右美沙芬和利鲁唑对甲基汞致神经毒性的影响及其机制的研究
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摘要
前言
甲基汞(methylmercury)是一种具有神经毒性的环境污染物,可通过食物链产生生物放大作用。对人类造成危害,损害的主要部位是大脑的枕叶(距状区视觉中枢)和小脑,对成人可导致记忆丧失,对儿童、婴幼儿可造成语言和记忆能力短缺等。目前,氯化甲基汞中毒致神经损伤机制多是对即早基因、肽类和单胺类神经递质进行研究。谷氨酸(glutamate,Glu)是哺乳动物脑内最重要的兴奋性氨基酸,有研究提出氯化甲基汞中毒可能与脑内谷氨酸代谢和传递异常有关,但其机制上不明确。虽然神经细胞内有谷氨酰胺合成酶(glutamine synthesis, GS)可将谷氨酸转变成谷氨酰胺(glutamine,Gln),但星形胶质细胞的高亲和力谷氨酸转运体对谷氨酸的再摄取、维持谷氨酸代谢平衡起到至关重要的作用,星形胶质细胞膜上主要表达2种高亲和性Glu转运体——GLAST(glutamate aspartate transporter, EAAT 1)和GLT—1(glutamate transporter 1, EAAT 2),二者均能逆浓度梯度将Glu从胞外转运至胞内,从而及时终止突触部位Glu的兴奋性传导并维持其胞外的稳态水平。神经胶质细胞生成的Gln,通过细胞间隙运送到神经元内,经磷酸活化谷氨酰胺酶(phosphate activated glutaminase,PAG)水解生成Glu。因此氯化甲基汞中毒引起的神经毒性很可能通过影响谷氨酸转运体、谷氨酰胺合成酶及经磷酸活化谷氨酰胺酶活性有关。同时,有研究表明,过量Glu可诱导的神经元Ca2+内流,而Ca2+超载可刺激星形胶质细胞中产生活力氧,因此氯化甲基汞神经毒性导致谷氨酸代谢异常和氧化损伤可能会有一定的关系。
右美沙芬是一种NMDA受体非竞争性拮抗剂,有研究表明其可阻断谷氨酸过量对NMDA受体的活化,抑制了钙离子内流。利鲁唑是一种钠离子通道抑制剂,又是谷氨酸释放抑制剂,有研究表明,利鲁唑的作用机制可能与促进谷氨酸摄取,增强其与谷氨酸转运体结合的作用有关。但是,目前应用右美沙芬和利鲁唑干预氯化甲基汞所致神经损伤的研究,鲜有报道。
本研究拟通过体内与体外实验相结合的方法,首先,研究右美沙芬和利鲁唑对氯化甲基汞致大鼠大脑皮质神经毒性及氧化损伤的影响,再研究利鲁唑对氯化甲基汞致星形胶质细胞谷氨酸代谢和转运障碍的影响。
材料与方法
一、体内动物实验
1、实验动物与分组
由中国医科大学实验动物中心提供实验用清洁级Wistar大鼠40只,体重180±10克,雌雄各半。动物室温度17-23℃,相对湿度45-55%,动物饲料由实验动物中心提供。正式实验前适应性喂养1周,按体重随机分成5组,每组8只。第1组为对照组,第2-3组分别为低、高剂量染氯化甲基汞组,第4组为右美沙芬预处理组,第5组为利鲁唑预处理组。第1-3组均皮下注射生理盐水,第4组皮下注射13.5μmol/kg右美沙芬,第5组皮下注射21.35μmol/kg利鲁唑。2小时后,第1组腹腔注射生理盐水,第2、3组分别腹腔注射4、12μmol/kg氯化甲基汞,第4、5组均腹腔注射12μmol/kg氯化甲基汞,注射容量均为5ml/kg。连续染毒4周,每周5次,每天1次,隔日干预1次。最后一次注射后24 h,将大鼠用乙醚麻醉,心脏放血后,切取大脑皮质。
2、测定指标
测汞含量用冷原子吸收法；Glu和Gln的含量按照南京建成试剂盒说明进行测定；GS活力测定用RENIS等描述的γ-谷氨酰转移酶的非生理学催化反应；PAG活力测定用CURI等的方法；丙二醛(malondialdehyde, MDA)含量测定用硫代巴比妥酸比色法；超氧化物歧化酶(superoxide dismutase, SOD)用黄嘌呤氧化酶法测定；谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)活性用DTNB比色法测定；蛋白含量测定用Folin-Lowry法。
二、体外神经胶质细胞培养
(一)神经胶质细胞培养及鉴定
参照Hertz等的方法进行星形胶质细胞培养,应用GFAP免疫细胞化学反应进行细胞鉴定,阳性率达95%后进行实验。
(二)测定指标
1、细胞活力测定及分组
通过MTT法分析,暴露于氯化甲基汞(0-40μM)4-24小时后细胞毒性的变化和利鲁唑(50-200μM)预处理4-24小时后的干预作用,并选择适当且有代表性的染毒和干预剂量和时间；使用倒置相差显微镜观察细胞形态学改变。
2、星形胶质细胞GS、GLAST和GLT-1mRNA和蛋白表达的测定
应用RT-PCR法检测GS、GLAST、GLT-1的基因表达,用G3PDH作内参；应用Western blot法检测GS、GLAST及GLT-1的蛋白表达,用β-actin作内参。
三、统计学分析
用SPSS 11.5软件进行数据处理,实验所得数据以平均值±标准差表示,采用单因素方差分析进行组间差异的显著性检验,两组间比较用Q检验(Students-Newman-Keuls, SNK)。
结果
一、体内动物实验
与对照组相比,随着染氯化甲基汞剂量的增加,大鼠大脑皮质中Hg、Glu和MDA含量增加,PAG活力逐渐增加；Gln含量下降,GSH-Px、SOD和GS活力下降。与12μmol/kg氯化甲基汞组相比,右美沙芬和利鲁唑预处理组中,大鼠大脑皮质Hg、Glu和MDA含量下降,PAG活力下降；Gln含量增加,GSH-Px、SOD和GS的活力增加。
