小脑以及脑干内总GSH含量。
3.4心理应激对大鼠各脑区HO-1表达的影响
WESTERNBLOT法检测两组大鼠皮层、海马以及纹状体内HO-1的表达情况。
3.5心理应激对大鼠各脑区MDA含量的影响
丙二醛(MDA)测定试剂盒测定心理应激组和对照组大鼠皮层、海马以及纹状体内MDA的含量。
4.心理应激引起大鼠脑铁代谢改变机制的探讨
4.1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±5)g,用于侧脑室注射α-CRF进行应激激素对大鼠脑铁代谢影响的研究。按体重随机分为ACF-心理应激组(P+ACF)、α-CRF-心理应激组(P+CRF)、ACF-对照组(P+ACF)和α-CRF-对照组(P+CRF),每组10只。
4.2心理应激模型的制作:同前
4.3细胞实验
选用原代培养的新生大鼠皮层神经细胞,通过给予糖皮质激素(1.0μmol/L)刺激建立应激细胞模型,同时在细胞培养基中加入一氧化氮合酶抑制剂L精氨酸和氨基胍(浓度分别为1.5μmol/L和1.2μmol/L)。
4.4大鼠脑组织NOS阳性神经元活性的测定
在第七天造模完成后即刻腹腔注射2%戊巴比妥进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏冷PBS冲洗,分离固定脑组织,冰冻切片,片厚40 um,收集于0.1 mol/L Tris-HC1 buffer(pH 7.6)中备用。染色方法为NADPH-d酶组织化学法:经Tris HC1 buffer漂洗5 min×3次,入孵育液37℃水浴1-2 h。对照液:无NADPH的孵育液。阳性神经元可分为强阳性、中等和弱阳性三种类型。强阳性NOS神经元染成深蓝色,弱阳性NOS神经元着色较淡,中等强度介于二者之间。统计NOS阳性细胞数,算出每个视野的均数。
4.5 CCK-8法测定神经元细胞的活性
4.6 Griess法测定神经元细胞内NO代谢物的含量
4.7电感耦合等离子体质谱(ICP-MS)分析法测定细胞内铁含量
4.8采用凝胶阻滞电泳法测定神经元内IRE反应元件结合活性
5.数据的统计与处理
实验数据采用SPSS10.0统计软件包采用one—way ANOVA检验进行数据分析,实验数据以平均数±标准差((?)±S)表示,显著性水平为P＜0.05,非常显著性水平为P＜0.01。
结果
1.心理应激对大鼠脑铁含量的影响
1.1大鼠血清CORT、ACTH含量以及下丘脑NE含量的测定结果
心理应激组大鼠血清CORT、ACTH含量与下丘脑NE含量较对照组明显升高(P＜0.05)。
1.2心理应激对大鼠全脑、皮层、海马、纹状体、小脑和脑干铁含量的影响
心理应激组大鼠全脑铁含量与对照组无差别,皮层、海马内铁含量较对照组比较有显著上升(P＜0.05),升高幅度分别达到50.88%和24.35%;心理应激组大鼠脑干铁含量显著低于对照组(P＜0.05),下降幅度达23.66%;心理应激组大鼠小脑铁含量与对照组无显著差异(P＞0.05)。
1.3心理应激对大鼠皮层、海马、纹状体、小脑和脑干NPBI含量的影响
心理应激组大鼠大脑皮层,海马以及纹状体内NPBI含量均显著高于对照组(P＜0.05);而脑干及小脑较对照组比较有下降的趋势,但没有统计学差异(P＞0.05)。
1.4心理应激对大鼠皮层中铁分布情况的影响
心理应激组大鼠大脑皮层运动区的神经元周围有大量铁颗粒的沉积,胶质细胞铁染色呈阳性。
2.心理应激对大鼠脑铁代谢机制的影响
2.1心理应激对大鼠皮层、海马以及纹状体内TfR1、Fn mRNA表达的影响
心理应激组大鼠海马及纹状体内TfR1 mRNA含量较对照组显著升高(P＜0.05),心理应激组皮层TfR1 mRNA含量有增加的趋势但没有显著性(P＞0.05);心理应激组大鼠皮层及纹状体内Fn mRNA含量较对照组明显降低(P＜0.05)。
2.2心理应激对大鼠皮层、海马以及纹状体内TfR1、Fn含量的影响
心理应激大鼠TfR1含量在皮层及海马内较对照组比较显著升高(P＜0.05);而心理应激组Fn含量在大脑、海马及纹状体较对照组比较显著下降(P＜0.05)。
2.3心理应激对大鼠皮层、海马以及纹状体内IRP1以及Lf表达的影响
心理应激导致大鼠皮层、海马以及纹状体内IRP1表达显著增加,而Lf在心理应激大鼠海马内表达显著增加。
3.心理应激对大鼠脑内抗氧化应激机制的影响
3.1心理应激对大鼠皮层、海马、纹状体、脑干和小脑内SOD活性的影响
心理应激组大鼠皮层、海马以及脑干内SOD活性均显著下降(P＜0.05);而SOD活性心理应激组大鼠纹状体内则显著上升(P＜0.05);心理应激组与对照组大鼠小脑内SOD活性没有明显的统计学差异(P＞0.05)。
3.2心理应激对大鼠皮层、海马、纹状体、脑干和小脑内总GSH含量的影响
心理应激组与对照组大鼠各脑区的总GSH含量没有明显的统计学差异(P＞0.05)。
3.3心理应激对大鼠皮层、海马以及纹状体内MDA含量的影响
心理应激组大鼠大脑皮层、海马以及纹状体内MDA含量较对照组比较均显著升高(P＜0.05)。
3.4心理应激对大鼠皮层、海马以及纹状体内HO-1的表达情况:
HO-1在心理应激大鼠皮层、海马以及纹状体内表达显著增加。
4.心理应激引起大鼠脑铁代谢改变的机制探讨
4.1应激激素对大鼠皮层、海马以及纹状体内铁含量的影响
侧脑室注射α-CRF的心理应激大鼠与注射ACF的心理应激大鼠、注射α-CRF和ACF的对照组大鼠在海马以及纹状体内铁含量均显著下降(P＜0.05);而侧脑室注射α-CRF的心理应激大鼠与注射ACF的心理应激大鼠、注射α-CRF和ACF的对照组大鼠皮层铁含量有降低的趋势:而侧脑室注射α-CRF的心理应激大鼠海马铁含量对照组比较铁含量显著升高(P＜0.05)。
4.2心理应激对大鼠脑组织NOS阳性神经元活性和数量的影响
心理应激组大鼠皮层、海马CA3区以及纹状体尾状核内NOS阳性神经元活性和数目较对照组显著升高(P＜0.05)。
4.3糖皮质激素和NOS抑制剂对大鼠皮层神经元NO含量的影响
糖皮质激素显著提高皮层神经元内NO代谢物的含量(P＜0.05);而CORT/L-NAME组和CORT/AG组皮层神经元之间NO代谢物的含量并没有明显的差别(P＞0.05),但较对照组比较含量则显著下降(P＜0.05)。
4.4糖皮质激素和NOS抑制剂对大鼠皮层神经元铁含量的影响
糖皮质激素具有显著提高皮层神经元内铁含量的作用(P＜0.05);而CORT/L-NAME组和CORT/AG组之间神经元铁含量并没有明显的差别,但较对照组比较含量显著下降(P＜0.05)。
4.5糖皮质激素和NOS抑制剂对大鼠皮层神经元内IRE反应元件结合活性的影响
糖皮质激素的刺激可显著提高神经元内IRE的结合活性,而两种NOS抑制剂则显著降低了神经元内IRE的结合活性。
结论
1.本研究所采用Communication Box System成功复制了大鼠的心理应激模型。心理应激在未改变全脑铁含量的情况下,大鼠皮层、纹状体以及海马内总铁含量以及毒性游离铁离子含量显著升高,铁染色的结果也发现铁在皮层组织间隙大量沉积,铁染色阳性神经元增多,表明心理应激可以导致铁元素在大鼠部分脑区含量增加。
2.导致心理应激大鼠某些脑区内铁沉积的原因是铁调控因素发生变化:虽然铁在部分脑区含量增加,但主要功能为储存铁的铁蛋白的含量并没有预期的增加反而是下降的,转铁蛋白受体1含量却是增加的,而铁调节蛋白1在心理应激组大鼠皮层、海马以及纹状体内表达升高,基于文献报道IRP1在脑区内的高表达将引起TfR1 mRNA和Fn mRNA含量升高。我们还发现心理应激导致海马内另一种重要的金属转运体乳铁蛋白的表达升高,而心理应激后海马铁含量的升高要比其他区域程度更高,同时也有研究发现长期的抑郁将导致海马体积的减少同时伴有大量神经元的空泡变性,因此海马极有可能是由心理应激所引起的铁代谢紊乱后引起氧化应激损伤的易受到攻击的部位。
