【作者】 赵敏；

【导师】 李敏；

【作者基本信息】 第二军医大学 ， 军事预防医学， 2008， 博士

【摘要】 铁作为人体必需的微量元素，广泛参与体内的代谢过程，其含量下降可影响血红蛋白的合成，进而影响红细胞的形成，甚至引起贫血。世界卫生组织报道全世界患贫血人数高达20亿，引发贫血的主要原因是铁摄入不足，但我们的前期研究发现在膳食铁供给充足的情况下，心理应激可以降低血清铁浓度并影响红细胞生成。而产生这种影响的机制仍需进一步研究。肝脏是机体主要的铁贮存部位，同时在铁稳态的调节中起着中心和枢纽作用，铁调素等许多重要的铁代谢相关蛋白都是肝脏特异性合成分泌的。铁缺乏和铁过量对机体都将产生不良影响，因此，机体需要具有严格的铁调控机制，既可提供足量的铁来发挥其生理功能，又可防止因过量而产生铁毒性，从而维持体内和细胞内的铁平衡。铁过负荷可以在组织、细胞以及细胞器水平引起脂质过氧化，非蛋白结合铁能通过Fenton反应产生羟自由基，攻击细胞膜上的不饱和脂链，铁蛋白中的三价铁被还原成为二价铁离子引起脂质过氧化反应，在氧自由基的形成中起着重要作用。国内外许多临床研究发现，某些疾病与肝铁代谢异常有密切的联系，如慢性炎症性贫血、酒精性肝炎、肝硬化、遗传性血色素沉着病均发生了不同程度的肝铁代谢紊乱。IL-6是炎症状态下通过诱导肝脏分泌铁调素增加引起低铁血症的必要条件，相反地中海贫血的患者肝脏分泌铁调素减少，而在心理应激情况下，肝脏铁代谢在血清铁下降的同时发挥怎样的作用，肝脏铁代谢发生了哪些改变以及发生这些改变的分子机制研究未见报道。现代社会生活和学习工作的负担常常使人们处于心理应激状态。通过进一步研究阐明心理应激对肝铁代谢的影响，对于全面了解贫血产生的原因，完善贫血的预防和治疗措施具有积极的意义。第一部分：心理应激对肝铁浓度和氧化应激反应的影响目的：观察心理应激对肝脏铁含量和氧化应激反应的影响。方法：心理应激模型制作：雄性SD大鼠按体重随机分为空白对照组、心理应激组和足底电击组。采用Communication Box System制作心理应激大鼠模型，每日一次，持续时间每次30min，实验周期分别为1d，3d、7d和14d。指标测定：应用原子吸收法测定肝组织铁浓度；BPS法测定非蛋白结合铁浓度，Perl’s法进行肝组织铁染色；应用酶联免疫法检测铁蛋白表达水平。图象分析及数据统计：实验数据用(x±s)表示，数据统计处理应用统计软件SPSS 12．0，统计方法采用方差分析。结果：1．心理应激对肝铁浓度的影响与对照组相比，7d和14d心理应激大鼠肝铁浓度，铁蛋白表达和非蛋白结合铁浓度均明显增加(P<0．05)；心理应激组和足底电击组相比，没有显著性差异(P>0．05)。Perl’s铁染色显示，与对照组比较，7d和14d心理应激组和足底电击组大鼠肝组织铁染色明显增加，铁沉积主要分布在肝细胞、内皮细胞和巨噬细胞。2．心理应激对氧化应激反应的影响与对照组相比，7天和14天心理应激组超氧化物歧化酶活性和丙二醛浓度明显升高(P<0．05)。心理应激组和足底电击组相比，没有显著性差异(P>0．05)。第二部分：心理应激影响肝铁代谢的分子机制目的：探讨心理应激影响肝脏铁获取和铁释放的分子调节机制。方法：指标测定方法：应用蛋白免疫印迹法检测铁调素(hepcidin)、膜铁转运蛋白(FP1)、转铁蛋白受体2(TfR2)蛋白表达；应用酶联免疫法检测转铁蛋白受体1表达水平。图象分析及数据统计：采用ImageJ图像分析系统进行灰度分析，实验数据用(x±s)表示，数据统计处理应用统计软件SPSS 12．0，统计方法采用方差分析和Pearson相关分析。结果：1．心理应激对肝脏铁代谢相关蛋白的影响与对照组相比，心理应激组3d后出现肝脏hepcidin和TFR2蛋白表达持续上调，FP1蛋白表达持续下调(P<0．05)。肝脏TfR1蛋白表达在7d和14d下调(P<0．05)。心理应激组和足底电击组相比，没有显著性差异(P>0．05)。2．心理应激大鼠肝铁浓度与hepcidin的关系肝脏hepcidin蛋白表达与肝脏铁浓度存在正相关(P<0．05)。Hepcidin升高先于肝铁浓度改变，提示心理应激诱导的肝脏hepcidin表达升高是引起机体铁代谢紊乱的重要原因。第三部分：心理应激大鼠hepcidin变化的调节因子研究目的：阐明IL-6-hepc轴在心理应激大鼠铁代谢紊乱中的作用。方法：实验动物处理：雄性SD大鼠按体重随机分为空白对照组、心理应激生理盐水组，足底电击生理盐水组，心理应激IL-6抗体组，足底电击IL-6抗体组。采用Communication Box System制作心理应激大鼠模型，每日一次，持续时间每次30min，实验周期分别为3d、7d。指标测定方法：应用原子吸收法测定血清铁和肝组织铁浓度；应用蛋白免疫印迹法检测肝铁调节机制中的重要调节蛋白hepcidin、FP1蛋白表达；应用酶联免疫法检测IL-6、TNF-α和IL-10表达水平。图象分析及数据统计：采用ImageJ图像分析系统进行灰度分析，实验数据用(x±s)表示，数据统计处理应用统计软件SPSS 12．0，统计方法采用方差分析和Pearson相关分析。结果：1．心理应激对细胞因子表达的影响与对照组相比，心理应激组3d开始出现肝脏IL-6和TNF-α蛋白表达持续升高(P<0．05)，IL-10蛋白表达没有显著性差异(P>0．05)。大鼠肝脏IL-6与hepcidin蛋白表达密切相关(P<0．05)。2．IL-6抗体抑制心理应激大鼠IL-6表达上调与生理盐水组相比，心理应激IL-6抗体组大鼠血清IL-6表达水平明显降低(P<0．05)，与对照组相比，心理应激IL-6抗体组和足底电击IL-6抗体组大鼠血清IL-6表达水平没有显著性差异(P>0．05)。3．IL-6抗体对心理应激大鼠铁分布的影响与生理盐水组相比，心理应激IL-6抗体组3d和7d血清铁浓度明显升高，7d肝铁浓度明显降低(P<0．05)，与对照组相比，心理应激IL-6抗体组和足底电击IL-6抗体组3d和7d血清铁、7d肝铁浓度没有显著性差异(P>0．05)。4．IL-6抗体对心理应激大鼠肝脏铁代谢相关蛋白表达的影响与生理盐水组相比，心理应激IL-6抗体组大鼠肝脏hepcidin蛋白表达降低，FP1蛋白表达升高(P<0．05)，与对照组相比，心理应激IL-6抗体组和足底电击IL-6抗体组大鼠肝脏hepcidin和FP1蛋白表达没有显著性差异(P>0．05)。结论：1．心理应激可导致肝脏铁储存升高和血清铁下降，肝脏以铁蛋白和含铁血黄素的形式储存铁增加；同时肝脏非蛋白结合铁浓度增加，并伴随脂质过氧化反应增强；随着应激时间延长，肝脏铁储存持续升高，血清铁浓度持续下降，肝铁浓度升高可能是血清铁浓度下降的原因之一。2．心理应激可诱导肝脏转铁蛋白受体2表达上调，而肝脏转铁蛋白受体1蛋白表达下调，提示心理应激条件下肝脏铁储存增加可能是通过转铁蛋白受体2摄取的；同时出现肝脏hepcidin蛋白表达上调与FP1表达下调，机体铁的循环再利用过程中巨噬细胞铁释放减少。3．肝脏hepcidin蛋白表达与肝铁浓度存在正相关，hepcidin表达上调先于肝铁浓度升高，提示心理应激所导致的肝脏hepcidin表达升高是引起了铁代谢的变化的原因；心理应激诱导IL-6和hepcidin蛋白表达持续上调，两者变化的时间和趋势一致，肝脏hepcidin与IL-6蛋白表达存在正相关。4．IL-6抗体使心理应激大鼠肝脏和血清铁分布趋于正常，并抑制应激大鼠肝脏hepcidin蛋白表达升高以及FP1蛋白表达的降低。IL-6-hepc轴是导致心理应激大鼠机体铁代谢紊乱的主要通路。更多还原

