【作者】 罗教华；

【导师】 舒为群；

【作者基本信息】 第三军医大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 前言及目的砷(Arsenic,As)是一种类金属,在自然界广泛分布于岩石、土壤和水环境中。世界上大约有20多个国家发现有地砷病病区或高砷区存在,饮用含砷超过0.05 mg/L水的人口在5 000万人以上,慢性砷中毒患者达数十万。孟加拉、印度和中国是全世界病情最重、病区面积最大、受危害人口最多的国家。在我国目前暴露于饮用水砷浓度等于或超过0.05 mg/L的人口为560万,暴露在饮用水砷浓度等于或超过0.01 mg/L的人口为1466万,这还不包括由于燃煤引起的燃煤型地方性砷中毒。砷对健康的危害是多方面的,可引发多器官的组织学和功能上的异常改变,严重者还可导致癌症,临床上以末梢神经炎、皮肤色素代谢异常、掌跖部皮肤角化、肢端缺血坏疽、皮肤癌变为主要表现。儿童每单位体重的日平均砷摄入量要高于人一生的日平均摄入量(特别是当砷来源于土壤时,因为儿童有近地游戏及爱吮吸手的习惯),而且由于儿童处于生理发育阶段对化学物质的毒作用更为敏感。流行病学调查显示,砷中毒对青少年的中枢神经系统损害主要表现为对学习记忆能力的损害。动物实验结果与流行病学调查一致。目前,砷致学习记忆能力损害的分子机理尚不十分清楚。海马是脑边缘系统中最重要的结构之一,是学习记忆等高级神经活动的重要部位。长时程增强(long-term potentiation,LTP)现被认为是海马学习记忆的细胞突触模式,在细胞水平检测学习记忆能力的最经典的指标。N-甲基-D-天门冬氨酸受体是兴奋型氨基酸特异性受体的一种、具有许多不同变构调控位点并对Ca2+高度通透的配体门控性离子通道,是由NR1、NR2(A-D)和NR3(A-B)亚单位组成的异聚体,近年来被认为是学习记忆中的关键物质而受到人们的关注。NMDA受体的亚基组成、受体胞内信号转导通路在LTP的诱导和维持中起重要作用。文献报道,急、慢性砷暴露可以抑制大鼠海马LTP的诱导和维持。因此,我们推测砷可能通过影响NMDA受体各亚基的表达水平及胞内信号传导通路,从而损害青少年的学习记忆能力。本研究以幼鼠为模型,通过饮水砷染毒,观察砷暴露对大鼠学习记忆能力、对NMDA受体各亚基的表达水平及胞内信号传导通路的影响,以探讨砷致学习记忆损害的分子机制,为防治砷中毒提供理论依据。方法1.选用断乳健康SPF级Sprague-Dawley雄性大鼠180只,体重40-50 g,在标准SPF级动物房内饲养。对照组大鼠饮用灭菌自来水,实验组大鼠分别饮用2.72 mg/L、13.6 mg/L和68 mg/L亚砷酸钠水溶液。饮水每两天更换一次。染毒时间分别为:1个月、2个月和3个月。用Morris水迷宫对大鼠进行行为学检测。实验终期,大鼠经10%水合氯醛腹腔麻醉,心脏采血后,断头分离脑组织,将脑组织在冰冷生理盐水中漂洗,用滤纸吸干,置于-70℃冰箱保存。2.采用微波消解-氢化物发生-原子荧光光谱法(HG-AFS)测定血砷、脑砷含量。分别取全血0.5 mL、脑组织0.2 g于聚四氟乙烯管,经微波消解仪消化。测量前,每个样品加5 ml 5%硫脲+5%抗坏血酸,然后用5%盐酸定容,混匀,用氢化物发生-原子荧光光谱法测定血砷和脑砷含量。砷的测定采用砷标准溶液进行质量控制。3.用Morris水迷宫检测大鼠的空间学习记忆能力改变,测试包括定位航行实验、空间探索实验和可视平台实验三个部分。4.大鼠经腹腔麻醉后,断头,在冰上快速分离海马,用双面刀片切取1 mm3海马CA1区组织块,2.5%的戊二醛溶液4℃固定24 h。漂洗,1%锇酸后固定,丙酮脱水,饱和醋酸铀电子染色,环氧树脂-618包埋。半薄切片(片厚1μm),TECNAI-10电子显微镜观察,照相。5.收集暴露3个月的大鼠海马,采用RT-PCR、Western-blot和免疫组化法检测海马中NMDA受体NR1、NR2A、NR2B亚基基因和蛋白表达。6.检测饮水砷暴露对大鼠海马NMDA受体信号传导通路的影响。用SignaTECT蛋白激酶检测试剂盒测定CaMKII活性,用Western-blot检测CaMKIIα的蛋白表达及磷酸化状态;用Western-blot检测PSD-95、SynGAP、ERK1/2蛋白表达。结果1.实验期间,实验组和对照组大鼠外观和一般情况良好,没有出现中毒症状。从第六周开始,68 mg/L剂量组大鼠体重增幅减慢,与对照组和其它两个实验组相比,差异有统计学意义(P<0.05)。这种差异一直维持到实验结束。2.在三个时间点,各实验组大鼠血砷、脑砷值均显著高于对照组,差异有统计学意义(P<0.05)。结果表明,砷在大鼠血液和脑组织中有不同程度的蓄积,我们成功的建立了砷中毒大鼠模型。3.染毒1个月和2个月的大鼠,空间学习记忆能力受损不明显;染毒3个月,砷暴露组大鼠逃逸潜伏期延长(P<0.05),而空间探索实验和可视平台实验数据差异均没有统计学意义。说明砷暴露组大鼠已经出现了学习记忆损害。4.透射电镜观察发现,砷暴露组能见到较多变性神经元,胞体皱缩变小,形态不规则;胞浆浓缩,基质染色变深;细胞器肿胀、空化,线粒体肿胀,脊减少或无脊,粗面内质网RER扩张。毛细血管周围结构明显水肿,血管管腔不规则,内皮细胞中部分粗面内质网、高尔基复合体、线粒体可见明显的扩张。突触前成分中突触小泡数量较少,轴突空化,轴突质淡。5.饮水砷暴露后,大鼠海马NMDA受体NR2A亚基的mRNA和蛋白表达都受到抑制,与对照相比具有统计学意义(P<0.05);而NR1和NR2B亚基的mRNA和蛋白表达没有受到明显影响。6.砷暴露后,大鼠海马CaMKII活性受到明显抑制,与对照相比具有统计学意义(P<0.05);CaMKIIα的蛋白表达及磷酸化状态没有改变。砷暴露后,PSD-95、ERK1/2蛋白表达下降(P<0.05),而SynGAP蛋白表达增高(P<0.05)。结论1.饮水砷暴露后,砷能进入血液循环,透过血脑屏障在脑组织内蓄积,引起脑组织超微结构改变,损害大鼠的空间学习记忆能力。2.砷暴露抑制NMDA受体NR2A亚基的表达,对NR1和NR2B亚基表达没有明显影响。NMDA受体亚基表达的改变,会影响LTP的诱导和维持。3.砷暴露改变NMDA受体表达、降低CaMKII的活性、抑制PSD-95和ERK1/2的蛋白表达、刺激SynGAP蛋白表达。这些改变都会抑制LTP的诱导和维持,进而影响学习记忆,这可能是砷致学习记忆损害的机制之一。更多还原