3.本研究发现抵御超氧自由基作为脑内抗氧化应激的第一道防御屏障的SOD活性在铁升高的那些脑区内的活性也是变化的,同时HO-1的表达以及脂质过氧化产物MDA含量也随之升高,提示心理应激导致部分脑区铁元素含量增加后引起了氧化应激损伤。
4.心理应激时HPA轴的激活是导致大鼠部分脑区铁含量升高的重要原因,因为我们在细胞实验中发现糖皮质激素对神经元内铁含量的增高有着重要的作用,糖皮质激素影响细胞铁含量则主要是通过促进细胞内NO的含量,导致铁反应元件活性增强,从而细胞内铁的吸收增强导致铁含量增加。而一氧化氮是导致心理应激大鼠局部脑区需铁量增加的一个重要因素:心理应激大鼠皮层、海马以及纹状体一氧化氮合酶阳性神经元活性和含量显著增加,基于我们的研究结果以及文献报道,心理应激的持续作用将导致NO从细胞内动员铁,造成铁的剥夺而激活IRP1结合活性,其结果导致Fn mRNA合成减少,TfR mRNA稳定性增加,从而引起心理应激大鼠部分脑区铁含量增加。
综上所述,本研究发现心理应激导致大鼠皮层、海马以及纹状体内总铁含量以及游离铁含量均增加,同时伴随着氧化应激损伤,由于心理应激导致局部脑区需铁量增加后激活铁调节因素的改变是导致心理应激大鼠脑铁含量增加的原因。因此我们认为应激作为一个全身性神经一内分泌反应,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 for vital cellular activities and as such is required to be present in a readily available form.However,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:oxidative damage is implied in the neurodegenerative diseases.Iron, especially non-protein-bound-iron(NPBI) catalyzes the formation of toxic hydroxyl radicals.Thus,iron toxicity can lead to the CNS irreversible pathological results.Previous researches including some experiments results our office did showed that motion sickness, sports and fatigue can make iron redistribution in the rat body,and we concluded previous researches and associated with modern social work tension,life pressure or study competition made people always be a more or less compressive stress.The exposure of animals to long-term stress produces deleterious effects on the brain,morphological neuronal damage in the CA2 and CA3 subfields of the hippocampus,an extensive literature demonstrate that glucocorticoids secreted during stress have a broad range of deleterious effects in the brain.Hence we guess:mental stress may be one of reasons for the brain iron metabolism disorders.
Objective
To study the characteristic effects of psychological stress on brain iron metabolism and to 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 iron concentrations in the rat brain 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±5 g 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.
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.
Measurement of serum CORT and ACTH,hypothalamus NE levels in rats under stress
The concentrations of serum CORT and ACTH were measured by the enzyme-linked immunoassays(ELISAs) kit;the hypothalamus NE levels were measured by the radioimmunofocus assays(RI) kit.
Measurement of brain iron concentrations in rats 1.4.1 Determination of total iron in rats
Iron concentrations were determined using a Varian SpectrAA-220G graphite furnace atomic absorption spectrometer equipped with a GTA 110 atomizer,programmable sample dispenser,and deuterium background correction.Standard addition method was used for calibration.Standards and control samples were prepared in an identical manner to the experimental samples.
1.4.2 Determination of NPBI levels in rats
NPBI levels were analyzed by a method using bathophcnanthroline disulfonate (BPS) to chelate ferrous iron,thus forming a complex that could be analyzed with spectrophotometry.Dissected brain tissues were homogenized in a glass homogenizer in 10 vol of 50 mmol/L phosphate buffer,pH 7.4.
1.4.3 Perl's iron staining
For Perl's staining,sections were processed through a series of graded alcohols,into xylene,and rehydrated back to water.Sections were counterstained with Neutral Red, dehydrated in increasing concentrations of ethanol,cleared in xylene,and mounted on slides.
