小胶质细胞对中脑多巴胺能神经元铁代谢的影响及其机制：体外细胞学实验方案

doi:10.3969/j.issn.2095-4344.2017.08.020

摘要/Abstract

摘要：

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。

摘要
背景：目前，铁代谢障碍已被公认为帕金森发病的一个关键因素，但是脑铁代谢的研究仍处于起步阶段，胶质细胞在脑铁代谢中所扮演的角色还不清楚。许多研究显示，小胶质细胞在帕金森发病中起重要的作用，且与铁的关系密切。
目的：在前期研究的基础上，深入探讨小胶质细胞与神经元在铁代谢领域的关系，阐明小胶质细胞在铁选择性损伤多巴胺能神经元致帕金森中的作用。
方法：实验为体外细胞学实验，在中国山东，青岛大学医学院完成。①改变小胶质细胞铁的水平，观察小胶质细胞条件培养基对中脑神经元的存活率及铁代谢的影响。用脂多糖激活不同铁水平的小胶质细胞，观察小胶质细胞条件培养基对中脑神经元存活率及铁代谢的影响；②应用小干涉RNA、ELASA方法观察小胶质细胞内不同的铁水平对小胶质细胞及其激活时所释放的炎症因子和乳铁蛋白的影响，观察小胶质细胞释放的炎症因子和乳铁蛋白对神经元存活率及铁代谢的影响；③改变中脑神经元的铁水平，然后加入上述小胶质细胞条件培养基，观察中脑神经元存活率及铁代谢的变化。实验方案经医院伦理委员会批准，批准号为01482 311234。大鼠的实验操作和取材遵循《关于善待实验动物的指导性意见》规定，并与美国国立卫生与健康研究院的指南一致。
结果与结论：实验证实了小胶质细胞的功能与黑质多巴胺能神经元铁代谢密切相关，如何调控小胶质细胞的功能使其向保护神经元的方向发展，这一方面的研究进展无疑将对帕金森病的防治有极大的推动作用。

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: 0000-0002-4235-796X(Shi Cheng-kui)

关键词:
组织构建, 组织工程, 小胶质细胞, 帕金森病, 铁, 神经元

Abstract:

BACKGROUND: Iron metabolism disorder has been proved to be a key factor of Parkinson’s disease, but research on brain iron metabolism is still immature, and the role of glial cells in the brain iron metabolism remains unclear. Many studies have pointed that microglia plays an important role in the pathogenesis of Parkinson’s disease, which closely related to iron.
OBJECTIVE: To further explore the relationship between microglia and neurons in iron metabolism based on our previous study, and to clarify the role of microglia in iron-induced selective damaged dopaminergic neurons of Parkinson’s disease.
METHODS: This was an in vitro cellular experiment, which was finished in the Medical College of Qingdao University, Shandong Province, China. The effects of conditioned medium of microglia on the survival rate of mesencephalic neurons and iron metabolism were observed by changing the iron levels in microglia. Lipopolysaccharide was used to activate different iron-loaded microglia, and then the effects of conditioned medium on the survival rate of mesencephalic neurons and iron metabolism were determined. The effects of different iron levels in microglia on microglia and inflammatory factors and lactoferrin released from the activated microglia were detected by siRNA and ELISA. The effects of these inflammatory factors and lactoferrin on the survival of mesencephalic neurons and iron metabolism were observed. The iron levels in mesencephalic neurons were changed, and then added into the above conditioned medium to observe the survival of neurons and iron metabolism. This experimental protocol was approved by the Hospital Ethics Committee (No. 01482 311234). All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals issued by the United States National Institutes of Health.
RESULTS AND CONCLUSION: Our study results show that the function of microglia is closely related to the iron metabolism in nigra dopaminergic neurons. Research on the rational use of microglia to prevent neuronal degeneration gives a strong impetus to the prevention and treatment of Parkinson’s disease.

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

Key words: Microglia, Parkinson Disease, Iron, Tissue Engineering

中图分类号:

R318

石成奎，陈志强. 小胶质细胞对中脑多巴胺能神经元铁代谢的影响及其机制：体外细胞学实验方案[J]. 中国组织工程研究, 2017, 21(8): 1262-1267.

Shi Cheng-kui, Chen Zhi-qiang. Effect of microglia on iron metabolism in midbrain dopaminergic neurons and the underlying mechanism: study protocol for an
in vitro cellular experiment[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(8): 1262-1267.

图/表（结果） 2

参考文献

[1] Mirza B, Hadberg H, Thomsen P, et al. The absence of reactive astrocytosis is indicative of a unique inflammatory process in Parkinson’s disease. Neuroscience. 2000;95: 425-432.

