心理应激大鼠肝脏SFXN1的表达变化及其对肝细胞铁代谢相关分子的影响
|
摘要
铁是机体含量最丰富的金属元素之一，含铁蛋白在体内发挥重要的生理功能，参与氧的转运，细胞呼吸、物质代谢、转录调节和DNA修复。机体铁缺乏会产生缺铁性贫血和多种代谢障碍，然而过量的铁在体内能通过Fenton反应引起脂质过氧化，进一步损伤蛋白质和（或）核酸。机体铁负荷与癌症、冠心病、帕金森病、糖尿病等多种疾病的发生有关。肝脏是机体储存铁的主要器官，也是铁过量损伤的主要器官，有大量研究资料证实，肝铁过负荷会导致肝细胞坏死，纤维化和肝肿瘤，还能引起胰岛素抵抗、增加胆固醇的合成和沉积。因此探讨铁过负荷的原因，寻找防治的潜在靶点成为医学研究的热点。目前已证实HFE、HJV、TfR2等基因突变导致遗传性血色素沉着病，反复输血、膳食铁摄入过多等因素都可以引起机体铁过负荷，但是对于大多数疾病存在铁过负荷的原因尚未完全阐明。
心理应激是指机体对外界有害物、威胁、挑战经认识评价后，知其将危害个人的生存和地位时，所产生的生理、心理和行为反应。现代生活中，人们在工作和生活中承受的负荷越来越大，精神紧张和心理压力已成为重要的应激源，心理因素对人体健康的影响正引起研究者广泛的关注。课题组前期的研究发现，心理应激可引起实验动物肝铁蓄积。其中部分原因可能是通过IL-6上调hepcidin及糖皮质激素对IRP1的调节，由于心理应激动物肝铁蓄积十分明显，是否还有其它的因素导致肝铁蓄积有待于进一步研究。
为了深入分析心理应激导致肝铁蓄积的可能原因，本研究采用基因芯片技术筛选心理应激大鼠肝脏的差异表达基因，并根据基因表达趋势分析与肝铁负荷变化趋势相一致的基因，寻找与肝铁代谢变化有关的分子。结果发现在心理应激情况下，hepcidin、TfR1等已知的与铁代谢有关的分子发生了变化，还发现SFXN1的表达增强。SFXN1也称为三羧基携带蛋白TCC，是sideroflexin家族成员之一，定位于线粒体膜上，是真核生物的一种进化保守蛋白。有研究资料表明，SFXN1可能具有转运有利于铁利用的某种成分进入线粒体的功能，flexed-tail小鼠SFXN1发生了移码突变，表现出红细胞线粒体内铁沉积的特征。线粒体是细胞电子传递和能量代谢最为旺盛的细胞器，线粒体内铁代谢紊乱会严重地影响到整个细胞的铁代谢。因此，SFXN1是否对细胞铁代谢相关分子产生一定的影响，从而参与心理应激反应肝铁负荷的发生，我们进行了进一步实验研究。
研究目的
利用基因芯片筛选心理应激情况下大鼠肝脏的差异表达基因，并在此基础上寻找可能参与心理应激铁代谢异常的基因，为阐明心理应激导致肝铁负荷提供新的研究思路。
研究方法
一、心理应激大鼠血清铁和肝铁含量变化
1、实验动物分组
健康雄性SD大鼠48只（体重120±10g），购自中英合资上海西普尔-必凯实验动物有限公司。按体重随机分为对照组、1天、3天、7天心理应激组4组，每组8只，其余作为电击组。自由饮食，动物实验室条件为：温度24℃士1℃，湿度50％～60％，光照自然昼夜节律变化。
2、制作心理应激模型
大鼠心理应激模型制作采用Communication box system。该装置由透明丙烯酸板制成，所有小室的一半铺绝缘板（A室），另一半不铺绝缘板（B室）。B室的大鼠每天足底电击30分钟，电压为80V，大鼠被电击后尖叫、跳跃、排便；相邻A室的大鼠通过听觉、视觉和嗅觉产生心理应激。
3、血清CORT、ACTH及NE测定
按检测试剂盒说明测定血清CORT、ACTH及NE的浓度。
4、测定肝铁、血清铁含量
湿法消化肝组织，原子吸收分光光度计火焰法测定肝铁和血清铁含量。
5、血清转铁蛋白饱和度的测定
采用血清铁和血清总铁结合力试剂盒测定。
二、心理应激大鼠肝脏SFXN1及铁代谢相关分子的变化
1、实验动物分组及处理
第一部分的实验动物，每组随机抽取三只大鼠，选择肝组织进行基因芯片分析。
2、基因芯片分析
采用Affymetrix公司的Rat Exon1.0STArray全转录组表达谱芯片，利用随机方差模型（RVM），以P﹤0.05和FDR﹤0.05为判断标准，进行差异基因筛选。用GO分类法查找与铁代谢有关的基因，并根据基因表达趋势分析与肝铁负荷变化趋势相一致的基因。
3、大鼠肝脏hepcidin、TfR1、sfxn1mRNA表达的变化
采用实时荧光定量PCR法测定大鼠肝脏内铁调素(hepcidin)、转铁蛋白受体1（transferrin receptor1，TfR1）、sideroflexin1（sfxn1）的mRNA表达变化。
4、测定大鼠肝组织hepcidin、TfR1、sfxn1蛋白含量
免疫印记法测定各组大鼠肝脏hepcidin、TfR1、sfxn1的蛋白含量。
三、 SFXN1对肝细胞铁代谢主要调节分子的影响
1、细胞培养
HepG2肝细胞株，含10%胎牛血清的DMEM培养液37℃、5%CO2培养。
2、Sfxn1siRNA转染细胞
按说明书将sfxn1siRNA转染HepG2细胞后，培养箱中培养48h。
3、肝细胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA测定
采用实时荧光定量PCR法测定HepG2细胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA的表达水平。
4、肝细胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白测定
采用Western blot法测定HepG2细胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白含量。
四、数据的统计与处理
采用SPSS16.0统计软件包对实验数据进行统计分析。两组间比较采用两独立样本t-检验；多组间比较采用单因素方差分析，方差齐时各组样本均数间两两比较采用LSD-t检验，各实验组间与对照组比较采用Dunnett法。方差不齐时采用Dunnett’s C检验。实验数据以平均数±标准差（x±s）表示，p<0.05认为有统计学意义。
结果
一、心理应激大鼠血清铁和肝铁含量变化
1、心理应激大鼠血清CORT、ACTH与NE含量变化
心理应激大鼠血清CORT、ACTH、NE含量较对照组明显升高，说明心理应激模型成功制作。
2、心理应激大鼠肝脏铁含量变化
随着应激时间的延长，大鼠肝组织铁含量逐渐升高，7天心理应激组大鼠肝脏铁含量与对照组比较上升了53.02%（P<0.05）。1天和3天心理应激组大鼠肝铁含量与对照组比较无统计学意义（P>0.05）。
3、心理应激大鼠血清铁含量变化
1天和3天心理应激组大鼠血清铁含量与对照组比较无统计学意义（P>0.05）,与对照组比较，7天心理应激组大鼠血清铁含量下降了29.21%，差异有统计学意义（P<0.05）。
4、心理应激大鼠血清转铁蛋白饱和度的变化
与对照组比较，7天心理应激组大鼠血清转铁蛋白饱和度下降了32.2%（P<0.05）；1天和3天心理应激组大鼠血清转铁蛋白饱和度较对照组分别下降了7.87%、19.85%，但无统计学意义（P>0.05）。
二、心理应激大鼠肝脏SFXN1及铁代谢相关分子的变化
1、基因芯片筛选心理应激大鼠肝脏铁代谢相关差异基因
在心理应激过程中，大鼠肝脏基因mRNA转录水平有明显差异的为2246个。对这些差异表达基因进行生物信息学分析，与铁代谢有关的差异基因有七个，分别为Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2。sfxn1基因的表达趋势分析显示其变化趋势与肝铁沉积变化趋势一致。
2、心理应激大鼠肝脏hepcidin、TfR1mRNA的表达变化
7天心理应激后大鼠肝脏hepcidin mRNA的表达水平较对照、1天、3天心理应激组均有显著升高（P<0.01）；心理应激第七天TfR1mRNA的表达水平比对照、1天、3天心理应激组升高，并有统计学意义。
3、心理应激大鼠肝脏hepcidin、TfR1的蛋白表达变化
7天心理应激组hepcidin蛋白的表达水平较对照、1天、3天心理应激组均有显著升高（P<0.01）。TfR1蛋白在应激后逐渐升高，7天心理应激组TfR1蛋白的表达水平与对照组比较有统计学意义（P<0.01）。
4、心理应激大鼠肝脏sfxn1的表达变化
应激后sfxn1mRNA的水平逐渐升高，3天应激组与对照组、1天应激组比较有统计学意义（P<0.01）；7天应激组sfxn1mRNA的表达水平比对照、1天、3天心理应激组升高，并有统计学意义（P<0.01）。
Sfxn1蛋白在应激后逐渐升高，7天心理应激组的表达水平较对照组升高66%（P<0.01）。
三、SFXN1对肝细胞铁代谢主要调节分子的影响
1、Sfxn1siRNA转染对HepG2细胞sfxn1mRNA表达的影响
HepG2细胞转染sfxn1siRNA后sfxn1mRNA的表达量较未转染的对照组降低（P<0.05）。
2、Sfxn1siRNA转染对HepG2细胞TfR1、 hepcidin mRNA表达的影响
Sfxn1siRNA转染HepG2细胞48小时后，转染组TfR1mRNA的表达量较未转染组降低了78%（P<0.01）。Hepcidin mRNA的表达量与对照组比较无区别。
3、Sfxn1siRNA转染对HepG2细胞IRP1、 IRP2mRNA表达的影响
Sfxn1siRNA转染HepG2细胞48小时后，转染组IRP1mRNA的表达量较未转染组降低了80%（P<0.05）。转染组IRP2mRNA的表达量与对照组比较无差别。
4、Sfxn1siRNA转染降低HepG2细胞sfxn1蛋白表达
Sfxn1siRNA转染HepG2细胞后使sfxn1蛋白表达下降（P<0.01）。
5、Sfxn1siRNA干扰对HepG2细胞TfR1、 hepcidin蛋白表达的影响
Sfxn1siRNA转染HepG2细胞后使TfR1蛋白表达下降（P<0.01）。hepcidin的蛋白表达不受影响。
6、Sfxn1siRNA干扰对HepG2细胞IRP1、 IRP2蛋白表达的影响
Sfxn1siRNA转染HepG2细胞后使IRP1蛋白表达下降（P<0.01）。IRP2的表达未发生改变。
结论
1.连续7天心理应激可以引起大鼠肝铁蓄积。
2.基因芯片发现心理应激过程中Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2共7个与铁代谢相关的基因发生了改变。hepcidin、TfR1、sfxn1的mRNA和蛋白水平随着应激天数的增加不断上升。
3. sfxn1能影响TfR1的表达
综上所述，心理应激时可能通过sfxn1引起转铁蛋白受体1表达增加，肝细胞摄入铁增加，导致肝铁蓄积。
Background
Iron is the most abundant transition metal in the body that is mainly present inprotein-bound forms such as heme and non-heme proteins, playing a major role in electrontransfer and oxygen utilization. Iron deficiency and iron overload can occur and may haveserious clinical consequences. The most common disorder associated with iron depletion isiron deficiency anemia,which affects more than30%of the world’s population.At the otherend of the spectrum,iron overload can occur.This transition metal promotes free radicalgeneration through Fenton and/or Haber–Weiss reactions, thus triggering secondary chainreactions in the oxidative modification of lipids, proteins, and DNA in different organs. Soiron metabolism is tightly regulated.
