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.     

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.     

Changes of ferrous iron and its transporters after intracerebral hemorrhage in rats

Objective: Ferrous iron is a major source inducing oxidative stress after intracerebral hemorrhage (ICH). Divalent metal transporter1 (DMT1) is the important and well-known plasma membrane transport protein which was proved to be involved in the transport of free ferrous iron in mammals. Ferroportin 1 (FPN1) is the unique exporter of ferrous iron from mammalian cells. The role of DMT1 and FPN1 in brain after ICH is still not elucidated. Therefore, we measure the expression of DMT1 and FPN1, to explore the correlations between ferrous iron and its specific transporters after ICH. Methods: Ninety-six Sprague-Dawley rats received intra-striatal infusions of 0.5 U type IV collagenase to establish ICH model. Ferrous iron content in brain was determined using Turnbull’s method. DMT1 and FPN1 expression were examined by immunohistochemical staining and Real-Time quantitative polymerase chain reaction (RT-PCR). With the use of confocal laser microscopy, we determined the colocalization of DMT1 and FPN1 at 1, 3, 7 and 14 days after ICH. Results: Ferrous iron deposition was shown in the perihematomal zone as early as 1 day after ICH; it reached a peak after 7 days and was not elevated within 14 days following ICH. The expression of the DMT1 upregulated and reached to peak at day 7 after ICH. FPN1 reached a plateau at 3 days post-ICH. Expression levels of DMT1 and FPN1 were in parallel with ferrous iron deposition. There was a positive correlation between FPN1 and DMT1. DMT1 mainly localized in the cytoplasm of glias and neurons. FPN1 were mostly distributed on the membrane of endothelial cells and glias. Confocal microscope showed that DMT1 colocalized with FPN1. Conclusions: DMT1 and FPN1 are positively influenced by ferrous iron status in brain after ICH. DMT1 and FPN1 attenuate iron overload after ICH via increasing transmembrane iron export.     

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.     

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.     

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.     

Ferrous and ferric iron accumulates in the brain of aged Long-Evans Cinnamon rats, an animal model of Wilson's disease

The Long-Evans Cinnamon (LEC) rat, which accumulates excess copper (Cu) in its liver, is an animal model of Wilson's disease. We evaluated and compared the distributions of Cu, ferrous (Fe2+), and ferric (Fe3+) iron in four-brain regions, namely, in the cerebral cortex, cerebellum, substantia nigra (SN), and striatum of LEC and Long-Evans Agouti rats at 30 and 55 weeks. Cu levels were elevated in the striatum of LEC rats, and Fe2+ and Fe3+ were higher in the striatum and SN of LEC rats. Ratios of Fe2+ to Fe3+ were > 1 in four regions, and were highest in the striatum and SN of LEC rats. Cu and iron levels were found to be augmented during aging, and we suggest that these accumulations may exert deleterious effects in aged LEC rats. This study is the first report that demonstrates regional differences of Fe2+ and Fe3+ accumulation in the brain of aged LEC rats. Further studies are required to elucidate the mechanisms of Cu and iron accumulations and of their effects.     

尼古丁处理对大鼠脑内多巴胺转运体和酪氨酸羟化酶的影响

目的 观察尼古丁处理大鼠脑内多巴胺转运体(DAT)和酪氨酸羟化酶(TH)的表达变化,探讨尼古丁处理对大鼠脑内多巴胺(DA)能神经体系的影响. 方法 选用雄性Wistar大鼠按每日 0.4 mg/kg 腹腔注射尼古丁 7d;利用免疫组织化学和免疫印迹法,检测尼古丁处理大鼠有关脑区DAT和TH的表达改变. 结果 与对照组相比:1. 免疫组织化学显示,尼古丁处理组大鼠伏核(NACC)和腹则被盖区(VTA)的DAT灰度值降低了12.43 %和12.85 %;TH的灰度值则降低了11.87 %和10.09 %.2. 免疫印迹法显示,尼古丁处理组大鼠尾壳核(CPu)-NACC、黑质(SN)-VTA的DAT与β-肌动蛋白(β-actin)条带相对吸光度比值增加了75.68 %和117.14 %;而TH的比值则分别增加了66.32 %和60.31 %. 结论 尼古丁处理增加大鼠脑内DAT和TH的表达,这可能与尼古丁的成瘾机制有关.     

