Effects of Intracerebroventricular Injection of Iron Dextran on the Iron Concentration and Divalent Metal Transporter 1 Expression in the Caudate Putamen and Substantia Nigra of Rats

The cellular localization of DMT1 and its functional characterization suggest that DMT1 may play an important role in the physiological brain iron transport. But the regulation of DMT1 expression by iron in the brain is still not clearly understood. In this study, both the contents of ferric and ferrous iron as well as DMT1 expression were evaluated in CPu and SN after ICV of 500 μg iron dextran/rat/day for 3 or 7 days. It was found that the iron levels in CPu and SN were not altered obviously until ICV for 7 days. Immunohistochemistry results indicated that the expression of DMT1 (?IRE) in CPu and SN was not altered significantly after 3 days of ICV. Whereas the expression of DMT1 (?IRE) decreased significantly after 7 days of ICV when ferrous iron was increased significantly. Contrary to that of DMT1 (?IRE) in the same regions, there were no significant alterations in DMT1 (+IRE) expression in CPu and SN in spite of the existence of the altered iron levels, compared with that of control groups. The results demonstrate that DMT1 (?IRE) expression was correlated probably with brain iron levels; especially, its regulation was correlated with ferrous iron (not ferric iron) in CPu and SN in adult rats, compared with those of saline‐injected control rats. The effect of ferrous iron on the expression of DMT1 (?IRE) in the brain also suggests that it might play a major physiological role in brain iron uptake and transport, but further studies are needed to clarify these issues. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.     

高铁对大鼠大脑皮层二价金属转运蛋白1表达的影响

目的 探讨在脑铁代谢中发挥重要生理作用的二价金属转运蛋白1(DMT1)的表达及其调控机制.方法 大鼠(n=6)侧脑室注射右旋糖酐铁3d和7d后,采用铁组织化学法检测脑内铁含量的变化,免疫组织化学技术检测大脑皮层中DMT1的两种亚型,即DMT1(+IRE)和DMT1(-IRE)蛋白表达的变化.结果 铁组织化学染色结果显示,大鼠侧脑室注射右旋糖酐铁500μg/(只·d)7d后,大脑皮层中二价铁和三价铁均显著增高.同时,免疫组织化学结果表明,与对照组相比,脑内达高铁状态时大脑皮层DMT1(+IRE)蛋白表达显著升高,而DMT1(-IRE)蛋白表达无显著变化.结论 在大鼠大脑皮层中,DMT1(+IRE)蛋白对铁水平的升高更为敏感,其表达与脑铁水平(尤其是二价铁)呈正相关.高铁对脑内不同区域内不同亚型DMT1表达的影响存在特异性.     

脂多糖对巨噬细胞铁代谢的影响

目的检测二价金属离子转运体(DMT1)和金属转运蛋白(FPN1)在原代培养巨噬细胞中的分布及脂多糖(LPS)对巨噬细胞铁代谢的影响。方法采用原代细胞培养、MTT显色、细胞化学染色和细胞免疫荧光等方法检测LPS对原代培养的巨噬细胞活性的作用及对DMT1和FPN1分布的影响。结果不带铁反应元件的二价金属离子转运体(DMT1-IRE)主要在细胞核中表达,在细胞质中分布较少。带铁反应元件的二价金属离子转运体(DMT1+IRE)主要分布于细胞质中,可以将吞噬小体中的铁向细胞质中转运。FPN1在巨噬细胞的细胞质和细胞膜均有表达,但主要分布于细胞质。结论巨噬细胞吞噬衰老的红细胞以后,其中的亚铁血红素在细胞质中分解,FPN1可能介导了亚铁血红素分解释放出来的铁在巨噬细胞质中的转运过程。LPS在低浓度时有促进巨噬细胞生长的作用,LPS浓度为10-5μg/L时达到高峰,随着浓度的增加,又开始抑制细胞生长。     