The liver is the major storage site for iron.But excessive iron deposition in liver willlead to further injury such as hepatocellular necrosis, inflammation, fibrosis and in somecases even to carcinoma. Hepatic iron overload could contribute to the development ofNAFLD or lipotoxicity.
Epidemiological studies have shown that psychological stress may lead to a variety ofdiseases, such as hypertension, ulcer disease, cerebrovascular accident, myocardialinfarction, diabetes, cancer, etc.
Our previous studies have shown that psychological stress can increase the liver irondeposition, but the exact mechanism is still not very clear. In this study, The technique ofmicroarray was used to analysis the liver gene expression profiles of psychological stressrat and to screen out iron related genes.We find out that psychological stress can increasesfxn1expression. Sfxn1also known as tricarboxylate carrier protein.It’s a member of thesideroflexin family. It’s located in the mitochondrial membrane. Sfxn1function might beinvolved in the transport of a component required for iron utilization into or out of themitochondria. But it is still unclear the exact role of the sfxn1. We study the sfxn1if it caneffect iron metabolism related molecules, in order to provide new ideas about thepsychological stress cause liver iron overload.
Objective
In this study, we use gene chip screen liver differentially expressed genes in PS rats.On this basis to find genes may be involved in iron metabolism of psychological stress, provide a new idea about the psychological stress cause liver iron overload. To clarify thepsychological stress how to lead to liver iron overload.
Methods
1. Effects of psychological stress on the rat serum iron and hepatic iron concentration
1.1Experimental animals
48male Sprague Dawley rat（s120±10g）were purchased from Shanghai-BK Ltd. Afteradaptation for5days, the rats were randomized into three groups: control group(n=8),psychological stress groups(futher divided into three subgroup as PS-1,PS-3,PS-7,with8rats in each subgroup),food-shocked group. Psychological stress rats were exposed tostress for30minutes every day. Animal were housed in a temperature-controlled room at24℃under a12-hour light/dark cycle. All animals used in this study received anutritionally-balanced rodent diet with “normal” iron level (35mg iron/kg diet) and waterad libitum. All animal procedures are carried out in accordance with establishedprocedures, Second Military Medical University Animal Care and Use Committee.
1.2Build psychological stress model of SD rats
The communication box was selected as the psychological stress apparatus. It dividedinto20compartments by transparent acrylic board with several small hole. The boardprevented each rat from physical contact but allowed them to receive cues such as visual,auditory and olfactory sensations from the neighboring animals. The compartments weredivided into two parts. Half of all small rooms spread insulation panels (A Room), and theother half do not shop insulated panels (B room). An electric generator with80voltageswas connected to the grid floor to generate electric foot shock.Rats in part B after theshock,exhibited a nociceptive stimulation-evoked response such as screaming,jumping,defecation. Rats in part A were not directly administered the foot electrical shock, butproduced psychological stress.
1.3Measurement of serum ACTH、CORT and NE concentrations
Serum ACTH concentration was measured by the enzyme-linked immunoassays(ELISAs) kit; the CORT and NE levels were measured by the radioimmunofocus assays(RI) kit.
1.4Measurement of liver iron concentration
Specimens were weighed and digested with concentrated nitric-perchloric acid mixture(4to1ratio). Iron estimated by atomic absorption spectrophotometry.
1.5Determination of serum iron
Serum iron concentration was determined in nonhemolyzed serum samples by atomicabsorption spectrophotometer.
1.6Determination of serum Transferrin Saturation
According to the manufacturer’s protocol,serum iron and total iron bindingcapacity(TIBC) were determined.
TS(%)=serum iron/TIBC×100%
2. Gene expression analysis of iron related genes in PS rat
2.1Experimental animals and treatment
Same as in part1.
2.2Gene expression profile analysis
The technique of cDNA microarray was used to screen out iron related genes onpsychological stress rats．Before and1，3，7day psychological stress，total liver RNA wereextracted and detected by Rat Exon1.0ST Array Gene Chip．We analysised the liver geneexpression profiles and then searched the iron related genes use gene ontology description.
2.3Real time PCR analysis for hepcidin、TfR1、sfxn1mRNA expression
Total RNA was extracted from liver tissue using TRIzol reagent and cDNA wasprepared. Real-Time PCR was performed for individual genes using SYBR green detectionsystem. The RT-PCR data were analyzed by standard curve method as described.
2.4Western blot analysis for hepcidin、TfR1、sfxn1protein levels
Livers were homogenized with lysis buffer. Proteins were quantified using the kanjiprotein assay kit based on the Bradford method. Aliquots of protein were loaded on10%sodium dodecyl sulfate polyarylamid gel electrophoresis under reducing conditions andsubsequently transferred to a PVDF membrane. The blot were blocked and probed withantibodies.The intensity of the specific bands was detected and analyzed.To ensure evenloading of the samples,the same membrane was proed with rabbit anti rat GAPDH polyclonal antibody.
3. The effect of sfxn1on hepatic cell iron-related molecules
3.1Cell Culture
HepG2(human liver hepatocellular carcinoma cell line) was purchased from TypeCulture Collection of the Chinese Academy of Sciences, Shanghai. HepG2cells weregrown at37°C with5%CO2in DMEM medium supplemented with10%heat-inactivatedfetal bovine serum (FBS),100U/ml penicillin and100ug/ml streptomycin.
3.2Sfxn1siRNA transfection
HepG2cells were transfected with sfxn1siRNA as protocol described.
3.3Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA levels in HepG2cells
Same as in part2.3.
3.4Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2protein level in HepG2cells
Experimental methods see part2.4.
4. Statistical analysis
Statistical analyses were performed using SPSS software for Windows (version16.0)(SPSS, Inc., Chicago, IL, USA). Data were presented as mean±S.D. All the experimentaldatas were analyzed by independent sample T test method or one-way ANOVA.Aprobability value of P <0.05was taken to be statistically significant.
Results
1. Effects of psychological stress on the rat serum iron and hepatic iron concentration
1.1Determination of the PS model establishment
After psychological stress exposure the levels of serum ACTH, corticosterone andNE were significantly higher than those in the control animals (P<0.05),indicating thePS model is establishment successfully.
1.2Effect of psychological stress on liver iron concentrations
We found that rat liver iron concentrations of7day PS group than the control groupincreased significantly (P <0.05), but1day、3day PS exposure groups no differencecompared with the control group.
1.3Effect of psychological stress on serum iron concentrations
Compared with the control group,1d-,3d-and7d psychological stress group serumiron decreased by11.32%,25.53%and29.21%respectivlely, the serum iron levels in7days PS exposure group significantly lower than the control group (P<0.05).
1.4Effect of psychological stress on serum Transferrin Saturation
Compared with control group,7days psychological stress group serum transferrinsaturation decreased32.2%(P<0.05),1and3days psychological stress group Serumtransferrin saturation fell7.87%,19.85%(P>0.05).
2. Gene expression analysis of iron related genes in PS rat
2.1Gene expression profile analysis
In the psychological stress rat liver, The significant difference genes number is2246.There are seven genes related with iron metabolism is changed.
2.2PS exposure caused changes in hepcidin、TfR1mRNA levels
After seven days of psychological stress, rat liver hepcidin mRNA expression levelswere significantly increased（P<0.01), but compared with the control group,1day and3days PS exposure did not change (P>0.05). TfR1mRNA levels were gradully increased in1day,3days and7days PS groups, but7days PS exposure group changed significantly(P<0.01).
2.3PS exposure changed hepcidin、TfR1protein expression
After7days PS exposure, rat liver hepcidin and TfR1protein levels were significantlyhigher than the control group(P<0.05)..
2.4PS exposure increase sfxn1expression in rat liver
Sfxn1mRNA levels were increased in3days、7days PS exposure compared with thecontrol group (P<0.01). After7days PS exposure, Sfxn1protein levels weresignificantly higher than the control group (P<0.05).
3. The effect of sfxn1on hepatic cell iron-related molecules
3.1Effect of sfxn1siRNA on sfxn1expression levels in HepG2cells
The mRNA expression of sfxn1was significantly decreased in siRNA transfection HepG2cell (P<0.05). Sfxn1protein level was significantly decreased in siRNAtransfection group (P<0.01).
3.2Sfxn1interference decrease mRNA expression of TfR1、IRP1
The TfR1、IRP1mRNA levels decreased in siRNA interference groups compared tocontrol group (P<0.05).But hepcidin、IRP2mRNA showed no difference in siRNAinterference group compared with control group (P>0.05).
3.3Effects of sfxn1interference on TfR1、IRP1protein levels
The TfR1、IRP1protein levels decreased in siRNA interference groups compared tocontrol group (P<0.05).
Conclusions
1.7days PS could cause iron accumulation in liver.
2. The gene chip found Hamp, Tfrc, sfxn1, GDF2,IREB2, NUBP1, NUBP2genes relatediron homeostasis is changed in the process of psychological stress.
3. Hepcidin,TfR1and sfxn1expression is rising with the increase in the number of stressdays.
4. sfxn1can affect the expression of TfR1
Taken together, we found psychological stress can increase transferrin receptor1expression may be by sfxn1,Result in liver cells intake of iron increased, made ironaccumulation in liver and even damage to liver.
