利福平调控小胶质细胞自噬抑制NLRP3炎症小体介导的神经炎症对多巴胺能神经元损伤的机制研究

Microglia-mediated neuroinflammation is acknowledged as one of the most important pathogenic factors in Parkinson's disease (PD). Our previous studies have found that rifampicin could protect dopamine neurons via inhibiting abnormal activation of microglia and reducing release of IL - 1 beta, but the mechanisms involved have not yet been fully elucidated. Our preliminary experimental results indicated that rifampicin suppressed activation of NLRP3 inflammasome induced by rotenone. And recent studies reported that cellular autophagy might inhibit activation of inflammasomes. Thus, we hypothesize that rifampicin inhibits NLRP3 inflammasome activation via mediating autophagy, thus leading to protection against injury of dopaminergic neurons induced by microglia-mediated neuroinflammation.. This project intends to adopt PD’s models induced by rotenone to clarify whether rifampicin inhibits pyroptosis and spread of inflammation resulting from NLRP3 inflammasome activation. Moreover, we will observe autophagy body formation using electron microscope and detect autophagy-related protein expression, such as beclin-1, p62, LC3 using immunohistochemical experiments to explore whether rifampicin inhibits NLRP3 inflammasome induced by rotenone via modulation of autophagy. We will find out the mechanisms involved in how rifampicin inhibits activation of NLRP3 inflammasome: whether rifampicin induces the formation of autophagy body to degrade components of inflammaomes directly or inhibit NLRP3 inflammasome activator-ROS generation via inducing autophagy, thus suppressing NLRP3 inflammasome indirectly. This study will provide new therapeutic targets for alleviating inflammatory damage to neurons, and offer new strategies for the prevention and treatment of PD.

小胶质细胞介导的神经炎症被公认为是帕金森病（PD）的重要致病因素。我们前期研究发现利福平可通过抑制小胶质细胞异常活化、减少IL-1β产生而保护多巴胺神经元，但具体机制尚未阐明。本课题预实验结果表明利福平可抑制鱼藤酮诱导的NLRP3炎症小体活化。而最近研究显示：细胞自噬可抑制炎症小体激活而避免过度的炎症反应。故我们猜测：利福平通过调控小胶质细胞自噬，抑制NLRP3炎症小体激活，而减轻PD中小胶质细胞炎症对多巴胺神经元的损伤。本研究拟采用鱼藤酮诱导的PD模型，阐明利福平是否通过抑制NLRP3炎症小体而减轻其所介导的细胞焦亡和炎症扩散；用电镜观察自噬体以及免疫组化方法检测beclin-1、p62、LC3等自噬相关蛋白的表达，阐明利福平是通过诱导自噬体降解炎症小体的直接通路还是通过激活线粒体自噬减少ROS产生的间接通路抑制炎症小体激活，为抑制帕金森病神经炎症提供新的治疗靶点，为PD防治提供新策略。

小胶质细胞介导的神经炎症被公认为是帕金森病(PD)的重要致病因素。所以使用安全有效的药物调控小胶质细胞炎症可能是防治PD的有效手段。我们猜测:利福平通过调控小胶质细胞自噬，抑制NLRP3炎症小体激活，而减轻PD中小胶质细胞炎症对多巴胺神经元的损伤。本研究项目采用鱼藤酮诱导的PD模型,主要进行以下研究：用PCR和WB检测炎症小体信号通路的表达，用DCFH-DA法和罗丹明123法检测细胞内ROS的产生和线粒体膜电位的改变，说明利福平通过抑制小胶质细胞NLRP3炎症小体信号通路，抑制其对多巴胺能神经元的炎症损伤。用电镜观察自噬体和线粒体以及免疫组化方法检测p62、LC3等自噬相关蛋白的表达，发现利福平通过调控线粒体自噬，减少ROS产生,从而抑制鱼藤酮诱导的NLRP3炎症小体激活。最后，用mRFP-GFP-LC3腺病毒载体法观察自噬流情况以及使用溶酶体荧光染色观察溶酶体功能，发现利福平可能主要通过恢复溶酶体功能而改善鱼藤酮引起的自噬流不畅。本研究为抑制帕金森病神经炎症提供新的治疗靶点,为PD防治提供新策略。