黄芩素干扰Rab11a/VPS4调控帕金森病α-突触核蛋白分泌的分子机制研究

α-synuclein (α-syn) is the pathological protein in Parkinson’s Disease (PD). α-syn aggregates in dopaminergic (DA) neurons, and transmits from cells to cells, which lead to immuno-inflammatory responses occur. Both ancient Chinese medical literature and experimental researches support treating PD based on heat-toxicity theory. Baicalein (BAI) alleviates α-syn aggregation and accumulation, and relieves subsequent immuno-inflammation and DA neurons degeneration as well. Previously, we showed that BAI inhibits α-syn secretion which is the vital link of α-syn transmission. However, the specific mechanism is still unclear. Since intracellular α-syn is secreted by two separate pathways that are recycling endosomes pathway and exosomes pathway. Rab11a and VPS4 are the key modulators of the two ways, respectively. All these promote us to ask whether BAI regulates the secretion of α-syn by interfering with Rab11a/VPS4 function. Here, we are going to systematically explore the interferential effects of BAI on Rab11a/VPS4 and sequential modification of α-syn secretion, aggregation and transmission, and immuno-inflammation in vitro. Then, we will apply BAI to PD mice to confirm the results in vitro via detecting the improvement of animals’ symptoms, as well as α-synucleinopathies, immunity disorders and inflammatory responses in the central nervous system, which is able to illuminate the mechanisms of BAI alleviating PD symptoms.

α-突触核蛋白（α-syn）是帕金森病（PD）的致病蛋白，在多巴胺能（DA）神经元聚集、传播并介导免疫炎症反应。中医古籍和现代实验均支持PD的“热毒”辨治。黄芩素（BAI）显著缓解α-syn聚集、沉积及抑制免疫炎症反应，延缓DA神经元变性。我们的前期工作发现，BAI可抑制α-syn传播的病理性分泌环节，但其机制未明。由于α-syn分泌通过循环内小体和外泌体2大途经介导，Rab11a和VPS4分别是这2条途经的调控因子，故推测BAI可能通过干扰Rab11a/VPS4功能而调控α-syn分泌。为此，本课题将通过离体细胞实验，系统观察BAI调控Rab11a/VPS4介导的α-syn分泌、聚集、传播及免疫炎症反应的分子机制。再以BAI处理模型小鼠，观察行为学变化，检测Rab11a/VPS4介导的α-syn病理变化及中枢免疫紊乱和炎症反应，对离体实验的结果加以验证，阐明BAI改善PD症状的作用机理。

作为帕金森病（PD）的致病蛋白，α-突触核蛋白（α-synuclein）在神经细胞间传播、沉积，导致PD进行性加重。中医古籍和现代实验均支持从“热毒”辨治PD。前期研究发现，黄芩素（BAI）可抑制α-synuclein传播的病理性分泌环节，但机制未明。α-synuclein的分泌受多种因子调控。我们推测，BAI通过干预上述调控因子而抑制α-synuclein分泌，缓解PD症状。本研究以BAI处理过表达α-synuclein MN9D细胞和小鼠，通过分子生物学、动物行为学及生物信息学等方法，分析和验证假说。细胞实验结果显示，BAI显著提高过表达α-synuclein MN9D细胞活性，抑制α-synuclein分泌，减少细胞内、外α-synuclein寡聚体的含量，对VPS4、Rab11a、Dnajc5、Snap23、Usp19等多种α-synuclein分泌调控因子mRNA表达均有干预作用。动物实验结果显示，BAI可显著改善过表达α-synuclein小鼠的运动功能障碍症状，减少黑质-纹状体多巴胺神经元丢失，抑制α-synuclein在小鼠的中枢神经系统内传播，降低黑质内VPS4含量。使用shRNA下调VPS4水平同样可降低过表达α-synuclein MN9D细胞内、外α-synuclein水平。网络药理学和分子对接分析结果显示，BAI可能对PD疾病过程中的炎症反应、氧化应激、细胞凋亡等及α-synuclein介导的神经炎症、细胞衰老、神经突触功能障碍、蛋白酶活性异常等病理生理过程具有改善作用。NFKB1、STAT3和CDKN1A可能是BAI干预α-synuclein病理过程的核心靶点。本项目研究结果为BAI在PD临床的应用及从“热毒”辨治PD提供了科学依据。