心理应激是指机体对外界有害物、威胁、挑战经认识评价后，知其将危害个人的生存和地位时，所产生的生理、心理和行为反应。现代生活中，人们在工作和生活中承受的负荷越来越大，精神紧张和心理压力已成为重要的应激源，心理因素对人体健康的影响正引起研究者广泛的关注。课题组前期的研究发现，心理应激可引起实验动物肝铁蓄积。其中部分原因可能是通过IL-6上调hepcidin及糖皮质激素对IRP1的调节，由于心理应激动物肝铁蓄积十分明显，是否还有其它的因素导致肝铁蓄积有待于进一步研究。
为了深入分析心理应激导致肝铁蓄积的可能原因，本研究采用基因芯片技术筛选心理应激大鼠肝脏的差异表达基因，并根据基因表达趋势分析与肝铁负荷变化趋势相一致的基因，寻找与肝铁代谢变化有关的分子。结果发现在心理应激情况下，hepcidin、TfR1等已知的与铁代谢有关的分子发生了变化，还发现SFXN1的表达增强。SFXN1也称为三羧基携带蛋白TCC，是sideroflexin家族成员之一，定位于线粒体膜上，是真核生物的一种进化保守蛋白。有研究资料表明，SFXN1可能具有转运有利于铁利用的某种成分进入线粒体的功能，flexed-tail小鼠SFXN1发生了移码突变，表现出红细胞线粒体内铁沉积的特征。线粒体是细胞电子传递和能量代谢最为旺盛的细胞器，线粒体内铁代谢紊乱会严重地影响到整个细胞的铁代谢。因此，SFXN1是否对细胞铁代谢相关分子产生一定的影响，从而参与心理应激反应肝铁负荷的发生，我们进行了进一步实验研究。
研究目的
利用基因芯片筛选心理应激情况下大鼠肝脏的差异表达基因，并在此基础上寻找可能参与心理应激铁代谢异常的基因，为阐明心理应激导致肝铁负荷提供新的研究思路。
研究方法
一、心理应激大鼠血清铁和肝铁含量变化
1、实验动物分组
健康雄性SD大鼠48只（体重120±10g），购自中英合资上海西普尔-必凯实验动物有限公司。按体重随机分为对照组、1天、3天、7天心理应激组4组，每组8只，其余作为电击组。自由饮食，动物实验室条件为：温度24℃士1℃，湿度50％～60％，光照自然昼夜节律变化。
2、制作心理应激模型
大鼠心理应激模型制作采用Communication box system。该装置由透明丙烯酸板制成，所有小室的一半铺绝缘板（A室），另一半不铺绝缘板（B室）。B室的大鼠每天足底电击30分钟，电压为80V，大鼠被电击后尖叫、跳跃、排便；相邻A室的大鼠通过听觉、视觉和嗅觉产生心理应激。
3、血清CORT、ACTH及NE测定
按检测试剂盒说明测定血清CORT、ACTH及NE的浓度。
4、测定肝铁、血清铁含量
湿法消化肝组织，原子吸收分光光度计火焰法测定肝铁和血清铁含量。
5、血清转铁蛋白饱和度的测定
采用血清铁和血清总铁结合力试剂盒测定。
二、心理应激大鼠肝脏SFXN1及铁代谢相关分子的变化
1、实验动物分组及处理
第一部分的实验动物，每组随机抽取三只大鼠，选择肝组织进行基因芯片分析。
2、基因芯片分析
采用Affymetrix公司的Rat Exon1.0STArray全转录组表达谱芯片，利用随机方差模型（RVM），以P﹤0.05和FDR﹤0.05为判断标准，进行差异基因筛选。用GO分类法查找与铁代谢有关的基因，并根据基因表达趋势分析与肝铁负荷变化趋势相一致的基因。
3、大鼠肝脏hepcidin、TfR1、sfxn1mRNA表达的变化
采用实时荧光定量PCR法测定大鼠肝脏内铁调素(hepcidin)、转铁蛋白受体1（transferrin receptor1，TfR1）、sideroflexin1（sfxn1）的mRNA表达变化。
4、测定大鼠肝组织hepcidin、TfR1、sfxn1蛋白含量
免疫印记法测定各组大鼠肝脏hepcidin、TfR1、sfxn1的蛋白含量。
三、 SFXN1对肝细胞铁代谢主要调节分子的影响
1、细胞培养
HepG2肝细胞株，含10%胎牛血清的DMEM培养液37℃、5%CO2培养。
2、Sfxn1siRNA转染细胞
按说明书将sfxn1siRNA转染HepG2细胞后，培养箱中培养48h。
3、肝细胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA测定
采用实时荧光定量PCR法测定HepG2细胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA的表达水平。
4、肝细胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白测定
采用Western blot法测定HepG2细胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白含量。
四、数据的统计与处理
采用SPSS16.0统计软件包对实验数据进行统计分析。两组间比较采用两独立样本t-检验；多组间比较采用单因素方差分析，方差齐时各组样本均数间两两比较采用LSD-t检验，各实验组间与对照组比较采用Dunnett法。方差不齐时采用Dunnett’s C检验。实验数据以平均数±标准差（x±s）表示，p<0.05认为有统计学意义。
结果
一、心理应激大鼠血清铁和肝铁含量变化
1、心理应激大鼠血清CORT、ACTH与NE含量变化
心理应激大鼠血清CORT、ACTH、NE含量较对照组明显升高，说明心理应激模型成功制作。
2、心理应激大鼠肝脏铁含量变化
随着应激时间的延长，大鼠肝组织铁含量逐渐升高，7天心理应激组大鼠肝脏铁含量与对照组比较上升了53.02%（P<0.05）。1天和3天心理应激组大鼠肝铁含量与对照组比较无统计学意义（P>0.05）。
3、心理应激大鼠血清铁含量变化
1天和3天心理应激组大鼠血清铁含量与对照组比较无统计学意义（P>0.05）,与对照组比较，7天心理应激组大鼠血清铁含量下降了29.21%，差异有统计学意义（P<0.05）。
4、心理应激大鼠血清转铁蛋白饱和度的变化
与对照组比较，7天心理应激组大鼠血清转铁蛋白饱和度下降了32.2%（P<0.05）；1天和3天心理应激组大鼠血清转铁蛋白饱和度较对照组分别下降了7.87%、19.85%，但无统计学意义（P>0.05）。
二、心理应激大鼠肝脏SFXN1及铁代谢相关分子的变化
1、基因芯片筛选心理应激大鼠肝脏铁代谢相关差异基因
在心理应激过程中，大鼠肝脏基因mRNA转录水平有明显差异的为2246个。对这些差异表达基因进行生物信息学分析，与铁代谢有关的差异基因有七个，分别为Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2。sfxn1基因的表达趋势分析显示其变化趋势与肝铁沉积变化趋势一致。
2、心理应激大鼠肝脏hepcidin、TfR1mRNA的表达变化
7天心理应激后大鼠肝脏hepcidin mRNA的表达水平较对照、1天、3天心理应激组均有显著升高（P<0.01）；心理应激第七天TfR1mRNA的表达水平比对照、1天、3天心理应激组升高，并有统计学意义。
3、心理应激大鼠肝脏hepcidin、TfR1的蛋白表达变化
7天心理应激组hepcidin蛋白的表达水平较对照、1天、3天心理应激组均有显著升高（P<0.01）。TfR1蛋白在应激后逐渐升高，7天心理应激组TfR1蛋白的表达水平与对照组比较有统计学意义（P<0.01）。
4、心理应激大鼠肝脏sfxn1的表达变化
应激后sfxn1mRNA的水平逐渐升高，3天应激组与对照组、1天应激组比较有统计学意义（P<0.01）；7天应激组sfxn1mRNA的表达水平比对照、1天、3天心理应激组升高，并有统计学意义（P<0.01）。
Sfxn1蛋白在应激后逐渐升高，7天心理应激组的表达水平较对照组升高66%（P<0.01）。
三、SFXN1对肝细胞铁代谢主要调节分子的影响
1、Sfxn1siRNA转染对HepG2细胞sfxn1mRNA表达的影响
HepG2细胞转染sfxn1siRNA后sfxn1mRNA的表达量较未转染的对照组降低（P<0.05）。
2、Sfxn1siRNA转染对HepG2细胞TfR1、 hepcidin mRNA表达的影响
Sfxn1siRNA转染HepG2细胞48小时后，转染组TfR1mRNA的表达量较未转染组降低了78%（P<0.01）。Hepcidin mRNA的表达量与对照组比较无区别。
3、Sfxn1siRNA转染对HepG2细胞IRP1、 IRP2mRNA表达的影响
Sfxn1siRNA转染HepG2细胞48小时后，转染组IRP1mRNA的表达量较未转染组降低了80%（P<0.05）。转染组IRP2mRNA的表达量与对照组比较无差别。
4、Sfxn1siRNA转染降低HepG2细胞sfxn1蛋白表达
Sfxn1siRNA转染HepG2细胞后使sfxn1蛋白表达下降（P<0.01）。
5、Sfxn1siRNA干扰对HepG2细胞TfR1、 hepcidin蛋白表达的影响
Sfxn1siRNA转染HepG2细胞后使TfR1蛋白表达下降（P<0.01）。hepcidin的蛋白表达不受影响。
6、Sfxn1siRNA干扰对HepG2细胞IRP1、 IRP2蛋白表达的影响
Sfxn1siRNA转染HepG2细胞后使IRP1蛋白表达下降（P<0.01）。IRP2的表达未发生改变。
结论
1.连续7天心理应激可以引起大鼠肝铁蓄积。
2.基因芯片发现心理应激过程中Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2共7个与铁代谢相关的基因发生了改变。hepcidin、TfR1、sfxn1的mRNA和蛋白水平随着应激天数的增加不断上升。
3. sfxn1能影响TfR1的表达
综上所述，心理应激时可能通过sfxn1引起转铁蛋白受体1表达增加，肝细胞摄入铁增加，导致肝铁蓄积。
Background
Iron is the most abundant transition metal in the body that is mainly present inprotein-bound forms such as heme and non-heme proteins, playing a major role in electrontransfer and oxygen utilization. Iron deficiency and iron overload can occur and may haveserious clinical consequences. The most common disorder associated with iron depletion isiron deficiency anemia,which affects more than30%of the world’s population.At the otherend of the spectrum,iron overload can occur.This transition metal promotes free radicalgeneration through Fenton and/or Haber–Weiss reactions, thus triggering secondary chainreactions in the oxidative modification of lipids, proteins, and DNA in different organs. Soiron metabolism is tightly regulated.
The liver is the major storage site for iron.But excessive iron deposition in liver willlead to further injury such as hepatocellular necrosis, inflammation, fibrosis and in somecases even to carcinoma. Hepatic iron overload could contribute to the development ofNAFLD or lipotoxicity.
Epidemiological studies have shown that psychological stress may lead to a variety ofdiseases, such as hypertension, ulcer disease, cerebrovascular accident, myocardialinfarction, diabetes, cancer, etc.
Our previous studies have shown that psychological stress can increase the liver irondeposition, but the exact mechanism is still not very clear. In this study, The technique ofmicroarray was used to analysis the liver gene expression profiles of psychological stressrat and to screen out iron related genes.We find out that psychological stress can increasesfxn1expression. Sfxn1also known as tricarboxylate carrier protein.It’s a member of thesideroflexin family. It’s located in the mitochondrial membrane. Sfxn1function might beinvolved in the transport of a component required for iron utilization into or out of themitochondria. But it is still unclear the exact role of the sfxn1. We study the sfxn1if it caneffect iron metabolism related molecules, in order to provide new ideas about thepsychological stress cause liver iron overload.
Objective
In this study, we use gene chip screen liver differentially expressed genes in PS rats.On this basis to find genes may be involved in iron metabolism of psychological stress, provide a new idea about the psychological stress cause liver iron overload. To clarify thepsychological stress how to lead to liver iron overload.
Methods
1. Effects of psychological stress on the rat serum iron and hepatic iron concentration
1.1Experimental animals
48male Sprague Dawley rat（s120±10g）were purchased from Shanghai-BK Ltd. Afteradaptation for5days, the rats were randomized into three groups: control group(n=8),psychological stress groups(futher divided into three subgroup as PS-1,PS-3,PS-7,with8rats in each subgroup),food-shocked group. Psychological stress rats were exposed tostress for30minutes every day. Animal were housed in a temperature-controlled room at24℃under a12-hour light/dark cycle. All animals used in this study received anutritionally-balanced rodent diet with “normal” iron level (35mg iron/kg diet) and waterad libitum. All animal procedures are carried out in accordance with establishedprocedures, Second Military Medical University Animal Care and Use Committee.
1.2Build psychological stress model of SD rats
The communication box was selected as the psychological stress apparatus. It dividedinto20compartments by transparent acrylic board with several small hole. The boardprevented each rat from physical contact but allowed them to receive cues such as visual,auditory and olfactory sensations from the neighboring animals. The compartments weredivided into two parts. Half of all small rooms spread insulation panels (A Room), and theother half do not shop insulated panels (B room). An electric generator with80voltageswas connected to the grid floor to generate electric foot shock.Rats in part B after theshock,exhibited a nociceptive stimulation-evoked response such as screaming,jumping,defecation. Rats in part A were not directly administered the foot electrical shock, butproduced psychological stress.
1.3Measurement of serum ACTH、CORT and NE concentrations
Serum ACTH concentration was measured by the enzyme-linked immunoassays(ELISAs) kit; the CORT and NE levels were measured by the radioimmunofocus assays(RI) kit.
1.4Measurement of liver iron concentration
Specimens were weighed and digested with concentrated nitric-perchloric acid mixture(4to1ratio). Iron estimated by atomic absorption spectrophotometry.
1.5Determination of serum iron
Serum iron concentration was determined in nonhemolyzed serum samples by atomicabsorption spectrophotometer.
1.6Determination of serum Transferrin Saturation
According to the manufacturer’s protocol,serum iron and total iron bindingcapacity(TIBC) were determined.
TS(%)=serum iron/TIBC×100%
2. Gene expression analysis of iron related genes in PS rat
2.1Experimental animals and treatment
Same as in part1.