二、体外神经胶质细胞培养
原代培养的星形胶质细胞,经GFAP免疫细胞化学染色鉴定后,阳性率95%。通过MTT法观察氯化甲基汞对星形胶质细胞有细胞毒性作用。与对照组相比,随着染氯化甲基汞剂量的增高(0-40μM)4-24小时后星形胶质细胞的形态损伤程度逐渐增加及膜的完整性逐渐下降,并表现出一定的剂量和时间依赖性的效应关系,选定氯化甲基汞染毒剂量为0、1、10、20μM,染毒时间为12小时；与20μM氯化甲基汞的剂量水平上相比,利鲁唑(50-200μM)预处理4-24小时后星形胶质细胞形态学损伤出现不同程度的减轻,确定利鲁唑干预剂量为100μM,干预时间为12小时；氯化甲基汞0-20μM染毒组之间,GSmRNA和蛋白表达差异没有显著性, GLAST及GLT-1的mRNA和蛋白表达逐渐下降。与氯化甲基汞(0-20μM)染毒组相比,利鲁唑100μM预处理12小时,细胞形态学损伤减轻程度明显,除GS外,GLAST及GLT-1的mRNA和蛋白表达有所增加。
结论
1、通过对大鼠腹腔注射氯化甲基汞溶液成功建立了氯化甲基汞中毒的大鼠模型,发现氯化甲基汞染毒可在大脑皮质中蓄积,破坏大脑皮质中“谷氨酸-谷氨酰胺循环”,导致大脑皮质谷氨酸代谢异常,产生神经毒性作用。同时其还可以引起大脑皮质的氧化损伤。
2、通过体内实验发现,右美沙芬和利鲁唑对氯化甲基汞引起的神经毒性和氧化损伤具有一定的拮抗作用。
3、通过体外星形胶质细胞实验发现,氯化甲基汞对神经胶质细胞具有明显的毒性,其可导致谷氨酸转运体功能及谷氨酸代谢障碍。
4、通过体外星形胶质细胞实验发现,利鲁唑对氯化甲基汞暴露造成的星形胶质细胞谷氨酸代谢和转运异常具有一定的拮抗作用。
Introduction
Methylmercury(MMC)is a kind of neurotoxicity of environmental pollutants. It can be harmful to human through the food chain by biomagnification.The main part of where it damages are occipital lobethe brain's(calcarine visual hub area) and the cerebellum. It causes the adult memory loss as well as the children a shortage of language and memory abilities.At present, the studies on the mechanisms of methylmercury neurotoxicity were mostly used to be on the immediate-early genes,peptides and monoaminergic transmitters. Glutamate is the most important excitatory amino acid in mammalian brains.Some studies showed that there were some relationships between methylmercury chloride and the disfuction of glutamate metablism and transporters. However, the mechansims about it are still unknown.Although there is no glutamate metablism-related enzyme in synaptic cleft, glutamate is converted to glutamine by glutamine synthesis.The high-affinity glutamate transporters in astrocytes play a crucial role in the re-uptake of glutamate and maintaining the balance of glutamate metabolism. Therefore, the neurotoxicity caused methylmercury is likely to effect the function of the transporters as well as the content and activity of glutamine synthetase.in the meantime, some studies have shown that excessive glutamate can induce Ca2＋influx and Ca2＋overload can stimulate the astrocytes to bring more active oxygen. Thus, there is some relationship between glutamate metabolism and oxidative damage which result from methylmercury.
