温肾养肝汤对MPTP所致帕金森病多巴胺能神经元损伤保护的分子机制研究
Molecular Mechanism Research of Wenshen Yanggan Decoction on Dopaminergic Neuron Injury in MPTP-Induced Parkinson's Disease
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摘要:目的 探讨温肾养肝汤保护多巴胺(Dopamine, DA)能神经元, 延缓帕金森病(Parkinson's disease, PD)进程的作用机制。方法 将50只小鼠随机分为正常组, 模型组, 温肾养肝汤高、低剂量组, 金刚烷胺组, 每组10只, 除正常组外, 其余组小鼠腹腔注射30 mg·kg-1 1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶(MPTP), 每周2次, 连续4周, 建立MPTP致小鼠PD模型, 灌胃温肾养肝汤，每日1次，连续3周。观察PD小鼠的行为学变化；免疫组化和尼氏染色法检测DA能神经元细胞数；HPLC法检测病理组织中DA、3, 4-二羟基苯乙酸(3, 4-Dihydroxyphenylacetic acid, DOPAC)和高香草酸(Homovanillic acid, HVA)含量；比色法测定小鼠黑质线粒体中ATP酶(ATPase)、线粒体呼吸链复合物Ⅰ(ComplexⅠ)活性；Western blot测定PINK1、Parkin、VDAC1、LC3Ⅱ/Ⅰ、P62蛋白相对表达量。结果 行为学检测显示, 与模型组相比, 温肾养肝汤能明显改善小鼠的肌肉力量和运动平衡(P < 0.01), 延长小鼠在滚筒上的时间(P < 0.01);免疫组化显示, 温肾养肝汤高剂量组可观察到大量酪氨酸氢化酶(Tyrosine hydrogenase，TH)免疫反应呈阳性的细胞, 明显的细胞突起, 与尼氏染色结果一致, 同时小鼠纹状体中DA和HVA含量明显提高(P < 0.05, P < 0.01);黑质线粒体酶活性和自噬通路相关蛋白研究显示, 高剂量温肾养肝汤能明显提高ATPase和ComplexⅠ活性(P < 0.01), 同时还能上调模型小鼠的PINK1(P < 0.05)、Parkin(P < 0.01)、LC3Ⅱ/Ⅰ(P < 0.01)蛋白表达, 抑制P62蛋白表达(P < 0.01)。结论 温肾养肝汤通过提高线粒体功能, 激活PINK1/Parkin介导的线粒体自噬过程来保护DA能神经元, 改善PD模型小鼠行为学功能障碍, 延缓PD进程。Abstract:OBJECTIVE To explore the potential mechanism of Wenshen Yanggan Decoction on protecting dopaminergic neurons and delaying the process of Parkinson's disease (PD).METHODS Fifty mice were randomly divided into 5 groups (n=10): control group, model group, Wenshen Yanggan Decoction high dose group, Wenshen Yanggan Decoction low dose group and amantadine group. Except for the control group, mice were injected intraperitoneally with 30 mg · kg-1 of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) twice a week for 4 consecutive weeks to establish a Parkinson's disease (PD) mouse model. The mice were administrated with Wenshen Yanggan Decoction by gavage once a day for 3 weeks. The behavioral effects of mice were observed. Immunohistochemistry and Nissl staining were used to detect the number of DA neurons. The contents of DA, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in pathological tissues were measured by HPLC. Colorimetric method was used to determine the activities of ATPase and mitochondrial respiratory chain ComplexⅠ in the substantia nigra. The protein expression levels of PINK1, Parkin, VDAC1, LC3Ⅱ/Ⅰ, and P62 were detected by Western blot.RESULTS The behavioral tests showed that compared with the model group, Wenshen Yanggan Decoction significantly improved the muscle strength and exercise balance of mice (P < 0.01), and prolonged the duration of mice on the roller (P < 0.01). The Results of the immunohistochemistry demonstrated that a large number of TH immunoreactive cells and obvious cell protrusions were observed in Wenshen Yanggan Decoction high dose group, which was consistent with the Results of the Nissl staining assay. In addition, Wenshen Yanggan Decoction significantly increased the levels of DA and HVA (P < 0.05, P < 0.01) in the striatum. Furthermore, treatment with the high dose Wenshen Yanggan Decoction profoundly promoted the activities of ATPase and ComplexⅠ(P < 0.01), up-regulated the protein levels of PINK1 (P < 0.05), Parkin (P < 0.01) and LC3Ⅱ/Ⅰ (P < 0.01), and inhibited P62 protein expression (P < 0.01) in MPTP-induced mice.CONCLUSION Wenshen Yanggan Decoction can improve mitochondrial function, and provide neuroprotective effect in PD by activating PINK1/Parkin mediated mitochondrial autophagy pathway.
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