2.2Gene expression profile analysis
The technique of cDNA microarray was used to screen out iron related genes onpsychological stress rats．Before and1，3，7day psychological stress，total liver RNA wereextracted and detected by Rat Exon1.0ST Array Gene Chip．We analysised the liver geneexpression profiles and then searched the iron related genes use gene ontology description.
2.3Real time PCR analysis for hepcidin、TfR1、sfxn1mRNA expression
Total RNA was extracted from liver tissue using TRIzol reagent and cDNA wasprepared. Real-Time PCR was performed for individual genes using SYBR green detectionsystem. The RT-PCR data were analyzed by standard curve method as described.
2.4Western blot analysis for hepcidin、TfR1、sfxn1protein levels
Livers were homogenized with lysis buffer. Proteins were quantified using the kanjiprotein assay kit based on the Bradford method. Aliquots of protein were loaded on10%sodium dodecyl sulfate polyarylamid gel electrophoresis under reducing conditions andsubsequently transferred to a PVDF membrane. The blot were blocked and probed withantibodies.The intensity of the specific bands was detected and analyzed.To ensure evenloading of the samples,the same membrane was proed with rabbit anti rat GAPDH polyclonal antibody.
3. The effect of sfxn1on hepatic cell iron-related molecules
3.1Cell Culture
HepG2(human liver hepatocellular carcinoma cell line) was purchased from TypeCulture Collection of the Chinese Academy of Sciences, Shanghai. HepG2cells weregrown at37°C with5%CO2in DMEM medium supplemented with10%heat-inactivatedfetal bovine serum (FBS),100U/ml penicillin and100ug/ml streptomycin.
3.2Sfxn1siRNA transfection
HepG2cells were transfected with sfxn1siRNA as protocol described.
3.3Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA levels in HepG2cells
Same as in part2.3.
3.4Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2protein level in HepG2cells
Experimental methods see part2.4.
4. Statistical analysis
Statistical analyses were performed using SPSS software for Windows (version16.0)(SPSS, Inc., Chicago, IL, USA). Data were presented as mean±S.D. All the experimentaldatas were analyzed by independent sample T test method or one-way ANOVA.Aprobability value of P <0.05was taken to be statistically significant.
Results
1. Effects of psychological stress on the rat serum iron and hepatic iron concentration
1.1Determination of the PS model establishment
After psychological stress exposure the levels of serum ACTH, corticosterone andNE were significantly higher than those in the control animals (P<0.05),indicating thePS model is establishment successfully.
1.2Effect of psychological stress on liver iron concentrations
We found that rat liver iron concentrations of7day PS group than the control groupincreased significantly (P <0.05), but1day、3day PS exposure groups no differencecompared with the control group.
1.3Effect of psychological stress on serum iron concentrations
Compared with the control group,1d-,3d-and7d psychological stress group serumiron decreased by11.32%,25.53%and29.21%respectivlely, the serum iron levels in7days PS exposure group significantly lower than the control group (P<0.05).
1.4Effect of psychological stress on serum Transferrin Saturation
Compared with control group,7days psychological stress group serum transferrinsaturation decreased32.2%(P<0.05),1and3days psychological stress group Serumtransferrin saturation fell7.87%,19.85%(P>0.05).
2. Gene expression analysis of iron related genes in PS rat
2.1Gene expression profile analysis
In the psychological stress rat liver, The significant difference genes number is2246.There are seven genes related with iron metabolism is changed.
2.2PS exposure caused changes in hepcidin、TfR1mRNA levels
After seven days of psychological stress, rat liver hepcidin mRNA expression levelswere significantly increased（P<0.01), but compared with the control group,1day and3days PS exposure did not change (P>0.05). TfR1mRNA levels were gradully increased in1day,3days and7days PS groups, but7days PS exposure group changed significantly(P<0.01).
2.3PS exposure changed hepcidin、TfR1protein expression
After7days PS exposure, rat liver hepcidin and TfR1protein levels were significantlyhigher than the control group(P<0.05)..
2.4PS exposure increase sfxn1expression in rat liver
Sfxn1mRNA levels were increased in3days、7days PS exposure compared with thecontrol group (P<0.01). After7days PS exposure, Sfxn1protein levels weresignificantly higher than the control group (P<0.05).
3. The effect of sfxn1on hepatic cell iron-related molecules
3.1Effect of sfxn1siRNA on sfxn1expression levels in HepG2cells
The mRNA expression of sfxn1was significantly decreased in siRNA transfection HepG2cell (P<0.05). Sfxn1protein level was significantly decreased in siRNAtransfection group (P<0.01).
3.2Sfxn1interference decrease mRNA expression of TfR1、IRP1
The TfR1、IRP1mRNA levels decreased in siRNA interference groups compared tocontrol group (P<0.05).But hepcidin、IRP2mRNA showed no difference in siRNAinterference group compared with control group (P>0.05).
3.3Effects of sfxn1interference on TfR1、IRP1protein levels
The TfR1、IRP1protein levels decreased in siRNA interference groups compared tocontrol group (P<0.05).
Conclusions
1.7days PS could cause iron accumulation in liver.
2. The gene chip found Hamp, Tfrc, sfxn1, GDF2,IREB2, NUBP1, NUBP2genes relatediron homeostasis is changed in the process of psychological stress.
3. Hepcidin,TfR1and sfxn1expression is rising with the increase in the number of stressdays.
4. sfxn1can affect the expression of TfR1
Taken together, we found psychological stress can increase transferrin receptor1expression may be by sfxn1,Result in liver cells intake of iron increased, made ironaccumulation in liver and even damage to liver.
引文
[1] M.W. Hentze, M.U. Muckenthaler, N.C Andrews. Balancing acts: molecular control of mammalianiron metabolism. Cell,2004,117:285–297.
[2] Bacon BR, Tavill AT, Brittenham GM, et al. Hepatic lipid peroxidation in vivo in rats with chroniciron overload. J Clin Invest,1983,71:429-439.
[3] Houglum K, Filip M, Witztum JL, et al. Malondialdehyde and4-hydroxynonenal protein adducts inplasma and liver of rats with iron overload. J Clin Invest,1990,86:1991-1998.
[4] Thakerngpol K, Fucharoen S, Boonyaphipat P, et al.Liver injury due to iron overload in thalassemia:histopathologic and ultrastructural studies. Biometals,1996,9:177–183.
[5] Purohit V,Russo D,Salin M.Role of iron in alcoholic liver disease:introduction and summary of thesymposium.Alcohol,2003,30(2):93-97.
[6] Guillaume Le Guenno，Emilie Chanse′aume，Marc Ruivard，et al. Study of iron metabolismdisturbances in an animal model of insulin resistance. Diabetes Research and Clinical Practice,2007(77):363–370.
[7] Ross M. Graham,Anita C. G. Chua,Kim W. Carter, et al. Hepatic Iron Loading in Mice IncreasesCholesterol Biosynthesis. HEPATOLOGY,2010,52:462-471.
[8] Min Zhao, Jianbo Chen, Min Li. Psychological stress induces hypoferremia through theIL-6-hepcidin axis in rats. BBRC,2008,373:90-93.
[1]A. Shander,M. D. Cappellini,L. T. Goodnough. Iron overload and toxicity: the hidden risk of multipleblood transfusions. Vox Sanguinis,2009,97:185–197
[2]JOHN W. EATON,MINGWEI QIAN. MOLECULAR BASES OF CELLULAR IRON TOXICITY.Free Radical Biology&Medicine,2002,(32)9:833–840.
[3]Yuling Zhao, Hailing Li, Zhonghong Gao, et al.Effects of dietary baicalin supplementation on ironoverload-induced mouse liver oxidative injury. European Journal of Pharmacology,2005,509:195–200.
[4]Wei C, Zhou J, Huang X, et al.Effects of psychological stress on serum iron and erythropoiesis. Int JHematol.2008Jul;88(1):52-6.
[5] Bacon BR, Tavill AT, Brittenham GM, et al. Hepatic lipid peroxidation in vivo in rats with chroniciron overload. J Clin Invest,1983,71:429-439.
[6] Houglum K, Filip M, Witztum JL, et al. Malondialdehyde and4-hydroxynonenal protein adducts inplasma and liver of rats with iron overload. J Clin Invest,1990,86:1991-1998.
[7] Thakerngpol K, Fucharoen S, Boonyaphipat P, et al.Liver injury due to iron overload in thalassemia:histopathologic and ultrastructural studies. Biometals,1996,9:177–183.
[8]Guillaume Le Guenno，Emilie Chanse′aume，Marc Ruivard，et al. Study of iron metabolismdisturbances in an animal model of insulin resistance. Diabetes Research and Clinical Practice,2007(77):363–370.
[9]Ross M. Graham,Anita C. G. Chua,Kim W. Carter, et al. Hepatic Iron Loading in Mice IncreasesCholesterol Biosynthesis. HEPATOLOGY,2010,52:462-471.
[10]Ogawa N, Kuwahara K. Psychophysiology of emotion-communication of emotion..J. Psychosom.Med,1966,6:352-357.
[11]贾克然，郭刚，刘开云,等.单纯心理应激小鼠模型的建立及对行为、内分泌免疫功能的影响.免疫学杂志,2009(3):18-22.
[1] Gregory J. Anderson, Deepak Darshan,Sarah J. Wilkins, et al. Regulation of systemic ironhomeostasis: how the bodyresponds to changes in iron demand. Biometals,2007(20):665–674.
[2]Robert E. Fleming, Mary C. Migas, Christopher C. Holden，et al. Transferrin receptor2:Continued expression in mouse liver in the face of iron overload and in hereditaryhemochromatosis. Proc Natl Acad Sci USA,2000,97(5),2214–2219.
[3] Enrico Rossi. Hepcidin-the Iron Regulatory Hormone. Clin Biochem Rev,2005,26,47-49.
[4]Lee P.Role of matriptase-2(TMPRSS6) in iron metabolism. ActaHaematol,2009,122(2-3):87-96.
[5]李瑶主编：基因芯片与功能基因组化学工业出版社北京2004，102-103.
[6]Pfaffl MW.A new mathematical model for relative quantification in real-timeRT-PCR．Nucleic Acids Res,2001,29(9):e45.
[7] Marino JH,Peyton C,Kenton SM.Accurate and statistically verified quantification ofrelative mRNA abundances using SYBR Green I and real-time RT-PCR.J LrnmunolMethods,2003,283(1-2):291—306.
[8]Li BW,Amy CR,Jie T,et a1.Quantitative analysis of gender-regulated transcripts infilarial nematode Brugia malayi by real-time RT-PCR．Molecular&BiochemicalParasitology．2004
[9] Bacon BR, Tavill AT, Brittenham GM, et al. Hepatic lipid peroxidation in vivo in ratswith chronic iron overload. J Clin Invest,1983,71:429-439.
[10]Peters TJ, O’Connell MJ, Ward RJ. Role of free-radical mediated lipid peroxidation inthe pathogenesis of hepatic damage by lysosomal disruption. In: Poli G, CheesmanHH, Dianzani MU, Slater TF, eds. Free Radicals in Liver Injury. Oxford, England:I.R.L.,1985:107-115.
[11] Houglum K, Filip M, Witztum JL, et al. Malondialdehyde and4-hydroxynonenalprotein adducts in plasma and liver of rats with iron overload. J ClinInvest,1990,86:1991-1998.
[12] Thakerngpol K, Fucharoen S, Boonyaphipat P, et al.Liver injury due to iron overloadin thalassemia: histopathologic and ultrastructural studies. Biometals,1996,9:177–183.
