神经毒素6-OHDA干预后星形胶质细胞铁转运蛋白表达的变化

doi:10.3969/j.issn.2095-4344.2016.07.016

摘要/Abstract

摘要：

文章快速阅读：

文题释义：

二价金属离子转运蛋白1(DMT1)：是哺乳类跨膜铁转运蛋白，是主要的铁转入蛋白，广泛分布于人体各组织。主要功能是介导小肠上皮细胞的铁吸收以及参与铁从内吞小体移位到胞浆的过程，也参与其他二价金属如Zn²⁺、Mn²⁺、Co²⁺、Cd²⁺、Cn²⁺、Ni²⁺和Pb²⁺的转运。在帕金森病患者的黑质发现DMT1表达异常增加，因而DMT1可能也与某些神经退行性疾病的形成有关。

膜铁转运蛋白Ferroportin1(FPN1)：又称铁调节转运体(IREG1)或金属转运蛋白(MTP1)，是一种跨膜的铁输出蛋白，是唯一的细胞铁流出通道，在成熟的十二指肠绒毛上皮细胞基底面、脾和肝的巨噬细胞、胎盘的合体滋养层细胞等都有表达，对其研究可能对铁超载或铁缺乏疾病的诊断和治疗有重要的医学应用前景。

背景：前期的研究已经证实，在神经元和小胶质细胞中，神经毒素6-OHDA能够增加铁转入蛋白DMT1的表达，减少铁转出蛋白FPN1的表达，可能导致帕金森病黑质铁的沉积。然而，6-OHDA能否在星形胶质细胞中发挥不同的作用尚不明确。

目的：观察6-OHDA作用于C6神经胶质瘤细胞(大鼠星形胶质细胞株)后，铁转运蛋白DMT1和FPN1表达变化。

方法：培养大鼠星形胶质细胞C6细胞，加入10 μmol/L 6-OHDA培养24 h，Western blots法检测6-OHDA干预后DMT1和FPN1蛋白表达。

结果与结论：用10 μmol/L 6-OHDA作用于大鼠C6星形胶质细胞24 h后，Western blots检测结果显示DMT1蛋白表达升高了2.5倍(P < 0.01)，FPN1蛋白表达升高了1倍(P < 0.05)。提示，6-OHDA通过同时增加铁转运蛋白DMT1和FPN1表达促进铁运速率，星形胶质细胞对6-OHDA的反应与神经元和小胶质细胞不同。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

ORCID: 0000-0002-1297-9477(Xu Man-man)

关键词: 组织构建, 组织工程, 铁转运蛋白, DMT1, FPN1, 星形胶质细胞, 6-OHDA, 帕金森病

Abstract:

BACKGROUND: Previous studies have confirmed that 6-hydroxydopamine is capable to increase the expression of divalent metal transporter-1 and reduce the expression of ferroportin-1 in the neurons and microglia, which may lead to iron deposition in the substantia nigra after Parkinson’s disease. However, it is unclear whether 6-hydroxydopamine can play diverse roles in astrocytes.
OBJECTIVE: To observe the effects of 6-hydroxydopamine on the expression of divalent metal transporter-1 and ferroportin-1 in rat C6 glioma cell lines.
METHODS: C6 glioma cell lines from rats were cultured in 10 μmol/L 6-hydroxydopamine for 24 hours. Then, protein expressions of divalent metal transporter-1 and ferroportiner-1 were measured by western blot method.
RESULTS AND CONCLUSION: The protein expressions of divalent metal transporter-1 and ferroportin-1 in C6 glioma cell lines were increased by 2.5 times (P < 0.01) and 1 time (P < 0.05), respectively, after treatment with 6-hydroxydopamine. These findings indicate that 6-hydroxydopamine can promote iron transport rate in astrocytes by increasing both divalent metal transporter-1 and ferroportin-1 expressions, and astrocytes has a different response to 6-hydroxydopamine from neurons and microglia.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

许曼曼，徐翎钰，杨晓，杜新星. 神经毒素6-OHDA干预后星形胶质细胞铁转运蛋白表达的变化[J]. 中国组织工程研究, 2016, 20(7): 1025-1030.

Xu Man-man, Xu Ling-yu, Yang Xiao, Du Xin-xing. 6-Hydroxydopamine up-regulates divalent metal transporter-1 and ferroportin-1 in C6 glioma cell lines[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(7): 1025-1030.

