锰干扰谷氨酸代谢和NMDA受体表达及MK-801对其防护作用的研究
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摘要
目的
锰(Manganese, Mn)作为机体必需的微量元素之一,在体内可以参与构成许多酶的活性基团或辅助因子,同时又是某些酶的激活剂,参与许多生物化学反应,但是过量的锰进入体内则会引起锰中毒。进入体内的锰可穿透血-脑屏障,蓄积在脑组织内,早期主要表现为神经行为功能的改变,长期过量接触锰主要表现为锥体外系损伤,出现类似帕金森氏综合征的症状。因此,关于锰的神经毒性研究备受广大学者的关注。对锰神经毒作用机理的研究至今尚未完全阐明,锰的神经毒作用涉及诸多方面：过量的锰可增加神经细胞的代谢,破坏线粒体,提高溶酶体活性,发生自消化作用,引起神经细胞退变及损伤,还可激活细胞色素氧化酶P-450活性而产生自由基。近年来研究表明,锰可干扰脑内的兴奋性神经递质谷氨酸的代谢而产生间接兴奋性神经毒作用。作为兴奋性氨基酸神经递质受体之一的N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor, NMDAR)具有独特的结构功能特点,它既是配体门控受体,又是电压依赖性门控受体,因而在神经生理、病理和毒理学研究中NMDA受体也受到了特别关注。MK-801是一种强有力NMDA受体非竞争性拮抗剂,易通过血脑屏障,可作用于NMDA受体通道内部的苯环哌啶位点进行别构调节,能有效阻滞谷氨酸与突触后膜受体结合,阻断NMDA受体偶联的ca2+通道激活,使Ca2+内流减少,从而使NMDA受体作用减弱。为了进一步了解锰对兴奋性神经递质代谢的影响与神经细胞损伤的关系,以及MK-801对锰神经毒性的防护作用。我们将从体内和体外实验两方面研究如下问题：①锰对谷氨酸代谢的影响,②锰对NR1、NR2A和NR2B亚单位表达的影响,③锰对原代培养神经元的毒性作用,④MK-801对锰致神经损伤的防护作用。这将对我们进一步了解锰神经毒性的具体机制有十分重要的意义,将为锰致神经细胞损伤的发病机理和防治提供重要理论和实验依据。
实验方法
一、体内动物实验
(一)实验动物与分组
由中国医科大学实验动物中心提供的实验用Wistar大鼠100只,体重170～190g,雌雄各半。正式实验前饲养7d,然后按体重随机分成5组,每组20只。第1组为对照组,腹腔注射0.9%氯化钠,第2-4组为锰染毒组,分别腹腔注射8,40,200μmol/kg的氯化锰溶液,第5组为MK-801预处理组,隔日皮下注射0.3μmol/kg的MK-801溶液,2h后腹腔注射200μmol/kg的氯化锰溶液,注射容量均为5 ml/kg。每周注射5次,共4周。
(二)测定指标
1、脑纹状体锰含量的测定
染毒4周后,每组处死8只大鼠,取出一定量的纹状体组织,用原子吸收分光光度火焰法测定Mn含量。
2、脑纹状体Glu和Gln含量的测定
染毒4周后,每组处死6只大鼠,取出一定量的纹状体组织,按南京建成试剂盒说明书操作,测定Glu和Gln含量。
3、脑纹状体PAG和GS活力的测定
染毒4周后,每组处死6只大鼠,取出一定量的纹状体组织,分别按Curi禾(?)Renis描述的方法测定PAG和GS活力。
4、脑纹状体NMDA受体mRNA和蛋白表达的测定
每组取4只大鼠纹状体组织,分别用RT-PCR和Western blot方法检测mRNA和蛋白的表达,按常规方法提取总RNA和总蛋白,检测NR1,NR2A和NR2B的mRNA表达,用G3PDH作内参；检测NR1, NR2A和NR2B的蛋白表达,用β-actin作内参,用凝胶成像分析系统照相,测定各条带的灰度值。
5、细胞凋亡的测定
每组4只大鼠取一定量纹状体组织,制成单细胞悬液后,用流式细胞仪检测细胞凋亡率；每组2只大鼠用4%的多聚甲醛灌流固定,取出纹状体进一步处理后,电镜观察神经元细胞凋亡。
二、体外神经元细胞培养
(一)神经元培养
取新生鼠脑,去脑膜后切碎,用胰蛋白酶消化后,将细胞悬液转移到200目筛网中过滤,以含有10%马血清的DMEM培养基稀释细胞悬液至1×106／1.5m1,接种于经多聚赖氨酸过夜处理的直径为60mm的培养皿中,置于37℃CO2孵箱中培养24 h后,吸去DMEM培养基,换成无血清的Neurobasal培养液继续培养。加入阿糖胞苷,抑制非神经元的增值和生长。
(二)神经元的鉴定
倒置显微镜观察神经元表面光滑,胞体大、饱满,呈锥体形、星形、多角形或不规则形,突起清晰,均匀细长,2-5个不等,突起之间相互交织成网,连结紧密。用免疫细胞化学的方法检测神经元特异性烯醇化酶(NSE)。
(三)测定指标
1、细胞活力测定
待细胞生长状态最佳时加入含锰的培养液,锰处理浓度分别为0,12.5,25,50,100,200,400μmol/L7个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,分别培养6、12、24、48 h后,用MTT法测定细胞活力。
2、培养液中LDH活力的测定
神经元细胞用锰处理浓度分别为0,12.5,25,50,100,200,400μmol/L7个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,分别培养6、12、24、48 h后,用比色法测定LDH活力。
3、TUNEL法检测细胞凋亡
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,按原位细胞凋亡检测试剂盒说明书进行凋亡细胞染色,分析细胞凋亡率。
4、神经元细胞NMDA受体mRNA和蛋白表达的测定
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L 4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,分别用RT-PCR和Western blot方法检测mRNA和蛋白的表达,按常规方法提取总RNA和总蛋白,检测NR1, NR2A和NR2B的mRNA表达,用G3PDH作内参；检测NR1, NR2A和NR2B的蛋白表达,用β-actin作内参,用凝胶成像分析系统照相,测定各条带的灰度值。
5、神经元细胞内Ca2+浓度的测定
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,用双波长荧光分光光度计检测。
6、神经元Na+-K+-ATPase和Ca2+-ATPase活力的测定
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,提取膜蛋白用比色法测定。
实验结果
一、体内动物实验
1、脑纹状体锰含量
随着染锰剂量的增加,纹状体Mn含量也逐渐升高。各染锰组纹状体Mn含量均明显高于对照组(P<0.05),200μmol/kg染锰组大鼠纹状体Mn含量达到最高值0.98±0.27μg／g组织湿重,是对照组的4.7倍(P<0.01)。MK-801预处理组Mn含量与200μmol/kg染锰组比较,无明显变化。
2、脑纹状体Glu和Gln含量
随着染锰剂量的增加,Glu含量逐渐升高,Gln含量逐渐下降。200μmol/kg染锰组与对照组比较,Glu含量明显升高2.5倍；Gln含量明显下降了39.4%(P<0.01)。MK-801预处理组与200μmol/kg染锰组比较,Glu含量下降不明显,Gln含量明显回升了36.8%(P<0.05)。
3、脑纹状体PAG和GS活力
随着染锰剂量的增加,PAG活力逐渐升高,GS活力逐渐下降。200μmol/kg染锰组与对照组比较,PAG活力明显升高了54.4%达到41.02±6.60μmol/min/g protein; GS活力明显下降了28.9%达到.44.47±6.93μmol/min/g protein(P＜0.01)。MK-801预处理组与200μmol/kg染锰组比较,PAG活力明显下降,GS活力明显回升。
4、脑纹状体NMDA受体mRNA和蛋白表达
随着染锰剂量的增加,NR1, NR2A, NR2B mRNA和蛋白的表达均有不同程度的下降,其中NR2A灰度值下降最明显。MK-801预处理组与200μmol/kg染锰组比较,NR2A的灰度值明显升高(P<0.05),NRl和NR2B灰度值均未见明显变化。
5、脑纹状体细胞凋亡
用流式细胞仪检测发现,随着染锰剂量的增加,细胞凋亡率逐渐升高。各染锰组大鼠纹状体细胞凋亡率均明显高于对照组(P<0.01)。MK-801预处理组与200μmol/kg染锰组比较,细胞凋亡率明显下降(P<0.01)。通过电镜观察染锰组大鼠神经元细胞胞体缩小,核染色体固缩,边聚,出现空泡变性。
二、体外神经元细胞培养
1、神经元细胞活力和LDH释放量
随锰浓度升高,细胞活力和LDH释放量均表现出明显的剂量-效应关系和时间-效应关系。根据MTT和LDH活力分析结果,我们将在后续试验中用25,100,400μmol/L锰处理浓度,作用神经元12h,这样不致于使细胞损伤过于严重,又能得到可靠的结果。