[13]Purohit V,Russo D,Salin M.Role of iron in alcoholic liver disease:introduction andsummary of the symposium.Alcohol,2003,30(2):93-97.
[14]Fischer JG,Glauert HP,Yin T,et al. Moderate iron overload enhances lipid peroxidationin livers of rats, but does not affect NF-kappaB activation induced by the peroxisomeproliferator, Wy-14,643. J Nutr,2002Sep;132(9):2525-31.
[15]Guillaume Le Guenno，Emilie Chanse′aume，Marc Ruivard，et al. Study of ironmetabolism disturbances in an animal model of insulin resistance. Diabetes Researchand Clinical Practice,2007(77):363–370.
[16]Ross M. Graham,Anita C. G. Chua,Kim W. Carter, et al. Hepatic Iron Loading in MiceIncreases Cholesterol Biosynthesis. HEPATOLOGY,2010,52:462-471.
[17]Tao Wen-ring,Mallard B． Diferentially expressed genes associated withStaphylococcus aureus mastitis of Canadian Holstein cows[J].Vet ImmunolImmunopathol,2007,120(3-4)：201—211.
[18]Towsend K，Trevino V，Falciani F，et a1．Identification of VDR-responsive genesignatures in breast cancer cells[J]．Oncology,2006,71(1-2):111-123．
[19]Smytb GK,Yang YH,Speed T． Statistical issues in cDNA microarray dataanalysis[J]．Methods Mol Biol,2003,224:111-136.
[20] Nemeth E,Tuttle MS,Powelson J,et al.Hepcidin regulates iron efflux by binding toferroportin and inducing its internalization. Science,2004,306(5704):2090-2093.
[21]Min Zhao, Jianbo Chen, Min Li. Psychological stress induces hypoferremia throughthe IL-6-hepcidin axis in rats. BBRC,2008,373:90-93.
[22] Tomas Ganz. Hepcidin and Its Role in Regulating Systemic Iron Metabolism.Hematology Am Soc Hematol Educ Program.2006:29-35,507.
[23] Rui-Hong Wang,Cuiling Li,Xiaoling Xu, et al. A role of SMAD4in iron metabolismthrough the positive regulation of hepcidin expression. CELL METABOLISM,2005(12),399-499.
[24]Kim R. Bridle, Ting-Kin Cheung, Therese L. Murphy. Hepcidin Is Down-regulated inAlcoholic Liver Injury:Implications for the Pathogenesis of Alcoholic Liver Disease.ALCOHOLISM CLINICAL AND EXPERIMENTAL RESEARCH,2006,30(1):102-112.
[25] Elizabeta Nemeth, Antonella Roetto, Giovanni Garozzo, et al. Hepcidin is decreasedin TFR2hemochromatosis. BLOOD,2005,15,(105):1803-1806.
[26]Kawabata H, Yang R, Hirama T, et al. Molecular cloning of transferrin receptor2. Anew member of the transferrin receptor-like family.J Biol Chem,1999,274(30):20826–20832.
[27] Kawabata H, Germain RS, Vuong PT,et a1．Transferrin receptor2-alpha supports cellgrowth both in iron-chelated cultured cells and in vivo.J BiolChem,2000,275(22):16618-25.
[28] Graham RM, Reutens GM, Herbison CE, et a1.Transferrin receptor2mediates uptakeof transferrin-bound and non-transferrin-bound iron. Journal of Hepatology,2008,48:327–334.
[29]Babitt JL,Huang FW,Wrighting DM,et al. Bone morphogenetic protein signaling byhemojuvelin regulates hepcidin expression. Nat Genet,2006,38:531–39.
[30]Babitt JL, Huang FW, Xia Y, et al. Modulation of bone morphogenetic proteinsignaling in vivo regulates systemic iron balance. J Clin Invest,2007,117,1933–39.
[31]Truksa J, Peng H, Lee P, et al. Bone morphogenetic proteins2,4, and9stimulatemurine Hepcidin1expression independently of Hfe, transferrin receptor2(Tfr2), andIL-6. Proc Natl Acad Sci U S A,2006,103(27):10289-10293.
[1]Xiaojing Li,Dandan Han,Richard Kin Ting Kam, et al. Developmental Expression ofsideroflexin Family Genes in Xenopus Embryos. DEVELOPMENTAL DYNAMICS,2010,239:2742–2747.
[2]Fleming MD, Campagna DR, Haslett JN, et al. A mutation in a mitochondrialtransmembrane protein is responsible for the pleiotropic hematological and skeletalphenotype of flexed-tail (f/f) mice. Genes Dev,2001Mar15,15(6):652-7.
[3]Roy CN, Andrews NC. Recent advances in disorders of iron metabolism: mutations,mechanisms and modifiers. Hum Mol Genet,2001,10(20):2181-6.
[4]Laurie E. Lenox, John M. Perry, Robert F. Paulson. BMP4and Madh5regulate theerythroid response to acute anemia. Blood.2005,105:2741-2748
[5]Palmieri F. The mitochondrial transporter family (SLC25): physiological andpathological implications.2004,Pflugers Arch,447:689–709.
[6]Haitina T, Lindblom J, Renstr m T, et al. Fourteen novel human members ofmitochondrial solute carrier family25(SLC25) widely expressed in the central nervoussystem. Genomics.2006Dec;88(6):779-90.
[7]Wohlrab H.Transport proteins (carriers) of mitochondria.IUBMBLife,2009,61(1):40-6.
[8]Gnoni GV, Priore P, Geelen MJ, et al. The mitochondrial citrate carrier: metabolic roleand regulation of its activity and expression. IUBMB Life,2009,61:987–994.
[9]Azzi A, Glerum M, Koller R, et al. The mitochondrial tricarboxylate carrier. JBioenerg Biomembr,1993,25(5):515-24.
[10]Shaw GC, Cope JJ, Li L, et al. Mitoferrin is essential for erythroid iron assimilation.Nature,2006,440(7080):96-100.
[11]Troadec MB, Warner D, Wallace J, et al. Targeted deletion of the mouse Mitoferrin1gene: from anemia to protoporphyria. Blood,2011,117(20):5494–5502.
[12]Paradkar PN, Zumbrennen KB, Paw BH, et al. Regulation of mitochondrial ironimport through differential turnover of mitoferrin1and mitoferrin2. Mol Cell Biol,2009,29(4):1007-16.
[13]Foury F, Roganti T. Deletion of the mitochondrial carrier genes MRS3andMRS4suppresses mitochondrial iron accumulation in a yeastfrataxin-deficient strain. J Biol Chem,2002,277(27):24475-83.
[14]Mühlenhoff U, Stadler JA, Richhardt N, et al.A specific role of the yeast mitochondrialcarriers MRS3/4p in mitochondrial iron acquisition under iron-limiting conditions. JBiol Chem.2003Oct17,278(42):40612-20.
[15]Zhang Y, Lyver ER, Knight SA, et al.Frataxin and mitochondrial carrier proteins,Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis. J Biol Chem.2005May20;280(20):19794-807.
[16]Nilsson R, Schultz IJ, Pierce EL, et al. Discovery of genes essential for hemebiosynthesis through large-scale gene expression analysis. Cell Metab,2009,10(2):119-30.
[17]Levi S, Corsi B, Bosisio M, et al. A human mitochondrial ferritin encoded by anintronless gene.J Biol Chem,2001,276(27):24437-40.
[18]Drysdale J, Arosio P, Invernizzi R,et al. Mitochondrial ferritin: a new player in ironmetabolism.Blood Cells Mol Dis.2002Nov-Dec;29(3):376-83.
[19]Yang H, Yang M, Guan H,et al. Mitochondrial ferritin in neurodegenerativediseases.Neurosci Res.2013Sep-Oct;77(1-2):1-7.
[20]Cazzola M, Invernizzi R, Bergamaschi G,et al. Mitochondrial ferritin expression inerythroid cells from patients with sideroblastic anemia. Blood,2003,101(5):1996–2000.
[21]Corsi B, Cozzi A, Arosio P, et al. Human mitochondrial ferritin expressed in HeLacells incorporates iron and affects cellular iron metabolism. J Biol Chem,2002,277(25):22430-7.
[22]Nie G, Sheftel AD, Kim SF, et al. Overexpression of mitochondrial ferritin causescytosolic iron depletion and changes cellular iron homeostasis. Blood,2005,105(5):2161-7.
[1]Evstatiev, Gasche,Christoph.Iron sensing and signalling.Gut,2012,61(6):933-952.
[2]Krause A,Neitz S,Magert HJ,et al. LEAP-1,a novel highly disulfide-bonded humanpeptide,exhibits antimicrobial activity. FEBS Lett,2000,480(2-3):147–150.
[3] Park CH, Valore EV, Waring AJ, et al. Hepcidin, a urinary antimicrobial peptidesynthesized in the liver. J Biol Chem,2001,76(11):7806–7810.
[4] Pigeon C, Ilyin G, Courselaud B, et al. A new mouse liver-specific gene, encoding aprotein homologous to human antimicrobial peptide hepcidin, is overexpressed duringiron overload. J Biol Chem,2001,276(11):7811–7819.
[5] Nicolas G, Bennoun M, Devaux I, et al. Lack of hepcidin gene expression and severetissue iron overload in upstream stimulatory factor2(USF2) knockout mice.Proc NatlAcad Sci USA,2001,98(15):8780–8785.
[6]Yves Deugnier,Pierre Brissot, Olivier Loreal. Iron and the liver: Update2008. Journal ofHepatology,2008,48: S113–S123.
[7] Roetto A, Papanikolaou G, Politou M, et al. Mutant antimicrobial peptide Hepcidin isassociated with severe juvenile hemochromatosis. Nat Genet.2003,33(1):21-22.
[8]Nemeth E,Tuttle MS,Powelson J,et al.Hepcidin regulates iron efflux by binding toferroportin and inducing its internalization. Science,2004,306(5704):2090-2093.
[9] Chang CK, Zhang X, Xiao C,et al. Research advances of hepcidin expression and itsregulation mechanism.Zhongguo Shi Yan Xue Ye Xue Za Zhi,2012Aug,20(4):1030-3.
[10] Enrico Rossi. Hepcidin-the Iron Regulatory Hormone. Clin Biochem Rev,2005,26:47-49.
[11]Wilkins SJ, Frazer DM, Millard KN, McLaren GD, Anderson GJ. Iron metabolism inthe hemoglobin-deficit mouse: correlation of diferric transferrin with hepcidinexpression. Blood.2006;107:1659–1664.
[12] R. Deicher,W. H. H rl. New insights into the regulation of iron homeostasis. EuropeanJournal of Clinical Investigation.2006,36:301–309.
[13]Trinder D, Baker E.Transferrin receptor2: a new molecule in iron metabolism.Int JBiochem Cell Biol.2003Mar;35(3):292-6.
[14] Kawabata H, Yang R, Hirama T, et al. Molecular cloning of transferrin receptor2. Anew member of the transferrin receptor-like family.J Biol Chem,1999,274(30):20826–20832.
[15] Kawabata H, Germain RS, Vuong PT,et a1．Transferrin receptor2-alpha supports cellgrowth both in iron-chelated cultured cells and in vivo.J BiolChem,2000,275(22):16618-25.
[16] Graham RM, Reutens GM, Herbison CE, et a1.Transferrin receptor2mediates uptakeof transferrin-bound and non-transferrin-bound iron. Journal of Hepatology,2008,48:327–334.
[17]Camaschella C, Roetto A, De Gobbi M, et al. Genetic haemochromatosis: genes andmutations associated with iron loading. Best Pract Res Clin Haematol.2002Jun;15(2):261-76.