Dextromethorphan is an antagonist of non-competitive NMDA receptor.It showed that dextromethorphan can prohibited the NMDA receptors from excessive activation by glutamate and inhibit calcium influx Riluzole is a sodium ion channel inhibitors and the inhibitor of glutamate release.The mechanisms of riluzole may be related to the promotion of glutamate uptake and the enhance of its integration with the glutamate transporters. However,the applications of dextromethorphan and riluzole to resist the nerve damage caused by methylmercury chloride have been reported rarely.
The in vivo and in vitro experiments were used to determine the effects of dextromethorphan and riluzole respectively on the methylmercury chloride-induced neurotoxicity of cerebral cortex and the disturption of astrocytic glutamate metablism and transporters after methylmercury chloride exposure.
Materials and Methods
1. In vivo animal experiments
The weight of 40 wistar rats from the Experimental Animal Center of China Medical University is 180±10 g.The number of female and male is the same.They were fed under the temperature of 17-23℃, relative humidity 45-55%.Before the experiment,they were fed one week and then were divided into 5 groups by weight at random, 8 animals for each group. The first group was the control group and the second and third groups were methyl-mercuric chloride (MMC) groups. The fourth and fifth groups were DM and riluzolegroup respectively.All the former three groups were subcutaneously (sc) injected with 0.9% NaCl.The fourth and fifth groups were sc injected with 13.5μmol/kg DM and 21.35μmol/kg respectively every other day. Two hours later, the animals in the control group were intraperitoneally(ip) given the injection of 0.9%NaCl, the second group was injected with 4.0μmol/kg MMC and from the third to the fifth groups were injected with 12.0μmol/kg MMC. The administration of MMC above was given five times a week, lasting 4 weeks as well as the administrati-on of DM and riluzole every other day before injected with MMC.24 hours after the last injection, 8 rats in each group sacrificed after paralyzed.Observe the contenrs of Hg, glutamate, glutamine and Malondialdehyde(MDA) as well as the activities of glutamine synthetase(GS), phosphate activated glutaminase(PAG), Superoxide Dismutase(SOD)and glutathione peroxidase(GSH-Px) in the rat cortex when they were injected the increasi-ng MMC dosage, DM and riluzole.