铁转运相关蛋白在肌萎缩性侧索硬化症转基因鼠脊髓中的表达变化

目的探讨肌萎缩性侧索硬化症(ALS)转基因鼠脊髓内铁转运相关蛋白表达变化与铁稳态失衡的关联。方法选取h SOD1G93A转基因鼠(ALS鼠)和同窝野生型鼠(WT鼠),分别于生后70、95和122 d分离脊髓,每时间点每组各9只实验动物。Western blotting检测脊髓组织内铁转运蛋白二价金属转运蛋白-1(DMT1)、铁转运蛋白-1(FPN1)及调节蛋白铁调节蛋白-1(IRP1)的表达;免疫荧光双重标记检测脊髓腰段前角内细胞共定位情况。结果 Western blotting显示,与WT鼠比较,各时间点ALS鼠脊髓内DMT1表达均显著降低(P<0.05,P<0.01);70 d FPN1表达升高(P<0.05),95 d和122 d表达下降(P<0.01); 95 d、122 d IRP1表达降低(P<0.01)。免疫荧光双重标记显示,在70 d WT鼠和ALS鼠腰段脊髓中DMT1主要与β-微管蛋白Ⅲ(β-tubulinⅢ)共表达。与WT组相比,95 d ALS鼠脊髓腰段前角神经元内DMT1免疫反应强,而FPN1荧光强度减弱。随疾病进展,DMT1、FP...     

Age-dependent and iron-independent expression of two mRNA isoforms of divalent metal transporter 1 in rat brain

The DMT1(Nramp2/DCT1) is a newly discovered proton-coupled metal-ion transport protein. The cellular localization and functional characterization of DMT1 suggest that it might play a role in physiological iron transport in the brain. In the study, we evaluated effects of dietary iron and age on iron content and DMT1 expression in four brain regions: cortex, hippocampus, striatum, substantia nigra. Total iron content in all regions was significantly lower in the low-iron diet rats and higher in the high-iron diet rats than that in the control animals, showing that dietary iron treatment for 6-weeks can alter brain iron levels. Contrary to our expectation, there was no significant alternation in DMT1(+IRE) and (-IRE) mRNA expression and protein content in all brain regions examined in spite of the existence of the altered iron levels in these regions after 6-weeks' diet treatment although TfR mRNA expression and protein level were affected significantly, as was expected. The data demonstrates that expression of DMT1(+IRE) and (-IRE) was not regulated by iron in these regions of adult rats. The lack of response of DMT1 to iron status in the brain suggests that the IRE of brain DMT1 mRNA might be not really iron-responsive and that DMT1-mediated iron transport might be not the rate-limiting step in brain iron uptake in adult rats. Our findings also showed that development can significantly affect brain iron and DMT1(+IRE) and (-IRE) expression but the effect varies in different brain regions, indicating a regionally specific regulation in the brain.     

脑缺血诱导大鼠皮层和海马二价金属离子转运体1表达增加的研究

目的:探讨脑缺血对大鼠皮层、海马二价金属离子转运体1(DMT1)表达的影响。方法:雄性Wistar大鼠随机分为脑缺血1、3、7、28 d和假手术组。结扎双侧颈总动脉建立脑缺血模型组,假手术组仅分离双侧颈总动脉但不结扎。采用RT-PCR测定DMT1+/-IRE mRNA的表达;采用免疫组化染色测定大鼠皮层及海马组织DMT1的表达。结果:大鼠皮层和海马DMT1+/-IRE mRNA的表达随缺血时间的延长逐渐增加。与假手术组比较,皮层DMT1+/-IRE mRNA的表达在缺血1、3 d时无差异(P>0.05);缺血7 d时表达增加(P<0.01),缺血28d时增加更明显(P<0.01)。海马DMT1-IRE mRNA表达除在缺血1 d时与假手术组无差异外(P>0.05),其余时间点DMT1+/-IRE mRNA表达均高于假手术组(P<0.01)。随缺血时间的延长,大鼠皮层、海马的锥体细胞、颗粒细胞及血管内皮细胞DMT1的表达逐渐增加。DMT1的表达除缺血1 d组与假手术组无差别外(P>0.05),其余各组均高于假手术组(P<0.05)。结论:脑缺血可诱导大鼠皮层及海马DMT1表达升高,DMT1表达的改变可能参与了脑缺血引起大鼠脑铁含量升高及神经元铁沉积过程。     