[2] Zhang X, Surguladze N, Slagle-Webb B, et al. Cellular iron status influences the functional relationship between microglia and oligodendrocytes. Glia. 2006;54(8):795-804.

[3] Wu DC, Jackson-Lewis V, Vila M, et al. Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci. 2002;22(5):1763-1771.

[4] Peng J, Stevenson FF, Oo ML, et al. Iron-enhanced paraquat-mediated dopaminergic cell death due to increased oxidative stress as a consequence of microglial activation. Free Radic Biol Med. 2009;46(2):312-320.

[5] O'Brien-Ladner AR, Nelson SR, Murphy WJ, et al. Iron is a regulatory component of human IL-1beta production. Support for regional variability in the lung. Am J Respir Cell Mol Biol. 2000;23(1):112-119.

[6] Helyar L, Sherman AR. Iron deficiency and interleukin 1 production by rat leukocytes. Am J Clin Nutr. 1987;46(2): 346-352.

[7] Smirnov IM, Bailey K, Flowers CH, et al. Effects of TNF-alpha and IL-1beta on iron metabolism by A549 cells and influence on cytotoxicity. Am J Physiol. 1999;277(2 Pt1):L257-263.

[8] Eisenstein RS, Tuazon PT, Schalinske KL, et al. Iron-responsive element-binding protein. Phosphorylation by protein kinase C. J Biol Chem. 1993;268:27363-27370.

[9] Schalinske KL, Eisenstein RS. Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL-60 cells. J Biol Chem. 1996;271:7168-7176.

[10] Kiemer AK, Förnges AC, Pantopoulos K, et al. ANP-induced decrease of iron regulatory protein activity is independent of HO-1 induction. Am J Physiol Gastrointest Liver Physiol. 2004;287:G518-6526.

[11] An L, Sato H, Konishi Y, et al. Expression and localization of lactotransferrin messenger RNA in the cortex of Alzheimer's disease. Neurosci Lett. 2009;452(3):277-280.

[12] Leveugle B, Faucheux BA, Bouras C, et al. Cellular distribution of the iron-binding protein lactotransferrin in the mesencephalon of Parkinson's disease cases. Acta Neuropathol. 1996;91(6):566-572.

[13] Wang J, Jiang H, Xie JX. Time dependent effects of 6-OHDA lesions on iron level and neuronal loss in rat nigrostriatal system. Neurochem Res. 2004;29(12): 2239-2243.

[14] Zhang S, Wang J, Song N, et al. Up-regulation of divalent metal transporter1 is involved in 1-methyl-4-phenylpyridinium (MPP(+))-induced apoptosis in MES23.5 cells. Neurobiol Aging. 2009;30(9):1466-1476.

[15] 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.

[16] Xu HM, Jiang H, Wang J, et al. Over-expressed human divalent metal transporter 1 is involved in iron accumulation in MES23.5 cells. Neurochem Int. 2008;52(6):1044-1051.

[17] Song N, Wang J, Jiang H, et al. Ferroportin 1 but not hephaestin contributes to iron accumulation in a cell model of Parkinson's disease. Free Radic Biol Med. 2010;48(2): 332-341.

[18] Youdim MB, Fridkin M, Zheng H. Bifunctional drug derivatives of MAO-B inhibitor rasagiline and iron chelator VK-28 as a more effective approach to treatment of brain ageing and ageing neurodegenerative diseases. Mech Ageing Dev. 2005;126(2):317-326.

[19] Hirsch EC. Altered regulation of iron transport and storage in Parkinson's disease. J Neural Transm Suppl. 2006;(71): 201-204.

引言

With the rapid development of neurosciences and its technology, microglia has been found to play a critical role in nervous system development, synaptic transmission, neural repair and regeneration and neuroimmunity; therefore, the role of glial cells in neurodegenerative diseases has attracted much attention. Iron has been confirmed to be involved in the pathogenesis of Parkinson’s disease, but study on the brain iron metabolism is till on initial stage. Exploring the role of microglia in the brain iron metabolism is an interesting challenge.

Large amounts of activated microglia are distributed in the substantia nigra pars compacta, which containing abundant iron deposition^[1].1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 6-hydroxydopamine, paraquat, and lipopolysaccharides (LPS) all can induce the microglia activation accomapanied by iron deposition prior to neuron injury^[2-3]. Iron accelerates the paraquat-induced injury of dopaminergic neurons with the assistance of microglia^[4], suggesting that microglia participate in the pathogenesis of Parkinson’s disease that closely related to iron. LPS can activate microglia in animal and human, which is closely related to the progressive degeneration of dopaminergic neurons. Microglia all can be activated after the injection of LPS into animal abdominal cavity and nigra or primary midbrain cells co-cultured with LPS, and a series of inflammatory factors (tumor necrosis factor α (TNF-α) and interleukin-1β

(IL-1β) appear. By a model of selective injury of dopaminergic neurons we successfully simulate the pathological progress of Parkinson’s disease and it has become a popularized method. Some studies reported that high concentration of iron inhibits IL-1β transcription in alveolar macrophages^[5], while some thought that low concentration of iron inhibits IL-1 synthesis^[6]. How dose iron level in microglia affect the release of inflammatory factors after its activation?