2. In vitro primary astrocytes
Primary cultured astrocytes isolated and cultured from cerebral cortex of newborn (1 day old) Sprague-Dawlery rats.The cells would be used after more than 95% of the cells stained positively for the astrocytic marker glial fibrillary acidic protein (GFAP) immunocytochemically.Cellular proliferation activity was detected by MTT method. we studied the cytotoxicity of methylmercury chloride to astrocytes and the effects of riluzole on disfunction of glutamate metabolism and transporters.And we chose the proper dosages and time during the experiments.The damage of fibroblasts was observed with inverted phase contrast microscope.This research made use of the RT-PCR to analyze the expression of GS, GLT-1, EAAC1 mRNA and the Western blotting to analyze the expression of GS,GLT-1, EAAC1 protein.The changes of intensity of mRNAs and proteins were normalized using the intensity obtained in the internal control bands(G3PDH andβ-actin respectively).
3. Statistical analysis
Mean and standard deviation were calculated for all measurements. All statistical analyses were performed using the SPSS software, version 11.5. Data were analyzed using one-way analysis of variance followed by the Students-Newman-Keuls, SNK. Statistical significance was set at P<0.05.
Results
1. In vivo animal experiments
Compared with the control group, in MMC group, the contents of Hg, Glu and MDA increased. The content of Gln was decreased. Moreover, the activities of GS and SOD,GSH-Px of the rat cortex were decreased. On the contrary,the activity of PAG increased. Compared with 12.0μmol/kg MMC group, in riluzole group, the contents of Hg,Glu,and MDA fell down.The content of Gln rose. Moreover, the activities of GS and SOD,GSH-Px of the rat cortex.
2. In vitro primary astrocytes
Compared with the control group, the level of the damage of the morphology of astrocyte and the destruction of membranous integrity are deteriorating with the increaing doses of methylmercury chloride (0-40μM,4-24h).There was no significant difference of GSmRNA and protein expression among the methylmercury chloride (0-20μM) treated group. Nevertheless, the EAAT1 and GLT-1 mRNA and protein expression of the methylmercury chloride (0-20μM) decreased.Observe the effects of riluzole (50-200μM) pretreatment 4 to 24 hours after destruction of astrocytes caused by 20μM methylmercury chloride exposure in cell morphology. Compare with the chloride methylmercury (0-20μM) treated group, riluzole group GLAST, and GLT-1 mRNA and protein expression increased which was treated with the chloride methylmercury (0-20μM).
Conclusions
1.The rat model of MMC was perfectly established by intraperitoneally injection by methylmercury chloride.The research showed that MMC could be conveyed through 'blood-brain barrier', disturb the 'glutamate-glutamine cycle' and indued oxidative damage.
2.By animal experiments,it was showed that dromethorphan and riluzole may have the certain antagonistic effect on exitotoxicity and oxidative damage of MMC.
3.By primary astrocytes cultures, we found that MMC could cause cytotoxicity,and induced the disorders of glutamate metabolism and transporters.
4.Riluzole may have the certain antagonistic effect on glutamate metabolism and transporters of astrocytes by methylmercury chloride exposure.