【Abstract】 Iron is essential for DNA synthesis and key metabolic processes in living organisms. The levels of iron in cell must be delicately balanced, as iron loading leads to free radical damage by the Fenton reaction. Iron mal-regulation may be induced by infection and inflammation, the reasons of which need to be studied. A series of iron-regulatory proteins, including iron transporters and soluble mediators have been identified, which have greatly enhanced our understanding of iron metabolism. Hepcidin has been shown to play a central role in the regulation of iron metabolism. Hepcidin knockout mice develop iron overload in the liver, pancreas and heart, whereas mice overexpressing hepcidin display severe iron deficiency and anemia. Iron overload increases and iron deficiency decreases the expression of hepcidin in the liver. Hepcidin is also regulated by inflammatory signals. However, the role of hepcidin in psychological stress is unknown.Recently, the burden of social life and work is so heavy that people suffer psychological stress frequently, which is considered to be the causes of many disorders, such as hypertension, gastric ulcer, and hyperthermia. Our previous findings have demonstrated that repeated psychological stress exposure could decrease the serum iron level and inhibit erythropoiesis [6]. Because the liver plays a central role in iron metabolism, the hepatic transferrin receptors tend to be responsible for the systemic physiologic changes. The disturbance of iron metabolism is related with many diseases, such as iron deficiency anemia and chronic hepatitis. So it is important to investigate the effect of psychological stress on liver and to understand the molecular mechanisms under iron metabolism.Design and MethodsAnimalsSprague-Dawley (SD) rats, male, aged 10 weeks (Shanghai-BK Co., Ltd) were caged individually at room temperature and 55±5% humidity. They were feed on with a standard diet and tap water freely. After 7 d adaptation, the rats were randomized into two groups: the psychological stress group and the control group, each group of which was subdivided into 1 d group, 3 d group, 7 d group and 14 d group. Pieces from the right part of liver were rapidly dissected and snap-frozen. For anti-IL-6 antibody experiments, rats were either received normal sodium or anti-rat IL-6 monoclonal antibody(2μg /d, R&D Systems Inc, USA) by intraperitoneal injection daily before psychological stress