2、TUNEL法检测细胞凋亡
随锰浓度的升高,神经元细胞凋亡率和积分光密度均逐渐上升。用MK-801预处理30 min后,400μmol/L锰处理神经元发现,与单纯用400μmol/L锰处理比较,细胞凋亡率和积分光密度均有所下降(P<0.05)。
3、神经元细胞NMDA受体mRNA和蛋白表达
随着锰处理浓度的增加,NR1、NR2A, NR2BmRNA和蛋白的表达均有不同程度的下降,其中NR2A灰度值下降最明显。MK-801预处理组与400μmol/L锰处理组比较,NR1和NR2A的灰度值明显升高(P<0.05), NR2B灰度值均未见明显变化。
4、神经元细胞内Ca2+浓度
随着锰处理浓度的增加,细胞内钙离子浓度也逐渐升高。不同浓度锰处理组,细胞内钙离子浓度均明显高于对照组(P<0.05),MK-801预处理组与400μmol/L锰处理组比较,细胞内钙离子浓度明显降低(P<0.01)。
5、神经元Na+-K+-ATPase和Ca2+-ATPase活力
随着锰处理浓度的增加,Na+-K+-ATPase和Ca2+-ATPase活力逐渐下降。MK-801预处理组与400μmol/L锰处理组,Na+-K+-ATPase和Ca2+-ATPase活力均明显升高。
结论
1、通过对大鼠腹腔注射氯化锰溶液成功建立了锰中毒大鼠模型。研究发现,锰可以在脑组织中聚积,损伤神经细胞,使大鼠产生神经毒性。
2、通过体内动物实验发现,锰可破坏鼠脑纹状体内“Glu-Gln循环通路”,使Glu含量升高,此机制与锰干扰Glu代谢相关酶有关,锰可使PAG活力升高,GS活力下降。
3、通过体内动物实验和体外神经元细胞培养发现,锰可以抑制NR1、NR2A和NR2B亚单位mRNA和蛋白的表达,并且NR2A亚单位对锰的抑制作用最敏感。
4、利用体外神经元培养技术发现,锰对体外培养的神经元细胞具有明显的毒性作用。
5、锰可以干扰体外培养神经元细胞内的钙稳态系统。
6、由于MK-801能有效阻滞谷氨酸与突触后膜受体结合,既可以阻断NMDA受体偶联的Ca2+通道,又可以阻断L型ca2+通道,还有抗氧化作用,所以它可以通过多种机制对锰的神经毒性有一定的防护作用。
Objective
Manganese (Mn) is one of essential trace elements found in a variety of biological tissues and constitutes many enzyme active groups or the accessory factor. Simultaneously, it is also certain enzyme activating agent and participates in many biochemical reactions. But excessive manganese enters the body to be able to cause manganism. Mn is possible to penetrate the blood-brain barrier and stores up in the brain organization. Early symptoms are main performance for nerve behavior function change. The long-term exposure Mn main performance for the extra pyramidal system is the damage, has similar symptoms resembling features of Parkinson's disease. Therefore, study on the neurotoxicology of Mn research the general scholar's attention. The mechanisms underlying the neurotoxicity of Mn are still incompletely understood and involve many aspects. The excessive manganese may increase the nerve cell metabolism, break mitochondria, and improve lysosomes activity, occurring self-digestion, causes the nerve cell to degenerative changes and damage. It also activate cytochrome oxidase P-450 to produce free radical. In recent years studied indicated, Mn may disturb excitatory neurotransmitter Glutamate metabolism to induce the indirect excitability neurotoxicity. N-methyl-D-aspartate receptor (NMDAR), an excitatory amino acid neurotransmitter receptor, possesses unique structure function characteristic and exerts essential physiological functions. It not only is the ligand gating acceptor, also is the voltage dependence gating acceptor. Thus in the neuro-physiology, the pathology and the toxicology research the NMDA acceptor has also received the special attention. MK-801 is one kind of powerful NMDA acceptor non-competitive antagonist. Through the blood-brain barrier, it may affect easily in the NMDA acceptor channel internal benzene ring pai-ding position spot carries on the construct adjustment and can effectively hinder the glutamate and postsynaptic membrane receptor combination. MK-801 may block NMDA the acceptor coupling the Ca2+ channel activation decreasing influx of extracellular Ca2+, thus weaken the NMDA receptor function. In order to further understand the relationships between effect of Mn on excitatory neurotransmitter metabolism and the nerve cell damage and protective effect of MK-801 on Mn neurotoxicity, we will test two aspects from in vivo and in vitro to study the following question:①Effect of Mn on Glutamate metabolism.②Effect of Mn on NR1, NR2A and NR2B subunit expression.③Toxic effects of Mn on primary cultured neurons.④Protective effect of MK-801 on Mn-induced nerve damage. It has the extremely vital significance to us further understood the certain mechanism of Mn neurotoxicity and provides the important theoretical and experimental basis for the pathogenesis and reventing and controlling of Mn-induced nerve cell damage.