甲基汞(methylmercury)是一种具有神经毒性的环境污染物,可通过食物链产生生物放大作用。对人类造成危害,损害的主要部位是大脑的枕叶(距状区视觉中枢)和小脑,对成人可导致记忆丧失,对儿童、婴幼儿可造成语言和记忆能力短缺等。目前,氯化甲基汞中毒致神经损伤机制多是对即早基因、肽类和单胺类神经递质进行研究。谷氨酸(glutamate,Glu)是哺乳动物脑内最重要的兴奋性氨基酸,有研究提出氯化甲基汞中毒可能与脑内谷氨酸代谢和传递异常有关,但其机制上不明确。虽然神经细胞内有谷氨酰胺合成酶(glutamine synthesis, GS)可将谷氨酸转变成谷氨酰胺(glutamine,Gln),但星形胶质细胞的高亲和力谷氨酸转运体对谷氨酸的再摄取、维持谷氨酸代谢平衡起到至关重要的作用,星形胶质细胞膜上主要表达2种高亲和性Glu转运体——GLAST(glutamate aspartate transporter, EAAT 1)和GLT—1(glutamate transporter 1, EAAT 2),二者均能逆浓度梯度将Glu从胞外转运至胞内,从而及时终止突触部位Glu的兴奋性传导并维持其胞外的稳态水平。神经胶质细胞生成的Gln,通过细胞间隙运送到神经元内,经磷酸活化谷氨酰胺酶(phosphate activated glutaminase,PAG)水解生成Glu。因此氯化甲基汞中毒引起的神经毒性很可能通过影响谷氨酸转运体、谷氨酰胺合成酶及经磷酸活化谷氨酰胺酶活性有关。同时,有研究表明,过量Glu可诱导的神经元Ca2+内流,而Ca2+超载可刺激星形胶质细胞中产生活力氧,因此氯化甲基汞神经毒性导致谷氨酸代谢异常和氧化损伤可能会有一定的关系。
右美沙芬是一种NMDA受体非竞争性拮抗剂,有研究表明其可阻断谷氨酸过量对NMDA受体的活化,抑制了钙离子内流。利鲁唑是一种钠离子通道抑制剂,又是谷氨酸释放抑制剂,有研究表明,利鲁唑的作用机制可能与促进谷氨酸摄取,增强其与谷氨酸转运体结合的作用有关。但是,目前应用右美沙芬和利鲁唑干预氯化甲基汞所致神经损伤的研究,鲜有报道。
本研究拟通过体内与体外实验相结合的方法,首先,研究右美沙芬和利鲁唑对氯化甲基汞致大鼠大脑皮质神经毒性及氧化损伤的影响,再研究利鲁唑对氯化甲基汞致星形胶质细胞谷氨酸代谢和转运障碍的影响。
材料与方法
一、体内动物实验
1、实验动物与分组
由中国医科大学实验动物中心提供实验用清洁级Wistar大鼠40只,体重180±10克,雌雄各半。动物室温度17-23℃,相对湿度45-55%,动物饲料由实验动物中心提供。正式实验前适应性喂养1周,按体重随机分成5组,每组8只。第1组为对照组,第2-3组分别为低、高剂量染氯化甲基汞组,第4组为右美沙芬预处理组,第5组为利鲁唑预处理组。第1-3组均皮下注射生理盐水,第4组皮下注射13.5μmol/kg右美沙芬,第5组皮下注射21.35μmol/kg利鲁唑。2小时后,第1组腹腔注射生理盐水,第2、3组分别腹腔注射4、12μmol/kg氯化甲基汞,第4、5组均腹腔注射12μmol/kg氯化甲基汞,注射容量均为5ml/kg。连续染毒4周,每周5次,每天1次,隔日干预1次。最后一次注射后24 h,将大鼠用乙醚麻醉,心脏放血后,切取大脑皮质。
2、测定指标
测汞含量用冷原子吸收法；Glu和Gln的含量按照南京建成试剂盒说明进行测定；GS活力测定用RENIS等描述的γ-谷氨酰转移酶的非生理学催化反应；PAG活力测定用CURI等的方法；丙二醛(malondialdehyde, MDA)含量测定用硫代巴比妥酸比色法；超氧化物歧化酶(superoxide dismutase, SOD)用黄嘌呤氧化酶法测定；谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)活性用DTNB比色法测定；蛋白含量测定用Folin-Lowry法。
二、体外神经胶质细胞培养
(一)神经胶质细胞培养及鉴定
参照Hertz等的方法进行星形胶质细胞培养,应用GFAP免疫细胞化学反应进行细胞鉴定,阳性率达95%后进行实验。
(二)测定指标
1、细胞活力测定及分组
通过MTT法分析,暴露于氯化甲基汞(0-40μM)4-24小时后细胞毒性的变化和利鲁唑(50-200μM)预处理4-24小时后的干预作用,并选择适当且有代表性的染毒和干预剂量和时间；使用倒置相差显微镜观察细胞形态学改变。
2、星形胶质细胞GS、GLAST和GLT-1mRNA和蛋白表达的测定
应用RT-PCR法检测GS、GLAST、GLT-1的基因表达,用G3PDH作内参；应用Western blot法检测GS、GLAST及GLT-1的蛋白表达,用β-actin作内参。
三、统计学分析
用SPSS 11.5软件进行数据处理,实验所得数据以平均值±标准差表示,采用单因素方差分析进行组间差异的显著性检验,两组间比较用Q检验(Students-Newman-Keuls, SNK)。
结果
一、体内动物实验
与对照组相比,随着染氯化甲基汞剂量的增加,大鼠大脑皮质中Hg、Glu和MDA含量增加,PAG活力逐渐增加；Gln含量下降,GSH-Px、SOD和GS活力下降。与12μmol/kg氯化甲基汞组相比,右美沙芬和利鲁唑预处理组中,大鼠大脑皮质Hg、Glu和MDA含量下降,PAG活力下降；Gln含量增加,GSH-Px、SOD和GS的活力增加。