Up-regulation of divalent metal transporter 1 is involved in 1-methyl-4-phenylpyridinium (MPP(+))-induced apoptosis in MES23.5 cells

Apoptosis has been identified as one of the important mechanisms involved in the degeneration of dopaminergic neurons in Parkinson's disease (PD). Our previous study showed increased iron levels in the substantia nigra as well as loss of dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse models. 1-Methyl-4-phenylpyridinium (MPP⁺) is commonly used to establish a cellular model of PD. Although intracellular iron plays a crucial role in MPP⁺-induced apoptosis, the molecular mechanism linking increased iron and MPP⁺-induced neurodegeneration is largely unknown. In the present study, we investigate the involvement of divalent metal transporter 1 (DMT1) that accounts for the ferrous iron transport in MPP⁺-treated MES23.5 cells. In the treated cells, a significant influx of ferrous iron was observed. This resulted in a decreased mitochondrial membrane potential. Additionally, an elevated level of ROS production and activation of caspase-3 were also detected, as well as the subsequent cell apoptosis. These effects could be fully abolished by iron chelator desferal (DFO). Increased DMT1 (−IRE) expression but not DMT1 (+IRE) accounted for the increased iron influx. However, there were no changes for iron regulatory protein 1 (IRP1), despite decreased expression of IRP2. Iron itself had no effect on IRP1 and IRP2 expression. Our data suggest that although DMT1 mRNA contains an iron responsive element, its expression is not totally controlled by this. MPP⁺ could up-regulate the expression of DMT1 (−IRE) in an IRE/IRP-independent manner. Our findings also show that MPP⁺-induced apoptosis in MES23.5 cells involves DMT1-dependent iron influx and mitochondria dysfunction.     

Expression of divalent metal transporter 1 (DMT1) isoforms in first trimester human placenta and embryonic tissues

BACKGROUND: Divalent metal transporter 1 (DMT1) is a transmembrane glycoprotein which mediates the proton-coupled transport of a variety of divalent metal ions. Two isoforms, which differ by the presence (DMT1-IRE) or absence (DMT1-nonIRE) of an iron-responsive element (IRE) in their 3' untranslated region, are implicated in apical iron transport and endosomal iron transport respectively. Although the expression pattern of DMT1 isoforms is tissue specific in adult, data regarding its expression in embryonic tissues are lacking. METHODS: Semiquantitative RT-PCR and immunohistochemistry were used to study the mRNA and protein expression of both DMT1 isoforms in embryonic tissues between 8 and 14 weeks gestational age. RESULTS: DMT1-IRE and DMT1-nonIRE expressions were ubiquitous in embryonic tissues examined. In the lung, statistically significant correlations were found between the levels of DMT1 isoform expression and gestational age. In the placenta, DMT1-IRE was the predominantly expressed isoform. Both isoform proteins were localized in embryonic epithelial cellular membrane. CONCLUSION: Both DMT1 isoforms are ubiquitously expressed in embryonic tissues in the first trimester. Predominant DMT1-IRE isoform expression in placenta suggests an iron-regulatory mechanism reminiscent of that in the adult duodenum. Epithelial distributions of both DMT1 isoforms are associated with the absorptive or excretory functions of the expressed tissues.     