Methods
一、In vivo animal experiments
(一) Experimental animal and treatment
100 Wistar rats obtained from the Laboratory Animal Center of Chinese Medicine University, weighing 170-190 gram. After an acclimation period of a week,100 rats were randomly divided into 5 groups with 20 animals in each group:control group, Mn-treated group (8,40, and 200μmol/kg) and MK-801 pre-treated group. The control group rats were intraperitoneally (i.p.) injected with 0.9%normal saline (group 1). Mn-treated rats were respectively i.p. injected with 8,40,200μmol/kg MnCl2 in sterile deionized water (group 2-4). MK-801 in sterile deionized water was given every two-day. The MK-801 pre-treated rats were subcutaneously (s.c.) injected with 0.3μmol/kg body weight/day, two hour before the i.p. administration with 200μmol MnCl2/kg body weight/day (group 5). The capacity of injection is all 5 ml/kg.4 weeks after administration, the rats were anatomized to get the tissue samples of the striatum.
(二) Measuring indexes
1. Measurement of Mn concentration in striatum
After administrated for 4 weeks, each group executes 8 rats. A certain amount striatum were separated and Mn concentration analysis was performed by HITACHI 180-80 atomic absorption spectrophotometer.
2. Measurements of Glu and Gln concentrations in striatum
After administrated for 4 weeks, each group executes 6 rats. A certain amount striatum were separated and Glu and Gln concentrations analysis were performed according to the kits'introduction.
3. Measurements of PAG and GS activities in striatum
After administrated for 4 weeks, each group executes 6 rats. A certain amount striatum were separated and PAG and GS activities analysis were performed according to the methods of Curi and Renis.
4. Measurements of NMDAR mRNA and proteins expression in striatum
After administrated for 4 weeks, each group executes 4 rats. Measurements of NMDAR mRNA and proteins expression were performed by RT-PCR ans Western Blotting analysis and collected total RNA and the total protein according to the conventional method. The changes of intensity of NR1, NR2A and NR2B mRNAs after Mn treatment were normalized using the intensity obtained in the internal control bands (G3PDH). The changes of intensity of NR1, NR2A and NR2B proteins after Mn treatment were normalized using the intensity obtained in the internal control bands (β-actin).
5. Apoptosis Detection
Each group 4 rats'striatum were dissected to use for the preparation of dissociated striatum cells, which were analyzed using flow cytometry. After administrated for 4 weeks, each group executes 2 rats. After the rats were perfused with 4% paraformaldehyde, a certain amount striatum were separated and viewed in a JEOL JSM-7300EX transmission electron microscope.
二、In vitro primary neuronal cultures
(一) Primary neuronal cultures
Cortices were isolated from the brains of neonatal Wister rats and chopped into 2 mm pieces under the light microscope. The cortical chunks were then suspended in trypsin solution. The whole solution was filtered through stainless steel. The cell pellets were resuspended in DMEM containing 10%(v/v) horse serum. The cell suspension was plated at 1×106 cells/1.5ml on six-well dishes pre-coated with poly-L-lysine and incubated at 37℃in humidified 95%air/5%CO2 for 24 h. After 24 h, the culture medium was changed to Phenol Red-free Neurobasal medium (without Phenol Red and estrogen-free) supplemented with B27,100 U/ml penicillin/streptomycin and L-glutamine (0.5 mM). These cells were then treated with cytosine arabinoside on the 3rd day for 48 h to eliminate dividing non-neuronal cells.
(二) Identification of neurons
Neuronal morphology was observed with an Olympus IX70 inverted phase-contrast microscope fitted with a digital camera system to capture images. Neuron-specific enolase (NSE) was immunostained by immunocytochemistry.
(三) Measuring indexes
1. Quantification of viability
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0-400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells were respectively treated for 6, 12,24,48 h, MTT assay were performed.
2. LDH assay in culture solution
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0-400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells were respectively treated for 6, 12,24,48 h, LDH assay were performed.
3. TdT-mediated dUTP nick-end labeling (TUNEL) assay
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, TUNEL assay was examined on paraformaldehyde-fixed cells using the Apop-tagTM in situ apoptosis detection kit.
4. Measurements of NMDAR mRNA and proteins expression in neurons
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, measurements of NMDAR mRNA and proteins expression were performed by RT-PCR ans Western Blotting analysis and collected total RNA and the total protein according to the conventional method. The changes of intensity of NR1, NR2A and NR2B mRNAs after Mn treatment were normalized using the intensity obtained in the internal control bands (G3PDH). The changes of intensity of NR1, NR2A and NR2B proteins after Mn treatment were normalized using the intensity obtained in the internal control bands (β-actin).
5. Measurement of intracellular free calcium
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, absolute values of [Ca2+]i in neurons were calibrated from the measured fluorescence signals by the use of F-4500 Fluorescence Spectrophotometer.
6. Na+-K+-ATPase and Ca2+-ATPase activities assay in neurons
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, Na+-K+-ATPase and Ca2+-ATPase activities assay were performed by spectrophotometer at 706 nm.