二、体外神经胶质细胞培养
原代培养的星形胶质细胞,经GFAP免疫细胞化学染色鉴定后,阳性率95%。通过MTT法观察氯化甲基汞对星形胶质细胞有细胞毒性作用。与对照组相比,随着染氯化甲基汞剂量的增高(0-40μM)4-24小时后星形胶质细胞的形态损伤程度逐渐增加及膜的完整性逐渐下降,并表现出一定的剂量和时间依赖性的效应关系,选定氯化甲基汞染毒剂量为0、1、10、20μM,染毒时间为12小时；与20μM氯化甲基汞的剂量水平上相比,利鲁唑(50-200μM)预处理4-24小时后星形胶质细胞形态学损伤出现不同程度的减轻,确定利鲁唑干预剂量为100μM,干预时间为12小时；氯化甲基汞0-20μM染毒组之间,GSmRNA和蛋白表达差异没有显著性, GLAST及GLT-1的mRNA和蛋白表达逐渐下降。与氯化甲基汞(0-20μM)染毒组相比,利鲁唑100μM预处理12小时,细胞形态学损伤减轻程度明显,除GS外,GLAST及GLT-1的mRNA和蛋白表达有所增加。
结论
1、通过对大鼠腹腔注射氯化甲基汞溶液成功建立了氯化甲基汞中毒的大鼠模型,发现氯化甲基汞染毒可在大脑皮质中蓄积,破坏大脑皮质中“谷氨酸-谷氨酰胺循环”,导致大脑皮质谷氨酸代谢异常,产生神经毒性作用。同时其还可以引起大脑皮质的氧化损伤。
2、通过体内实验发现,右美沙芬和利鲁唑对氯化甲基汞引起的神经毒性和氧化损伤具有一定的拮抗作用。
3、通过体外星形胶质细胞实验发现,氯化甲基汞对神经胶质细胞具有明显的毒性,其可导致谷氨酸转运体功能及谷氨酸代谢障碍。
4、通过体外星形胶质细胞实验发现,利鲁唑对氯化甲基汞暴露造成的星形胶质细胞谷氨酸代谢和转运异常具有一定的拮抗作用。
Introduction
Methylmercury(MMC)is a kind of neurotoxicity of environmental pollutants. It can be harmful to human through the food chain by biomagnification.The main part of where it damages are occipital lobethe brain's(calcarine visual hub area) and the cerebellum. It causes the adult memory loss as well as the children a shortage of language and memory abilities.At present, the studies on the mechanisms of methylmercury neurotoxicity were mostly used to be on the immediate-early genes,peptides and monoaminergic transmitters. Glutamate is the most important excitatory amino acid in mammalian brains.Some studies showed that there were some relationships between methylmercury chloride and the disfuction of glutamate metablism and transporters. However, the mechansims about it are still unknown.Although there is no glutamate metablism-related enzyme in synaptic cleft, glutamate is converted to glutamine by glutamine synthesis.The high-affinity glutamate transporters in astrocytes play a crucial role in the re-uptake of glutamate and maintaining the balance of glutamate metabolism. Therefore, the neurotoxicity caused methylmercury is likely to effect the function of the transporters as well as the content and activity of glutamine synthetase.in the meantime, some studies have shown that excessive glutamate can induce Ca2＋influx and Ca2＋overload can stimulate the astrocytes to bring more active oxygen. Thus, there is some relationship between glutamate metabolism and oxidative damage which result from methylmercury.