Differences in the uptake of iron from Fe(II) ascorbate and Fe(III) citrate by IEC-6 cells and the involvement of ferroportin/IREG-1/MTP-1/SLC40A1

Dietary iron is present in the intestine as Fe(II) and Fe(III). Since enterocytes take up Fe(II) by the divalent metal transporter (DMT1), Fe(III) must be reduced. Whether other Fe(III) transport processes are present is unknown. Release of iron from the enterocyte into the plasma involves the iron-regulated transporter-1/metal transporter protein-1 (IREG-1/MTP-1, ferroportin) but ferroportin is also found on the apical membrane. We compared the uptake of iron from Fe(II):ascorbate and Fe(III):citrate using the rat intestinal enterocyte cell line-6 (IEC-6), in the presence of ferrous chelators, a blocking antibody to ferroportin, at different pH and during the over-expression of DMT1. Firstly, surface ferrireduction was absent. Secondly, blocking ferroportin partly and totally reduced Fe(II) and Fe(III) uptake, respectively. Thirdly, optimal Fe(II) uptake occurred at pH5.5 but Fe(III) uptake was unaffected by pH and, fourthly, over-expression of DMT1 increased uptake of Fe(II) and Fe(III). This indicates that an increased extracellular H⁺ concentration facilitates DMT1-mediated Fe(II) uptake at the cell membrane. However, since Fe(III) uptake required DMT1, but not cell surface ferrireduction, and was independent of variations in extracellular pH, it appears that Fe(III) is internalised before ferrireduction and transport by DMT1. Ferroportin may function as a modulator of DMT1 activity and play a role in Fe(III) uptake, possibly by affecting the number or affinity of citrate binding sites.     

Distribution of divalent metal transporter-1 in the monkey basal ganglia

An accumulation of iron occurs in the brain with age, and it is thought that this may contribute to the pathology of certain neurodegenerative diseases, including Parkinson's disease. In this study, we elucidated the distribution of divalent metal transporter-1 (DMT1) in the monkey basal ganglia by immunocytochemistry, and compared it with the distribution of ferrous iron in these nuclei by Turnbull's Blue histochemical staining. We observed a general correlation between levels of DMT1, and iron staining. Thus, regions such as the caudate nucleus, putamen, and substantia nigra pars reticulata contained dense staining of DMT1 in astrocytic processes, and were also observed to contain large numbers of ferrous iron granules. The exceptions were the globus pallidus externa and interna, which contained light DMT1 staining, but large numbers of ferrous iron granules. The thalamus, subthalamic nucleus, and substantia nigra pars compacta contained neurons that were lightly stained for DMT1, but few or no iron granules. The high levels of DMT1 expression in some of the nuclei of the basal ganglia, particularly the caudate nucleus, putamen, and substantia nigra pars reticulata, may account for the high levels of iron in these regions.     

NF-κB及ICAM-1在脑出血大鼠及过氧化氢损伤大鼠脑微血管内皮细胞中的表达

目的： 探讨脑出血后炎症反应机制及核因子-κB(NF-κB)与细胞间粘附分子-1(ICAM-1)间表达的关系。方法： 建立大鼠脑出血模型及过氧化氢损伤大鼠脑微血管内皮细胞的模型，分别采用免疫组化、原位杂交、免疫细胞化学及Western blotting方法观察NF-κB和ICAM-1的表达。结果： 大鼠脑出血后NF-κB 及ICAM-1表达均增强，ICAM-1的表达高峰（1 d）先于NF-κB（4 d），但在体外实验中，过氧化氢损伤后即刻大鼠脑微血管内皮细胞中NF-κB表达即增加，2 h后ICAM-1表达也上调，NF-κB的抑制剂吡咯烷二硫氨基甲酸（PDTC)可下调NF-κB及ICAM-1表达。结论: 在活性氧损伤中NF-κB作为ICAM-1的活化因子，可上调ICAM-1表达，但在脑出血这一复杂的病理生理变化中，尚有其它因素参与对NF-κB 及ICAM-1表达的调控。     

脑血康片对急性脑出血大鼠PAR-1表达及动态演变的影响(英)

