a7nAchR/miR210信号通路在中枢胆碱能抗炎通路调控免疫性炎症的作用及机制研究

Immune abnormal mediated inflammation may deteriorate dopaminergic neurons damage of Parkinson's disease (PD). However, effective strategies are absent currently. It has been reported that an endogenous cholinergic anti-inflammatory pathway existed in brain, similar to that in the peripheral system, which was charactered by activation of a7 nicotinic acetylcholine receptor (a7nAchR). a7nAchR and miR210 are also associated with inflammation and T cell differentiation, respectively. In our previous studies, we found that activation of a7nAchR within the brain could upregulate the expression of miR210, suppress immune-inflammatory response and reduce neuron death.T cell differentiation is closely associated with neuroinflammation.Therefore, we speculated that miR210 might participate in the process that the central cholinergic anti-inflammatory pathway regulated the differentiation of T cells, and reduced the immune-inflammatory injury of PD. In this study, we adopted the 6-hydroxydopamine to make the PD models, also cultivated CD4+ T cells and induced its differentiation in vitro. We investigated the effectiveness of the cholinergic anti-inflammatory pathway on the regulation of immune-inflammation from the whole, tissue, cell and molecular levels, which would provide new direction and new target for PD treatment.

免疫功能异常介导的炎症反应可加重帕金森病（Parkinson's disease，PD）多巴胺能神经元损伤，目前仍缺乏有效调控措施。研究发现脑内存在类似外周的胆碱能抗炎通路，并且a7烟碱型乙酰胆碱能受体（a7 nicotinic acetylcholine receptor,a7nAchR）、miR210与炎症、T淋巴细胞分化相关；我们前期研究发现激活脑内细胞表面a7nAchR可增加miR210表达，同时抑制缺血后炎症反应，减少神经元凋亡。T淋巴细胞分化与神经炎症密切相关。因此，我们推测miR210参与中枢胆碱能抗炎通路调控T淋巴细胞分化，减少PD免疫性炎症损伤。本实验采用6-羟基多巴胺诱导PD模型及体外CD4+T淋巴细胞分化培养模型，从整体、组织、细胞水平改变a7nAchR、miR210表达研究中枢胆碱能抗炎通路调控免疫性炎症反应及分子机制，为PD治疗提供新方向和靶点。

帕金森病 (Parkinson’s disease, PD) 是目前世界第二大神经退行性疾病，给家庭和社会带来沉重经济负担。PD发病机制复杂，免疫性炎症反应加重PD进程，但确切发病机制仍不明。本课题以 a7nAchR/miR210 信号通路为切入点，采用脑立体定位注射6-OHDA构建PD大鼠模型，旋转实验筛选成功模型，进行迈步实验、转棒实验等行为学检测，发现PD大鼠较正常对照组大鼠迈步时间明显延长(p < 0.05)，转棒停留时间缩短(p < 0.05)，给予PD组大鼠电刺激迷走神经耳支（auricular vagus nerve stimulation，aVNS）（激活脑内中枢胆碱能抗炎通路）干预后，迈步时间缩短( p < 0.05)，转棒停留时间延长( p < 0.05)，初步证实激活中枢胆碱能抗炎通路对PD大鼠行为学改善有一定作用。免疫组化方法定量检测各组大鼠中脑黑质区酪氨酸羟化酶 (Tyrosine hydroxylase，TH)阳性细胞数。发现PD大鼠中脑TH阳性细胞数较正常组明显减少( p < 0.05)， aVNS干预后，TH阳性细胞数增加( p < 0.05)。Elisa法检测大鼠中脑炎性因子表达（TNF-a、IL-1β），提示激活脑内中枢胆碱能通路可以明显降低PD中脑炎性因子水平。免疫荧光双染色法检测大鼠中脑免疫细胞 CD4+/Th17 和 CD4+/Treg 细胞，发现PD大鼠中脑存在CD4淋巴细胞亚群浸润，Th17增加，aVNS治疗组Th17细胞数下降，Treg细胞数增加，证实PD中脑存在CD4+T 淋巴细胞浸润且失衡现象。沉默miR210表达PD大鼠转棒停留时间较PD组缩短，平衡木通过时间延长(p < 0.05)。中枢胆碱能抗炎通路神经保护作用减弱，炎性因子抑制作用减弱，CD4亚型分化漂移，提示miR210介导中枢胆碱能抗炎通路对PD免疫炎症调控作用。体外无菌分离小鼠脾脏细胞，免疫磁珠分离CD4 T细胞，miR210 inhibitor干预，发现miR210抑制后HIF-1a、Foxp3蛋白表达较对照组下降，RoRγt表达上升（p < 0.05），流式检测发现Foxp3、RoRγt数量变化趋势一致，证实miR210调控CD4亚型分化，有望成为 PD 治疗提供新视角和新靶点。aVNS具有无创性、简易性，为其临床应用提供一定基础研究和依据。