CBS-H2S硫巯基化修饰caspase-1对小胶质细胞NLRP3炎症小体的调控及其在帕金森病炎症中的作用

Chronic neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD) and is considered to be the key contributor to the death of dopaminergic neurons in the substantia nigra. Activation of microglial NLRP3 inflammasome is a critical event in PD-related inflammatory cascade. In previous studies, we found that both hydrogen sulfide (H2S) and overexpression of its synthase cystathionine beta synthase (CBS) inhibited the NLRP3 activation and IL-1β production, while cbs knockdown increased NLRP3 protein expression in microglia. Moreover, CBS expression in the midbrain of PD mice and microglia was reduced. These data indicate that CBS-H2S system regulates microglia NLRP3 inflammasome activation and may be involved in the pathogenesis of PD inflammation. In addition, inflammasome-related protein caspase-1 is rich in cysteine residues in its active site. Thus, we propose that H2S may inhibit inflammasome activity via S-sulfhydration modification of the cysteine residues of caspase-1. Therefore, based on these data, this project will use microglia specific cbs knockout mice and a variety of other approaches to study the role and underlying mechanisms of CBS-H2S in NLRP3 inflammasome activity and to clarify its function in PD-related neuroinflammation, from the molecular, cellular and animal levels. The achievements of this study will be helpful for identifying the role of CBS in PD -related inflammation.

神经炎症在帕金森病(PD)发病机制中占有重要地位，是黑质多巴胺神经元死亡关键机制。小胶质细胞NLRP3炎症小体激活是PD炎症级联反应核心事件。既往研究我们发现硫化氢(H2S)是重要抑炎分子，并对PD鼠发挥保护作用，但具体参与分子及相关调控机制尚不清楚。近期我们发现，H2S及其合成酶胱硫醚-β-合成酶(CBS)过表达均抑制NLRP3炎症小体激活及IL-1β等生成，敲减cbs时NLRP3表达增加；PD小鼠中脑和小胶质细胞CBS表达降低，提示CBS-H2S可能调控小胶质细胞炎症小体及PD炎症。炎症小体蛋白caspase-1活性位点富含半胱氨酸残基，因此我们推测H2S经硫巯基化修饰caspase-1而抑制炎症小体活性。本项目拟在此基础上，利用小胶条件性cbs敲除鼠等多种资源与技术，从分子、细胞到动物水平研究CBS-H2S对小胶质NLRP3炎症小体的调控作用与机制，揭示CBS在PD炎症中的作用。

神经炎症是帕金森病(PD)病理过程中黑质多巴胺神经元死亡的重要机制之一，而小胶质细胞NLRP3炎症小体激活是PD相关神经炎症的核心事件。既往我们及国内外多个团队报道硫化氢(H2S)是内源性炎症抑制分子，适量地吸入H2S气体或H2S释放剂在多个PD动物及细胞模型中发挥保护作用，但具体的细胞与分子机制尚未完全阐明。在本课题资助下，我们研究发现多种病理性刺激（LPS、α-Syn）后、以及衰老小鼠脑内小胶质细胞CBS转录表达降低，与细胞因子表达增加呈负相关。构建小胶质细胞CBS条件性敲除小鼠Cbsflox/flox；Cx3cr1CreER，通过动物及细胞培养等实验发现CBS是小胶质细胞甚至脑内调控氧化还原稳态的关键分子，CBS表达或活性降低是小胶质细胞对炎症刺激易感性增加的基础。CBS敲减细胞中线粒体ROS水平增加，而CBS激动剂及H2S释放剂均抑制ATP等诱导的线粒体ROS生成。而且，体内外实验表明CBS过表达或活性增加及H2S释放均能通过抑制线粒体ROS而减轻小胶质细胞NLRP3炎症小体激活、IL-1β生成，缓解炎症诱导的多巴胺能神经元损伤缺失。综上，本课题揭示了CBS-H2S对小胶质细胞NLRP3炎症小体的抑制作用及分子机制，为PD治疗提供了新靶点，完成了课题研究目标，相关成果已发表SCI论文5篇，尚有部分成果正在修稿中。基于这上述研究，我们提出提高小胶质细胞CBS表达及活性有望成为PD药物研发的策略之一。团队将继续以减缓神经炎症和PD病理为目标、寻找提高CBS表达或活性的小分子化合物。