目的：探讨脑出血后蛋白酶激活的受体-1（PAR-1）的动态表达及脑血康片(NXKT)的干预作用。 方法： 将72只Wistar大鼠随机分为正常组、脑出血模型6 h、24 h、3 d、7 d组和脑血康片6 h、24 h、3 d、7 d组。用Ⅶ-S型胶原酶诱导大鼠脑出血模型。免疫组化方法测定各时间点大鼠脑出血后血肿周围水肿组织PAR-1蛋白的表达；RT-PCR检测PAR-1 mRNA的表达。 结果： 正常组大鼠大脑PAR-1蛋白和PAR-1 mRNA表达轻度阳性, 模型组6 h时PAR-1表达强度开始增强，24 h PAR-1表达进一步增加，于3 d达到高峰，然后开始下降，7 d时明显下降。在6 h、24 h、3 d及7 d各时点，模型组、NXKT组PAR-1 mRNA吸光度比值及PAR-1阳性细胞数升高与正常组比较均有显著差异(P<0.05或P<0.01)，NXKT组PAR-1阳性细胞数、PAR-1 mRNA吸光度比值降低与模型组比较各时点均有显著差异(P＜0.05或P＜0.01)。 结论： 脑出血后PAR-1受到凝血酶的持续活化，脑出血后凝血酶的作用可能是通过PAR-1介导的; NXKT可抑制PAR-1活化，从而使行为学得到改善,这可能是NXKT促进神经功能修复的主要机制之一。     

Quinacrine attenuates increases in divalent metal transporter-1 and iron levels in the rat hippocampus,after kainate-induced neuronal injury

The present investigation was carried out to elucidate the effect of the antimalarial drug quinacrine on levels of expression of the non-heme iron transporter, divalent metal transporter-1 (DMT1) and iron, in the hippocampus of rats after kainate treatment. The untreated hippocampus was lightly stained for DMT1, while an increase in DMT1 staining in astrocytes in the degenerating cornu ammonis (CA) fields, after kainate lesions. The increased DMT1 immunoreactivity was correlated with increased levels of Fe3+ and Fe2+ staining in the CA fields, as demonstrated by iron histochemistry (Perl's and Turnbull's blue stain for Fe3+ and Fe2+). The increases in DMT1 and iron staining were significantly attenuated by quinacrine. Rats injected with kainate and daily i.p. injections of quinacrine (5 mg/kg) for 7 days or 2 weeks showed significantly lower levels of DMT1 immunoreactivity and iron staining, compared with rats injected with kainate and saline. These results show that DMT1 expression is closely linked to iron levels, and provide further support for a crucial role that DMT1 plays in iron accumulation in the degenerating hippocampus.     

骨髓间充质干细胞移植脑出血模型大鼠血肿周围轴突生长抑制因子MAG及OMgp的表达*

背景：中枢神经系统损伤后轴突再生困难与损伤灶周围高表达轴突生长抑制因子Nogo-A、MAG、OMgp有关。 目的：观察骨髓间充质干细胞移植对脑出血模型大鼠神经功能恢复及血肿周围轴突生长抑制因子MAG、OMgp表达的影响。 方法：全骨髓贴壁法分离培养大鼠骨髓间充质干细胞,使用立体定向纹状体注入胶原酶法制作大鼠脑出血模型。SD大鼠分为假手术组、脑出血模型组及骨髓间充质干细胞干预组,骨髓间充质干细胞干预组于造模后24 h经尾静脉注射1×107的骨髓间充质干细胞悬液,各组于造模后1,3,7,14 d进行神经功能评分,并在各时间点处死大鼠行免疫组织化学法检测MAG、OMgp的表达情况。 结果与结论：假手术组术后各个时间点均未见明显神经功能缺损;脑出血模型组神经功能缺损术后1 d时即达高峰,14 d时有进一步的恢复;骨髓间充质干细胞干预组神经功能评分在3,7,14 d各个时间点均较模型组明显降低(P < 0.05)。各个时间点模型组及骨髓间充质干细胞干预组血肿周围神经元及胶质细胞MAG、OMgp的表达均较假手术组明显升高,骨髓间充质干细胞干预组较模型组表达均有降低(P < 0.05)。提示骨髓间充质干细胞移植可明显促进大鼠脑出血后神经功能的恢复,其机制可能与下调血肿周围MAG、OMgp的表达有关。      