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。     

Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

Iron acquisition is critical to the growth and virulence of Legionella pneumophila. Previously, we found that L. pneumophila uses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted by L. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability of L. pneumophila and other species of Legionella to take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing of L. pneumophila culture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis of L. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.     

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.     

DMT1 polymorphism and risk of Parkinson's disease

Growing evidence suggests that iron accumulation in the substantia nigra (SN) is involved in the pathology of Parkinson’s diseases (PD). Divalent metal transporter 1 (DMT1) is an endogenous transporter for ferrous iron, the levels of which are significantly increased in the SN in postmortem PD brains. To study the possible association of DMT1 gene with PD occurrence, one mutation (1303C/A) and two single nucleotide polymorphisms (SNPs) (1254T/C and IVS4 + 44C/A) in DMT1 gene were investigated in 192 PD patients in a Han Chinese population and 193 healthy controls by method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Direct sequencing was performed in 10% of the samples to validate the genotyping results. Our results failed to find any significant association between the tested genotypes, alleles or mutation and PD, however, a haplotype (C alleles of 1254T and IVS4 + 44C/A polymorphisms) occurred at greater frequencies in PD subjects compared with that of control (18.2% versus 11.4%, OR = 1.72, 95% CI = 1.15-2.59, P = 0.01). These results suggest that CC haplotype in DMT1 gene is a possible risk factor for PD in this Han Chinese population.     

Racial disparities in children’s blood lead levels: Possible implication of divalent metal transporter 1

Adverse effects of lead exposure at low-dose (<10 μg/dL) in children showed a growing interest over the last decades. Black ethnicity is usually associated with elevated blood lead levels (BLLs), independently of age and socioeconomic conditions. The gastrointestinal uptake of lead represents a key step in the process of lead kinetic and toxicity. The involvement of divalent metal transporter 1 (DMT1) in the lead absorption has been previously presumed and reported. I postulate that inter-ethnic differences in DMT1 expression may explain a large part of the racial disparity in children’s BLLs, and suggest a few analyses to test this hypothesis. The hypothesis rests on some observations from previous researches. The inverse association between BLLs and iron intake has been reported in both cross-sectional and follow-up studies. It appears that no study specifically addressed the modifying effect of ethnicity in this association. Previous reports suggest that DMT1 is the primary mechanism for gastrointestinal iron absorption. There are four forms of DMT1 expressed in the enterocytes, which did not respond similarly to iron changes. It is not excluded that some children be more likely to uptake ingested lead depending on DMT1 isoform expressed. I hypothesize that the expression of the more active DMT1 isoform (+1A/+IRE) is more common in Non-Hispanic Black compared with Non-Hispanic White children, and I suggest how to test this hypothesis. If the hypothesis is confirmed, this would suggest that the prevention of iron-deficiency must be included in the primary programs for preventing increase of BLLs in Non-Hispanic Black children, rather than as part of secondary prevention. Moreover, thorough studies would be useful to characterize the interaction between environmental lead levels and DMT1 expression in relation to BLLs in young children.     

Acupuncture inhibits ferric iron deposition and ferritin-heavy chain reduction in an MPTP-induced parkinsonism model

This study investigated the effect of acupuncture on iron-related oxidative damage in a mouse model designed as a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism model. To generate the chronic parkinsonism model, mice were intraperitoneally injected with MPTP (20 mg/kg, one daily injection) for 30 days and acupuncture was performed at acupoints LR3 (Taichong) and GB34 (Yanglingquan) at 48 h intervals. Acupuncture inhibited decreases in the immunoreactivities of tyrosine hydroxylase (TH) and dopamine transporter (DAT) that occurred as a result of MPTP neurotoxicity. The presence of ferric iron (Fe³⁺), but not ferrous iron (Fe²⁺), was strongly increased in the substantia nigra (SN) as a result of chronic loading of MPTP, whereas the ferritin-heavy chain (F-H) was significantly decreased. However, acupuncture treatment inhibited the increase in ferric iron and the decrease in the F-H that was induced by MPTP. Additionally, treatment with MPTP and acupuncture caused no changes in the presence of ferrous iron and ferritin-light chain (F-L) as a result of the treatments. The mRNA of F-H was also not affected. These results suggest that acupuncture may inhibit iron-related oxidative damage and may prevent the deleterious alteration of iron metabolism in the MPTP model.     

