鱼藤酮对大鼠中脑神经元多巴胺转运、储存和代谢的影响

目的探讨鱼藤酮对多巴胺(DA)神经元选择性毒性作用的可能机制。方法大鼠背部sc鱼藤酮(1.0或1.5mg.kg-1.d-1)28d,观察大鼠的外观和行为变化。然后麻醉处死大鼠,取中脑黑质组织。分别用免疫组织化学法、Western蛋白印迹法和RT-PCR法检测鱼藤酮对大鼠中脑黑质DA神经元突触囊泡单胺转运体(VMAT2)表达的影响;以卞胺为底物进行比色,检测组织单胺氧化酶(MAO)的活性;高效液相色谱-荧光检测器流速梯度法测定黑质组织内DA及其代谢产物3,4-二羟基苯乙酸(DOPAC)和高香草酸(HVA)的含量。结果注射鱼藤酮28d后,大鼠发生外观和行为改变,出现类帕金森病症候群特征;中脑黑质DA神经元VMAT2蛋白免疫深染的细胞数量减少;鱼藤酮1.0和1.5mg.kg-1组VMAT2蛋白和基因表达与正常对照组比较明显降低,MAO活性分别从对照组的(23.6±7.6)升高至(37.8±5.0)和(40.5±4.3)kU.h-1.g-1蛋白,均具有统计学意义;实验组大鼠中脑黑质组织内DA及其代谢产物DOPAC和HVA含量明显减少。结论鱼藤酮可抑制大鼠中脑黑质DA神经元VMAT2的表达,增强DA代谢相关酶MAO的活性,进而影响DA的转运、储存和代谢,这可能是鱼藤酮DA神经元选择性毒性作用机制之一。

Effects of rotenone on transportation, storage and metabolism of dopamine in rat middle brain

AIM To investigate the effects of rotenone on vesicular monoamine transporter 2 (VMAT₂) expression and monoamine oxidase (MAO) acitivity of dopamine(DA) neurons in the substantia nigra. METHODS The rats were subcutaneously injected with rotenone 1.0 and 1.5 mg·kg^-1·d^-1 for 28 d. The behavioral symptoms of the rats were observed carefully. At 24 h after the last injection the rats were anesthetized and sacrificed. Then the substantia nigra was dissected on ice. Immunohistochemistry, Western blot and RT-PCR were employed to detect VMAT₂ expression in the substantia nigra. MAO activity of the brain tissues was also measured biochemically with benzylamine as a substrate. DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the tissues were determined by using high performance liquid chromatograph with a fluorescence detector. RESULTS The behaviors similar to Parkinsonism were observed in the rats treated with rotenone. The number of VMAT₂ immunstained-obviously cells decreased in the substantia nigra of the rats treated with rotenone in contrast with the control group. Protein and mRNA expressions of VMAT₂ were demonstrated to be signifcantly decreased in the substantia nigra tissues of the rats administrated with rotenone at the doses of 1.0 or 1.5 mg·kg^-1 compared to the control group. The activity of MAO, however, was increased from (23.6±7.6) to (37.8±5.0) and (40.5±4.3) kU·h^-1·g^-1 protein, respectively. DA and its metabolites DOPAC and HVA in the tissue were decreased. CONCLUSION The inhibition of rotenone on VMAT₂ expression and MAO activaty, leading to dysfunction of dopamine transportation, storage and metabolism, may be one of the mechanisms of the selective toxicology of rotenone on dopamine neurons.