2.Effects of Psychological Stress on Brain Iron Metabolism
2.1 Determination of TfR1 and Fn mRNA levels
Real time Q-RT-PCR was performed using IQ5 Real-Time PCR Detection System. Two step RT-PCR method was performed using Real Time PCR Master Mix.Primers used to analyze all the transcripts have been reported else where.The Q-RT-PCR data were analyzed by 2-~(ΔΔ)AACT method as described
2.2 Determination of TfR1 and Fn levels
The concentrations of TfR1 and Fn in the cortex,hippocampus and striatum samples were assessed using a commercially available ELISA kitswith the absorbance read on a microplate reader at a wavelength of 450 nm.
2.3 Western blotting analysis of IRP-1 and Lf expression
Dissected tissues from the cortex,hippocampus and striatum were homogenized separately by a dounce homogenizer in lysis buffer.Proteins were incubated overnight at 4℃with a primary antibody against IRP-1(monoclonal,1:1000,Santa),Lf(rabbit polyclonal,1:500,Santa),orβ-actin(rabbit polyclonal,1:10000,Sigma).The blots were developed by incubation in ECL chemiluminescence reagent and subsequently exposed to BioMax Light Film.
3.Effects of Psychological Stress on Brain oxidative status
3.1 Measurement of SOD activity in rat brain
Tissue samples were preserved in 50μl of 5 mM butylated hydroxytoluene to prevent further lipid peroxidation.SOD activity was measured using WST-1 kit with the absorbance read on a microplate reader at a wavelength of 450 nm.SOD activity of each region were normalized to wet tissue weight(mg) and expressed asμmol/mg.
3.2 Measurement of GSH levels in rat brain
GSH level was measured using kit with the absorbance read on a microplate reader at a wavelength of 490 nm.GSH concentration of each region were normalized to wet tissue weight(mg) and expressed asμmol/mg.
3.3 Measurement of MDA concentrations in rat cerebral cortex,hippocampus and striatum
MDA concentration was assessed using a MDA assay kit with the absorbance read on a microplate reader at a wavelength of 586 nm.MDA concentration of each region were normalized to wet tissue weight(mg) and expressed asμg/mg.
3.4 Western blotting analysis of HO-1 expression
Dissected tissues from the cortex,hippocampus and striatum were homogenized separately by a dounce homogenizer in lysis buffer.Proteins were incubated overnight at 4℃with a primary antibody against HO-1(monoclonal,1:1000,Santa) orβ-actin(rabbit polyclonal,1:10000,Sigma).
4.Effects of molecular mechanisms of psychological stress on brain iron metabolism
4.1 Effects stress hormones on brain iron contents
Male SD rats(120±5g body weight) were divided into the ACF-psychological stress group(P+ACF),α-CRF-psychological stress group(P+CRF),ACF-control group (P+ACF) andα-CRF-control group(P+CRF).Each rat was exposed to stress for 30 minutes every day.Iron concentrations were also determined using a Varian SpectrAA-220G graphite furnace atomic absorption spectrometer.
4.2 Measurement of the number and activity of nitric oxide synthase(NOS) positive neurons
The sections were processed using the NADPH-diaphorase(NADPH-d) histochemical method.NOS positive neurons were counted in 4 fields of the cerebral cortex,hippocampal CA3 and caudate putamen.Data were analyzed by One-way ANOVA.
4.3 Cell preparation for intraceUular NO and iron analysis
The primarily cultured cortical neurons were plated into in a poly 1-lysine-coated 6-well plates at a density of about 1×106 cells/ml.The cells were treated with corticosterone(CORT,1μmol/L) or corticosterone/ L-NAME or corticosterone/ AG (CORT,1μmol/l;L-NAME 1.5μmol/L)(CORT,1μmol/l;AG 1.2μmol/L) for 24 h. Untreated neurons served as control.Counting Kit-8 was used to count the cells in three groups.
4.4 NO measurement
NO production was assayed by measuring the nitrite concentrations with the Griess assays.Plates wcrc incubated at 25℃for 10 min,and the absorbance at 550 nm was measured with a microplatc reader.Nitrite concentrations wcrc calculated with sodium nitrite standard curve as a reference.
4.5 Analysis of intracellular iron
The experiments were carried out with a quadrupole ICP-MS X7 equipped with collision cell technology.A blank sample was included for baseline check for every run. Standards and control samples were prepared in an identical manner to the samples.
a) Measurement of IRE binding activity
We used gel retardation to detect the IRE binding activity in the neuron,and the autoradiographic images were analyzed by immunosorbent assay method. Statistical analysis
All results were expressed as mean±SE.Statistical analysis was carried out by using SPSS 11.0.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.PS exposure increased the iron concentrations in some brain regions
1.1 Determination of the PS model estabolishment
We found that the levels of serum ACTH,corticosterone and hypothalamus noradrenalin in the model animals were significantly higher than those in the control animals(P＜0.05),indicating the successful of model establishment.
1.2 Effects of psychological stress on iron concentrations
We found that the iron levels in the right frontal cortex,hippocampus and striatum were significantly higher in the PS exposure group than in the control group(P＜0.05); however,no significant difference was observed in iron levels in the whole brain and the cerebellum between the two groups.Moreover,the iron level in the brain stem of the PS exposure group was significantly lower than that of the control group(P＜0.05)
1.3 Effects of psychological stress on NPBI levels
We also found that the concentrations of NPBI in the right frontal cortex, hippocampus and striatum were significantly higher in the PS exposure group than in the control group(P＜0.05,P＜0.01 for hippocampus),and no significant difference was observed in NPBI concentration in the brain stem and cerebellum between the two groups.
1.4 Iron staining results in the cortex
Perl's iron staining revealed weak staining for iron in the cortex of control group, and very strong staining in the cortex of PS rats.
2.Effects of Psychological Stress on Brain Iron Metabolism
2.1 PS exposure caused changes in TfR1 and Fn mRNA
Real time-PCR analysis showed that PS exposure increased TfR1 mRNA levels in the cortex,hippocampus and striatum(P＜0.05 for hippocampus and striatum),though the increase in the cortex was not significant;the Fn mRNA level of PS exposure group was significantly lower than that in the control group(P＜0.01 for cortex,P＜0.05 for striatum), though the decrease was not significant in the hippocampus.