图/表（结果） 2

参考文献

[1] Haddad D, Nakamura K. Understanding the susceptibility of dopamine neurons to mitochondrial stressors in Parkinson’s disease. FEBS Lett. 2015; 589(24 Pt A):3702-3713.
[2] Zeineddine R, Yerbury JJ. The role of macropinocytosis in the propagation of protein aggregation associated with neurodegenerative diseases. Front Physiol. 2015;6:277.
[3] Goldman JG, Aggarwal NT, Schroeder CD. Mild cognitive impairment: an update in Parkinson's disease and lessons learned from Alzheimer's disease. Neurodegener Dis Manag. 2015;5(5):425-443.
[4] Youdim MB, Stephenson G, Ben Shachar D. Ironing iron out in Parkinson's disease and other neurodegenerative diseases with iron chelators: a lesson from 6-hydroxydopamine and iron chelators, desferal and VK-28. Ann N Y Acad Sci. 2004; 1012:306-325.
[5] Devos D, Moreau C, Devedjian JC, et al. Targeting chelatable iron as a therapeutic modality in Parkinson’s disease. Antioxid Redox Signal. 2014;21(2):195-210.
[6] Singh N, Haldar S, Tripathi AK, et al. Iron in neurodegenerative disorders of protein misfolding: a case of prion disorders and Parkinson’s disease. Antioxid Redox Signal. 2014;21(3):471-484.
[7] Weinreb O, Mandel S, Youdim MB, et al. Targeting dysregulation of brain iron homeostasis in Parkinson’s disease by iron chelators. Free Radic Biol Med. 2013; 62:52-64.
[8] Martin WR, Wieler M, Gee M. Midbrain iron content in early Parkinson disease: a potential biomarker of disease status. Neurology. 2008;70(16 Pt 2):1411-1417.
[9] Wieler M, Gee M, Martin WR. Longitudinal midbrain changes in early Parkinson’s disease: iron content estimated from R2*/MRI. Parkinsonism Relat Disord. 2015; 21(3):179-183.
[10] Qian ZM, Shen X. Brain iron transport and neurodegeneration. Trends Mol Med. 2001;7(3): 103-108.
[11] Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997;388(6641):482-488.
[12] Garrick MD, Dolan KG, Horbinski C, et al. DMT1: a mammalian transporter for multiple metals. Biometals. 2003;16(1):41-54.
[13] Ke Y, Qian ZM. Divalent metal transporter-1: a newly discovered important ferroportin. Shengwu Huaxue yu Shengwu Wuli Jinzhan. 2002;29(2):184-188.
[14] Yu X, Du T, Song N, et al. Decreased iron levels in the temporal cortex in postmortem human brains with Parkinson disease. Neurology. 2013;80(5):492-495.
[15] Kano O, Ikeda K, Iwasaki Y, et al. Decreased iron levels in the temporal cortex in postmortem human brains with Parkinson disease. Neurology. 2013; 81(13):1181-1182.
[16] Jiang H, Song N, Xu H, et al. Up-regulation of divalent metal transporter 1 in 6-hydroxydopamine intoxication is IRE/IRP dependent. Cell Res. 2010;20(3):345-356.
[17] Li W, Jiang H, Song N, et al. Oxidative stress partially contributes to iron-induced α-synuclein aggregation in SK-N-SH cells. Neurotox Res. 2011;19(3):435-442.
[18] Sangchot P, Sharma S, Chetsawang B, et al. Deferoxamine attenuates iron-induced oxidative stress and prevents mitochondrial aggregation and alpha-synuclein translocation in SK-N-SH cells in culture. Dev Neurosci. 2002; 24(2-3):143-153.
[19] Taniguchi R, Kato HE, Font J, et al. Outward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportin. Nat Commun. 2015;6:8545.
[20] Li J, Qian ZM, Chang YZ, et al. Recent progress in the studies of ferroportin 1. Sheng Li Ke Xue Jin Zhan. 2004;35(4):349-351.
[21] Wang J, Jiang H, Xie JX.Ferroportin1 and hephaestin are involved in the nigral iron accumulation of 6-OHDA-lesioned rats. Eur J Neurosci. 2007;25(9): 2766-2772.
[22] Knyihár-Csillik E, Chadaide Z, Mihály A, et al. Effect of 6-hydroxydopamine treatment on kynurenine aminotransferase-I (KAT-I) immunoreactivity of neurons and glial cells in the rat substantia nigra. Acta Neuropathol. 2006;112(2):127-137.
[23] Wang J, Song N, Jiang H, et al. Pro-inflammatory cytokines modulate iron regulatory protein 1 expression and iron transportation through reactive oxygen/nitrogen species production in ventral mesencephalic neurons. Biochim Biophys Acta. 2013;1832(5):618-625.
[24] Zhang HY, Wang ND, Song N, et al. 6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes. Biometals. 2013;26(5):705-714.