两种脑出血动物模型的AQP4表达与磁共振成像的比较研究

目的比较两种脑出血(ICH)动物模型磁共振成像(MRI)的差异,并对其分子机制进行探讨。方法将胶原酶和自体血注入大鼠尾状核建立脑出血模型,分别于术后6h、12h、1d、2d、3d和7d进行磁共振成像扫描,观察两组脑出血模型血肿体积的大小;运用干湿重法、伊文思蓝(EB)测定法和免疫印迹分别检测两组脑出血模型的脑含水量(BWC)、血脑屏障(BBB)通透性和AQP4的表达变化。结果胶原酶模型组的血肿体积在脑出血后1d达到最大并持续至3d,自体血组的血肿体积在12h达到最大;胶原酶模型组的BWC在1d达到高峰并持续至3d,而自体血组BWC在2d逐渐降低;胶原酶模型组的EB含量在12h达到高峰并持续至2d,而自体血组EB含量在1d逐渐下降;两组模型的AQP4表达量在6h开始升高,1d达到高峰,2d逐渐降低。两组模型比较具有统计学差异(P<0.05)。结论胶原酶对BBB和细胞外基质的破坏以及AQP4的表达变化是两组脑出血模型血肿体积出现差异的重要原因。     

Augmented internalisation of ferroportin to late endosomes impairs iron uptake by enterocyte-like IEC-6 cells

Absorption of iron occurs by duodenal enterocytes, involving uptake by the divalent metal transporter-1 (DMT1) and release by ferroportin. Ferroportin responds to the hepatocyte-produced 25-amino-acid-peptide hepcidin-25 by undergoing internalisation to late endosomes that impair iron release. Ferroportin is also expressed on the apical membrane of polarised Caco-2 cells, rat intestinal cells and in IEC-6 cells (an intestinal epithelial cell line). A blocking antibody to ferroportin also impairs the uptake, but not the release, of iron. In this study IEC-6 cells were used to study the mechanism of impairment or recovery from impairment produced by the blocking antibody and the fate of DMT1 and ferroportin. Uptake of 1 M Fe(II) was studied by adding the antibody from time 0 and after adding or removing the antibody once a steady state had been reached. Surface binding, maximum iron transport rate V_max and transporter affinity (K_m) were measured after impairment of iron uptake. Ferroportin and DMT1 distribution were assessed by immunofluorescence microscopy. Antibody-mediated impairment, or recovery from impairment, of Fe(II) uptake occurred within minutes. Impairment was lost when the antibody was combined with the immunizing peptide. DMT1 and ferroportin undergo internalisation to late endosomes and, in the presence of the antibody, augmented internalisation of DMT1 and ferroportin caused swelling of late endosomes. Surface binding of Fe(II) and iron transport V_max were reduced by 50%, indicating that the antibody removed membrane-bound DMT1. The ferroportin antibody induced rapid turnover of membrane ferroportin and DMT1 and its internalisation to late endosomes, resulting in impaired Fe(II) uptake.     

铁调素在小鼠脑内的表达及其对膜铁转运蛋白1和二价金属离子转运体1表达的影响

目的 探讨铁调素(hepcidin)在小鼠脑内的表达及其对膜铁转运蛋白1(ferroportir 1)和二价金属离子转运体1(DMT1)表达的调节作用.方法 应用RT-PCR技术检测铁调素在正常小鼠各脑区的表达分布,并观察了脑室内注射铁调素对DMT1、膜铁转运蛋白1表达的影响 结果 铁调素在小鼠脑内有广泛表达,且不同脑区表达程度不同,脉络丛部分表达较高.结论 侧脑室内注射铁调素后,能够显著影响DMT1、膜铁转运蛋白1表达,且具有明显的区域特异性.