2.2 PS exposure caused changes in TfR1 and Fn levels
TfR1 levels in the cortex,stfiatum and hippocampus were significantly higher than those in the control group(P＜0.05);Fn concentrations in the cortex and hippocampus were significantly lower in PS exposure group than in the control group(P＜0.05);and the Fn concentration in the striatum was also lower than that of the control group,but the difference was not significant.
2.3 PS exposure increased IRP1 and Lf expression in different parts of rat brain
Western blot analysis showed that PS exposure increased IRP-1 immunoreactivity in the cortex,hippocampus and striatum in the rat brain;and the expression of Lf was also increased in the hippocampus of the PS-exposed rats.
3.PS exposure intensified the oxidative reaction in rat brain
3.1 SOD activities in rat brain of two groups
SOD activities in the cortex and hippocampus in the PS group were significantly lower than those in the control group(P＜0.01 for cortex,P＜0.05 for hippocampus);SOD activity in the striatum was significantly higher in the PS exposure group than in the control group(P＜0.01).No significant difference in SOD activities in the cerebellums and brain stem was observed between the two groups.
3.2 MDA levels in rat brain of two groups
We also found that MDA levels were significantly increased in the cortex, hippocampus and striatum of the PS exposure group compared to the control group (P＜0.01).
4.Effects of molecular mechanisms of psychological stress on brain iron metabolism
4.1 PS exposure increased NOS positive neurons in different brain regions
The number of NOS positive neurons in the cerebral cortex,hippocampal CA3 and caudate putamen were significantly higher in PS-exposed rats than in the control animals(P＜0.05).
4.2 Effects of CORT and NOS inhibitors on NO concentrations in primarily cultured nerve cells
The NO production of the CORT cultured nerve cells was increased significantly compared to the control group and L-NAME cultured cells(P＜0.05);and the NO production of the NOS inhibitor cultured nerve cells was decreased significantly compared to the control group(P＜0.05).
4.3 Effects of CORT and NOS inhibitors on iron concentrations in primarily cultured nerve cells
Iron concentrations of the CORT cultured nerve cells were increased significantly compared to the control group and the NOS inhibitor group(P＜0.05);and the iron concentrations of the NOS inhibitor cultured nerve cells were decreased significantly compared to the control group(P＜0.05).
Conclusions
1.we speculate that stress might have caused change in normal iron metabolism,and our speculation was confirmed in this study:We found that,although PS did not alter the total content of brain iron under normal dietary iron levels,the iron contents were increased in the cerebral cortex,striatum,and hippocampus,which happen to be the regions involved in degeneration diseases.The staining result of iron also revealed iron deposition in the cerebral cortex.Therefore,it can be concluded that the iron concentrations are increased in some specific regions of the brain after PS exposure.
2.We believe that PS induces iron deposition in certain cerebral regions by changing the iron regulation factors.Although we found that there was iron accumulation in some areas of the PS rats brain,there was no compensatory increase in Fn protein.More interestingly,there was an increase in TfR1 levels as would not be expected under these conditions.When TfR1 levels were increased,the cell can increase uptake of iron from extra cellular transferrin.Increased IRP1 expression in the cortex,hippocampus and striatum of PS-exposed rats might have caused the changes in TfR1 mRNA and Fn mRNA.In addition,we noticed that PS exposure also caused higher expression of Lf, another important metal transporter,in the hippocampus;and the iron deposition in the hippocampus after PS exposure was significantly higher than those of other regions. Studies have found that long-term depression could lead to decreased volume and neuron death in the hippocampus.Hence we speculate that the hippocampus is most vulnerable brain parts to the oxidative stress induced by PS-associated disorder of iron metabolism, which deserves further study in the future.
3.Our data showed that PS did induced oxidative damages in some regions of rat brain:SOD activity,which catalyzes the breakdown of superoxide radicals and provides the first line defense against oxygen toxicity,had undergone changes,and the MDA level, which is a by-product of the lipid peroxidation process.
4.We believe that NO is an important factor for the increased iron demand in local cerebral regions of PS-exposed rats.The increased number of NOS positive neurons will unavoidably lead to the increase of NO secretion.Glucocorticoids can greatly influence NO diffusion to different brain areas and NO is very important to protect the neurons under stress.Our results showed that corticosterone increased the NO production in cortex nerve cells,and NOS inhibitor decreased the NO production in the corticosterone cultured cells.Meanwhile,we also found that iron concentrations were significantly increased in the corticosterone treated cells and significantly decreased in NOS inhibitor treated ceils.Therefore,we believe that the activation NO induced by glucocorticoids under background of stress is an important reason for the upregulation of iron in some brain areas.
In conclusion,we found in the present study that the contents of iron and NPBI were both increased in the cerebral cortex,hippocampus,and striatum of rats exposed to PS,accompanied by intense oxidative stress response,which is caused by PS-induced increase of local iron demand and the subsequent activation of iron regulation system. We believe that PS-induced location iron deposition and subsequent intensification of oxidative stress response is one of the important reasons for neurodegenerative disease.
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1.Caltagirone A,Weiss G,Pantopoulos K.Modulation of cellular iron metabolism by hydrogen peroxide.Effects of H_2O_2 on the expression and function of iron-responsive element-containing mRNAs in B6 fibroblasts.J Biol Chem,2001(276):19738-19745.
2.Nunez-Millacura C,Tapia V,Munoz P,Maccioni RB,Nunez MT.An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells.J.Neurochem 2002(82):240-248.
3.Bush,AI.The metallobiology of Alzheimer's disease.Trends Neurosci,2003(26):207-214.
4.Andersen JK.Iron dysregulation and Parkinson's disease.J Alzheimers Dis,2004(6 Suppl):S47-52.
5.Berg D,Youdim MB.Role of iron in neurodegenerative disorders.Top Magn Reson Irnaging,2006(17):5-17.
6.Hochstein P,Ernster L ADP-activated lipid peroxidation coupled to the TPNH oxidase system of microsomes.Biochera.Biophys.Res.Common,1963(12):388-394.
7.Hochstein P,Nordenbrand K,Ernster LEvidence for the involvement of iron in the ADP-activated peroxidation of lipids in microsomes and mitochondria.Biochem.Biophys.Res.Cornmon,1964(14):323-328.