Results
一、In vivo animal experiments
1. Mn concentration in striatum
There was a significant increase in Mn concentration in the striatum of Mn-treated rats compared to control rats. In 200μmol/kg Mn-treated group, the Mn concentration significantly increased 4.7-fold in striatum. However, the Mn concentration in MK-801 pre-treated rats was not different compared with 200μmol/kg Mn-treated group.
2. Glu and Gln concentrations in striatum
With the increase of administered-MnCl2 dosage, Glu concentration was increased and Gln concentration was decreased. Glu concentration was approximate 2.5-fold increased in 200 umol/kg Mn-treated rats. Gln concentration was decreased in 200μmol/kg Mn-treated rats by 39.4%, relative to saline treated controls. MK-801 pre-treatment did not affect Glu concentration in the striatum. However, MK-801 pre-treatment increased Gln concentration compared with 200μmol/kg Mn-treated group (P＜0.05).
3. PAG and GS activities in striatum
With the increase of administered-MnCl2 dosage, GS activity was decreased and PAG activity was increased. A signiffcant (P<0.01) decrease in GS activity was observed in 200μmol/kg Mn-treated group (44.47±6.93μmol/min/g pro). PAG activity was significantly increased by 54.4%in 200μmol/kg Mn-treated rats (41.02±6.60μmol/min/g pro) compared with control animals (P＜0.01). Compared with 200μmol/kg Mn-treated animals, MK-801 pre-treatment blocked GS activity decrease and PAG activity increase.
4. NMDAR mRNA and proteins expression in striatum
With the increase of administered-MnCl2 dosage, mRNA and protein levels of NR1, NR2A and NR2B1 were reduced in different extent compared with those of control group. Expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. Compared with 200μmol/kg Mn-treated animals, MK-801 pre-treatment increase the levels of NR2A mRNA and protein. However, the levels of NR1 and NR2B mRNA and protein were not found the obvious change.
5. Cell apoptosis in striatum
Total apoptosis rate was significantly increased with the increase of administered-MnCl2 dosage using flow cytometry. However, there were significantly reduced apoptotic cells in the MK-801 pre-treated group compared with 200μmol/kg Mn-treated group. Under transmission electron microscopy, in 200μmol/kg Mn-treated group, neurons showed features of apoptosis, characterized by nucleus shrinkage, dense aggregation of chromatin and chromatin margination.
二、In vitro primary neuronal cultures
1. Viability and LDH assay
With the increase of Mn treatment concentration, viability of neurons and LDH levels in the culture medium displayed the obvious dosage-effect relations and the time-effect relations. According to MTT and LDH analysis resulting, Mn exposures in subsequent experiments were restricted to concentrations of 25,100,400μmol/L Mn for 12 h, which ensure that Mn treatment levels were representative and appropriate concentrations that did not cause severe cytotoxicity and the sufficient concentrations to induce measurable neurologic deficits.
2. TUNEL assay
With the increase of Mn treatment concentration, there was a significant increase of apoptotic cell death and Integrated Optical Density (IOD) in a concentration-dependent manner. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment reduced the cell apoptosis rate and IOD.
3. NMDAR mRNA and proteins expression in neurons
With the increase of Mn treatment concentration, mRNA and protein levels of NR1, NR2A and NR2B were reduced in different extent compared with those of control group. Expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment increase the levels of NR1 and NR2A mRNA and protein. However, the levels of NR2B mRNA and protein were not found the obvious change.
4. [Ca2+]i in primary cultured neurons
With the increase of Mn treatment concentration, [Ca2+]; increased evidently. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment reduced the increase of [Ca2+]i.
5. Na+-K+-ATPase and Ca2+-ATPase activities in primary cultured neurons
With the increase of Mn treatment concentration, Na+-K+-ATPase and Ca2+-ATPase activities were decreased. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment raised Na+-K+-ATPase and Ca2+-ATPase activities.
Conclusions
1. The rat model of manganism was successfully established by intraperitoneally injection with MnCl2 The research discovered Mn level was obviously increased in rat striatum of manganism. Mn may cause the nerve cell to occur apoptosis by transmission electron microscopy and flow cytometry.
2. By animal experiments, we discovered Mn could disrupt "Glu-Gln cyclic pathway" in rat striatum, which cause PAG activity increase and GS activity decrease.
3. By animal experiments and primary neuronal cultures, we discovered Mn could inhibit expression of NR1, NR2A and NR2B mRNAs and proteins in rat striatum and expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. NR2A and NR2B subunits were associated with the different intracellular signal pathways, which might be one of Mn-induced cell apoptosis reasons.
4. By primary neuronal cultures, we discovered Mn could cause obviously cytotoxicity in primary cultured neurons. Main performance:the cellular transparence decreased, the cell bodies were shrinkage, the neuraxon of neurons shortened, the network disappeared, and some had already been ruptured and suspended. The viability of neurons was significantly decreased and LDH release significantly increased. Our data demonstrated that significant apoptotic cell death were evident in Mn-treated neurons using apoptotic TUNEL assay.
5. Mn can disrupt intracellular calcium homeostasis. Mn treatment increased intracellular [Ca2+]; evidently and inhibited Na+-K+-ATPase and Ca2+-ATPase activities in primary cultured neurons.
6. Because MK-801 can effectively hinder the glutamate and postsynaptic membrane receptor combination and not only block NMDA the acceptor coupling the Ca2+channel activation but also L-type calcium channels decreasing influx of extracellular Ca+and antioxidation, MK-801 has certain protective function on Mn-induced neurotoxicity through many kinds of mechanisms.