[18]Fleming RE,Ahmann JR, Migas MC,et al. Targeted mutagenesis of the murinetransferrin receptor-2gene produces hemochromatosis. Proc Natl Acad Sci U S A.2002,99:10653-10658.
[19]Bartnikas TB, Wildt SJ, Wineinger AE, et al. A novel rat model of hereditaryhemochromatosis due to a mutation in transferrin receptor2.Comp Med.2013Apr;63(2):143-55.
[20]Wallace DF, Summerville L, Crampton EM, et al. Combined deletion of Hfe andtransferrin receptor2in mice leads to marked dysregulation of hepcidin and ironoverload. Hepatology.2009Dec;50(6):1992-2000.
[21]Wallace DF, Summerville L, Subramaniam VN, Targeted Disruption of the HepaticTransferrin Receptor2Gene in Mice Leads to Iron Overload. Gastroenterology.2007Jan,132(1):301-10.
[22]Martha B,Johnson,Caroline A. Enns. Diferric transferrin regulates transferrin receptor2protein stability. BLOOD,2004,104(13):4287-4293.
[23]Robb A,Wessling-Resnick M. Regulation of transferrin receptor2protein levels bytransferrin. Blood,2004,104:4294-4299.
[24]Chen J, Wang J, Meyers KR,, et al. Transferrin-directed internalization and cycling oftransferrin receptor2.Traffic，2009Oct;10(10):1488-501.
[25] Juxing Chen, Caroline A. Enns. The Cytoplasmic Domain of Transferrin Receptor2Dictates Its Stability and Response to Holo-transferrin in Hep3B Cells. THEJOURNAL OF BIOLOGICAL CHEMISTRY,2007,282(9):6201–6209.
[26]Ehrke SG,Kulaksiz H,Herrmann T,et al. Expression of hepcidin in hereditaryhemochromatosis: evidence for a regulation in response to the serum transferrinsaturation and to non-transferrin-bound iron. Blood,2003,102:371-376.
[27]Gao J,Chen J,Kramer M,et al.Interaction of the hereditary hemochromatosis proteinHFE with transferrin receptor2is required for transferrin-induced hepcidinexpression.Cell Metab,2009,9:217–227.
[28]Wallace DF,Trinder D,Subramaniam VN.Hepcidin regulation by HFE and TFR2: is itenough to give a hepatocyte a complex? Gastroenterology,2009,137(3):1173-5.
[29]Tapasree Goswami,Nancy C. Andrews. Hereditary Hemochromatosis Protein, HFE,Interaction with Transferrin Receptor2Suggests a Molecular Mechanism forMammalian Iron Sensing. THE JOURNAL OF BIOLOGICAL CHEMISTRY,2006,281(39):28494–28498.
[30]Juxing Chen, Maja Chloupkova, Junwei Gao, et al. HFE Modulates TransferrinReceptor2Levels in Hepatoma Cells via Interactions That Differ from TransferrinReceptor1-HFE Interactions. THE JOURNAL OF BIOLOGICAL CHEMISTRY,2007,282(51):36862–36870.
[31]Robert E. Fleming. Iron Sensing as a Partnership:HFE and Transferrin Receptor2. CellMetabolism,2009,9(3):211-2.
[32]Abdul Waheed, Robert S. Britton, Jeffrey H. Grubb, et al. HFE association withtransferrin receptor2increases cellular uptake of transferrin-bound iron. Archives ofBiochemistry and Biophysics,2008,474:193–197.
[33] Alessia Calzolari, Carla Raggi, Silvia Deaglio, et al. TfR2localizes in lipid raftdomains and is released in exosomes to activate signal transduction along the MAPKpathway. J Cell Sci,2006,119(21):4486-98.
[34]Ramey G, Deschemin JC, Vaulont S. Cross-talk between the mitogen activated proteinkinase and bone morphogenetic protein/hemojuvelin pathways is required for theinduction of hepcidin by holotransferrin in primary mouse hepatocytes.Haematologica,2009,94:765–72
[35]Meyer C, Godoy P, Bachmann A,et a1. Distinct role of endocytosis for Smad andnon-Smad TGF-β signaling regulation in hepatocytes.J Hepatol.2011,55(2):369-78.
[36]Wang RH,Li C,Xu X,A role of Smad4in iron metabolism through the positiveregulation of hepcidin expression.Cell Metab,2005,2:399–409.
[37]Mleczko-sanecka K,Casanovas G,Ragab A,et a1.SMAD7controls iron metabolism as apotent inhibitor of hepcidin expression.Blood,2010,115:2657–2665.
[38]Zhang AS, Yang F, Wang J, et al. Hemojuvelin-neogenin interaction is required forbone morphogenic protein-4-induced hepcidin expression. J Biol Chem.2009Aug21;284(34):22580-9.
[39]Yann Gibert, Victoria J. Lattanzi, et al. BMP Signaling Modulates Hepcidin Expressionin Zebrafish Embryos Independent of Hemojuvelin. PLoS One.2011;6(1): e14553.
[40]Truksa J, Peng H, Lee P, et al. Bone morphogenetic proteins2,4, and9stimulatemurine Hepcidin1expression independently of Hfe, transferrin receptor2(Tfr2), andIL-6. Proc Natl Acad Sci U S A,2006,103(27):10289-10293.
[41]Meynard D,Kautz L,Darnaud V,et al. Lack of the bone morphogenetic protein BMP6induces massive iron over-load.Nat Genet,2009,41:478–481.
[42]Andriopoulos B Jr,Corradini E,Xia Y,et al. BMP-6is a key endogenous regulator ofhepcidin expression and iron metabolism.Nat Genet,2009,41:482–487.
[43] Kautz L,meyrard D,MonnierA,et al. Iron regulates phosphorylation of Smadl／5／8and gene expression of Bmp6， Smad7， ldl， and Atoh8in the mouseliver.Blood,2008,112:1503–1509.
[44]Atndt S,Maegdefrau U,Dorn C,et a1.Iron-induced expression of bone morphogenicprotein6in intestinal cells is the main regulator of hepatic hepcidin expression invivo.Gastroenterology,2010,138:372–382.
[45]Kautz L,Meynard D,Besson-Fournier C,et a1.Iron overload induces BMP6expressionin the liver but not in the duodenum.Haematologica,2011Feb,96(2):199-203.
[46]Brakensiek K, Fegbeutel C, M lzer M, et al. Juvenile hemochromatosis due tohomozygosity for the G320V mutation in the HJV gene with fatal outcome. ClinGenet,2009Nov,76(5):493-5.
[47]Papanikolaou G, Tzilianos M, Christakis JI, et al. Hepcidin in iron overloaddisorders.Blood,2005,105:4103–5.
[48]Huang FW, Pinkus JL, Pinkus GS, et al. A mouse model of juvenile hemochromatosis.J Clin Invest,2005,115(8):2187-2191.
[49] Niederkofler V, Salie R, Arber S. Hemojuvelin is essential for dietary iron sensing,and its mutation leads to severe iron overload. J Clin Invest,2005Aug,115(8):2180-6.
[50] Malyszko J..Hemojuvelin: the hepcidin story continues.Kidney Blood Press Res.2009,32(2):71-6.
[51] Kuninger D, Kuns-Hashimoto R, Kuzmickas R, et al. Complex biosynthesis of themuscle-enriched iron regulator RGMc. J. Cell Sci,2006,119:3273–83.
[52] Zhang AS, Enns CA. Iron homeostasis: recently identified proteins provide insightinto novel control mechanisms. J Biol Chem.2009Jan9,284(2):711-5.
[53]McMahon S, Grondin F, McDonald PP, et al. Hypoxia enhanced expression of theproprotein convertase furin is mediated by hypoxia-inducible factor-1: impact on thebioactivation of proproteins. J Biol Chem,2005,280(8):6561-6569.
[54]Lee P.Role of matriptase-2(TMPRSS6) in iron metabolism.Acta Haematol.2009,122(2-3):87-96..
[55]Folgueras AR,de Lara FM,Pendás AM,et al.Membrane-bound serine proteasematriptase-2(Tmprss6) is an essential regulator of iron homeostasis. Blood,2008Sep15,112(6):2539-45.
[56]Pellegrino RM, Coutinho M, D'Ascola D, et al. Two novel mutations in the tmprss6gene associated with iron-refractory iron-deficiency anaemia (irida) and partialexpression in the heterozygous form. Br J Haematol,2012Sep,158(5):668-72.
[57] SM, Strouse JJ, Markianos K, et al. Mutations in TMPRSS6cause iron-refractory irondeficiency anemia (IRIDA). Nat Genet,2008May,40(5):569-71.
[58] Silvestri L,Pagani A,Nai A, et al.The serine protease matriptase-2(TMPRSS6) inhibitshepcidin activation by cleaving membrane hemojuvelin. Cell Metab,2008Dec,8(6):502-11.
[2] Bacon BR, Tavill AT, Brittenham GM, et al. Hepatic lipid peroxidation in vivo in rats with chroniciron overload. J Clin Invest,1983,71:429-439.
[3] Houglum K, Filip M, Witztum JL, et al. Malondialdehyde and4-hydroxynonenal protein adducts inplasma and liver of rats with iron overload. J Clin Invest,1990,86:1991-1998.
[4] Thakerngpol K, Fucharoen S, Boonyaphipat P, et al.Liver injury due to iron overload in thalassemia:histopathologic and ultrastructural studies. Biometals,1996,9:177–183.
[5] Purohit V,Russo D,Salin M.Role of iron in alcoholic liver disease:introduction and summary of thesymposium.Alcohol,2003,30(2):93-97.
[6] Guillaume Le Guenno，Emilie Chanse′aume，Marc Ruivard，et al. Study of iron metabolismdisturbances in an animal model of insulin resistance. Diabetes Research and Clinical Practice,2007(77):363–370.
[7] Ross M. Graham,Anita C. G. Chua,Kim W. Carter, et al. Hepatic Iron Loading in Mice IncreasesCholesterol Biosynthesis. HEPATOLOGY,2010,52:462-471.
[8] Min Zhao, Jianbo Chen, Min Li. Psychological stress induces hypoferremia through theIL-6-hepcidin axis in rats. BBRC,2008,373:90-93.
[1]A. Shander,M. D. Cappellini,L. T. Goodnough. Iron overload and toxicity: the hidden risk of multipleblood transfusions. Vox Sanguinis,2009,97:185–197
[2]JOHN W. EATON,MINGWEI QIAN. MOLECULAR BASES OF CELLULAR IRON TOXICITY.Free Radical Biology&Medicine,2002,(32)9:833–840.
[3]Yuling Zhao, Hailing Li, Zhonghong Gao, et al.Effects of dietary baicalin supplementation on ironoverload-induced mouse liver oxidative injury. European Journal of Pharmacology,2005,509:195–200.
[4]Wei C, Zhou J, Huang X, et al.Effects of psychological stress on serum iron and erythropoiesis. Int JHematol.2008Jul;88(1):52-6.
[5] Bacon BR, Tavill AT, Brittenham GM, et al. Hepatic lipid peroxidation in vivo in rats with chroniciron overload. J Clin Invest,1983,71:429-439.
[6] Houglum K, Filip M, Witztum JL, et al. Malondialdehyde and4-hydroxynonenal protein adducts inplasma and liver of rats with iron overload. J Clin Invest,1990,86:1991-1998.
[7] Thakerngpol K, Fucharoen S, Boonyaphipat P, et al.Liver injury due to iron overload in thalassemia:histopathologic and ultrastructural studies. Biometals,1996,9:177–183.
[8]Guillaume Le Guenno，Emilie Chanse′aume，Marc Ruivard，et al. Study of iron metabolismdisturbances in an animal model of insulin resistance. Diabetes Research and Clinical Practice,2007(77):363–370.