8.Nunez-Millacura C,Tapia V,Munoz P,Maccioni RB,Nunez MT.An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells.J.Neurochem,2002(82):240-248.
9.Millan-Plano S,Garcia JJ,Martinez-Ballarin E,Reiter RJ,Orteqa-Gutierrez S,Lazaro RM,Escanero JF.Melatonin and pinoline prevent aluminium-induced lipid peroxidation in rat synaptosomes.J Trace Elem Med Biol,2003(17):39-44.
10.Topal T,Oter S,Korkmaz A,Sadir S,Metinyurt G,Korkmazhan ET,Serdar MA,Bilgic H,Reiter RJ.Exogenously administered and endogenously produced melatonin reduce hyperbaric oxygen-induced oxidative stress in rat lung.Life Sci,2004(75):461-467.
11.Ischiropoulos H,Beckman JS.Oxidative stress and nitration in neurodegeneration:cause,effect,or association? J Clin Invest,2003(111):163-169.
12.Schipper HM.Heme oxygenase-1:role in brainaging and neurodegeneration.Exp Gerontol,2000(35):821-830.
13.高谦.血红素加氧酶系统的分子特性及其在脑中的作用[J].国外医学生理、病理科学与临床分册,2002(22):174-176.
14.Atwood CS,Obrenovich ME,Liu T,Chan H,Perry G,Smith MA,Martins RN.Amyloid-beta:a chameleon walking in two worlds:a review of the trophic and toxic properties of amyloid-beta.Brain Res Brain Res Rev,2003(43):1-16.
15.Bush,AI.The metallobiology of Alzheimer's disease.Trends Neurosci,2003(26):207-214.
1.Pu YM,Wang Q,Tang PL,Qian ZM.Effect of iron and lipid peroxidation on development of cerebellar granule cell in vitro.Neuroscience,1999(89):855-861.
2.Qian ZM,Shen X.Brain iron transport and neurodegeneration.Trends Mol Med,2001(7):103-108.
3.Heather MY,Brien AJ,Fumes JB.Relationships between NADPH diaphorase staining and neuronal endothelial,and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissue.Histochem Cell Bio,1997(1107):19-29.
4.包新民,舒斯云.大鼠脑立体定位图谱.北京:人民卫生出版社,1991:33-41.
5.刘辉,陈俊抛,田时雨,谭盛,高曲文.神经元型一氧化氮合酶在学习记忆过程中的变化和作用.中国神经免疫学和神经病学杂志.2000(7):116-121.
7.Chrousos GP,Gold PW.The concepts of stress and stress system disorders.JAMA,1992(267):1244-1252.
8.Irwin M.Stress-induced immune suppression.Ann N Y Acad Sci,1993(697):203-218.
9.Baram TZ,Hatalski CG.Neuropeptide-mediated excitability:a key triggering mechanism for seizure generation in the developing brain.Trends Neurosci,1998(21):471-476.
10.Leibold EA,GuoB.Iron-dependent regulation of ferritin and transferrin receptor expression by the iron-responsive element binding proteins.Ann Rev Nutr,1992(12):345-368.
11.Kira S,Ponka P.Control of transferrin receptor expression via nitric oxide-mediated modulation of iron-regulatory protein 2.J Biol Chem 1999(274):33035-33042.
12.Kim S,Ponka P.Effects of interferon- γ and lipopolysaccharide on macrophage iron metabolism are mediatded by nitric oxide-induced degradation of iron regulatory protein 2.J Biol Chem,2000(275):6220-6226.
13.Wardrop SL,Watts RN,Richardson DR.Nitrogen monoxide activates iron regulatory protein 1,RNA-binding activity by two possible mechanisms:effect an the[4Fe-4s]cluster and iron mobilization from cells.Biochemistry,2000(39):2748-2758.
14.金玉祥,陈嘉峰,胡波,韩三春,王绍.应激性防御反应过程中脑内一氧化氮合酶阳性神经元分布的变化.神经解剖学杂志,2001(16):29-32.
[1]Qian ZM,Wang Q.(1998) Expression of iron transport proteins and excessive iron accumulation,in the brain in neurodegenerative disorders.Brain Res Rev,27:257-267.
[2]Richardson DR,Ponka P(1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells.Biochim Biophys Acta 1331:1-40.
[3]Andersen JK(2004) Iron dysregulation and Parkinson's disease.J Alzheimers Dis.12:6(6 Suppl):S47-52.
[4]Berg D,Youdim MB(2006) Role of iron in neurodegenerative disorders.Top Magn Reson Imaging 17:5-17.
[5]Qian ZM,Pu YM,Tang PL,Wang Q(1998)Transferrin-bound iron uptake by the cultured cerebellar granule cells.Neurosci Lett 251:9-12.
[6]Qian ZM,Shen X(2001) Brain iron transport and neurodegeneration.Trends Mol Med 7:103-108.
[7]Pantopoulos K(2004) Iron metabolism and the IRE/IRP regulatory system:an update.Ann N Y Acad Sci,1012:1-13.
[8]Lee DW,Andersen JK,Kaur D(2006) Iron dysregulation and neurodegeneration:the molecular connection.Mol Interv 6:89-97.
[9]Caltagirone A,Weiss G,Pantopoulos K(2001) Modulation of cellular iron metabolism by hydrogen peroxide.Effects of H_2O_2 on the expression and function of iron-responsive element-containing mRNAs in B6 fibroblasts. J Biol Chem 276:19738-19745.
[10] Rouault TA (2001) Iron on the brain. Nat Genet 28:299-300.
[11] Gutteridge JM (1992) Iron and oxygen radicals in brain. Ann Neural 32:S16-21.
[12] Fredriksson A, Schroder N, Eriksson P, Izquierdo I, Archer T (2001) Neonatal iron potentiates adult MPTP-induced neurodegenerative and functional deficits. Parkinsonism Relat Disord 7: 97-105.
[13] Richardson DR, Ponka P (1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta 1331:1-40.
[14] Zhou B, Westaway SK, Levinson B, Johnson MA, Gitschier J, Hayflick SJ (2001) A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nat Genet 28:345-349.
[15] Hellman NE, Kono S, Miyajima H, Gitlin JD. Biochemical analysis of a missense mutation in aceruloplasminemia.J Biol Chem 2002; 277:1375-80.