Dextromethorphan is an antagonist of non-competitive NMDA receptor.It showed that dextromethorphan can prohibited the NMDA receptors from excessive activation by glutamate and inhibit calcium influx Riluzole is a sodium ion channel inhibitors and the inhibitor of glutamate release.The mechanisms of riluzole may be related to the promotion of glutamate uptake and the enhance of its integration with the glutamate transporters. However,the applications of dextromethorphan and riluzole to resist the nerve damage caused by methylmercury chloride have been reported rarely.
The in vivo and in vitro experiments were used to determine the effects of dextromethorphan and riluzole respectively on the methylmercury chloride-induced neurotoxicity of cerebral cortex and the disturption of astrocytic glutamate metablism and transporters after methylmercury chloride exposure.
Materials and Methods
1. In vivo animal experiments
The weight of 40 wistar rats from the Experimental Animal Center of China Medical University is 180±10 g.The number of female and male is the same.They were fed under the temperature of 17-23℃, relative humidity 45-55%.Before the experiment,they were fed one week and then were divided into 5 groups by weight at random, 8 animals for each group. The first group was the control group and the second and third groups were methyl-mercuric chloride (MMC) groups. The fourth and fifth groups were DM and riluzolegroup respectively.All the former three groups were subcutaneously (sc) injected with 0.9% NaCl.The fourth and fifth groups were sc injected with 13.5μmol/kg DM and 21.35μmol/kg respectively every other day. Two hours later, the animals in the control group were intraperitoneally(ip) given the injection of 0.9%NaCl, the second group was injected with 4.0μmol/kg MMC and from the third to the fifth groups were injected with 12.0μmol/kg MMC. The administration of MMC above was given five times a week, lasting 4 weeks as well as the administrati-on of DM and riluzole every other day before injected with MMC.24 hours after the last injection, 8 rats in each group sacrificed after paralyzed.Observe the contenrs of Hg, glutamate, glutamine and Malondialdehyde(MDA) as well as the activities of glutamine synthetase(GS), phosphate activated glutaminase(PAG), Superoxide Dismutase(SOD)and glutathione peroxidase(GSH-Px) in the rat cortex when they were injected the increasi-ng MMC dosage, DM and riluzole.
2. In vitro primary astrocytes
Primary cultured astrocytes isolated and cultured from cerebral cortex of newborn (1 day old) Sprague-Dawlery rats.The cells would be used after more than 95% of the cells stained positively for the astrocytic marker glial fibrillary acidic protein (GFAP) immunocytochemically.Cellular proliferation activity was detected by MTT method. we studied the cytotoxicity of methylmercury chloride to astrocytes and the effects of riluzole on disfunction of glutamate metabolism and transporters.And we chose the proper dosages and time during the experiments.The damage of fibroblasts was observed with inverted phase contrast microscope.This research made use of the RT-PCR to analyze the expression of GS, GLT-1, EAAC1 mRNA and the Western blotting to analyze the expression of GS,GLT-1, EAAC1 protein.The changes of intensity of mRNAs and proteins were normalized using the intensity obtained in the internal control bands(G3PDH andβ-actin respectively).
3. Statistical analysis
Mean and standard deviation were calculated for all measurements. All statistical analyses were performed using the SPSS software, version 11.5. Data were analyzed using one-way analysis of variance followed by the Students-Newman-Keuls, SNK. Statistical significance was set at P<0.05.
