α7-nAChRs对脑内免疫细胞的调控及其与帕金森病的相关性研究

Parkinson's disease (PD) is the second most common neurodegenerative disease, after Alzheimer's disease. The potential causes of PD remain uncertain but recent studies suggest neuroinflammation and glia activation play important roles in PD pathogenesis. This inflammatory response initiates a cascade of events resulting in the concentration and activation of innate immune cells at the site of tissue injury. The release of immunoactive substances such as cytokines, neurotrophic factors, and chemokines initiate local actions and can result in a more generalized immune response. The resultant neuroinflammatory environment can cause activation of glial cells located in the brain, which appear to play a prominent role in PD. Recently considerable evidence suggest that the prevalence of PD is lower in smokers than in non-smokers, the mechanisms of nicotine-induced neuroprotection remain unclear. Recent studies suggest that the nicotinic anti-inflammatory pathway may have important clinical implications, as treatment with nicotinic agonists can potentially modulate the production of pro-inflammatory cytokines from immune cells via interactions at α7-containing nicotinic acetylcholine receptors (α7-nAChRs). Thus, α7-nAChRs may serve as a crucial link between inflammation and neurodegeneration in PD and could represent a pharmacological target for potential induction of neuroprotection. Our previous data have demonstrated that systemic administration of nicotine alleviated MPTP-induced behavioral symptoms, improved motor coordination, protected against dopaminergic neuron loss and activated astrocytes and microglia in the substantia nigra in the acute and chronic PD mouse model. In mouse primary cultured astrocytes, pretreatment with nicotine suppressed MPP+- or LPS-induced astrocyte activation evidenced by both decreased production of TNF-α and inhibition of Erk1/2 and p38 activation in astrocytes, and the protective effects of nicotine were abolished by the α7-nAChR-selective antagonist methyllycaconitine (MLA). Those data suggest that α7-nAChR-mediated inhibition of astrocyte activation is an important mechanism underlying the protective effects of nicotine. Therefore, the objective of the present study was to determine whether nicotine, acting at α7-nAChRs, could regulate immunocyte-mediated disfunction, and may provide some insight into changes in cholinergic signaling associated with PD pathogenesis and present valuable information for new drug therapies. We test hypothesis about α7-nAChRs providing a new therapeutic strategy for treatment of neurological disorders. Nicotinic agonists that specifically target the α7-nAChR might be developed and serve as potential therapeutic agents for PD and other neuroinflammation-related central nervous system diseases.

帕金森病（Parkinson's disease, PD）病理生理机制复杂，免疫炎症反应学说一直是PD主要病理机制研究和关注的热点。流行病学统计显示，吸烟者患PD的概率远低于不吸烟者，nAChRs或与之相关。α7-nAChRs在胆碱能抗炎通路中发挥关键作用，该通路被认为是机体最重要的神经免疫抗炎机制之一。我们的预实验结果提示烟碱可通过激活α7-nAChRs抑制小鼠急、慢性PD模型黑质区星形胶质细胞、小胶质细胞的活化，阻止多巴胺神经元的损伤，其机制与MAPK信号通路有关。因此，我们提出假说："α7-nAChRs激动剂可对脑内免疫细胞功能调控，从而在PD中发挥神经保护作用"。为了验证这一假说，本项目拟在前期研究工作基础上，应用制备的α7-nAChRs敲除鼠, 在整体、细胞和分子水平研究、阐明α7-nAChRs对脑内免疫细胞功能的调控作用及其机制，为探索靶向特定烟碱受体的调节剂、防治PD提供学术依据。

帕金森病（Parkinson's disease, PD）是发病率仅次于老年痴呆症的神经系统退行性疾病，但对其确切的病因机制目前仍不清楚。免疫炎症反应学说作为PD的主要机制成为近年来研究和关注的热点。星形胶质细胞，小胶质细胞等脑内主要免疫细胞可能为PD的神经保护治疗提供新策略。α7-nAChRs（α7-Nicotinic acetylcholine receptors, α7-nAChRs）在“胆碱能抗炎通路”中发挥关键作用，α7-nAChRs是免疫神经调节中的重要环节，在整体、细胞和分子水平系统研究α7-nAChRs对PD脑内免疫细胞的调节机制，阐明α7-nAChRs在PD发生、发展中的作用，研究成果不仅有助于深化对PD病理机制的认识，也为研发理想的多潜能神经保护剂提供新的靶标。首先，我们培养小鼠原代中脑星形胶质细胞，并外源性给予过氧化氢（H2O2）、烟碱和甲基牛扁亭（methyllycaconitine, MLA）刺激，发现H2O2可浓度依赖性地促进原代星形胶质细胞凋亡，应用烟碱预保护有效地抑制了H2O2诱导的星形胶质细胞凋亡，该作用可被 α7-nAChRs 特异性拮抗剂 MLA取消，提示烟碱抑制H2O2诱导的星形胶质细胞凋亡主要是通过 α7-nAChRs调控的。其次，应用野生型（Wildtype, WT）小鼠建立慢性 MPTP/p PD小鼠模型，在体给予烟碱和MLA，在整体动物阐明烟碱可通过α7-nAChRs调节星形胶质细胞功能，发挥DA能神经元的保护作用，减轻MPTP所诱导的小鼠运动功能障碍。第三，进一步探讨PD发病机制中α7-nAChRs的相关信号转导机制，烟碱能够通过Wnt/β-catenin信号通路改善MPTP诱导的运动功能障碍、多巴胺神经元的死亡、星形胶质细胞的激活、小胶质细胞的激活和纹状体多巴胺含量及转运体的减少。烟碱也可以通过Wnt/β-catenin信号通路改善由MPP+损伤的SH-SY5Y细胞，并且烟碱的这种治疗作用能够被选择性α7-nAChR拮抗剂MLA或siRNA消除。最后，进一步探讨α7-nAChRs对下丘脑 POMC 神经元兴奋性和异质性突触重塑的影响，为探索α7-nAChRs在PD患者可能出现的血糖紊乱的下丘脑调节机制中的作用打下学术基础。