[16] Rebeck GW, Harr SD, Strickland DK, Hyman BT. (1995) Multiple, diverse senileplaque-associated proteinsare ligands of an apolipoprotein E receptor, thealpha-macroglobulin receptor/low densitylipoprotein receptor-related protein. Ann Neurol 37: 211-217.
[17] Schipper HM (2000) Heme oxygenase-1: role in brain aging and neurodegeneration. Exp Gerontol 35: 821-830.
[18] Cairo G, Pietrangelo A (2000) Iron regulatory proteins in pathobiology. Biochem. J 352:241-250.
[19] Mehlhase J, Sandig G, Pantopoulos K, Grune T (2005) Oxidation-induced ferritin turnover in microglial cells: role of proteasome. Free Radical Biol Med 38:276-285.
[20] Martins, EA, Robalinho, RL, Meneghini, R (1995) Oxidative stress induces activation of a cytosolic protein responsible for control of iron uptake. Arch. Biochem. Biophys 316:128-134.
[21] Nunez-Millacura C, Tapia V, Munoz P, Maccioni RB, Nunez MT (2002) An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells. J. Neurochem 82:240-248.
[22]Nunez MT,Nunez-Millacura C,TapiaV,Munoz P,Mazariegos D,Arredondo M,Mura C,Maccioni RB(2003) Iron-activated iron uptake:a positive feedback loop mediated by iron regulatory protein 1.Biometals 16:83-90.
[23]Pinero DJ,Hu J,Cormor JR(2000) Alternations in the interaction between iron regulatory proteins and their responsive element in normal and Alzeimer's diseased brains.Cell Mol Biol 46:761-776.
[24]Faucheux BA,Martin ME,Beaumont C,Hunot S,Hauw JJ,Agid Y,Hirsch EC(2002)Lack of up-regulation of ferdtin is associated with sustained iron regulatory protein-1binding activity in the substantia nigra of patients with Parkinson's disease.J.Neurochem 83:320-330.
[25]Jellinger KA(1999) The role of iron in neurodegeneration.Drugs Aging 14:115-140.
[26]Ke Y,Ming Qian Z(2003) Iron misregulation in the brain:a primary cause of neurodegenerative disorders.Lancet Neurol 2:246-253.
[27]Beard JL,Connor JR,Jones BC(1993) Iron in the brain.Nutr.Rev.51:157-170.
[28]吉雁鸿、郭俊生、李敏(2003)机体代谢变化与晕船的关系.中国公共卫生19:147-178.
[29]李敏、赵法Ji(2000) 生脉散对热应激大鼠血浆锌、铜、铁等微量元素含量的影响.卫生研究 29:45-46.
[30]Smith SM,Zwart SR,Block G,Davis-Street JE(2005) The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station.J Nutr 135:437-443.
[31]Nikolova-Todorova Z,Troic T(2003) Effect of surgical trauma on patient nutritional status.Med Arh 57(4 Suppl 1):29-31.
[32]Anna MR,Gregory DB,Gregory DB(1998) Iron Status and Non-Specific Symptoms of Female Students.American College of Nutrition 17:351-355.
2.Zhou B,Westaway SK,Levinson B,Johnson MA,Gitschier J,Hayflick SJ.A novel pantothenate kinase gene(PANK2) is defective in Hallervorden-Spatz syndrome.Nat Genet,2001(28):345-349.
3.Qian ZM,Shen X.Brain iron transport and neurodegeneration.Trends Mol Med,2001(7):103-108.
4.Rouault TA.iron on the brain.Nat Genet,2001(28):299-300.
5.Ke Y,Ming Qian Z.Iron misregulation in the brain:a primary cause of neurodegenerative disorders.Lancet Neurol,2003(2):246-253.
6.Smith SM,Zwart SR,Block G,Davis-Street JE.The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station.J Nutr,2005(35):437-443.
7.Nikolova-Todorova Z,Troic T.Effect of surgical trauma on patient nutritional status.Meal,4rh 2003(57-4 Suppl 1):29-31.
8.Anna MR,Gregory DB,Gregory DB.Iron Status and Non-Specific Symptoms of Female Students.Journal of the American College of Nutrition,1998(17):351-355.
9.Kaufer D,Soreq H.Tracking cholinergic pathways from psychological and chemical stressors to variable neurodeterioration paradigms.Curt Opin Neurol,1999(12):739-43.
10.Miyashita T,Yamaguchi T,Motoyama K,Unno K,Nakano Y,Shimoi K.Social stress increases biopyrrins,oxidative metabolites of bilirnbin,in mouse urine.Biochem Biophys Res Commun,2006(349):775-780.
11.Abe H,Hidaka N,Kawagoe C,Odagiti K,Watanabe Y,Ikeda T,Ishizuka Y,Hashiguchi H,Takeda R,Nishimod T,Ishida Y.Prenatal psychological stress causes higher emotionality,depression-like behavior,and elevated activity in the hypothalamo-pituitary-adrenal axis.Neurosci Res,2007(59):145-151.
12.Cui R,Suemaru K,Li B,Araki H.Tbe effects of atropine on changes in the sleep patterns induced by psychological stress in rats.Eur J Pharmacol,2008(579):153-159.
13.Beard JL,Connor JR,Jones BC.Iron in the brain.Nutr Rev,1993(51):157-170.
14.Atwood CS,Obrenovich ME,Liu T,Chan H,Perry G,Smith MA,Martins RN.Amyloid-beta:a chameleon walking in two worlds:a review of the trophic and toxic properties of amyloid-beta.Brain Res Brain Res Rev,2003(43):1-16.
15.Bush,AI.Tbe metallobiology of Alzbeimer's disease.Trends Neurosci,2003(26):207-214.
16.吉雁鸿、郭俊生、李敏.机体代谢变化与晕船的关系.中国公共卫生,2003(19):147-178.
17.李敏、赵法几生脉散对热应激大鼠血浆锌、铜、铁等微量元素含量的影响、卫生研究2000(29):45-46
18.Sheline YI,Sanghavi M,Mintun MA,Gado MH.Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression.J Neurosci,1999(19):5034-5043.
1.Qian ZM,Wang Q.Expression of iron transport proteins and excessive iron accumulation in the brain in neurodegenerative disorders.Brain Res Rev,1998(27):257-267.
2.Hentze MW,Muckenthaler MU,Andrews NC.Balancing acts:molecular control of mammalian iron metabolism.Cell 2004(117):285-297.