锰(Manganese, Mn)作为机体必需的微量元素之一,在体内可以参与构成许多酶的活性基团或辅助因子,同时又是某些酶的激活剂,参与许多生物化学反应,但是过量的锰进入体内则会引起锰中毒。进入体内的锰可穿透血-脑屏障,蓄积在脑组织内,早期主要表现为神经行为功能的改变,长期过量接触锰主要表现为锥体外系损伤,出现类似帕金森氏综合征的症状。因此,关于锰的神经毒性研究备受广大学者的关注。对锰神经毒作用机理的研究至今尚未完全阐明,锰的神经毒作用涉及诸多方面：过量的锰可增加神经细胞的代谢,破坏线粒体,提高溶酶体活性,发生自消化作用,引起神经细胞退变及损伤,还可激活细胞色素氧化酶P-450活性而产生自由基。近年来研究表明,锰可干扰脑内的兴奋性神经递质谷氨酸的代谢而产生间接兴奋性神经毒作用。作为兴奋性氨基酸神经递质受体之一的N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor, NMDAR)具有独特的结构功能特点,它既是配体门控受体,又是电压依赖性门控受体,因而在神经生理、病理和毒理学研究中NMDA受体也受到了特别关注。MK-801是一种强有力NMDA受体非竞争性拮抗剂,易通过血脑屏障,可作用于NMDA受体通道内部的苯环哌啶位点进行别构调节,能有效阻滞谷氨酸与突触后膜受体结合,阻断NMDA受体偶联的ca2+通道激活,使Ca2+内流减少,从而使NMDA受体作用减弱。为了进一步了解锰对兴奋性神经递质代谢的影响与神经细胞损伤的关系,以及MK-801对锰神经毒性的防护作用。我们将从体内和体外实验两方面研究如下问题：①锰对谷氨酸代谢的影响,②锰对NR1、NR2A和NR2B亚单位表达的影响,③锰对原代培养神经元的毒性作用,④MK-801对锰致神经损伤的防护作用。这将对我们进一步了解锰神经毒性的具体机制有十分重要的意义,将为锰致神经细胞损伤的发病机理和防治提供重要理论和实验依据。
实验方法
一、体内动物实验
(一)实验动物与分组
由中国医科大学实验动物中心提供的实验用Wistar大鼠100只,体重170～190g,雌雄各半。正式实验前饲养7d,然后按体重随机分成5组,每组20只。第1组为对照组,腹腔注射0.9%氯化钠,第2-4组为锰染毒组,分别腹腔注射8,40,200μmol/kg的氯化锰溶液,第5组为MK-801预处理组,隔日皮下注射0.3μmol/kg的MK-801溶液,2h后腹腔注射200μmol/kg的氯化锰溶液,注射容量均为5 ml/kg。每周注射5次,共4周。
(二)测定指标
1、脑纹状体锰含量的测定
染毒4周后,每组处死8只大鼠,取出一定量的纹状体组织,用原子吸收分光光度火焰法测定Mn含量。
2、脑纹状体Glu和Gln含量的测定
染毒4周后,每组处死6只大鼠,取出一定量的纹状体组织,按南京建成试剂盒说明书操作,测定Glu和Gln含量。
3、脑纹状体PAG和GS活力的测定
染毒4周后,每组处死6只大鼠,取出一定量的纹状体组织,分别按Curi禾(?)Renis描述的方法测定PAG和GS活力。
4、脑纹状体NMDA受体mRNA和蛋白表达的测定
每组取4只大鼠纹状体组织,分别用RT-PCR和Western blot方法检测mRNA和蛋白的表达,按常规方法提取总RNA和总蛋白,检测NR1,NR2A和NR2B的mRNA表达,用G3PDH作内参；检测NR1, NR2A和NR2B的蛋白表达,用β-actin作内参,用凝胶成像分析系统照相,测定各条带的灰度值。
5、细胞凋亡的测定
每组4只大鼠取一定量纹状体组织,制成单细胞悬液后,用流式细胞仪检测细胞凋亡率；每组2只大鼠用4%的多聚甲醛灌流固定,取出纹状体进一步处理后,电镜观察神经元细胞凋亡。
二、体外神经元细胞培养
(一)神经元培养
取新生鼠脑,去脑膜后切碎,用胰蛋白酶消化后,将细胞悬液转移到200目筛网中过滤,以含有10%马血清的DMEM培养基稀释细胞悬液至1×106／1.5m1,接种于经多聚赖氨酸过夜处理的直径为60mm的培养皿中,置于37℃CO2孵箱中培养24 h后,吸去DMEM培养基,换成无血清的Neurobasal培养液继续培养。加入阿糖胞苷,抑制非神经元的增值和生长。
(二)神经元的鉴定
倒置显微镜观察神经元表面光滑,胞体大、饱满,呈锥体形、星形、多角形或不规则形,突起清晰,均匀细长,2-5个不等,突起之间相互交织成网,连结紧密。用免疫细胞化学的方法检测神经元特异性烯醇化酶(NSE)。
(三)测定指标
1、细胞活力测定
待细胞生长状态最佳时加入含锰的培养液,锰处理浓度分别为0,12.5,25,50,100,200,400μmol/L7个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,分别培养6、12、24、48 h后,用MTT法测定细胞活力。
2、培养液中LDH活力的测定
神经元细胞用锰处理浓度分别为0,12.5,25,50,100,200,400μmol/L7个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,分别培养6、12、24、48 h后,用比色法测定LDH活力。
3、TUNEL法检测细胞凋亡
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,按原位细胞凋亡检测试剂盒说明书进行凋亡细胞染色,分析细胞凋亡率。
4、神经元细胞NMDA受体mRNA和蛋白表达的测定
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L 4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,分别用RT-PCR和Western blot方法检测mRNA和蛋白的表达,按常规方法提取总RNA和总蛋白,检测NR1, NR2A和NR2B的mRNA表达,用G3PDH作内参；检测NR1, NR2A和NR2B的蛋白表达,用β-actin作内参,用凝胶成像分析系统照相,测定各条带的灰度值。
5、神经元细胞内Ca2+浓度的测定
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,用双波长荧光分光光度计检测。
6、神经元Na+-K+-ATPase和Ca2+-ATPase活力的测定
神经元细胞用锰处理浓度分别为0,25,100,400μmol/L4个组,MK-801预处理组,用10μmol/L MK-801预处理30 min后,再用400μmol/L氯化锰处理神经元,培养12h后,提取膜蛋白用比色法测定。
实验结果
一、体内动物实验
1、脑纹状体锰含量
随着染锰剂量的增加,纹状体Mn含量也逐渐升高。各染锰组纹状体Mn含量均明显高于对照组(P<0.05),200μmol/kg染锰组大鼠纹状体Mn含量达到最高值0.98±0.27μg／g组织湿重,是对照组的4.7倍(P<0.01)。MK-801预处理组Mn含量与200μmol/kg染锰组比较,无明显变化。
2、脑纹状体Glu和Gln含量
随着染锰剂量的增加,Glu含量逐渐升高,Gln含量逐渐下降。200μmol/kg染锰组与对照组比较,Glu含量明显升高2.5倍；Gln含量明显下降了39.4%(P<0.01)。MK-801预处理组与200μmol/kg染锰组比较,Glu含量下降不明显,Gln含量明显回升了36.8%(P<0.05)。
3、脑纹状体PAG和GS活力
随着染锰剂量的增加,PAG活力逐渐升高,GS活力逐渐下降。200μmol/kg染锰组与对照组比较,PAG活力明显升高了54.4%达到41.02±6.60μmol/min/g protein; GS活力明显下降了28.9%达到.44.47±6.93μmol/min/g protein(P＜0.01)。MK-801预处理组与200μmol/kg染锰组比较,PAG活力明显下降,GS活力明显回升。
4、脑纹状体NMDA受体mRNA和蛋白表达
随着染锰剂量的增加,NR1, NR2A, NR2B mRNA和蛋白的表达均有不同程度的下降,其中NR2A灰度值下降最明显。MK-801预处理组与200μmol/kg染锰组比较,NR2A的灰度值明显升高(P<0.05),NRl和NR2B灰度值均未见明显变化。
5、脑纹状体细胞凋亡
用流式细胞仪检测发现,随着染锰剂量的增加,细胞凋亡率逐渐升高。各染锰组大鼠纹状体细胞凋亡率均明显高于对照组(P<0.01)。MK-801预处理组与200μmol/kg染锰组比较,细胞凋亡率明显下降(P<0.01)。通过电镜观察染锰组大鼠神经元细胞胞体缩小,核染色体固缩,边聚,出现空泡变性。
二、体外神经元细胞培养
1、神经元细胞活力和LDH释放量
随锰浓度升高,细胞活力和LDH释放量均表现出明显的剂量-效应关系和时间-效应关系。根据MTT和LDH活力分析结果,我们将在后续试验中用25,100,400μmol/L锰处理浓度,作用神经元12h,这样不致于使细胞损伤过于严重,又能得到可靠的结果。