[25] Bushong EA, Martone ME, Ellisman MH.Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development. Int J Dev Neurosci. 2004;22(2):73-86.
[26] Saavedra A, Baltazar G, Santos P, et al. Selective injury to dopaminergic neurons up-regulates GDNF in substantia nigra postnatal cell cultures: role of neuron-glia crosstalk. Neurobiol Dis. 2006;23(3):533-542.
[27] Mena MA, García de Yébenes J. Glial cells as players in parkinsonism: the “good”, the “bad”, and the “mysterious” glia. Neuroscientist. 2008;14(6):544-560.
[28] Halassa MM, Fellin T, Takano H, et al. Synaptic islands defined by the territory of a single astrocyte. J Neurosci. 2007;27(24):6473-6477.
[29] Kimelberg HK. Functions of mature mammalian astrocytes: a current view.Neuroscientist. 2010;16(1): 79-106.
[30] Zeinstra E, Wilczak N, Chesik D, et al. Simvastatin inhibits interferon-gamma-induced MHC class II up-regulation in cultured astrocytes. J Neuroinflammation. 2006;3:16.
[31] Abboud S, Haile DJ.A novel mammalian iron-regulated protein involved in intracellular iron metabolism.J Biol Chem. 2000;275(26):19906-19912.
[32] Jakel RJ, Kern JT, Johnson DA, et al. Induction of the protective antioxidant response element pathway by 6-hydroxydopamine in vivo and in vitro. Toxicol Sci. 2005;87(1):176-186.
[33] [33] Mladenka P, Sim?nek T, Hübl M, et al. The role of reactive oxygen and nitrogen species in cellular iron metabolism. Free Radic Res. 2006;40(3):263-272.
[34] Zhao R, WAng GQ. The effect of deferiprone on excess free iron accumulation, free radicals and functional outcome in intracerebral hemorrhage rats. Zhongfeng yu Shenjing Jibing Zazhi. 2015;32(4):344-346.
[35] Wang J, Song N, Xu HM, et al. The Role of Iron and Alpha-synuclein Interacting in Parkinson’s Disease. Sheng Li Ke Xue Jin Zhan. 2015;46(3):180-184.
[36] Junxia X, Hong J, Wenfang C, et al. Dopamine release rather than content in the caudate putamen is associated with behavioral changes in the iron rat model of Parkinson’s disease. Exp Neurol. 2003;182(2):483-489.
[37] Youdim MB. What have we learnt from CDNA microarray gene expression studies about the role of iron in MPTP induced neurodegeneration and Parkinson’s disease? J Neural Transm Suppl. 2003; (65):73-88.
[38] Salazar J, Mena N, Hunot S, et al. Divalent metal transporter 1 (DMT1) contributes to neurodegeneration in animal models of Parkinson’s disease. Proc Natl Acad Sci U S A. 2008;105(47):18578-18583.
[39] Zhang S, Wang J, Song N, et al. Up-regulation of divalent metal transporter 1 is involved in 1-methyl-4-phenylpyridinium (MPP(+))-induced apoptosis in MES23.5 cells. Neurobiol Aging. 2009; 30(9):1466-1476.

引言

Parkinson’s disease is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, giving rise to dopamine depletion in the striatum, which occurs mainly in the middle-aged and elderly with the clinical manifestations of akinesia, muscular rigidity, resting tremor and postural reflex disorder^[1-3]. Although heredity, environment and oxidative stress play a certain role, the exact pathogenesis of Parkinson’s disease is not fully understood.

