TLR信号通路调节因子ß-arrestin 2 对多巴胺能神经元的保护作用及机制研究

In recent years, major research efforts have focused on the neuroprotective interventions based on the pathogenesis of Parkinson’s disease (PD). Accumulating evidence has shown that microglial cell-mediated neuroinflammation plays a pivotal role in the onset and progression of PD, and Toll-like receptors (TLRs) are critical factors for the initiation of microglial activation. TRAF6 and its downstream molecule NF-κB, which are involved in the onset of PD, are important mediators in TLR-mediated proinflammatory signaling. ß-arrestin 2 is a ubiquitously expressed cytosolic protein. It has been demonstrated that ß-arrestin 2 exerts the ability of interacting with TRAF6 and NF-κB inhibitor alpha (IκBα), and consequently preventing activation of NF-κB. Our preliminary data have shown that after TLR stimulation, ß-arrestin 2 also binds with p38, which promotes p38 activation and subsequent expression of anti-inflammatory cytokine IL-10. Accordingly, we speculate that ß-arrestin 2 might exert a neuroprotective effect on dopaminergic neurons in PD by modulating TLR signaling and inhibiting microglial activation. In this project, we will employ laboratory techniques including Western blot, EMSA, coimmunoprecipitation, and gene transfection. We intend to assess whether ß-arrestin 2 could inhibit microglial activation and protect dopaminergic neurons in PD cellular and animal models, providing a novel relevant target for neuroprotective treatments of PD patients.

近年来，寻找针对帕金森病（PD）发病环节的神经保护剂是PD相关研究的热点之一。小胶质细胞介导的神经炎症在PD发生和发展中起重要作用，而TLRs是PD小胶质细胞活化信号启动的关键因子。TLRs活化后，可激活TRAF6及其下游NF-κB，参与PD发病。ß-arrestin 2是一种细胞内可溶性蛋白，能与TRAF6和IκBα结合，抑制NF-κB的活性。申请人前期研究发现ß-arrestin 2还可与TLR信号通路中p38结合并促使其磷酸化，诱导抗炎因子IL-10表达。据此我们设想：ß-arrestin 2可通过调节TLR信号通路，抑制小胶质细胞活化，对PD多巴胺能神经元发挥保护作用。本课题拟建立PD细胞模型和动物模型，采用Western blot、EMSA、免疫共沉淀、基因转染等技术，探讨ß-arrestin 2能否抑制PD小胶质细胞活化保护多巴胺能神经元，为PD神经保护治疗提供新线索。

近年来，寻找针对帕金森病（PD）发病环节的神经保护剂是PD相关研究的热点之一。小胶质细胞介导的神经炎症在PD发生和发展中起重要作用，而TLRs是PD小胶质细胞活化信号启动的关键因子。ß-arrestin 2是一种细胞内可溶性蛋白，对TLRs信号通路具有明确的调节作用。本研究将ß-arrestin 2高表达慢病毒(LV5-Arrb2)转染至原代培养的中脑小胶质细胞，LPS处理细胞，观察ß-arrestin 2对小胶质细胞活化的抑制作用。将高表达ß-arrestin 2的小胶质细胞与多巴胺能神经元共培养，观察ß-arrestin 2对多巴胺能神经元的保护作用。研究结果发现，ß-arrestin 2显著抑制了多巴胺能神经元的凋亡及细胞数目的减少；能显著抑制IL-1β、IL-6和TNF-α的转录和表达；降低小胶质细胞NO水平和iNOS的转录水平；抑制小胶质细胞内活性氧产生；抑制NF-κB 的 DNA 结合能力；通过免疫共沉淀法发现，ß-arrestin 2能与TRAF6结合。本研究结果提示了ß-arrestin 2对PD模型中炎症反应的抑制及多巴胺能神经元的保护作用，为PD的神经保护治疗提供新线索和新思路。