2、TUNEL法检测细胞凋亡
随锰浓度的升高,神经元细胞凋亡率和积分光密度均逐渐上升。用MK-801预处理30 min后,400μmol/L锰处理神经元发现,与单纯用400μmol/L锰处理比较,细胞凋亡率和积分光密度均有所下降(P<0.05)。
3、神经元细胞NMDA受体mRNA和蛋白表达
随着锰处理浓度的增加,NR1、NR2A, NR2BmRNA和蛋白的表达均有不同程度的下降,其中NR2A灰度值下降最明显。MK-801预处理组与400μmol/L锰处理组比较,NR1和NR2A的灰度值明显升高(P<0.05), NR2B灰度值均未见明显变化。
4、神经元细胞内Ca2+浓度
随着锰处理浓度的增加,细胞内钙离子浓度也逐渐升高。不同浓度锰处理组,细胞内钙离子浓度均明显高于对照组(P<0.05),MK-801预处理组与400μmol/L锰处理组比较,细胞内钙离子浓度明显降低(P<0.01)。
5、神经元Na+-K+-ATPase和Ca2+-ATPase活力
随着锰处理浓度的增加,Na+-K+-ATPase和Ca2+-ATPase活力逐渐下降。MK-801预处理组与400μmol/L锰处理组,Na+-K+-ATPase和Ca2+-ATPase活力均明显升高。
结论
1、通过对大鼠腹腔注射氯化锰溶液成功建立了锰中毒大鼠模型。研究发现,锰可以在脑组织中聚积,损伤神经细胞,使大鼠产生神经毒性。
2、通过体内动物实验发现,锰可破坏鼠脑纹状体内“Glu-Gln循环通路”,使Glu含量升高,此机制与锰干扰Glu代谢相关酶有关,锰可使PAG活力升高,GS活力下降。
3、通过体内动物实验和体外神经元细胞培养发现,锰可以抑制NR1、NR2A和NR2B亚单位mRNA和蛋白的表达,并且NR2A亚单位对锰的抑制作用最敏感。
4、利用体外神经元培养技术发现,锰对体外培养的神经元细胞具有明显的毒性作用。
5、锰可以干扰体外培养神经元细胞内的钙稳态系统。
6、由于MK-801能有效阻滞谷氨酸与突触后膜受体结合,既可以阻断NMDA受体偶联的Ca2+通道,又可以阻断L型ca2+通道,还有抗氧化作用,所以它可以通过多种机制对锰的神经毒性有一定的防护作用。
Objective
Manganese (Mn) is one of essential trace elements found in a variety of biological tissues and constitutes many enzyme active groups or the accessory factor. Simultaneously, it is also certain enzyme activating agent and participates in many biochemical reactions. But excessive manganese enters the body to be able to cause manganism. Mn is possible to penetrate the blood-brain barrier and stores up in the brain organization. Early symptoms are main performance for nerve behavior function change. The long-term exposure Mn main performance for the extra pyramidal system is the damage, has similar symptoms resembling features of Parkinson's disease. Therefore, study on the neurotoxicology of Mn research the general scholar's attention. The mechanisms underlying the neurotoxicity of Mn are still incompletely understood and involve many aspects. The excessive manganese may increase the nerve cell metabolism, break mitochondria, and improve lysosomes activity, occurring self-digestion, causes the nerve cell to degenerative changes and damage. It also activate cytochrome oxidase P-450 to produce free radical. In recent years studied indicated, Mn may disturb excitatory neurotransmitter Glutamate metabolism to induce the indirect excitability neurotoxicity. N-methyl-D-aspartate receptor (NMDAR), an excitatory amino acid neurotransmitter receptor, possesses unique structure function characteristic and exerts essential physiological functions. It not only is the ligand gating acceptor, also is the voltage dependence gating acceptor. Thus in the neuro-physiology, the pathology and the toxicology research the NMDA acceptor has also received the special attention. MK-801 is one kind of powerful NMDA acceptor non-competitive antagonist. Through the blood-brain barrier, it may affect easily in the NMDA acceptor channel internal benzene ring pai-ding position spot carries on the construct adjustment and can effectively hinder the glutamate and postsynaptic membrane receptor combination. MK-801 may block NMDA the acceptor coupling the Ca2+ channel activation decreasing influx of extracellular Ca2+, thus weaken the NMDA receptor function. In order to further understand the relationships between effect of Mn on excitatory neurotransmitter metabolism and the nerve cell damage and protective effect of MK-801 on Mn neurotoxicity, we will test two aspects from in vivo and in vitro to study the following question:①Effect of Mn on Glutamate metabolism.②Effect of Mn on NR1, NR2A and NR2B subunit expression.③Toxic effects of Mn on primary cultured neurons.④Protective effect of MK-801 on Mn-induced nerve damage. It has the extremely vital significance to us further understood the certain mechanism of Mn neurotoxicity and provides the important theoretical and experimental basis for the pathogenesis and reventing and controlling of Mn-induced nerve cell damage.
