基于经典Wnt信号通路与氧化应激关联与调控对孤独症的影响机制研究

Autism is a complex,pervasive developmental disorder.The etiology of autism remains unclear.Our studies and others have shown that the canonical Wnt signaling pathway and oxidative stress are involved in the pathogenesis of autism. Whereas,it is unclear whether a relationship exists between the Wnt/β-catenin pathway and oxidative stress in autism and how they infulence the process of autism. In our preliminary studies, we have shown that 1) Oxidative homeostasis occurs in rats with autism,2) the activity of the canonical Wnt pathway was enhanced in autism-like rats,3) oxidative stress markers levels were lowered, while antioxidant stress markers levels were increased following the downregulation of the Wnt pathay by sulindac, and 4) sulindac attenuated autism-like behavioral abnormalities. Using an established model of autism in rats, we will carry out a series of experiments to accomplish three specific aims. In Specific aim 1, we will examine the relationship of the canonical Wnt pathway and oxidative stress in autism.In Specific aim 2, we will explore the key time window of treatment intervention of autism. In Specific aim 3, we will find a valuable and potential therapeutic drug target. The data from this project will guide us to explore a detailed cellular mechanism regarding the interaction between the canonical Wnt pathway and oxidative stress in autism. The outcome will also be used to support translational clinical studies to evaluate the effectiveness of sulindac in the treatment of patients with autism.

孤独症（Autism）是一种广泛的神经发育障碍性疾病，其发病机制目前还是一个谜。我们的研究与既有研究结果表明，氧化应激与经典Wnt信号通路均参与孤独症的发病。然而，两者是否相互作用进而影响孤独症的发生发病进程目前尚缺乏文献支持。本题预实验显示：1）孤独症大鼠氧化应激失调；2）孤独症大鼠经典Wnt信号通路活性增加；3)舒林酸下调经典Wnt信号通路以后，氧化应激标记物减少，抗氧化应激标记物增加；4）舒林酸下调经典Wnt信号通路以后，孤独症样的行为学异常有所改善。本题拟用孤独症动物模型，研究1)孤独症发生过程中氧化应激与经典Wnt信号通路的关系，2）探索孤独症治疗干预的时间窗，3)寻求孤独症治疗的靶药物。该项目的成果将指导我们1）孤独症发生过程中氧化应激与经典Wnt信号通路的关系机制，2）评估舒林酸治疗孤独症患者的可行性。

研究表明，氧化应激与经典Wnt 信号通路均参与孤独症的发生。经典Wnt信号通路与氧化应激是否相互作用影响孤独症发生进程目前尚缺乏文献支持。为此，本项目采用丙戊酸盐（VPA）孤独症模型，系统研究经典Wnt信号通路活性及氧化应激变化，进而通过经典Wnt 信号通路或氧化应激干预研究上述干预对孤独症模型的效应。.本项目实验结果显示，.1. 经典Wnt信号通路活性增加及氧化稳态异常：与对照组相比，VPA组GSK-3β mRNA及β-catenin磷酸化表达降低，而β-catenin mRNA及GSK-3β磷酸化升高；同时，c-myc及cyclinD1 mRNA升高。氧化应激指标检测结果显示，与Control组相比，VPA组4-羟壬烯醛（4-HNE）升高，而硫氧还原蛋白（Trx）1、Trx2 mRNA降低。.2. 经典 Wnt 信号通路调控氧化稳态：舒林酸（sulindac）处理后，Western blot结果显示，与VPA组相比，VPA与sulindac同时处理组GSK-3β表达升高，而β-catenin 表达降低；同时，与Control组相比，VPA组GSK-3β表达降低，而β-catenin 升高。另外，VPA与sulindac同时处理组4-HNE表达低于VPA组，而VPA组 4-HNE表达高于Control组。用细胞培养同样证实，经典Wnt信号通路调控VPA处理的原代神经元氧化应激水平。.3. NAC改善孤独症样行为学异常并非通过经典Wnt信号通路：NAC处理后，旷场实验结果显示，与VPA组相比，VPA与NAC同时处理组重复呆板样行为持续的时间及次数均减少。比色法结果显示，与Control组相比，VPA组丙二醛（MDA）含量增加，而谷胱甘肽（GSH）减少。但与VPA组相比，VPA与NAC同时处理组MDA含量水平减少，而GSH增加。Western blot研究结果显示，与Control组相比，VPA组GSK-3β磷酸化及β-catenin胞浆与胞核水平表达升高，而β-catenin磷酸化水平表达均低于对照组，但与VPA组相比，VPA与NAC同时处理组β-catenin胞浆与胞核水平表达变化无统计学差异。 .以上提示，经典Wnt信号通路活性增加可能导致对孤独症易感性增加；经典 Wnt 信号通路调控氧化稳态影响孤独症发生，而舒林酸有可能成为临床治疗孤独症的靶药物。