exposure. All animal studies were in accordance with institutional animal care guidelines and were approved by the animal research committee at the Second Military Medicine University, Shanghai, China.Psychological stress exposureThe communication box paradigm was used equipped with a grid floor composed of stainless steel rods. Plastic plates were placed on half of the grid floors for electric insulation. The rats (foot shock group) in compartments without plastic plates received foot shock without a warning signal. The rats (psychological stress group) in compartments with plastic plates did not receive foot shock, but received emotional stimuli from the rats in the foot shock group. All rats were exposed to the psychological stress for 30 min every morning, at 10:00-10:30. Compared with the stressed rats, the control rats were placed individually in the compartments with plastic plates and without exposure to any stress.Iron analysisBlood was sampled by retro-orbital phlebotomy into heparinized tubes before the rats were humanely killed. Quantification of serum and liver iron content was measured using a Varian SpectrAA-220G graphite furnace atomic absorption spectrophotometer, equipped with a GTA 110 atomizer, programmable sample dispenser, and deuterium background correction.ELISA and Western blot analysisHepatic tissue was homogenized and lysed for ELISA and western blots. TFR1 and ferritin (R&D Systems Inc, USA) were analyzed using commercially available ELISA kits. Western blotting was performed with rabbit polyclonal anti-mouse TFR2 (Santa Cruz, Inc.). Identical samples were blotted with anti-β-actin (Sigma) polyclonal antibody to keep the amount of loading protein equal. Immunoreactive bands were detected by goat polyclonal anti-rabbit-HRP antibodies (Santa Cruz, Inc.).Measurement of superoxide dismutase (SOD) activity, malondialdehyde (MDA) and NTBI concentrationsThe thiobarbiruric acid (TBA) method was used to measure the liver MDA level with MDA-586 kit (Nanjing Jiancheng Bioengineering Institute). SOD activity was measured with kit-WST (Dojindo Laboratories). For NTBI determination, the liver tissue homogenates were analyzed using bathophenanthroline disulfonate (BPS) to chelate ferrous iron, thus forming a complex which could be analyzed with spectrophotometry. Commercially available BPS (4, 7-diphenyl-1,10-phenanthroline disulfonate) and ferrous ammonium sulfate [(NH4)2 Fe(SO4)2 ] of the highest purity (Sigma) were used in the measurement.Perl’s stainingSamples were fixed in aqueous formaldehyde solution (buffered 4% vol/vol) and were embedded in paraffin. The liver sections were stained with Perls’ Prussian blue to assess the non-heme iron deposition.Statistical AnalysisSPSS 12.0 software (SPSS institute, Chicago, IL, USA) was used for statistical analysis. Student’s t-test was