Methods
一、In vivo animal experiments
(一) Experimental animal and treatment
100 Wistar rats obtained from the Laboratory Animal Center of Chinese Medicine University, weighing 170-190 gram. After an acclimation period of a week,100 rats were randomly divided into 5 groups with 20 animals in each group:control group, Mn-treated group (8,40, and 200μmol/kg) and MK-801 pre-treated group. The control group rats were intraperitoneally (i.p.) injected with 0.9%normal saline (group 1). Mn-treated rats were respectively i.p. injected with 8,40,200μmol/kg MnCl2 in sterile deionized water (group 2-4). MK-801 in sterile deionized water was given every two-day. The MK-801 pre-treated rats were subcutaneously (s.c.) injected with 0.3μmol/kg body weight/day, two hour before the i.p. administration with 200μmol MnCl2/kg body weight/day (group 5). The capacity of injection is all 5 ml/kg.4 weeks after administration, the rats were anatomized to get the tissue samples of the striatum.
(二) Measuring indexes
1. Measurement of Mn concentration in striatum
After administrated for 4 weeks, each group executes 8 rats. A certain amount striatum were separated and Mn concentration analysis was performed by HITACHI 180-80 atomic absorption spectrophotometer.
2. Measurements of Glu and Gln concentrations in striatum
After administrated for 4 weeks, each group executes 6 rats. A certain amount striatum were separated and Glu and Gln concentrations analysis were performed according to the kits'introduction.
3. Measurements of PAG and GS activities in striatum
After administrated for 4 weeks, each group executes 6 rats. A certain amount striatum were separated and PAG and GS activities analysis were performed according to the methods of Curi and Renis.
4. Measurements of NMDAR mRNA and proteins expression in striatum
After administrated for 4 weeks, each group executes 4 rats. Measurements of NMDAR mRNA and proteins expression were performed by RT-PCR ans Western Blotting analysis and collected total RNA and the total protein according to the conventional method. The changes of intensity of NR1, NR2A and NR2B mRNAs after Mn treatment were normalized using the intensity obtained in the internal control bands (G3PDH). The changes of intensity of NR1, NR2A and NR2B proteins after Mn treatment were normalized using the intensity obtained in the internal control bands (β-actin).
5. Apoptosis Detection
Each group 4 rats'striatum were dissected to use for the preparation of dissociated striatum cells, which were analyzed using flow cytometry. After administrated for 4 weeks, each group executes 2 rats. After the rats were perfused with 4% paraformaldehyde, a certain amount striatum were separated and viewed in a JEOL JSM-7300EX transmission electron microscope.
二、In vitro primary neuronal cultures
(一) Primary neuronal cultures
Cortices were isolated from the brains of neonatal Wister rats and chopped into 2 mm pieces under the light microscope. The cortical chunks were then suspended in trypsin solution. The whole solution was filtered through stainless steel. The cell pellets were resuspended in DMEM containing 10%(v/v) horse serum. The cell suspension was plated at 1×106 cells/1.5ml on six-well dishes pre-coated with poly-L-lysine and incubated at 37℃in humidified 95%air/5%CO2 for 24 h. After 24 h, the culture medium was changed to Phenol Red-free Neurobasal medium (without Phenol Red and estrogen-free) supplemented with B27,100 U/ml penicillin/streptomycin and L-glutamine (0.5 mM). These cells were then treated with cytosine arabinoside on the 3rd day for 48 h to eliminate dividing non-neuronal cells.
(二) Identification of neurons
Neuronal morphology was observed with an Olympus IX70 inverted phase-contrast microscope fitted with a digital camera system to capture images. Neuron-specific enolase (NSE) was immunostained by immunocytochemistry.
(三) Measuring indexes
1. Quantification of viability
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0-400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells were respectively treated for 6, 12,24,48 h, MTT assay were performed.
2. LDH assay in culture solution
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0-400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells were respectively treated for 6, 12,24,48 h, LDH assay were performed.
3. TdT-mediated dUTP nick-end labeling (TUNEL) assay
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, TUNEL assay was examined on paraformaldehyde-fixed cells using the Apop-tagTM in situ apoptosis detection kit.
4. Measurements of NMDAR mRNA and proteins expression in neurons
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, measurements of NMDAR mRNA and proteins expression were performed by RT-PCR ans Western Blotting analysis and collected total RNA and the total protein according to the conventional method. The changes of intensity of NR1, NR2A and NR2B mRNAs after Mn treatment were normalized using the intensity obtained in the internal control bands (G3PDH). The changes of intensity of NR1, NR2A and NR2B proteins after Mn treatment were normalized using the intensity obtained in the internal control bands (β-actin).
5. Measurement of intracellular free calcium
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, absolute values of [Ca2+]i in neurons were calibrated from the measured fluorescence signals by the use of F-4500 Fluorescence Spectrophotometer.
6. Na+-K+-ATPase and Ca2+-ATPase activities assay in neurons
Treatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, Na+-K+-ATPase and Ca2+-ATPase activities assay were performed by spectrophotometer at 706 nm.
Results
一、In vivo animal experiments
1. Mn concentration in striatum
There was a significant increase in Mn concentration in the striatum of Mn-treated rats compared to control rats. In 200μmol/kg Mn-treated group, the Mn concentration significantly increased 4.7-fold in striatum. However, the Mn concentration in MK-801 pre-treated rats was not different compared with 200μmol/kg Mn-treated group.
2. Glu and Gln concentrations in striatum
With the increase of administered-MnCl2 dosage, Glu concentration was increased and Gln concentration was decreased. Glu concentration was approximate 2.5-fold increased in 200 umol/kg Mn-treated rats. Gln concentration was decreased in 200μmol/kg Mn-treated rats by 39.4%, relative to saline treated controls. MK-801 pre-treatment did not affect Glu concentration in the striatum. However, MK-801 pre-treatment increased Gln concentration compared with 200μmol/kg Mn-treated group (P＜0.05).
