补肾平肝法对A53T转基因帕金森病大鼠α-突触核蛋白降解网络的影响研究

Background：Parkinson's disease (PD) is the most common form of neurodegenerative disease that cause movement disorder in human being without early stage treatment, dopamine therapy is only a downstream intervention. α-synuclein not only participate the early stage PD pathogensis, but also closely related with non-motor symptoms. The death of dopamine-generating cells in the substantia nigra and α-synuclein positive inclusions in neurons are two core pathological characters in PD pathogenesis. Many researchers found that such pathological protein depositions in the brain are related with the dysregulation of degradation net of α-synuclein recently. while, PD is recognized as ChanZheng in Chinese medicine, deficiency of kidney and liver Yin is the most common Chinese Zheng syndrome; And herbs with einforcing kidney and suppressing hyperactive liver function is the most commonly used method in clinic. Aim: Present research aims to observe the influence of Chinese herbal formula with reinforcing kidney and suppressing hyperactive liver function on behavioral and substantia nigra(ultra-structure of neurons andα-synuclein depositions) pathological changes inA53T mutation transgenic rat model of Parkinson’s disease, which possesses age-related α-synuclein deposition and dopamine-generating cells death in its brain. And further investigate influences of our herbal formula in ubiquitin proteasome system and autophagy-lysosome system of A53T mutation transgenic rat. Methods: Rotating rod and gait analysis will be used to check rat movements; electron microscope will be applied to observe ultra-structure of neurons; immunohistochemisty, Western blot and ELISA will be used to test expressions of α-synuclein and key proteins in both ubiquitin proteasome system and autophagy-lysosome system. Application: The whole picture of α-synuclein degradation net in A53T rat will be depicted and pharmaceutical targets of the herbal formula with reinforcing kidney and suppressing hyperactive liver function will be revealed, and new theraputic methods will be provided.

背景：帕金森病（PD）是最常见的导致人类运动障碍的神经变性病，目前仍缺乏针对PD早期的治疗，现有多巴胺治疗仅干预PD下游机制。α-突触核蛋白(α-Syn)参与PD早期发病过程，与PD非运动症状关系密切，α-syn的堆积主要与脑内蛋白质降解网络异常、降解不足有关。补肾平肝法中药具有改善非运动症状的作用，但效应靶点不明确。目的：评价补肾平肝中药对PD转基因大鼠模型α-突触核蛋白及降解通路的影响。方法：以A53T转基因帕金森病大鼠为对象，比较大中小不同剂量中药组及对照组的运动功能、黑质区神经元超微结构的差异，并通过免疫组化、Western blot和ELISA方法对α-syn、泛素-蛋白酶体系统和自吞噬-溶酶体系统的关键蛋白进行检测。意义：发现补肾平肝法对PD转基因大鼠α-syn降解网络的影响，有望从PD早期病生理环节角度获得突破，改变目前PD多巴胺治疗局限，提供可同时改善非运动症状的治疗策略。

背景：帕金森病(PD)是最常见的导致人类运动障碍的神经变性病，目前仍缺乏针对PD早 期的治疗，现有多巴胺治疗仅干预PD下游机制。α-突触核蛋白(α-Syn)参与PD早期发病过程 ，与PD非运动症状关系密切，α-syn的堆积主要与脑内蛋白质降解网络异常、降解不足有关。 补肾平肝法中药具有改善非运动症状的作用，但效应靶点不明确。方法: 基于网络药理学研究方法，分析补肾平肝方中各个药物发挥作用的核心成分与PD疾病的核心靶标。运用转棒实验测评A53T转基因帕金森病大鼠的运动功能，并通过免疫组化、Western blot方法对α-syn、泛素-蛋白酶体系统和自吞噬-溶酶体系统的关键蛋白进行检测。结果：使用网络药理学方法发现补肾平肝方与PD疾病核心靶标共1150个，其中SNCA、TH、LAMP2等为目前具有较大研究意义的核心靶标。进一步构建出补肾平肝方治疗PD的通路共258条，根据PValue排名前二十位的通路均有相关文献验证与Autophagy关系密切，同时KEGG Pathway富集到Parkinson’s disease(4.59E-14)、Autophagy-animal(6.09E-12)和Mitophagy-animal(2.03E-09)。补肾平肝方对PD α-synuclein降解机制的研究发现，通过行为学Rotarod test补肾平肝方中药组与模型组比较转棒持续时间明显较多；组织形态学IHC检测中发现补肾平肝方中药组与模型组比较，可以增多TH的阳性表达，减少α-synuclein的阳性表达；在WB检测相关蛋白的相对含量表达方面，补肾平肝方中药组与模型组比较，能够通过调控提高PD A53T转基因模型大鼠脑内黑质TH、LAMP-2A、LC3Ⅱ/Ⅰ及P62相对蛋白含量表达，降低α-synuclein的表达。意义：补肾平肝方与PD核心靶标包括SNCA、TH及LAMP-2。中药复方可能是通过LAMP-2的作用靶点调控自噬溶酶体相关生物学过程，提高对大鼠中脑黑质α-synuclein的降解作用。评价补肾平肝中药对 PD转基因大鼠模型α-突触核蛋白及降解通路的影响，为中医药治疗PD提供科学依据。