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.     

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。     

革兰氏阳性细菌铁摄取机制的研究进展

铁是大多数细菌生长存活的基本营养物质,细菌能否从宿主体内摄取足够的铁对于其毒力和致病起着关键的作用.铁在宿主体内主要以游离铁、含铁蛋白和血红素三种形式存在.细菌可以通过复杂的铁摄取机制来获得宿主体内的铁进而感染宿主.因此,探究细菌的铁摄取机制有助更好的了解细菌的生长和致病过程.     

谷氨酸钠、γ-氨基丁酸注入黑质对大鼠尾壳核二价金属离子转运体1和膜铁转运辅助蛋白表达的影响

目的 探讨谷氨酸、γ-氨基丁酸(GABA)对大鼠尾壳核铁代谢的影响.方法 大鼠立体定位后,向大脑黑质分别注射谷氨酸钠(MSG)和GABA,观察大鼠尾壳核铁含量,黑质多巴胺能神经元酪氨酸羟化酶(TH)的变化以及尾壳核的无铁反应元件结构的二价金属离子转运体1(DMT1-IRE)、膜铁转运辅助蛋白(HP)含量的变化.结果 与对照组相比,MSG组大鼠尾壳核铁含量显著增加,GABA组与对照组相比没有显著差异;谷氨酸钠组和GABA组大鼠黑质TH免疫阳性细胞平均吸光度(AA)与对照组相比均无显著差异;与对照组相比,谷氨酸钠组大鼠尾壳核DMT1-IRE表达均显著增加,而GABA组DMT1-IRE表达有明显降低;谷氨酸钠组大鼠尾壳核HP表达显著降低,GABA组HP表达显著增高.结论 黑质的谷氨酸和GABA可能通过影响尾壳核DMT1-IRE和HP的表达影响纹状体尾壳核的铁代谢.     

DMT1在APP/PS1转基因小鼠大脑皮层老年斑内的定位研究

目的研究二价金属离子转运体1(divalent metal transporter 1,DMT1)在APP/PS1转基因小鼠大脑皮层内的定位分布,探讨DMT1异常表达影响脑铁代谢平衡从而参与AD发病的可能机制。方法应用免疫组织化学方法观察DMT1在9月龄APPsw/PS1小鼠大脑皮层的阳性分布;应用免疫荧光双标技术和共聚焦激光扫描显微镜观察DMT1蛋白和β淀粉样蛋白(β-amyloid peptide,Aβ)在APP/PS1转基因小鼠大脑皮层老年斑内的一致性分布和位置关系。结果APP/PS1转基因小鼠大脑皮层老年斑内均有DMT1阳性表达;DMT1和Aβ免疫双标发现DMT1免疫阳性产物与Aβ共存于老年斑,二者分布具有一致性。结论DMT1在APP/PS1转基因小鼠大脑皮层老年斑内大量表达,其分布与Aβ具有一致性,提示DMT1可能参与AD脑内Aβ沉积和老年斑形成。     

DMT1在多巴胺能神经元变性中的作用研究

目的: 研究二价金属离子转运蛋白1(DMT1)在乳胞素(lactacystin)诱导的SH-SY5Y细胞中的表达改变,从而进一步了解DMT1在帕金森病(PD)神经元损伤中的可能作用机制。方法: 建立 lactacystin 损伤的SH-SY5Y细胞模型,用免疫荧光、Western blotting等方法检测细胞DMT1表达水平的变化;在高亚铁环境下,荧光探针DCFH-DA检测胞内氧化应激水平的变化,免疫组织化学法、Western blotting检测胞内α-突触核蛋白(α-SYN)聚合体的改变。结果: Lactacystin处理后,细胞活力呈浓度依赖性降低。与正常对照组相比,lactacystin处理组DMT1表达增加(P<0.01)。正常对照组、lactacystin处理组及Fe²⁺处理组3组比较,其细胞活力逐渐降低,胞内氧化应激反应逐渐增强,胞浆α-SYN低聚体(43-55 kD)表达量逐渐增多(P<0.05)。结论: Lactacystin诱导SH-SY5Y细胞高表达DMT1,增强细胞摄铁能力,这可能是铁直接或者通过氧化应激反应促进胞内α-SYN的错误折叠和聚集、最终导致PD神经元损伤的关键因素。     

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...     

血红素加氧酶-1在铁代谢中的作用

铁是机体必需的微量元素,有着广泛的生物学作用,包括氧化运输和细胞呼吸作用,缺铁会引起细胞生长停滞或死亡,铁过载会使细胞发生氧化应激进而损伤细胞膜、蛋白质、核酸.血红素加氧酶-1(HO-1)分解血红素产生铁是铁重复利用最主要的来源,在铁代谢中的作用至关重要.     