Iron is one of the essential trace elements in the human body, which is widely involved in the body metabolic processes. The iron as an essential component involved in the synthesis of myelin sheath and neurotransmitters is particularly important for the brain. Existing evidence has shown that during the brain development, iron deficiency leads to irreversible behavioral and cognitive disorders in children, and excessive iron can cause damage to nerve cells, and thereby becomes one of the initial reasons for neurodegenerative diseases^[4]. In this century, excessive iron-reduced damage to dopaminergic neurons in the substantia nigra-corpus striatum system is becoming an issue of attentions. Due to its cytotoxicity and its ability to promote hydroxyl radical production, iron has a non-negligible role in the pathogenesis of Parkinson’s disease^[5-7]. It is confirmed that iron accumulation is involved in the pathogenesis of Parkinson’s disease. Selective deposition of irons exists in the substantia nigra of Parkinson’s disease patients and animal models. We can observe an increase in iron content in the substantia nigra of patients at early stage of Parkinson’s disease, and moreover, the iron content is correlated with the progression of Parkinson’s disease^[8-9].

Studies have shown that an abnormal increase in iron content in the brain may be due to some iron transporters out of control. Divalent metal transporter-1 (DMT1) is the major iron into protein widely expressed in the brain^[10-12], which has a similar structure to Nramp1 that consists of 12 transmembrane domains (Figure 1)^[13]. DMT1 is widely expressed in the brain and highly expressed in the iron-enriched substantia nigra. Additionally, DMTI is also highly expressed in cells with demands for a lot of irons. Excessive iron deposition is likely to cause a series of pathological changes, and thereby destroy nerve cells, eventually leading to a variety of brain diseases, such as neurodegenerative diseases (including Parkinson’s disease and Alzheimer’s disease^[14-15]. It has been reported that there is an excessive expression of DMTI in the substantia nigra neurons of Parkinson’s disease patients, and DMT1 excessive expression may cause selective accumulation of irons and in turn result in an increase of reactive oxygen species, eventually leading to death of neurons^[16-18].

Ferroportin-1 (FPN1) is currently the only known transmembrane protein for cellular iron efflux, which is also known as Fe-regulated transport protein 1 (IREG1) or metal transport protein 1 (MTP1)^[19]. FPN1 consists of 571 amino acids and has at least 10 transmembrane domains (Figure 2), with a relative molecular mass of about 62 000^[20]. Northernblot analysis shows that FPN1 is widely distributed in the body tissues, such as the small intestine, placenta, spleen, liver, kidney, heart, muscle, lung and brain. Brain studies in rats have shown that FPN1 is highly expressed in the hippocampus, cerebral cortex, cerebellum, thalamus and striatum, especially in the cortical pyramidal neurons and the apical dendrites. In addition, FPN1 is often found to highly express in the hippocampal pyramidal cells and granule cells as well as in the habenular nucleus. As previously reported, FPN1 is considered to be involved in iron deposition in the substantia nigra of Parkinson’s disease by reducing the expression^[21].

6-Hydroxydopamine (6-OHDA) is a widely used neurotoxic agent that can selectively damage dopaminergic neurons in vivo and in vitro. The toxicity of 6-OHDA is generated through reactive oxygen species-mediated oxidative damage to mitochondria^[22]. In our previous studies, we have demonstrated that 6-OHDA can increase DMT1 but reduce FPN1 in neurons and microglia^[23-24]. However, it is unclear whether 6-OHDA can play different roles in astrocytes. In this study, we aimed to investigate the changes of both DMT1 and FPN1 expressions in the C6 glioma cell lines after treatment with 10 μmol/L 6-OHDA for 24 hours.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

材料方法

Design

In vitro cytological study.

Time and setting

Experiments were completed at the National Key Laboratory of Physiology, Qingdao University Medical School, China in July 2015.

Materials

Rat C6 glioma cell lines were provided by Shanghai Cell Bank of the Chinese Academy of Sciences. Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis., MO, USA). The primary antibodies against FPN1 and DMT1 were separately from the Sigma Chemical Co. and the Alpha Diagnostic (ADI, San Antonio, TX, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco (Grand Island, NY, USA).

Methods

Cell cultures

C6 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin at 37 ℃, in a humid atmosphere of 5% CO₂ and 95% air. For experiments, cells at 70%-80% confluence were sub-cultured and seeded at a density of 10⁵ cells/cm².