3.Pantopoulos K.Iron metabolism and the IRE/IRP regulatory system:an update.Ann N Y Acad Sci,2004(1012):1-13.
4.Qian ZM,Pu YM,Tang PL,Wang Q.Transferrin-bound iron uptake by the cultured cerebellar granule cells.Neurosci Lett,1998(251):9-12.
5.Qian ZM,Liao QK,To Y,Ke Y,Tsoi YK,Wang GF,Ho KP.Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.Cell Mol Biol(Noisy-le-grand),2000(46):541-548.
6.Ke Y,Qian ZM.Iron misregulation in the brain:a primary cause of neurodegenerative disorders.Lancet Neurol,2003(2):246-253.
7.Gelman BB.Iron in CNS disease.J Neuropathol Exp Neurol,1995(54):477-486.
8.Andersen JK.Iron dysregulation and Parkinson's disease.J Alzheimers Dis,2004(6 Suppl):S47-52.
9.Honda K,Smith MA,Zhu X,Baus D,Merrick WC,Tartakoff AM,Hattier T,Harris PL,Siedlak SL,Fujioka H,Liu Q,Moreira PI,Miller FP,Nunomura A,Shimohama S,Perry G.Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron.J Biol Chem,2005(4):76-91.
10.Cairo G,Pietrangelo A.Iron regulatory proteins in pathobiology.Biochern J,2000(352):241-250.
11.Qian ZM,Tang PL,Wang Q.Iron crosses the endosomal membrane by a carrier-mediated process.Prog Biophys Mol Biol,1997(67):1-15.
12.Qian ZM,Shen X.Brain iron transport and neurodegeneration.Trends Mol Med,2001(7):103-108.
13. Bremner J, Narayan M, Anderson E, Staib L, Miller H, Charney D. Hippocampal volume reduction in major depression. Am J Psychiatry, 2000(157):115-118.
14. Sheline Y, Sanghavi M, Mintun M, Gado M. Depression duration but not age predicts hippocampal volume loss in medical healthy women with recurrent major depression. J Neurosci, 1999(19):5034-5043.
1.Caltagirone A,Weiss G,Pantopoulos K.Modulation of cellular iron metabolism by hydrogen peroxide.Effects of H_2O_2 on the expression and function of iron-responsive element-containing mRNAs in B6 fibroblasts.J Biol Chem,2001(276):19738-19745.
2.Nunez-Millacura C,Tapia V,Munoz P,Maccioni RB,Nunez MT.An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells.J.Neurochem 2002(82):240-248.
3.Bush,AI.The metallobiology of Alzheimer's disease.Trends Neurosci,2003(26):207-214.
4.Andersen JK.Iron dysregulation and Parkinson's disease.J Alzheimers Dis,2004(6 Suppl):S47-52.
5.Berg D,Youdim MB.Role of iron in neurodegenerative disorders.Top Magn Reson Irnaging,2006(17):5-17.
6.Hochstein P,Ernster L ADP-activated lipid peroxidation coupled to the TPNH oxidase system of microsomes.Biochera.Biophys.Res.Common,1963(12):388-394.
7.Hochstein P,Nordenbrand K,Ernster LEvidence for the involvement of iron in the ADP-activated peroxidation of lipids in microsomes and mitochondria.Biochem.Biophys.Res.Cornmon,1964(14):323-328.
8.Nunez-Millacura C,Tapia V,Munoz P,Maccioni RB,Nunez MT.An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells.J.Neurochem,2002(82):240-248.
9.Millan-Plano S,Garcia JJ,Martinez-Ballarin E,Reiter RJ,Orteqa-Gutierrez S,Lazaro RM,Escanero JF.Melatonin and pinoline prevent aluminium-induced lipid peroxidation in rat synaptosomes.J Trace Elem Med Biol,2003(17):39-44.
10.Topal T,Oter S,Korkmaz A,Sadir S,Metinyurt G,Korkmazhan ET,Serdar MA,Bilgic H,Reiter RJ.Exogenously administered and endogenously produced melatonin reduce hyperbaric oxygen-induced oxidative stress in rat lung.Life Sci,2004(75):461-467.
11.Ischiropoulos H,Beckman JS.Oxidative stress and nitration in neurodegeneration:cause,effect,or association? J Clin Invest,2003(111):163-169.
12.Schipper HM.Heme oxygenase-1:role in brainaging and neurodegeneration.Exp Gerontol,2000(35):821-830.
13.高谦.血红素加氧酶系统的分子特性及其在脑中的作用[J].国外医学生理、病理科学与临床分册,2002(22):174-176.
14.Atwood CS,Obrenovich ME,Liu T,Chan H,Perry G,Smith MA,Martins RN.Amyloid-beta:a chameleon walking in two worlds:a review of the trophic and toxic properties of amyloid-beta.Brain Res Brain Res Rev,2003(43):1-16.
15.Bush,AI.The metallobiology of Alzheimer's disease.Trends Neurosci,2003(26):207-214.
1.Pu YM,Wang Q,Tang PL,Qian ZM.Effect of iron and lipid peroxidation on development of cerebellar granule cell in vitro.Neuroscience,1999(89):855-861.
2.Qian ZM,Shen X.Brain iron transport and neurodegeneration.Trends Mol Med,2001(7):103-108.
3.Heather MY,Brien AJ,Fumes JB.Relationships between NADPH diaphorase staining and neuronal endothelial,and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissue.Histochem Cell Bio,1997(1107):19-29.
4.包新民,舒斯云.大鼠脑立体定位图谱.北京:人民卫生出版社,1991:33-41.
5.刘辉,陈俊抛,田时雨,谭盛,高曲文.神经元型一氧化氮合酶在学习记忆过程中的变化和作用.中国神经免疫学和神经病学杂志.2000(7):116-121.
7.Chrousos GP,Gold PW.The concepts of stress and stress system disorders.JAMA,1992(267):1244-1252.
8.Irwin M.Stress-induced immune suppression.Ann N Y Acad Sci,1993(697):203-218.
9.Baram TZ,Hatalski CG.Neuropeptide-mediated excitability:a key triggering mechanism for seizure generation in the developing brain.Trends Neurosci,1998(21):471-476.
10.Leibold EA,GuoB.Iron-dependent regulation of ferritin and transferrin receptor expression by the iron-responsive element binding proteins.Ann Rev Nutr,1992(12):345-368.