Is it related to the regulation of NF-kB and AP-1? In peripheral tissue, TNF-α and IL-1β can increase the uptake of non-transferrin-bound iron in A549 cells, but the underlying mechanism remains unclear^[7]. Protein kinase C (PKC) participated in signal transduction after IL-1 binding to receptors, and PKC can stimulate the level of phosphorylation of iron-responsive element-binding protein(IRE), thus resulting in high affinity IRE RNA binding activity^[8-9], indicating that extracellular factors affect intracellular iron metabolism by regulating the level of IRE^[9]. However, PKC activators and inhibitors in different cells expose no effect on the IRE RNA binding activity, showing that different species and cells hold different iron metabolism regulation^[10].

Lactoferrin (LF) is a multifunctional protein of the transferrin family, which binds to LF receptor to form LF-LFR complex that enters into cells to release iron through endocytosis. In nigra, only microglia is positive for LF mRNA expression, and LFR mainly distributed in neurons^[11]. The expression levels of LF and LFR in surviving neurons in the substantia nigra were increased, and showed no change in the non-affected area of Parkinson’s disease, suggesting that its expression is associated with the injury of dopaminergic neurons^[12]. Whether the syhthesis and release of LF and LFR are regulated by microglia and iron, and the underlying mechanism is not fully understood. LF indeed is a bridge that connects microglia with iron metabolism.

To summarize, microglia function is closely related to iron metabolism in dopaminergic neurons in the substantia nigra. Does intracellular iron level affect microglia function and activation? Whether microglia act on iron homeostasis in dopaminergic neurons by regulating inflammatory factors (TNF-α and IL-1β) and LF? Research on the iron metabolism in microglia is immature, but its relationship is beyond all doubt. How to transform the microglia function into neuroprotection is an important hot spot.

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

MATERIALS AND METHODS

Study design

An in vitro cellular experiment.

Study setting

The National Key Laboratory for Physiology of the Medical College of Qingdao University, Shandong Province, China.

Objective

To explore the relationship of microglia and neurons in iron metabolism, and to clarify the role of microglia in iron-induced selective damaged dopaminergic neurons of Parkinson’s disease. (1) Microglia related to Parkinson’s disease can affect the iron metabolism in dopaminergic neurons, and the function of microglia is associated with the iron levels. (2) Microglia acts on the iron metabolism in dopaminergic neurons by releasing inflammatory factors and LF. (3) The susceptibility of dopaminergic neurons to microglia depends on the iron levels in dopaminergic neurons.

Methods

Culture of ventral midbrain neurons and microglia

Neurons were isolated from the ventral midbrain of fetal rats and microglia isolated from the Wistar rat brain born within 24 hours, both of which were then cultured.

Effect of different iron levels in microglia on midbrain neurons

Effect of different iron levels in the inactivated microglia on midbrain neurons: microglia were respectively incubated with iron (FAC) and iron chelator (DFO) for 24 hours, and intracellular iron levels were then measured with Calcein to confirm whether these reagents resulted in intracellular high or low iron status. Afterwards, the cells were washed three times with CMF-HBSS, and the special culture medium for neurons was added, the culture medium was collected at 24 hours and centrifuged. The supernatant was stored using 0.45 μm filter at -20 ℃, and exhausted within 1 week. After ventral midbrain neurons cultured for 7 days, the conditioned medium (CM) was added and cultured for 48 hours. The survival of neurons, iron regulatory protein expression and iron levels in neurons were detected. There were four groups, including control (special medium), CM(DFO), CM and CM(FAC) groups.

Effect of different iron levels in the activated microglia on midbrain neurons: Microglia were respectively incubated with FAC and DFO for 24 hours, and intracellular iron levels were then measured with Calcein to confirm whether these reagents resulted in intracellular high or low iron status. Afterwards, the cells were washed three times with CMF-HBSS, and the special culture medium for neurons and 80 μg/L LPS were added, the culture medium was collected at 48 hours and centrifuged. The supernatant was stored using 0.45 μm filter at -20 ℃, and exhausted within 1 week. After ventral midbrain neurons cultured for 7 days, the CM was added and cultured for 48 hours. The survival of neurons, iron regulatory protein expression and iron levels in neurons were detected. There were five groups, including control (special medium for neurons), LPS, CM(DFO+LPS), CM(LPS) and CM(FAC+LPS) groups.