used to determine whether differences were statistically significant in groups. Data were expressed as (X|-)±SD. P <0.05 represents statistically significant difference.ResultsPsychological stress leads to hepatic iron accumulationPsychological stress exposure increased hepatic ferritin and decreased TFR1 expression as early as 7 days (p<0.05) in rats. No significant difference was detected on 3 day between control group and psychological stress group. On contrary, TFR2 expression was up-regulated by psychological stress exposure in liver as early as 3 days. As displayed in figure 2, we revealed that liver tissue SOD activity and MDA level in the psychological stress group were higher than that in control group (p<0.05); simultaneously, the hepatic NTBI levels in the psychological stress group were increased obviously compared to that in the control one (p<0.05). Control group and 3 days psychological stress group did not show iron overload, whereas at 7 and 14 days some iron deposition was observed, principally in hepatocytes and macrophages. Liver structure was normal.Cytokine and iron metabolism response to psychological stressIL-6 and TNF-αproduction increased since 3 days after psychological stress exposure in rat liver. There was no significant difference in levels of IL-10. Western blot analysis documented that rat liver displayed an up-regulation of hepcidin and down-regulation of ferroportin on 3 days and 7 days in response to psychological stress. Furthermore, rat duodenal displayed a down-regulation of ferroportin and apparent absorptivity of iron decreased on 3 days and 7 days in response to psychological stress.Anti-IL-6 antibody inhibits the effect of psychological stress on iron distribution and iron metabolismAfter rats received intraperitoneal injection of anti-rat IL-6 antibody, psychological stress exposure didn’t significantly changed liver iron store or serum iron level on 3 days and 7 days compared with control group. Compared with NS-treated group, anti-rat IL-6 antibody significantly decreased liver iron store on 7 days and increased serum iron level of psychological stress rats on 3 days. Anti-rat IL-6 antibody down-regulated hepcidin and up-regulated ferroportin expression in liver induced by psychological stress on 3 days and 7 days.The present study suggests that psychological stress leads to hypoferremia, which is regulated by IL-6-hepcidin axis. Inhibition of iron absorption and increased liver iron store contribute to hypoferremia. The hepatic TFR2 was up-regulated by psychological stress in rats which may contribute to the increased hepatic iron accumulation as haemosiderin, ferritin and NTBI, and increasing products of lipid peroxidation. However, the mechanism that how the psychological stress induces iron mal-regulation still needs to be investigated.更多还原