Western blot analysis

Cells were treated with 10 μmol/L 6-OHDA for 24 hours and then collected after supernatant removal. After three washes with TBST, cells were digested directly on culture plates with RIPA lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet-40, 0.5% sodium deoxycholate, 1 mmol/L ethylene diamine tetraacetie acid, 1 mmol/L phenylmethylsulfonyl fluoride) with protease inhibitors (pepstatin 1g/mL, aprotinin 1g/mL, leupeptin 1g/mL) for 30 minutes on ice. The insoluble material was removed by centrifugation

(1 2 000 r/min, 20 minutes, 4 ℃). 30 μg total proteins were extracted and separated using 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene fluoride membranes. Blots were probed with anti-DMT1 antibody (1:800, ADI) and anti-FPN1 antibody (1:800, Sigma). Blots were also probed with anti-β-actin monoclonal antibody (1:10 000, Sigma) as a loading control. Cross-reactivity was visualized using UVP gel imaging system and photographed. The DMT1 and FPN1 expressions were represented as the ratio of DMT1/β-actin and FPN1/β-actin. The experiment was repeated six times.

Main outcome measures

Changes in DMT1 and FPN1 expressions after 6-OHDA intervention.

Statistical analysis

Data are presented as mean±SEM and were statistically analyzed using SPSS 17.0 software. The intergroup comparisons were made by one-way analysis of variance and Tukey test. P < 0.05 and

P < 0.01 were considered statistically or greatly statistically significant.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

讨论

Astrocytes as the major glial cell type in the central nervous system are widely distributed in the brain gray and white matters, and have multiple functions, including cell support during central nervous system development. Astrocytes participate in the various physiological or pathological processes, including the formation and maintenance of blood-brain barrier, neuronal development and synaptic remodeling, glutamate metabolism, and stable maintenance of extracellular K⁺, and are generally accepted as a principle contributor for the uptake of a variety of nutrients such as transition metals crossing the blood-brain barrier^[25-28]. Additionally, astrocytes have a close relationship with central nervous system inflammation and immunoreactions^[29-30]. DMT1 is mainly expressed in the end feet of astrocytes around the blood capillaries, indicating a major role of astrocytes in brain iron uptake. These cells have a strong ability to accumulate irons and have a higher tolerance to iron. Iron overloading can trigger neuronal cell and microglia death, and however, activate astrocyte proliferation^[31]. Thus, astrocytes play an important role in regulating brain iron homeostasis and protecting other brain cells from iron-mediated oxidative stress.

6-OHDA, a norepinephrine homolog, can create oxidative stress through the production of free radicals and reactive oxygen to destroy dopaminergic neurons in the substantia nigra, and damage dopamine synthesis in the substantia nigra and the transport path to the striatum, thereby resulting in catecholamine and acetylcholine neurotransmitter imbalance and causing a series of symptom and pathological changes similar to human Parkinson’s disease, such as unilateral rotation. 6-OHDA is now commonly used to establish animal models of Parkinson’s disease and cell model of Parkinson’s disease nerve gas, which is a kind of selective neurotoxins. Its toxicity on the one hand can happen outside the cells from oxidation, intracellular H₂O₂ and OH• indirect oxidative stress; on the other hand, 6-OHDA can directly enter into the cells by the dopamine transporter uptake and start to play a toxic role of oxidative stress^[32-33]. In the 6-OHDA induced Parkinson’ disease model and PD patients, glial cells in the substantia nigra pars compacta appear to have obvious responses. During the generation process, activated astrocytes and DA nerve endings produce a large number of “cross-talk”, and reactive astrocytes in DA neuron degeneration play an important role.

Recent studies have shown that brain iron metabolism can cause a lot of brain diseases^[34]. Iron is found a higher expression with the presence of oxidative stress in the brain basal ganglia and senile plaques of Alzheimer’s disease and Parkinson’s disease patients^[35-36]. Increase in iron concentration in the brain can lead to iron oxidative stress and deficiency of antioxidant protection, which may be one reason for neuronal degenerative death. In recent years, studies have shown that the degree of oxidative damage in the brain tissue is directly related to the local brain iron, and iron-induced oxidative damage has been increasingly attracting attentions^[37-38]. Accumulating evidence has demonstrated that the generation of reactive oxygen can be involved in the regulation of iron and subsequently regulate the iron-related proteins. Iron deposition in the substantia nigra is actively involved in the pathogenesis of Parkinson’s disease.

Our previous studies have shown that increased expression of DMT1 and decreased expression of FPN1 can account for the iron deposition in both neurons and microglia with 6-OHDA treatment^{[23-24, 39]}. In this study, we found that both DMT1 and FPN1 expressions were increased in C6 glioma cell lines treated with 10 μmol/L 6-OHDA for 24 hours. Experimental findings indicate that 6-OHDA promotes iron transport rate in astrocytes by increasing both DMT1 and FPN1 expressions, and astrocytes has a different response compared with neurons and microglia.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程