锰中毒性帕金森综合征大鼠脑中DMT1、FP1的研究

A study of divalent metal transporter 1 and ferroportin 1 in brain of rats with manganese-induced parkinsonism

目的 研究锰中毒性帕金森综合征大鼠脑黑质二价金属离子转运体（DMT1）、铁转运蛋白（FP1）的表达变化.方法 将80只SD大鼠随机分成4组,对照组腹腔注射生理盐水,低、中、高剂量组分别腹腔注射5、15、20 mg/kg的MnCl2溶液,连续16周.第16周进行3项行为学测试.电感耦合等离子原子发射光谱法（ICP-AES）测试中脑黑质锰含量,免疫组织化学染色法观察黑质酪氨酸羟化酶（TH）阳性表达量,判定锰中毒性帕金森综合征大鼠模型是否成功;采用免疫组化及荧光定量PCR的方法测定大鼠脑黑质中DMT1、FP1的表达量.结果 该实验方法成功制备锰中毒性帕金森综合征大鼠模型.5、15、20 mg/kg染锰组大鼠中脑黑质锰含量分别为（1.72±0.33）、（2.92±0.77）、（5.65± 1.60） μg/g,均高于对照组[（0.56±0.20） μ/g],差异有统计学意义（P＜0.01）.5、15、20 mg/kg染锰组中脑黑质TH阳性细胞平均吸光度值（0.054±0.008、0.016±0.004、0.003±0.001）,均低于对照组（0.109±0.019）,差异有统计学意义（P＜0.01）.15、20 mg/kg染锰组中脑黑质DMT1阳性细胞平均吸光度值（0.062±0.004、0.116±0.064）均高于对照组（0.015±0.007）,差异有统计学意义（P＜0.01）.5、15、20 mg/kg染锰组中脑黑质FP1阳性细胞平均吸光度值（0.092±0.011、0.048±0.008、0.008±0.002）均低于对照组（0.306±0.081）,差异有统计学意义（P＜0.01） 15、20 mg/kg染锰组大鼠黑质DMTl mRNA表达量（0.052±0.0126、0.124±0.0299）均高于对照组（0.001±0.000 4）,差异有统计学意义（P＜0.05）;5、15、20 mg/kg染锰组中脑黑质FP1 mRNA表达量（0.059±0.007 6,0.033±0.009 4,0.002±0.000 7）均低于对照组（0.162±0.046 3）,差异有统计学意义（P＜0.05）.结论 锰中毒性帕金森综合征大鼠模型中,DMT1表达增加及FP1表达降低,可能参与脑黑质锰积聚及多巴胺能神经元缺失的发生发展过程.

Objective To study the changes in the expression of divalent metal transporter 1 （DMT1） and ferroportin 1 （FP1） in the substantia nigra （SN） of rats with manganese-induced parkinsonism.Methods Eighty Sprague-Dawley rats were randomly divided into four groups.Rats in the control group were injected intraperitoneally with saline solution.Rats in the low-dose,medium-dose,and high-dose groups were injected intraperitoneally with 5,15,and 20 mg/kg MnCl2 solution,respectively,for 16 weeks.Three behavioral tests were performed at the 16th week.The concentration of Mn2＋ in the SN was determined by inductively coupled plasma-atomic emission spectrometry （ICP-AES）,and the positive expression of tyrosine hydroxylase （TH） was measured by immunohistochemical staining to determine whether rats with manganese-induced parkinsonism were successfully produced.The expression of DMT1 and FP1 in SN was measured by immunohistochemical staining and fluorescent quantitative polymerase chain reaction.Results Rats with manganese-induced parkinsonism were successfully produced using the above method.Compared with that in the control group,the concentrations of Mn-2＋ in the SN of rats exposed to 5,15,and 20 mg/kg Mn2＋ were significantly higher （1.72±0.33 vs 0.56±0.20 μg/g,P〈0.01;2.92±0.77 vs 0.56±0.20 μg/g,P〈0.01;5.65±1.60 vs 0.56±0.20 μg/g,P〈0.01）.The mean ODs of TH-positive cells in the SN of rats exposed to 5,15,and 20 mg/kg Mn-2＋ were significantly lower than that in the control group （0.054±0.008 vs 0.109±0.019,P〈0.01;0.016±0.004 vs 0.109±0.019,P〈0.01;0.003±0.001 vs 0.109±0.019,P〈0.01）.Compared with that in the control group,the mean optical densities （ODs） of DMT1-positive cells in the SN of rats exposed to 15.and 20 mg/kg Mn2＋ were significantly higher （0.062±0.004 vs 0.015±0.007,P〈0.01：0.116±0.064 vs 0.015±0.007,P〈0.01）.The mean ODs of FPl-positive cells in the SN of rats exposed to 5,15,and 20 mg/kg Mn2＋ were significantly lower than that in the control group （0.092±0.011 vs 0.306±0.081,P〈0.01;0.048±0.008 vs 0.306±0.081,P〈0.01：0.008±0.002 vs 0.306 ±0.081,P〈 0.01）.Rats exposed to 15 and 20 mg/kg Mn2＋ had significantly higher expression of DMT1 mRNA in the SN than those in the control group （0.052±0.0126 vs 0.001±0.0004,P〈0.05;0.124±0.0299 vs 0.001±0.0004,/〈0.05）.However,rats exposed to 5,15,and 20 mg/kg Mn2＋ had significantly lower expression of FP1 mRNA in the SN than those in the control group （0.059±0.0076 vs 0.162±0.0463,P〈0.05;0.033±0.0094 vs 0.162±0.0463.P〈 0.05;0.002±0.0007 vs 0.162±0.0463,P〈0.05）.Conclusion The increased expression of DMT1 and reduced expression of FP1 may be involved in the processes of Mn2＋ accunulation in the SN and dopaminergic nuron loss in rats with manganese-induced parkinsonism.