铅暴露对大鼠海马不同亚区铜转运蛋白表达影响的研究

Expression of copper transport protein in different regions of hippocampus of lead-exposed rats

目的探讨铅暴露对大鼠海马不同亚区(CA1、CA3和DG区)铜转运蛋白表达的影响,为铅神经毒性的机制研究提供依据。方法选取45只SPF级SD雄性健康大鼠,随机均分为对照组(双蒸水)、低铅暴露组(0. 3 g/L乙酸铅)和高铅暴露组(0. 6 g/L乙酸铅),每组15只。采用自由饮水方式染毒,每周5 d,连续20周。Morris水迷宫测试大鼠的空间学习和记忆能力;电感耦合等离子质谱仪(ICP-MS)测定大鼠海马不同亚区铅和铜含量;苏木素-伊红(HE)染色法观察大鼠海马组织病理学变化;实时PCR检测大鼠海马铜转运蛋白mRNA表达水平。结果与对照组相比,低和高铅暴露组大鼠的逃逸潜伏期明显延长,穿台次数明显减少,差异有统计学意义(P<0. 05或P<0. 01)。随着染毒剂量增加,大鼠海马不同亚区神经元排列紊乱,大量细胞死亡,细胞核固缩、破裂,其中CA1区损伤程度最小。低和高铅暴露组大鼠海马CA1、CA3和DG区中铅的含量明显高于对照组,且高铅暴露组CA3区高于低铅暴露组(P<0. 01);CA3和DG区中铜的含量分别是对照组的1. 08和1. 10倍、1. 12和1. 09倍(P<0. 01);CA1区铜转入蛋白CTR1、DMT1和铜转出蛋白ATP7A mRNA表达比对照组增加(P<0. 05或P<0. 01),CA3区内CTR1和DMT1 mRNA表达分别是对照组的1. 95和2. 84倍、2. 74和1. 20倍,ATP7A mRNA表达是对照组的0. 89和0. 51倍(P<0. 01),且与低铅暴露组相比,高铅暴露组CTR1 mRNA表达增加,ATP7A mRNA表达降低(P<0. 01);DG区CTR1和DMT1比对照组分别增加了0. 71和2. 17倍、4. 23和4. 32倍,ATP7A mRNA表达增加了3. 78和2. 01倍(P<0. 05或P<0. 01),上述差异均有统计学意义。结论铅暴露可引起大鼠海马不同亚区损伤,这可能与海马CA1、CA3和DG区铜含量增加及铜转运蛋白的异常表达有关。

Objective To investigate the effects of the expression of copper transporters in different regions in hippocampus(CA1,CA3,and DG) of rats exposed to lead,and to provide the theoretical basis for the mechanism of lead neurotoxicity. Methods Forty-five SPF grade male SD rats were randomly divided into three groups,control group(double distilled water),low dose Pb exposed group(0. 3 g/L) and high dose Pb exposed group(0. 6 g/L),which were exposed by free drinking water. The rats were exposed with lead acetate water solution of Pb dose groups by free drinking water for 20 weeks,and five days per week. The Morris water maze was used to test the spatial learning and memory ability of rats. The lead and copper contents of different regions on hippocampus were determined by inductively coupled plasma mass spectrometry(ICP-MS). Histopathological changes of CA1,CA3 and DG regions were observed by HE staining. Real-time PCR was used to detect the effect of lead on the mRNA expression changes of the copper transport-related mRNA(CTR1,DMT1,ATP7 A) in CA1,CA3 and DG regions of rats. Result There were no significant differences in terms of body weight among three groups of rats. Compared with the control group,the escape latency was significantly increased,while less times to cross the platform were clearly decreased in other groups(P<0. 05 or P<0. 01).The neurons in the each hippocampus regions of low and high Pb dose groups rats were showed disorder,a large number of cells died,and the nuclei were pyknotic and ruptured,and the other two regions suffered more damage than CA1 region. Lead content in CA1,CA3 and DG regions was significantly higher in the low and high dose groups than in the control group(P < 0. 01). The copper contents in CA3 and DG regions of rats of low and high dose groups were respectively 1. 08 and 1. 10,1. 12 and 1. 09 folds,higher than those in the control group(P < 0. 01). While compared with the control group of CA1 region in rats,the mRNA expression of CTR1,DMT1,ATP7 A were increased of dose groups rats(P<0. 05 or P<0. 01). The mRNA expression of the CTR1 and DMT1 of the low and high dose groups were increased 0. 95 and 1. 84 folds,1. 74 and 0. 20 folds in the CA3 region respectively,while the mRNA expression of ATP7 A mRNA was decreased 0. 89 and 0. 51 folds respectively(P<0. 01).It was found that the CTR1 mRNA expression of the high dose group was increased 0. 46 folds than that of the low Pb dose group,and the mRNA expression of ATP7 A was decreased 0. 75 folds(P < 0. 01). The mRNA expression of the CTR1 and DMT1 increased 0. 71 and 2. 17 folds,4. 23 and 4. 32 folds than control groups in DG region of rats,at the same time,the mRNA expression of the ATP7 A was increased 3. 78 and 2. 01 folds(P<0. 05 or P<0. 01). Conclusion Lead exposure could cause the impairment in hippocampus different regions of rats,which may be related to the increase of copper content in hippocampal CA1,CA3 and DG areas and abnormal expression of copper transporter.