[9]Ross M. Graham,Anita C. G. Chua,Kim W. Carter, et al. Hepatic Iron Loading in Mice IncreasesCholesterol Biosynthesis. HEPATOLOGY,2010,52:462-471.
[10]Ogawa N, Kuwahara K. Psychophysiology of emotion-communication of emotion..J. Psychosom.Med,1966,6:352-357.
[11]贾克然，郭刚，刘开云,等.单纯心理应激小鼠模型的建立及对行为、内分泌免疫功能的影响.免疫学杂志,2009(3):18-22.
[1] Gregory J. Anderson, Deepak Darshan,Sarah J. Wilkins, et al. Regulation of systemic ironhomeostasis: how the bodyresponds to changes in iron demand. Biometals,2007(20):665–674.
[2]Robert E. Fleming, Mary C. Migas, Christopher C. Holden，et al. Transferrin receptor2:Continued expression in mouse liver in the face of iron overload and in hereditaryhemochromatosis. Proc Natl Acad Sci USA,2000,97(5),2214–2219.
[3] Enrico Rossi. Hepcidin-the Iron Regulatory Hormone. Clin Biochem Rev,2005,26,47-49.
[4]Lee P.Role of matriptase-2(TMPRSS6) in iron metabolism. ActaHaematol,2009,122(2-3):87-96.
[5]李瑶主编：基因芯片与功能基因组化学工业出版社北京2004，102-103.
[6]Pfaffl MW.A new mathematical model for relative quantification in real-timeRT-PCR．Nucleic Acids Res,2001,29(9):e45.
[7] Marino JH,Peyton C,Kenton SM.Accurate and statistically verified quantification ofrelative mRNA abundances using SYBR Green I and real-time RT-PCR.J LrnmunolMethods,2003,283(1-2):291—306.
[8]Li BW,Amy CR,Jie T,et a1.Quantitative analysis of gender-regulated transcripts infilarial nematode Brugia malayi by real-time RT-PCR．Molecular&BiochemicalParasitology．2004
[9] Bacon BR, Tavill AT, Brittenham GM, et al. Hepatic lipid peroxidation in vivo in ratswith chronic iron overload. J Clin Invest,1983,71:429-439.
[10]Peters TJ, O’Connell MJ, Ward RJ. Role of free-radical mediated lipid peroxidation inthe pathogenesis of hepatic damage by lysosomal disruption. In: Poli G, CheesmanHH, Dianzani MU, Slater TF, eds. Free Radicals in Liver Injury. Oxford, England:I.R.L.,1985:107-115.
[11] Houglum K, Filip M, Witztum JL, et al. Malondialdehyde and4-hydroxynonenalprotein adducts in plasma and liver of rats with iron overload. J ClinInvest,1990,86:1991-1998.
[12] Thakerngpol K, Fucharoen S, Boonyaphipat P, et al.Liver injury due to iron overloadin thalassemia: histopathologic and ultrastructural studies. Biometals,1996,9:177–183.
[13]Purohit V,Russo D,Salin M.Role of iron in alcoholic liver disease:introduction andsummary of the symposium.Alcohol,2003,30(2):93-97.
[14]Fischer JG,Glauert HP,Yin T,et al. Moderate iron overload enhances lipid peroxidationin livers of rats, but does not affect NF-kappaB activation induced by the peroxisomeproliferator, Wy-14,643. J Nutr,2002Sep;132(9):2525-31.
[15]Guillaume Le Guenno，Emilie Chanse′aume，Marc Ruivard，et al. Study of ironmetabolism disturbances in an animal model of insulin resistance. Diabetes Researchand Clinical Practice,2007(77):363–370.
[16]Ross M. Graham,Anita C. G. Chua,Kim W. Carter, et al. Hepatic Iron Loading in MiceIncreases Cholesterol Biosynthesis. HEPATOLOGY,2010,52:462-471.
[17]Tao Wen-ring,Mallard B． Diferentially expressed genes associated withStaphylococcus aureus mastitis of Canadian Holstein cows[J].Vet ImmunolImmunopathol,2007,120(3-4)：201—211.
[18]Towsend K，Trevino V，Falciani F，et a1．Identification of VDR-responsive genesignatures in breast cancer cells[J]．Oncology,2006,71(1-2):111-123．
[19]Smytb GK,Yang YH,Speed T． Statistical issues in cDNA microarray dataanalysis[J]．Methods Mol Biol,2003,224:111-136.
[20] Nemeth E,Tuttle MS,Powelson J,et al.Hepcidin regulates iron efflux by binding toferroportin and inducing its internalization. Science,2004,306(5704):2090-2093.
[21]Min Zhao, Jianbo Chen, Min Li. Psychological stress induces hypoferremia throughthe IL-6-hepcidin axis in rats. BBRC,2008,373:90-93.
[22] Tomas Ganz. Hepcidin and Its Role in Regulating Systemic Iron Metabolism.Hematology Am Soc Hematol Educ Program.2006:29-35,507.
[23] Rui-Hong Wang,Cuiling Li,Xiaoling Xu, et al. A role of SMAD4in iron metabolismthrough the positive regulation of hepcidin expression. CELL METABOLISM,2005(12),399-499.
[24]Kim R. Bridle, Ting-Kin Cheung, Therese L. Murphy. Hepcidin Is Down-regulated inAlcoholic Liver Injury:Implications for the Pathogenesis of Alcoholic Liver Disease.ALCOHOLISM CLINICAL AND EXPERIMENTAL RESEARCH,2006,30(1):102-112.
[25] Elizabeta Nemeth, Antonella Roetto, Giovanni Garozzo, et al. Hepcidin is decreasedin TFR2hemochromatosis. BLOOD,2005,15,(105):1803-1806.
[26]Kawabata H, Yang R, Hirama T, et al. Molecular cloning of transferrin receptor2. Anew member of the transferrin receptor-like family.J Biol Chem,1999,274(30):20826–20832.
[27] Kawabata H, Germain RS, Vuong PT,et a1．Transferrin receptor2-alpha supports cellgrowth both in iron-chelated cultured cells and in vivo.J BiolChem,2000,275(22):16618-25.
[28] Graham RM, Reutens GM, Herbison CE, et a1.Transferrin receptor2mediates uptakeof transferrin-bound and non-transferrin-bound iron. Journal of Hepatology,2008,48:327–334.
[29]Babitt JL,Huang FW,Wrighting DM,et al. Bone morphogenetic protein signaling byhemojuvelin regulates hepcidin expression. Nat Genet,2006,38:531–39.
[30]Babitt JL, Huang FW, Xia Y, et al. Modulation of bone morphogenetic proteinsignaling in vivo regulates systemic iron balance. J Clin Invest,2007,117,1933–39.
[31]Truksa J, Peng H, Lee P, et al. Bone morphogenetic proteins2,4, and9stimulatemurine Hepcidin1expression independently of Hfe, transferrin receptor2(Tfr2), andIL-6. Proc Natl Acad Sci U S A,2006,103(27):10289-10293.
[1]Xiaojing Li,Dandan Han,Richard Kin Ting Kam, et al. Developmental Expression ofsideroflexin Family Genes in Xenopus Embryos. DEVELOPMENTAL DYNAMICS,2010,239:2742–2747.
[2]Fleming MD, Campagna DR, Haslett JN, et al. A mutation in a mitochondrialtransmembrane protein is responsible for the pleiotropic hematological and skeletalphenotype of flexed-tail (f/f) mice. Genes Dev,2001Mar15,15(6):652-7.
[3]Roy CN, Andrews NC. Recent advances in disorders of iron metabolism: mutations,mechanisms and modifiers. Hum Mol Genet,2001,10(20):2181-6.
[4]Laurie E. Lenox, John M. Perry, Robert F. Paulson. BMP4and Madh5regulate theerythroid response to acute anemia. Blood.2005,105:2741-2748
[5]Palmieri F. The mitochondrial transporter family (SLC25): physiological andpathological implications.2004,Pflugers Arch,447:689–709.
[6]Haitina T, Lindblom J, Renstr m T, et al. Fourteen novel human members ofmitochondrial solute carrier family25(SLC25) widely expressed in the central nervoussystem. Genomics.2006Dec;88(6):779-90.
[7]Wohlrab H.Transport proteins (carriers) of mitochondria.IUBMBLife,2009,61(1):40-6.
[8]Gnoni GV, Priore P, Geelen MJ, et al. The mitochondrial citrate carrier: metabolic roleand regulation of its activity and expression. IUBMB Life,2009,61:987–994.
[9]Azzi A, Glerum M, Koller R, et al. The mitochondrial tricarboxylate carrier. JBioenerg Biomembr,1993,25(5):515-24.
[10]Shaw GC, Cope JJ, Li L, et al. Mitoferrin is essential for erythroid iron assimilation.Nature,2006,440(7080):96-100.
[11]Troadec MB, Warner D, Wallace J, et al. Targeted deletion of the mouse Mitoferrin1gene: from anemia to protoporphyria. Blood,2011,117(20):5494–5502.
[12]Paradkar PN, Zumbrennen KB, Paw BH, et al. Regulation of mitochondrial ironimport through differential turnover of mitoferrin1and mitoferrin2. Mol Cell Biol,2009,29(4):1007-16.
[13]Foury F, Roganti T. Deletion of the mitochondrial carrier genes MRS3andMRS4suppresses mitochondrial iron accumulation in a yeastfrataxin-deficient strain. J Biol Chem,2002,277(27):24475-83.
[14]Mühlenhoff U, Stadler JA, Richhardt N, et al.A specific role of the yeast mitochondrialcarriers MRS3/4p in mitochondrial iron acquisition under iron-limiting conditions. JBiol Chem.2003Oct17,278(42):40612-20.
[15]Zhang Y, Lyver ER, Knight SA, et al.Frataxin and mitochondrial carrier proteins,Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis. J Biol Chem.2005May20;280(20):19794-807.
[16]Nilsson R, Schultz IJ, Pierce EL, et al. Discovery of genes essential for hemebiosynthesis through large-scale gene expression analysis. Cell Metab,2009,10(2):119-30.
[17]Levi S, Corsi B, Bosisio M, et al. A human mitochondrial ferritin encoded by anintronless gene.J Biol Chem,2001,276(27):24437-40.
[18]Drysdale J, Arosio P, Invernizzi R,et al. Mitochondrial ferritin: a new player in ironmetabolism.Blood Cells Mol Dis.2002Nov-Dec;29(3):376-83.
[19]Yang H, Yang M, Guan H,et al. Mitochondrial ferritin in neurodegenerativediseases.Neurosci Res.2013Sep-Oct;77(1-2):1-7.
[20]Cazzola M, Invernizzi R, Bergamaschi G,et al. Mitochondrial ferritin expression inerythroid cells from patients with sideroblastic anemia. Blood,2003,101(5):1996–2000.
[21]Corsi B, Cozzi A, Arosio P, et al. Human mitochondrial ferritin expressed in HeLacells incorporates iron and affects cellular iron metabolism. J Biol Chem,2002,277(25):22430-7.
[22]Nie G, Sheftel AD, Kim SF, et al. Overexpression of mitochondrial ferritin causescytosolic iron depletion and changes cellular iron homeostasis. Blood,2005,105(5):2161-7.
[1]Evstatiev, Gasche,Christoph.Iron sensing and signalling.Gut,2012,61(6):933-952.
[2]Krause A,Neitz S,Magert HJ,et al. LEAP-1,a novel highly disulfide-bonded humanpeptide,exhibits antimicrobial activity. FEBS Lett,2000,480(2-3):147–150.
[3] Park CH, Valore EV, Waring AJ, et al. Hepcidin, a urinary antimicrobial peptidesynthesized in the liver. J Biol Chem,2001,76(11):7806–7810.
