艾地苯醌对帕金森病小鼠行为学改善的作用及其机制

Effect and Mechanism of Idebenone on Behavioral Improvement in Mice with Parkinson's Disease

关键词:  艾地苯醌  帕金森病  行为学  线粒体

Key words:  idebenone  Parkinson's disease  behaviourology  mitochondria

摘要:

目的 研究艾地苯醌对于1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）诱导的帕金森病小鼠行为学改善的作用及可能的机制。方法 将小鼠分为对照组，模型组，艾地苯醌高、低剂量组。除对照组外，其余组别连续1周注射20 mg·kg^-1的MPTP诱导小鼠帕金森病，低剂量组小鼠每日给予艾地苯醌5 mg·kg^-1，高剂量组小鼠每日给予艾地苯醌10 mg·kg^-1，模型组给予等体积的生理盐水，连续干预14 d，对照组小鼠常规饲养。小鼠行为学实验采用旷场实验、爬杆实验、转轮实验、游泳实验、悬挂实验。免疫组化法检测小鼠黑质酪氨酸羟化酶（tyrosine hydroxylase，TH）的表达，Nissl染色检测小鼠中脑蛋白合成能力，MDA、SOD试剂盒检测小鼠黑质氧化损伤的水平，TUNEL染色检测小鼠黑质神经元的凋亡，免疫荧光法检测小胶质细胞IBA-1的表达，Western blot检测小鼠黑质中Bax、Bcl-2、caspase-9的表达。RT-QPCR检测线粒体DNA拷贝数mt-ND1以及线粒体DNA复制关键转录因子POLG1、POLG2、TFAM的mRNA表达水平。ELISA法检测小鼠外周血中炎症因子IL-1β、TNF-α、IL-6的表达水平。结果 模型组小鼠的运动能力相比对照组显著下降，且平衡感受到严重影响。艾地苯醌干预后，小鼠行为学得到显著改善，相比模型组具有显著性差异（P<0.05）。相比对照组，模型组小鼠黑质中TH、SOD的表达显著下调（P<0.05）、神经元凋亡率和MDA、IBA-1的水平显著上调（P<0.05），外周血中炎症因子IL-1β、TNF-α、IL-6的表达水平显著上调（P<0.05），Bax、caspase-9表达上调，DNA拷贝数以及相关转录因子表达下调。与模型组相比，艾地苯醌可以提高黑质中TH和SOD的表达，下调黑质中神经元凋亡率、IBA-1的表达以及外周血炎症因子水平，下调凋亡相关蛋白Bax、caspase-9，上调Bcl-2的表达，提高DNA的拷贝数和相关转录因子的表达。结论 艾地苯醌可以改善帕金森病小鼠的行为学，其机制和抗氧化应激损伤、抑制线粒体凋亡信号、改善线粒体损伤有关。

Abstract:

OBJECTIVE To study the effect and possible mechanism of idebenone on behavior improvement of Parkinson's disease(PD) mice induced by MPTP. METHODS The mice were divided into control group, model group, high and low dose of idebenone groups. The mice were injected with 20 mg·kg^-1 MPTP for one week to induce Parkinson's disease expect those in control group. The mice in low dose of idebenone group were given idebenone 5 mg·kg^-1 daily while high dose of idebenone group with 10 mg·kg^-1 daily for 14 d. The model group was given saline of equal volume and control group were normally reared. The behavioral tests of mice were performed by open field test, rod climbing test, runner test, swimming test and suspension test. The expression of tyrosine hydroxylase(TH) in substantia nigra of mice was detected by immunohistochemistry, the protein synthesis ability of mice was detected by Nissl staining, the oxidative damage of substantia nigra was detected by MDA and SOD kits, and the apoptosis of substantia nigra neurons was detected by TUNEL staining. Immunohistochemistry detected the expression of IBA-1 in microglia while Western blot detected the expression of Bax, Bcl-2 and caspase-9 in the substantia nigra. RT-qPCR was used to detect mt-ND1 and mRNA expression levels of key transcription factors POLG1, POLG2 and TFAM. The expression of IL-1β, TNF-α, and IL-6 in the peripheral blood of mice was detected by ELISA. RESULTS The movement ability of mice in model group was significantly lower than that of control group, the balance was seriously affected. After the intervention of idebenone, the behavior of mice was significantly improved, which was significantly different from that of model group(P<0.05). Compared with the control group, the expression of TH and SOD in substantia nigra of the model group was significantly down-regulated(P<0.05), the apoptosis rate of neurons and the levels of MDA and IBA-1 were significantly up-regulated(P<0.05), the expression levels of inflammatory factors IL-1β, TNF-α, IL-6 in peripheral blood were significantly up-regulated(P<0.05), the expression of Bax and caspase-9 were up-regulated, the DNA copy number and the related transcription factors were significantly down-regulated(P<0.05). Compared with model group, idebenone could increase the expression of TH and SOD, down-regulate the apoptosis rate, expression of IBA-1 in substantia nigra and inflammatory factors in peripheral blood, and down-regulate the apoptosis related proteins Bax and caspase-9, up-regulate the expression of Bcl-2. Idebenone could also increase the copy number of DNA and expression of related transcription factors. CONCLUSION Idebenone can improve the behavior of Parkinson's disease mice, and its mechanism is related to anti-oxidative stress injury, inhibiting mitochondrial apoptosis signal and improving mitochondrial damage.