【Abstract】 Background and purposeArsenic is a naturally occurring element widely present in the environment. Worldwide, more than 20 industrialized and less industrialized countries have drinking water contaminated with arsenic. There are more than 50 million people exposed to groundwater arsenic concentrations above the World Health Organization maximum permissible limits 50μg/L, many thousands of people developed arsenicalism. The three most affected areas in the world are Bangladesh, India and China. In both these areas, most of the source of arsenic is geological in origin, contaminating aquifers which provide water for over one million tube wells. In China, more than 5.6 million people are exposed to groundwater arsenic concentrations above 50μg/L, and more than 14.66 million people are exposed to groundwater arsenic concentrations above 10μg/L. Moreover, there are many people exposed to high levels of arsenic from burning coal.Chronic arsenic toxicity results in multisystem disease, such as peripheral neuritis, skin lesions (Hyperpigmentation, palmar and solar keratosis), peripheral vascular disease, hypertension, Blackfoot disease, and high risk of cancers. Young children (i.e., ages 0–6 years) may receive a much higher daily dose of chemical per-unit-body-weight than a lifetime average daily dose, particularly for chemicals in soil, because of their greater hand-to-mouth behavior than adults. Young children may also be more sensitive to the toxic effects of some chemicals than older age groups because of developmental and physiological differences. Epidemiological investigations found that arsenic toxicity can cause central nervous system impairment in adolescents, and the main manifestations of it are impairment of learning and memory. Animal experiments also found arsenic toxicity can impair the ability of learning and memory in rats. Up to now, the mechanism of the learning and memory impairment are poorly understood.The hippocampus is a part of the limbic system that is crucial to memory function and spatial navigation. Long-term potentiation (LTP) is widely accepted as a cellular and molecular model of information processing and storage by neural network. N-methyl-D-aspartate receptor (NMDAR) is one member of the glutamate-gated ion-channel receptor, with a high permeability to Ca2+ ions. To date, three main families of NMDAR subunits have been identified: NR1, NR2 (A-D) and NR3 (A-B). Many of the important NMDAR properties are influenced by the subunits composing the receptor assembly. NMDAR widely expresses in the central nervous system, and plays key roles in excitatory synaptic transmission. NMDAR subunit composition and its postsynaptic signaling pathway have important role in the induction and maintenance of LTP. Literatures have reported that acute and chronic arsenic exposure can significantly inhibit the induction and maintenance of LTP. So, we presume that arsenic exposure can impair the ability of learning and memory in adolescent through influencing the NMDAR subunit composition and its postsynaptic signaling pathway.The purpose of this study is to investigate the possible mechanism of arsenic neurotoxicity. We chose weaning Sprague-Dawley male rats as experimental model. The rats were assigned to four groups randomly (namely control, group A, group B and group C) and fed with water respectively containing 0, 2.72, 13.6 and 68 mg/L sodium arsenite for 1, 2, 3 months respectively. We will focus on the effects of arsenic exposure on learning and memory, the expression of NMDAR subunits and its postsynaptic signaling pathway.Methods1. A total of 180 weaning Sprague-Dawley male rats (40–50 g) were obtained from the