Pathways of microglia affecting the iron metabolism in midbrain neurons

Microglia were induced by DFO, FAC, LPS, DFO+LPS, CM+LPS and FAC+LPS, respectively to prepare the CM. The levels of TNF-α and IL-1β in the culture medium were observed. The expression levels of NF-kB and AP-1 in the microglial nucleus were observed to reveal the iron level in microglia inducing the secretion of inflammatory factors. The neurons were preincubated with TNF-α and IL-1β antibody, and the effects of each CM on the survival of neurons, iron regulatory protein expression and iron levels in neurons were detected. 100 μg/L TNF-α, 10 μg/L IL-1β and the combination of these two inflammatory factors and CM were used to treat neurons, and the neuronal survival rate, iron regulatory protein expression and iron level in neurons were detected to observe whether iron level in microglia s affect the effect of inflammatory factors on neurons. There were ten groups, such as control, CM, CM(FAC), TNF-α, TNF-α+CM, TNF-α+CM(FAC), TNF-α+CM(DFO), IL-1β+CM, IL-1β+CM(FAC), and IL-1β+CM(DFO) groups. After the pretreatment of neurons with FAC and DFO, the changes of all above indexes were observed to show whether the iron levels in neurons could affect the effect of cytokines released by microglia on mesencephalic neurons.

Microglia were treated with DFO, FAC, LPS, LPS+DFO and LPS+FAC, respectively, and then the expression level of LF in the medium was detected by western blot assay. The CM was added into the mesencephalic neurons, and the survival of neurons, the expression of LF-R, the iron transport and the iron levels were observed. SiRNA technique was used to observe the changes of the above indexes after interfering with LF. SiRNA sequences for lactoferrin: Sense: 5’-GCA AGA GAT GCT GAA CAA ATT CAA GAG ATT TGT TCA GCA TCT CTT GCT TTT TT-3’; Antisense: 5’-AAT TAA AAA AGC AAG AGA TGC TGA ACA AAT CTC TTG AAT TTG TTC AGC ATC TCT TGC GGC C-3’. The vector was pSilencer 1.0-U6.

The effects of the above CM on the survival rate of midbrain neurons, iron transport and iron levels were observed after the treatment of midbrain neurons with LF-R antibody. After the pretreatment of neurons with FAC and DFO, the changes of all above indexes were observed.

Study procedures

The aim of this study is to clarify (1) whether intracellular iron levels affect the activation of microglia and the release of cytokines; (2) whether and how microglia affects the iron metabolism in dopaminergic neurons; (3) whether the iron levels in neurons promote the effect of cytokines on neurons (Figure1).

Outcome measures

Primary outcome measures

Neuronal survival rate; expression level of iron regulation protein in neurons; iron levels in neurons; LF-R expression level in neurons.

Secondary outcome measures

Levels of inflammatory factors (TNF-α and IL-1β) in neurons; NF-kB and AP-1 expression levels in microglia nuclear; LF level.

Table 1 Outcome measures

The measures are shown in Table 1.

Index	CM group	CM(FAC) group	CM(DFO) group	CM(LPS) group	CM(LPS+ FAC) group	CM(LPS+ DFO) group
Neuronal survival rate	√	√	√	√	√	√
Iron regulation protein	√	√	√	√	√	√
Iron level	√	√	√	√	√	√
Lactoferrin receptor	√	√	√	√	√	√
Tumor necrosis factor-α	√	√	√	√	√	√
Interleukin-1β	√	√	√	√	√	√
Lactoferrin	√	√	√	√	√	√
Nuclear factor-κB	√	√	√	√	√	√
AP-1	√	√	√	√	√	√

Note: CM: Conditioned medium; FAC: iron; DFO: iron chelator: LPS: lipopolysaccharides.

Statistical analysis

All hypothesis tests were bilateral test, and P < 0.05 was considered statistically significant. Data analysis was performed on SPSS 20.0 software (IBM Corp, Armonk, NY, USA). All data are expressed as the mean±SD, and one-way analysis of variance was used analysis of differences of the means among groups.

Data collection, management, and analysis

All data will be recorded electronically, and saved in a dedicated computer. Continuous variables from each record will be collected for descriptive statistical analysis, to allow real-time review and identify any potential deviation. The validity of the data will be assessed by the censor once every 6 months through a random sampling of 10% of the database. Only the researchers participating in the study will have the right to query the database, which will not be modified. Statistical analysis will be completed by professional statisticians, who will produce a statistical report. The statistical results will be given to the project manager, who will be responsible for writing the research report.

Ethical approval

This experimental protocol was approved by the Hospital Ethics Committee (No. 01482 311234). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals formulated by the National Institutes of Health, USA.

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