3. PAG and GS activities in striatum
With the increase of administered-MnCl2 dosage, GS activity was decreased and PAG activity was increased. A signiffcant (P<0.01) decrease in GS activity was observed in 200μmol/kg Mn-treated group (44.47±6.93μmol/min/g pro). PAG activity was significantly increased by 54.4%in 200μmol/kg Mn-treated rats (41.02±6.60μmol/min/g pro) compared with control animals (P＜0.01). Compared with 200μmol/kg Mn-treated animals, MK-801 pre-treatment blocked GS activity decrease and PAG activity increase.
4. NMDAR mRNA and proteins expression in striatum
With the increase of administered-MnCl2 dosage, mRNA and protein levels of NR1, NR2A and NR2B1 were reduced in different extent compared with those of control group. Expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. Compared with 200μmol/kg Mn-treated animals, MK-801 pre-treatment increase the levels of NR2A mRNA and protein. However, the levels of NR1 and NR2B mRNA and protein were not found the obvious change.
5. Cell apoptosis in striatum
Total apoptosis rate was significantly increased with the increase of administered-MnCl2 dosage using flow cytometry. However, there were significantly reduced apoptotic cells in the MK-801 pre-treated group compared with 200μmol/kg Mn-treated group. Under transmission electron microscopy, in 200μmol/kg Mn-treated group, neurons showed features of apoptosis, characterized by nucleus shrinkage, dense aggregation of chromatin and chromatin margination.
二、In vitro primary neuronal cultures
1. Viability and LDH assay
With the increase of Mn treatment concentration, viability of neurons and LDH levels in the culture medium displayed the obvious dosage-effect relations and the time-effect relations. According to MTT and LDH analysis resulting, Mn exposures in subsequent experiments were restricted to concentrations of 25,100,400μmol/L Mn for 12 h, which ensure that Mn treatment levels were representative and appropriate concentrations that did not cause severe cytotoxicity and the sufficient concentrations to induce measurable neurologic deficits.
2. TUNEL assay
With the increase of Mn treatment concentration, there was a significant increase of apoptotic cell death and Integrated Optical Density (IOD) in a concentration-dependent manner. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment reduced the cell apoptosis rate and IOD.
3. NMDAR mRNA and proteins expression in neurons
With the increase of Mn treatment concentration, mRNA and protein levels of NR1, NR2A and NR2B were reduced in different extent compared with those of control group. Expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment increase the levels of NR1 and NR2A mRNA and protein. However, the levels of NR2B mRNA and protein were not found the obvious change.
4. [Ca2+]i in primary cultured neurons
With the increase of Mn treatment concentration, [Ca2+]; increased evidently. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment reduced the increase of [Ca2+]i.
5. Na+-K+-ATPase and Ca2+-ATPase activities in primary cultured neurons
With the increase of Mn treatment concentration, Na+-K+-ATPase and Ca2+-ATPase activities were decreased. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment raised Na+-K+-ATPase and Ca2+-ATPase activities.
Conclusions
1. The rat model of manganism was successfully established by intraperitoneally injection with MnCl2 The research discovered Mn level was obviously increased in rat striatum of manganism. Mn may cause the nerve cell to occur apoptosis by transmission electron microscopy and flow cytometry.
2. By animal experiments, we discovered Mn could disrupt "Glu-Gln cyclic pathway" in rat striatum, which cause PAG activity increase and GS activity decrease.
3. By animal experiments and primary neuronal cultures, we discovered Mn could inhibit expression of NR1, NR2A and NR2B mRNAs and proteins in rat striatum and expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. NR2A and NR2B subunits were associated with the different intracellular signal pathways, which might be one of Mn-induced cell apoptosis reasons.
4. By primary neuronal cultures, we discovered Mn could cause obviously cytotoxicity in primary cultured neurons. Main performance:the cellular transparence decreased, the cell bodies were shrinkage, the neuraxon of neurons shortened, the network disappeared, and some had already been ruptured and suspended. The viability of neurons was significantly decreased and LDH release significantly increased. Our data demonstrated that significant apoptotic cell death were evident in Mn-treated neurons using apoptotic TUNEL assay.
5. Mn can disrupt intracellular calcium homeostasis. Mn treatment increased intracellular [Ca2+]; evidently and inhibited Na+-K+-ATPase and Ca2+-ATPase activities in primary cultured neurons.
6. Because MK-801 can effectively hinder the glutamate and postsynaptic membrane receptor combination and not only block NMDA the acceptor coupling the Ca2+channel activation but also L-type calcium channels decreasing influx of extracellular Ca+and antioxidation, MK-801 has certain protective function on Mn-induced neurotoxicity through many kinds of mechanisms.
引文
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1 Zoni S, Albini E, Lucchini R. Neuropsychological testing for the assessment of manganese neurotoxicity:a review and a proposal[J]. Am J Ind Med.2007; 50(11):812-830.
2 Bouchard M, Mergler D, Baldwin M, et al. Neuropsychiatric symptoms and past manganese exposure in a ferro-alloy plant[J]. Neurotoxicology.2007; 28(2):290-297.
3 Fitsanakis VA, Au C, Erikson KM, et al. The effects of manganese on glutamate, dopamine and gamma-aminobutyric acid regulation[J]. Neurochem Int.2006; 48(6-7):426-433.
4 Cai T, Yao T, Li Y, et al. Proteasome inhibition is associated with manganese-induced oxidative injury in PC12 cells[J]. Brain Res.2007; 1185:359-365.
5 Yazbeck C, Moreau T, Sahuquillo J, et al. Effect of maternal manganese blood levels on erythrocyte calcium-pump activity in newborns[J]. Sci Total Environ.2006; 354(1):28-34.
6 Zhang F, Xu Z, Gao J, et al. In vitro effect of manganese chloride exposure on energy metabolism and oxidative damage of mitochondria isolated from rat brain [J]. Environmental Toxicology and Pharmacology.2008; 26(2):232-236.
7 Crooks DR, Welch N, Smith DR. Low-level manganese exposure alters glutamate metabolism in GABAergic AF5 cells[J]. Neurotoxicology.2007; 28(3):548-554.
8 Kalivas PW. The glutamate homeostasis hypothesis of addiction[J]. Nat Rev Neurosci.2009; 10(8):561-572.
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