Experimental Animal Center of this university, and kept in SPF laboratory animal room. The rats were assigned to four groups randomly and fed with water respectively containing 0, 2.72, 13.6 and 68 mg/L sodium arsenite for 1, 2, 3 months respectively. The As-containing water was freshly prepared every 2 days. Water used was free from bacterial contamination. Rats had free access to food.After arsenic exposure, samples of hippocampus were collected from two rats in each group for transmission electron microscopic study, and 10 rats from each group were tested in Morris water maze according to a modified procedure of Morris. At the end of spatial learning task, these rats were anesthetized with sodium pentobarbital by i.p. injection. Blood was collected by heart puncture; brains were separated and immediately transferred into liquid nitrogen, then stored at -70℃.2. The level of arsenic was determined using hydride generation atomic fluorescence spectrometry. Briefly, 0.2 g brain samples or 0.5ml blood were digested with 3 ml of concentrated HNO3 and 2 ml of concentrated H2O2 in microwave digestion system. Before measurement, added 10 ml 5% ascorbic acid and 5% sulfocarbamide to the digested samples then diluted to 50 ml with 5% hydrochloric acid. A blank digest was carried out in the same way. The concentration in diluted samples was determined in an atomic fluorescence spectrometer. The accuracy of the method was verified by standard reference materials.3. The acquisition of spatial learning and memory was assessed in the Morris water maze on three different components: hidden platform acquisition, probe trial and subsequent visible platform test.4. The hippocampus samples of 1 mm3 cube were dissected from CA1 area under anatomical microscope. After fixed in 2.5% glutaraldehyde, the specimens were sequentially processed with 1% osmium tetroxide, graded ethyl alcohols and embedded in EPON 618. The thin sections on copper mesh grids were stained with the heavy metals, uranyl acetate and lead citrate for contrast. After drying, the grids were then viewed on a transmission electron microscope.5. The expression of NMDAR subunits (NR1、NR2A、NR2B) in hippocampus were detected by the means of RT-PCR, Western-blot and immunohistochemistry. The hippocampi were collected from the rats exposed to arsenic for three months.6. To investigate the effect of arsenic exposure on the postsynaptic signaling pathway, we detected several important molecular. The activity of CaMKII was assayed with the SignaTECT Protein Kinase Assay Systems. Protein expression of CaMKIIα, p-CaMKIIα, PSD-95, SynGAP and ERK1/2 were detected by the means of Western-blot.Results1. General appearance and physical condition of arsenic-exposed and control rats were closely observed and no obvious difference was noticed. There were no differences in food intake between groups during the study. Compared with the rats in other three groups, rats in group C gained less body weights from the 6th week to the end of the study (P<0.05). Rats in group A and B did not show significant differences in body weight comparing with control rats. 2. Blood arsenic content increased with the concentrations of arsenic in drinking water, and there