鼻咽癌CNE1细胞微波吸收谱测量及其生物效应研究初步

电磁生物学是一门实验科学，主要通过实验方法的建立和实验数据的获得开展电磁生物效应的研究。本文进行鼻咽癌细胞系CNE1对微波辐照的吸收谱测量,搭建以HP83630扫频信号源．标量网络分析仪AV3617为主要器件的程控自动化测试系统．在此基础上设定了可重复操作的实验方法。通过实验发现：CNE1对扫频源微波的最大功率吸收峰（即频率窗）出现在37．257GHz附近，重复性良好；而且对连续微波辐照在几个峰值吸收频率点处均有明显时间窗效应。     

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.     

Regulation of DMT1 on Bone Microstructure in Type 2 Diabetes

Diabetic osteoporosis is gradually attracted people''s attention. However, the process of bone microstructure changes in diabetic patients, and the exact mechanism of osteoblast iron overload are unclear. Therefore, the present study aimed to explore the function of DMT1 in the pathological process of diabetic osteoporosis. We build the type two diabetes osteoporosis models with SD rats and Belgrade rats, respectively. Difference expression of DMT1 was detected by using the method of immunohistochemistry and western blotting. Detection of bone microstructure and biomechanics and iron content for each group of samples. We found that DMT1 expression in type 2 diabetic rats was higher than that in normal rats. The bone biomechanical indices and bone microstructure in the rat model deficient in DMT1 was significantly better than that in the normal diabetic model. The loss of DMT1 can reduce the content of iron in bone. These findings indicate that DMT1 expression was enhanced in the bone tissue of type 2 diabetic rats, and plays an important role in the pathological process of diabetic osteoporosis. Moreover, DMT1 may be a potential therapeutic target for diabetic osteoporosis.     

The Capsule-Encoding viaB Locus Reduces Intestinal Inflammation by a Salmonella Pathogenicity Island 1-Independent Mechanism

Salmonella enterica serotype Typhimurium elicits acute neutrophil influx in the human intestinal mucosa within 1 or 2 days after infection, resulting in inflammatory diarrhea. In contrast, no overt symptoms are observed within the first 1 or 2 weeks after infection with S. enterica serotype Typhi. Here we show that introduction of the capsule-encoding viaB locus of serotype Typhi reduced the ability of serotype Typhimurium to elicit acute intestinal inflammation in a streptomycin-pretreated mouse model. Serotype Typhimurium requires a functional invasion-associated type III secretion system (type III secretion system 1 [T3SS-1]) to elicit cecal inflammation within 48 h after infection of streptomycin-pretreated mice, and the presence of the viaB locus reduced its invasiveness for human intestinal epithelial cells in vitro. However, a reduced activity of T3SS-1 could not account for the ability of the viaB locus to attenuate cecal inflammation, because introduction of the viaB locus into an invasion-deficient serotype Typhimurium strain (invA mutant) resulted in a significant reduction of pathology and inflammatory cytokine expression in the cecum 5 days after infection of mice. We conclude that a T3SS-1-independent mechanism contributes to the ability of the viaB locus to reduce intestinal inflammation.Salmonella enterica serotype Typhimurium causes acute gastroenteritis, characterized by exudative inflammatory infiltrates in the intestine that are dominated by neutrophils (34). Exudative inflammation and diarrhea develop within 48 h after ingesting the organisms. The orchestration of these rapid intestinal inflammatory responses can be studied using the streptomycin-pretreated mouse model (11). In this model, motility and chemotaxis contribute to the induction of inflammatory responses in the cecum at early time points, between 10 and 24 h after infection, but become dispensable at later stages, between 48 and 120 h after infection (32). Mutants that lack a functional invasion-associated type III secretion system (type III secretion system 1 [T3SS-1]) are unable to elicit cecal inflammation within the first 48 h after infection, but marked inflammatory changes with neutrophil recruitment still develop in the cecal mucosa between 72 and 120 h after infection (5). These data suggest that flagella and T3SS-1 are important for eliciting early inflammatory changes in the ceca of mice. Induction of T3SS-1-independent inflammatory changes at later stages of infection (72 to 120 h after inoculation) require the presence of a functional myeloid differentiation primary response protein 88 (MyD88), an intracellular adaptor required for signaling through bacterium-specific Toll-like receptors (TLRs) (13).In contrast to serotype Typhimurium, S. enterica serotype Typhi does not elicit neutrophil recruitment in the intestinal mucosa but instead is associated with a systemic infection termed typhoid fever (34). This disease has an incubation period of approximately 2 weeks (24), which suggests that the pathogen initially prevents the generation of marked host responses. Severe interstitial inflammation in the intestine may develop late, 2 to 3 weeks after the onset of symptoms, and may result in hemorrhage, ulceration, and intestinal perforation at areas of Peyer''s patches (4). However, neutrophils remain scarce in intestinal infiltrates of typhoid fever patients (18, 22, 23, 31). The clinical presentation of typhoid fever suggests that serotype Typhi can prevent the rapid recruitment of neutrophils that characterizes the exudative inflammatory changes observed during serotype Typhimurium-induced gastroenteritis (27).Recently, the viaB locus has been implicated in reducing the amount of neutrophil chemoattractants (CXC chemokines) produced by cultured human epithelial cells, macrophages, or colonic tissue explants in response to serotype Typhi infection (26, 38, 39). Introduction of the cloned viaB locus into serotype Typhimurium reduces neutrophil recruitment and CXC chemokine expression in bovine ligated ileal loops (28). The viaB locus contains genes involved in the regulation (tviA), biosynthesis (tviBCDE), and export (vexABCDE) of the serotype Typhi Vi capsular polysaccharide (36). The TviA regulatory protein represses expression of flagella (39), and the presence of the viaB locus reduces T3SS-1-mediated invasion (1, 41), two factors important for inducing early intestinal inflammatory responses in the streptomycin-pretreated mouse model (10, 32) and in bovine ligated ileal loops (30, 40). Flagella contribute to inflammation in the ceca of streptomycin-pretreated mice largely by conferring motility and chemotaxis (32), which in turn promotes invasion of the intestinal epithelium by increasing bacterial contact with host cells (14, 15). Collectively, these observations raise the possibility that the viaB locus may reduce intestinal inflammation in vivo because the efficiency of T3SS-1-mediated invasion is reduced. However, this idea has not been tested experimentally, in part because no animal model is available for the strictly human-adapted serotype Typhi.Here we constructed a serotype Typhimurium strain stably expressing the Vi capsular antigen to test the role of the viaB locus in attenuating cecal inflammation in the streptomycin-pretreated mouse model. Our results suggest a mechanism for viaB-mediated attenuation of inflammatory responses in vivo that is independent of the serotype Typhimurium T3SS-1.     