Effect of timing of iron supplementation on maternal and neonatal growth and iron status of iron-deficient pregnant rats

We have previously shown that maternal iron (Fe) deficiency not only reduces fetal size, but also increases blood pressure in the offspring when they are adults. In this paper we examine whether there are critical periods when supplementation reverses or fails to reverse the effect both on size and on expression of genes of Fe metabolism. We made dams Fe deficient, mated them and provided supplements of Fe in the diet from the beginning of gestation (0.5 days), from 7.5 days or from 14.5 days. Within 12 h of birth, dams and neonates were killed and tissues taken and examined. Fe deficiency throughout pregnancy reduces neonatal size. Supplementation from the beginning of the first, second or third week all reduced the effect. Maternal haematocrit was restored to normal levels only in animals given supplements for at least 2 weeks. In contrast, the neonates' Fe levels were normal in all supplemented groups. These results were mirrored in liver Fe levels and in transferrin receptor mRNA. Iron-responsive element (IRE)-regulated divalent metal transporter 1 (DMT1) increased in maternal and neonatal liver. Non-IRE-regulated DMT1 levels did not change in the maternal liver, but decreased in the neonatal liver. H and L ferritin mRNA levels also showed different patterns in the mother and her offspring. Finally, the neonatal size correlated with maternal Fe stores, and not with those of the fetus. The data demonstrate that Fe supplementation during pregnancy is most effective when given early, rather than later, in gestation.     

Iron and aluminum deposition in the meninges of the lamprey: identification of an aluminum-ferritin inclusion body

The meningeal tissue of the brain and spinal cord of larval and juvenile adults of lampreys (Petromyzon marinus) was examined by routine electron microscopy, electron microscopic histochemistry, and electron-probe x-ray microanalysis to locate sites of iron deposition. A magnetometer was used for identification of ferromagnetic iron. Ferritin particles, representing ferric iron, are present in abundance within the cytoplasmic matrices and in dense bodies of meningeal cells of both the brain and spinal cord of larvae and juveniles. These round cells of the meninges also contain abundant glycogen and lipid. Small quantities of ferrous iron are associated to the latter inclusion. Aluminum deposits are present within an electron-dense material of many ferritin-containing inclusions of meningeal cells of the larval brain. Ferromagnetic material was not detected in larval and upstream-migrant lampreys. The deposition of iron and aluminum in the meninges of lampreys may be related to physiological and environmental factors, respectively, and/or to an important interaction between the two metals.     

Acquisition of Iron by Gardnerella vaginalis

Six Gardnerella vaginalis strains were examined for the ability to utilize various iron-containing compounds as iron sources. In a plate bioassay, all six strains acquired iron from ferrous chloride, ferric chloride, ferrous sulfate, ferric ammonium citrate, ferrous ammonium sulfate, bovine and equine hemin, bovine catalase, and equine, bovine, rabbit, and human hemoglobin. All six strains also acquired iron from human lactoferrin, but not from human transferrin, as determined by a liquid broth growth assay. Siderophore production was detected in eight G. vaginalis strains by the chrome azurol S universal chemical assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytoplasmic membrane proteins isolated from G. vaginalis 594 grown under iron-replete and iron-restricted conditions revealed several iron-regulated proteins ranging in molecular mass from 33 to 94 kDa. These results indicate that G. vaginalis may acquire iron from iron salts and host iron compounds.     

Increased uptake of divalent metals lead and cadmium into the brain after kainite-induced neuronal injury

An increase in iron level, number of iron positive cells and ferritin expression has been observed in the rat hippocampus after neuronal injury induced by the excitotoxin, kainate. This is accompanied by an increased expression of divalent metal transporter-1 (DMT1) in the lesioned hippocampus, suggesting that the transporter may be partially responsible for the iron accumulation. DMT1 has a broad substrate range that includes other divalent metals such as lead (Pb) and cadmium (Cd), and the present study was carried out to elucidate the uptake of these metals in the kainate-injected brain. The technique of atomic absorption spectroscopy was used for analyses. Significantly higher lead and cadmium levels were detected in the hippocampus and other brain areas of intracerebroventricular kainate-injected rats treated with lead and cadmium in the drinking water, compared to intracerebroventricular saline-injected rats treated with lead and cadmium in the drinking water. Since very low levels of lead and cadmium are present in the normal animal, these results indicate increased uptake of lead and cadmium into brain areas as a result of the kainate injections. Increased iron levels were also detected in the hippocampus of the kainate-injected rats. The above results show increased uptake of divalent metals into brain areas undergoing neurodegeneration.