Results
1. In vivo animal experiments
Compared with the control group, in MMC group, the contents of Hg, Glu and MDA increased. The content of Gln was decreased. Moreover, the activities of GS and SOD,GSH-Px of the rat cortex were decreased. On the contrary,the activity of PAG increased. Compared with 12.0μmol/kg MMC group, in riluzole group, the contents of Hg,Glu,and MDA fell down.The content of Gln rose. Moreover, the activities of GS and SOD,GSH-Px of the rat cortex.
2. In vitro primary astrocytes
Compared with the control group, the level of the damage of the morphology of astrocyte and the destruction of membranous integrity are deteriorating with the increaing doses of methylmercury chloride (0-40μM,4-24h).There was no significant difference of GSmRNA and protein expression among the methylmercury chloride (0-20μM) treated group. Nevertheless, the EAAT1 and GLT-1 mRNA and protein expression of the methylmercury chloride (0-20μM) decreased.Observe the effects of riluzole (50-200μM) pretreatment 4 to 24 hours after destruction of astrocytes caused by 20μM methylmercury chloride exposure in cell morphology. Compare with the chloride methylmercury (0-20μM) treated group, riluzole group GLAST, and GLT-1 mRNA and protein expression increased which was treated with the chloride methylmercury (0-20μM).
Conclusions
1.The rat model of MMC was perfectly established by intraperitoneally injection by methylmercury chloride.The research showed that MMC could be conveyed through 'blood-brain barrier', disturb the 'glutamate-glutamine cycle' and indued oxidative damage.
2.By animal experiments,it was showed that dromethorphan and riluzole may have the certain antagonistic effect on exitotoxicity and oxidative damage of MMC.
3.By primary astrocytes cultures, we found that MMC could cause cytotoxicity,and induced the disorders of glutamate metabolism and transporters.
4.Riluzole may have the certain antagonistic effect on glutamate metabolism and transporters of astrocytes by methylmercury chloride exposure.
引文
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3 Hu B, Sun S, Induction of increased intracellular calcium in astrocytes by glutamate through activating NMDA and AMPA receptors[J]. Huazhong Univ Sci Technolog Med Sci.2003; 23(3):254-257.
4 Claudia Allritz, Stefanie Bette, Maciej Figiel, et al. Comparative structural and functional analysis of the GLT-1/EAAT-2 promoter from man and rat[J]. Journal of Neuroscience Research,2009; 88(6):1234-1241.
5 Albrecht J,Talbot M, Kimelberg H, et al. The role of sulfhydrlgroups and calcium in the mercuric chloride-induced inhibition of glutamate uptake in rat primary astrocyte cultures. Brain Res,1993; 607:249-254.
6李小刚,朱克,李楠,等.谷氨酸对神经元钙离子内流和凋亡的影响及谷氨酸受体拮抗剂保护作用的研究[J].中华老年心脑血管病杂志,2001;3(2)：122-125.
7 Antonio Gonzalez, Jose A. Parientea, Gines M. Salidoa.Ethanol stimulates ROS generation by mitochondria through Ca2＋ mobilization and increases GFAP content in rat hippocampal astrocytes[J]. Brain Research,2007; 1178:28-37.
8 Eun-Joo Shina, Seung-Yeol Nahb, Jong Seok Chaea, et al.Dextromethorphan attenuates trimethyltin-induced neurotoxicity via σ 1 receptor activation in rats[J]. Neurochemistry International,2007; 50(6):791-799.
9 Renis M, Cardile V, Russo A, et al. Glutamine synthetase activity and HSP70 levels in cultured rat astrocytes:effect of 1-octadecyl-2methyl-rac-glycero-3phosphocholine[J]. Brain Res,1998; 783:143-150.
10 Gosselin, R.D., O'onnor, R.M., Tramullas, M., et al. Riluzole Normalizes Early-LifeStress-induced Visceral Hypersensitivity in Rats: Role of Spinal Glutamate Reuptake Mechanisms [J]. Gastroenterology.2010; article in press.
11 Frizzo, M.E., Dall'Onder, L.P., Dalcin, K.B., Souza, D.O.,. Riluzole enhances glutamate uptake in rat astrocyte cultures. Cell. Mol. Neurobiol.2004; 24:123-128.
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