Cellular Interactions in the Intestinal Stem Cell Niche

Archivum Immunologiae et Therapiae Experimentalis - Epithelial cells are one of the most actively cycling cells in a mammalian organism and therefore are prone to malignant transformation. Already...     

Temozolomide Drives Ferroptosis via a DMT1-Dependent Pathway in Glioblastoma Cells

PurposeTemozolomide is used in first-line treatment for glioblastoma. However, chemoresistance to temozolomide is common in glioma patients. In addition, mechanisms for the anti-tumor effects of temozolomide are largely unknown. Ferroptosis is a form of programmed cell death triggered by disturbed redox homeostasis, overloaded iron, and increased lipid peroxidation. The present study was performed to elucidate the involvement of ferroptosis in the anti-tumor mechanisms of temozolomide.Materials and MethodsWe utilized the CCK8 assay to evaluate cytotoxicity. Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), iron, and glutathione (GSH) were measured. Flow cytometry and fluorescence microscope were used to detect the production of reactive oxygen species (ROS). Western blotting, RT-PCR and siRNA transfection were used to investigate molecular mechanisms.ResultsTemozolomide increased the levels of LDH, MDA, and iron and reduced GSH levels in TG905 cells. Furthermore, we found that ROS levels and DMT1 expression were elevated in TG905 cells treated with temozolomide and were accompanied by a decrease in the expression of glutathione peroxidase 4, indicating an iron-dependent cell death, ferroptosis. Our results also showed that temozolomide-induced ferroptosis is associated with regulation of the Nrf2/HO-1 pathway. Conversely, DMT1 knockdown by siRNA evidently blocked temozolomide-induced ferroptosis in TG905 cells.ConclusionTaken together, our findings indicate that temozolomide may suppress cell growth partly by inducing ferroptosis by targeting DMT1 expression in glioblastoma cells.     

SLC11 family of H+-coupled metal-ion transporters NRAMP1 and DMT1

NRAMP1 (natural resistance-associated macrophage protein-1) and DMT1 (divalent metal-ion transporter-1) make up the SLC11 gene family of metal-ion transporters that are energized by the H⁺ electrochemical gradient. Long known to confer resistance to bacterial infection, NRAMP1 functions at the phagolysosomal membrane of macrophages and neutrophils. NRAMP1 most likely contributes to macrophage antimicrobial function by extruding essential metal ions (including Mn²⁺) from the phagolysosome via H⁺/metal-ion cotransport. An alternative hypothesis in the literature proposes that NRAMP1 concentrate Fe²⁺ within the phagolysosome by means of H⁺/Fe²⁺ antiport, resulting in the generation of toxic free radicals. DMT1 is expressed widely and accepts as substrates a broad range of transition metal ions, among which Fe²⁺ is transported with high affinity (K _0.52 M). DMT1 accounts both for the intestinal absorption of free Fe²⁺ and for transferrin-associated endosomal Fe²⁺ transport in erythroid precursors and many other cell types. DMT1 is up-regulated dramatically in the intestine by dietary iron restriction and, despite high serum iron levels, is not appropriately down-regulated in hereditary hemochromatosis.