11.Kira S,Ponka P.Control of transferrin receptor expression via nitric oxide-mediated modulation of iron-regulatory protein 2.J Biol Chem 1999(274):33035-33042.
12.Kim S,Ponka P.Effects of interferon- γ and lipopolysaccharide on macrophage iron metabolism are mediatded by nitric oxide-induced degradation of iron regulatory protein 2.J Biol Chem,2000(275):6220-6226.
13.Wardrop SL,Watts RN,Richardson DR.Nitrogen monoxide activates iron regulatory protein 1,RNA-binding activity by two possible mechanisms:effect an the[4Fe-4s]cluster and iron mobilization from cells.Biochemistry,2000(39):2748-2758.
14.金玉祥,陈嘉峰,胡波,韩三春,王绍.应激性防御反应过程中脑内一氧化氮合酶阳性神经元分布的变化.神经解剖学杂志,2001(16):29-32.
[1]Qian ZM,Wang Q.(1998) Expression of iron transport proteins and excessive iron accumulation,in the brain in neurodegenerative disorders.Brain Res Rev,27:257-267.
[2]Richardson DR,Ponka P(1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells.Biochim Biophys Acta 1331:1-40.
[3]Andersen JK(2004) Iron dysregulation and Parkinson's disease.J Alzheimers Dis.12:6(6 Suppl):S47-52.
[4]Berg D,Youdim MB(2006) Role of iron in neurodegenerative disorders.Top Magn Reson Imaging 17:5-17.
[5]Qian ZM,Pu YM,Tang PL,Wang Q(1998)Transferrin-bound iron uptake by the cultured cerebellar granule cells.Neurosci Lett 251:9-12.
[6]Qian ZM,Shen X(2001) Brain iron transport and neurodegeneration.Trends Mol Med 7:103-108.
[7]Pantopoulos K(2004) Iron metabolism and the IRE/IRP regulatory system:an update.Ann N Y Acad Sci,1012:1-13.
[8]Lee DW,Andersen JK,Kaur D(2006) Iron dysregulation and neurodegeneration:the molecular connection.Mol Interv 6:89-97.
[9]Caltagirone A,Weiss G,Pantopoulos K(2001) Modulation of cellular iron metabolism by hydrogen peroxide.Effects of H_2O_2 on the expression and function of iron-responsive element-containing mRNAs in B6 fibroblasts. J Biol Chem 276:19738-19745.
[10] Rouault TA (2001) Iron on the brain. Nat Genet 28:299-300.
[11] Gutteridge JM (1992) Iron and oxygen radicals in brain. Ann Neural 32:S16-21.
[12] Fredriksson A, Schroder N, Eriksson P, Izquierdo I, Archer T (2001) Neonatal iron potentiates adult MPTP-induced neurodegenerative and functional deficits. Parkinsonism Relat Disord 7: 97-105.
[13] Richardson DR, Ponka P (1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta 1331:1-40.
[14] Zhou B, Westaway SK, Levinson B, Johnson MA, Gitschier J, Hayflick SJ (2001) A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nat Genet 28:345-349.
[15] Hellman NE, Kono S, Miyajima H, Gitlin JD. Biochemical analysis of a missense mutation in aceruloplasminemia.J Biol Chem 2002; 277:1375-80.
[16] Rebeck GW, Harr SD, Strickland DK, Hyman BT. (1995) Multiple, diverse senileplaque-associated proteinsare ligands of an apolipoprotein E receptor, thealpha-macroglobulin receptor/low densitylipoprotein receptor-related protein. Ann Neurol 37: 211-217.
[17] Schipper HM (2000) Heme oxygenase-1: role in brain aging and neurodegeneration. Exp Gerontol 35: 821-830.
[18] Cairo G, Pietrangelo A (2000) Iron regulatory proteins in pathobiology. Biochem. J 352:241-250.
[19] Mehlhase J, Sandig G, Pantopoulos K, Grune T (2005) Oxidation-induced ferritin turnover in microglial cells: role of proteasome. Free Radical Biol Med 38:276-285.
[20] Martins, EA, Robalinho, RL, Meneghini, R (1995) Oxidative stress induces activation of a cytosolic protein responsible for control of iron uptake. Arch. Biochem. Biophys 316:128-134.
[21] Nunez-Millacura C, Tapia V, Munoz P, Maccioni RB, Nunez MT (2002) An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells. J. Neurochem 82:240-248.
[22]Nunez MT,Nunez-Millacura C,TapiaV,Munoz P,Mazariegos D,Arredondo M,Mura C,Maccioni RB(2003) Iron-activated iron uptake:a positive feedback loop mediated by iron regulatory protein 1.Biometals 16:83-90.
[23]Pinero DJ,Hu J,Cormor JR(2000) Alternations in the interaction between iron regulatory proteins and their responsive element in normal and Alzeimer's diseased brains.Cell Mol Biol 46:761-776.
[24]Faucheux BA,Martin ME,Beaumont C,Hunot S,Hauw JJ,Agid Y,Hirsch EC(2002)Lack of up-regulation of ferdtin is associated with sustained iron regulatory protein-1binding activity in the substantia nigra of patients with Parkinson's disease.J.Neurochem 83:320-330.
[25]Jellinger KA(1999) The role of iron in neurodegeneration.Drugs Aging 14:115-140.
[26]Ke Y,Ming Qian Z(2003) Iron misregulation in the brain:a primary cause of neurodegenerative disorders.Lancet Neurol 2:246-253.
[27]Beard JL,Connor JR,Jones BC(1993) Iron in the brain.Nutr.Rev.51:157-170.
[28]吉雁鸿、郭俊生、李敏(2003)机体代谢变化与晕船的关系.中国公共卫生19:147-178.
[29]李敏、赵法Ji(2000) 生脉散对热应激大鼠血浆锌、铜、铁等微量元素含量的影响.卫生研究 29:45-46.
[30]Smith SM,Zwart SR,Block G,Davis-Street JE(2005) The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station.J Nutr 135:437-443.
[31]Nikolova-Todorova Z,Troic T(2003) Effect of surgical trauma on patient nutritional status.Med Arh 57(4 Suppl 1):29-31.
[32]Anna MR,Gregory DB,Gregory DB(1998) Iron Status and Non-Specific Symptoms of Female Students.American College of Nutrition 17:351-355.
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