[4] Pigeon C, Ilyin G, Courselaud B, et al. A new mouse liver-specific gene, encoding aprotein homologous to human antimicrobial peptide hepcidin, is overexpressed duringiron overload. J Biol Chem,2001,276(11):7811–7819.
[5] Nicolas G, Bennoun M, Devaux I, et al. Lack of hepcidin gene expression and severetissue iron overload in upstream stimulatory factor2(USF2) knockout mice.Proc NatlAcad Sci USA,2001,98(15):8780–8785.
[6]Yves Deugnier,Pierre Brissot, Olivier Loreal. Iron and the liver: Update2008. Journal ofHepatology,2008,48: S113–S123.
[7] Roetto A, Papanikolaou G, Politou M, et al. Mutant antimicrobial peptide Hepcidin isassociated with severe juvenile hemochromatosis. Nat Genet.2003,33(1):21-22.
[8]Nemeth E,Tuttle MS,Powelson J,et al.Hepcidin regulates iron efflux by binding toferroportin and inducing its internalization. Science,2004,306(5704):2090-2093.
[9] Chang CK, Zhang X, Xiao C,et al. Research advances of hepcidin expression and itsregulation mechanism.Zhongguo Shi Yan Xue Ye Xue Za Zhi,2012Aug,20(4):1030-3.
[10] Enrico Rossi. Hepcidin-the Iron Regulatory Hormone. Clin Biochem Rev,2005,26:47-49.
[11]Wilkins SJ, Frazer DM, Millard KN, McLaren GD, Anderson GJ. Iron metabolism inthe hemoglobin-deficit mouse: correlation of diferric transferrin with hepcidinexpression. Blood.2006;107:1659–1664.
[12] R. Deicher,W. H. H rl. New insights into the regulation of iron homeostasis. EuropeanJournal of Clinical Investigation.2006,36:301–309.
[13]Trinder D, Baker E.Transferrin receptor2: a new molecule in iron metabolism.Int JBiochem Cell Biol.2003Mar;35(3):292-6.
[14] Kawabata H, Yang R, Hirama T, et al. Molecular cloning of transferrin receptor2. Anew member of the transferrin receptor-like family.J Biol Chem,1999,274(30):20826–20832.
[15] Kawabata H, Germain RS, Vuong PT,et a1．Transferrin receptor2-alpha supports cellgrowth both in iron-chelated cultured cells and in vivo.J BiolChem,2000,275(22):16618-25.
[16] Graham RM, Reutens GM, Herbison CE, et a1.Transferrin receptor2mediates uptakeof transferrin-bound and non-transferrin-bound iron. Journal of Hepatology,2008,48:327–334.
[17]Camaschella C, Roetto A, De Gobbi M, et al. Genetic haemochromatosis: genes andmutations associated with iron loading. Best Pract Res Clin Haematol.2002Jun;15(2):261-76.
[18]Fleming RE,Ahmann JR, Migas MC,et al. Targeted mutagenesis of the murinetransferrin receptor-2gene produces hemochromatosis. Proc Natl Acad Sci U S A.2002,99:10653-10658.
[19]Bartnikas TB, Wildt SJ, Wineinger AE, et al. A novel rat model of hereditaryhemochromatosis due to a mutation in transferrin receptor2.Comp Med.2013Apr;63(2):143-55.
[20]Wallace DF, Summerville L, Crampton EM, et al. Combined deletion of Hfe andtransferrin receptor2in mice leads to marked dysregulation of hepcidin and ironoverload. Hepatology.2009Dec;50(6):1992-2000.
[21]Wallace DF, Summerville L, Subramaniam VN, Targeted Disruption of the HepaticTransferrin Receptor2Gene in Mice Leads to Iron Overload. Gastroenterology.2007Jan,132(1):301-10.
[22]Martha B,Johnson,Caroline A. Enns. Diferric transferrin regulates transferrin receptor2protein stability. BLOOD,2004,104(13):4287-4293.
[23]Robb A,Wessling-Resnick M. Regulation of transferrin receptor2protein levels bytransferrin. Blood,2004,104:4294-4299.
[24]Chen J, Wang J, Meyers KR,, et al. Transferrin-directed internalization and cycling oftransferrin receptor2.Traffic，2009Oct;10(10):1488-501.
[25] Juxing Chen, Caroline A. Enns. The Cytoplasmic Domain of Transferrin Receptor2Dictates Its Stability and Response to Holo-transferrin in Hep3B Cells. THEJOURNAL OF BIOLOGICAL CHEMISTRY,2007,282(9):6201–6209.
[26]Ehrke SG,Kulaksiz H,Herrmann T,et al. Expression of hepcidin in hereditaryhemochromatosis: evidence for a regulation in response to the serum transferrinsaturation and to non-transferrin-bound iron. Blood,2003,102:371-376.
[27]Gao J,Chen J,Kramer M,et al.Interaction of the hereditary hemochromatosis proteinHFE with transferrin receptor2is required for transferrin-induced hepcidinexpression.Cell Metab,2009,9:217–227.
[28]Wallace DF,Trinder D,Subramaniam VN.Hepcidin regulation by HFE and TFR2: is itenough to give a hepatocyte a complex? Gastroenterology,2009,137(3):1173-5.
[29]Tapasree Goswami,Nancy C. Andrews. Hereditary Hemochromatosis Protein, HFE,Interaction with Transferrin Receptor2Suggests a Molecular Mechanism forMammalian Iron Sensing. THE JOURNAL OF BIOLOGICAL CHEMISTRY,2006,281(39):28494–28498.
[30]Juxing Chen, Maja Chloupkova, Junwei Gao, et al. HFE Modulates TransferrinReceptor2Levels in Hepatoma Cells via Interactions That Differ from TransferrinReceptor1-HFE Interactions. THE JOURNAL OF BIOLOGICAL CHEMISTRY,2007,282(51):36862–36870.
[31]Robert E. Fleming. Iron Sensing as a Partnership:HFE and Transferrin Receptor2. CellMetabolism,2009,9(3):211-2.
[32]Abdul Waheed, Robert S. Britton, Jeffrey H. Grubb, et al. HFE association withtransferrin receptor2increases cellular uptake of transferrin-bound iron. Archives ofBiochemistry and Biophysics,2008,474:193–197.
[33] Alessia Calzolari, Carla Raggi, Silvia Deaglio, et al. TfR2localizes in lipid raftdomains and is released in exosomes to activate signal transduction along the MAPKpathway. J Cell Sci,2006,119(21):4486-98.
[34]Ramey G, Deschemin JC, Vaulont S. Cross-talk between the mitogen activated proteinkinase and bone morphogenetic protein/hemojuvelin pathways is required for theinduction of hepcidin by holotransferrin in primary mouse hepatocytes.Haematologica,2009,94:765–72
[35]Meyer C, Godoy P, Bachmann A,et a1. Distinct role of endocytosis for Smad andnon-Smad TGF-β signaling regulation in hepatocytes.J Hepatol.2011,55(2):369-78.
[36]Wang RH,Li C,Xu X,A role of Smad4in iron metabolism through the positiveregulation of hepcidin expression.Cell Metab,2005,2:399–409.
[37]Mleczko-sanecka K,Casanovas G,Ragab A,et a1.SMAD7controls iron metabolism as apotent inhibitor of hepcidin expression.Blood,2010,115:2657–2665.
[38]Zhang AS, Yang F, Wang J, et al. Hemojuvelin-neogenin interaction is required forbone morphogenic protein-4-induced hepcidin expression. J Biol Chem.2009Aug21;284(34):22580-9.
[39]Yann Gibert, Victoria J. Lattanzi, et al. BMP Signaling Modulates Hepcidin Expressionin Zebrafish Embryos Independent of Hemojuvelin. PLoS One.2011;6(1): e14553.
[40]Truksa J, Peng H, Lee P, et al. Bone morphogenetic proteins2,4, and9stimulatemurine Hepcidin1expression independently of Hfe, transferrin receptor2(Tfr2), andIL-6. Proc Natl Acad Sci U S A,2006,103(27):10289-10293.
[41]Meynard D,Kautz L,Darnaud V,et al. Lack of the bone morphogenetic protein BMP6induces massive iron over-load.Nat Genet,2009,41:478–481.
[42]Andriopoulos B Jr,Corradini E,Xia Y,et al. BMP-6is a key endogenous regulator ofhepcidin expression and iron metabolism.Nat Genet,2009,41:482–487.
[43] Kautz L,meyrard D,MonnierA,et al. Iron regulates phosphorylation of Smadl／5／8and gene expression of Bmp6， Smad7， ldl， and Atoh8in the mouseliver.Blood,2008,112:1503–1509.
[44]Atndt S,Maegdefrau U,Dorn C,et a1.Iron-induced expression of bone morphogenicprotein6in intestinal cells is the main regulator of hepatic hepcidin expression invivo.Gastroenterology,2010,138:372–382.
[45]Kautz L,Meynard D,Besson-Fournier C,et a1.Iron overload induces BMP6expressionin the liver but not in the duodenum.Haematologica,2011Feb,96(2):199-203.
[46]Brakensiek K, Fegbeutel C, M lzer M, et al. Juvenile hemochromatosis due tohomozygosity for the G320V mutation in the HJV gene with fatal outcome. ClinGenet,2009Nov,76(5):493-5.
[47]Papanikolaou G, Tzilianos M, Christakis JI, et al. Hepcidin in iron overloaddisorders.Blood,2005,105:4103–5.
[48]Huang FW, Pinkus JL, Pinkus GS, et al. A mouse model of juvenile hemochromatosis.J Clin Invest,2005,115(8):2187-2191.
[49] Niederkofler V, Salie R, Arber S. Hemojuvelin is essential for dietary iron sensing,and its mutation leads to severe iron overload. J Clin Invest,2005Aug,115(8):2180-6.
[50] Malyszko J..Hemojuvelin: the hepcidin story continues.Kidney Blood Press Res.2009,32(2):71-6.
[51] Kuninger D, Kuns-Hashimoto R, Kuzmickas R, et al. Complex biosynthesis of themuscle-enriched iron regulator RGMc. J. Cell Sci,2006,119:3273–83.
[52] Zhang AS, Enns CA. Iron homeostasis: recently identified proteins provide insightinto novel control mechanisms. J Biol Chem.2009Jan9,284(2):711-5.
[53]McMahon S, Grondin F, McDonald PP, et al. Hypoxia enhanced expression of theproprotein convertase furin is mediated by hypoxia-inducible factor-1: impact on thebioactivation of proproteins. J Biol Chem,2005,280(8):6561-6569.
[54]Lee P.Role of matriptase-2(TMPRSS6) in iron metabolism.Acta Haematol.2009,122(2-3):87-96..
[55]Folgueras AR,de Lara FM,Pendás AM,et al.Membrane-bound serine proteasematriptase-2(Tmprss6) is an essential regulator of iron homeostasis. Blood,2008Sep15,112(6):2539-45.
[56]Pellegrino RM, Coutinho M, D'Ascola D, et al. Two novel mutations in the tmprss6gene associated with iron-refractory iron-deficiency anaemia (irida) and partialexpression in the heterozygous form. Br J Haematol,2012Sep,158(5):668-72.
[57] SM, Strouse JJ, Markianos K, et al. Mutations in TMPRSS6cause iron-refractory irondeficiency anemia (IRIDA). Nat Genet,2008May,40(5):569-71.
[58] Silvestri L,Pagani A,Nai A, et al.The serine protease matriptase-2(TMPRSS6) inhibitshepcidin activation by cleaving membrane hemojuvelin. Cell Metab,2008Dec,8(6):502-11.