was significant difference among the treatment groups for three exposure time respectively (P<0.05). The arsenic content in brain increased with the concentration of arsenic in drinking water. The statistical significances were present between individual experiment group and control for three exposure time respectively (P<0.05). The results indicated that we have successfully raised the arsenic poisoning rats.3. There was no obvious impairment of spatial learning in rats exposed to arsenic for 1 and 2 months. After 3 months arsenic exposure, the rats in group C exhibited significant deficit in hidden platform acquisition compared with control and group A (P<0.05). Rats in group A and B did not show significant deficit comparing with control rats at the end of arsenic exposure. There were no significant difference in the spatial probe test and visible platform trial among the four groups. The results indicated the impairment of spatial memory in rats of group C.4. In the hippocampus of arsenic-exposed rat, individual shrunk cells could be seen with condensed cytoplasm and nucleus. The mitochondrion became swollen and vacuolized along with the cristae disorder and less in number. Rough endoplasmic reticulum presented sacculated distension. Vascular endothelial cells presented swollen mitochondria and the lumen of blood vessel irregular in the hippocampus of arsenic exposed rat. Edema around the capillary was obvious. The quantity of synaptic vesicle in synapse was decreased, and edema inside cells and organelles were confirmed. These ultra-structural changes became worse with increasing dose of arsenic exposure.5. A decreasing trend of NR2A mRNA and protein levels in hippocampus was observed after arsenic exposure. Decreases of NR2A mRNA and protein expression in hippocampus of rats treated with arsenic was in a dose-dependent manner (P<0.05). The expressions of NR1 and NR2B in hippocampus were not affected by arsenic toxicity.6. The activity of CaMKII in hippocampus was decreased significantly after arsenic exposure (P<0.05), while the protein expressions of CaMKIIαand p-CaMKIIαwere not affected. After arsenic exposure, the protein expressions of PSD-95, ERK1/2 decreased significantly (P<0.05). On the contrary, the protein level of SynGAP increased significantly (P<0.05). Conclusion1. After arsenic exposure from drinking water, arsenic can be rapidly absorbed into the blood circulation. Arsenic can penetrate the blood-brain barrier and accumulate in the brain. Its presence is associated with a series of ultra-structural pathological changes of the brain, and the impairment of spatial learning and behavioral tasks.2. Arsenic exposure can significantly inhibit the mRNA and protein expression of the NMDAR subunit NR2A. This change in NR2A was presented with the absence of changes in NR1and NR2B expression.3. Changed NMDAR subunit expression, lower CaMKII activity, lower expression of PSD-95 and ERK1/2 protein, together with higher expression of SynGAP protein after arsenic exposure, these results could inhibit the induction and maintenance of LTP. These changes maybe partly account for the molecular mechanism of learning and memory impairment caused by arsenic exposure.更多还原