课题组前期研究证实心脏停搏心肺复苏在激活NADPH氧化酶同时，也激活抗氧化（天然免疫）信号Nrf2。免疫功能失调是心脏骤停后综合征的核心致病机制之一。NADPH氧化酶/ROS信号通路,Keap1/Nrf2/H0-1天然免疫信号通路,TLR4/MyD88/NFκB炎症信号通路三者之间的相互作用对复苏后免疫内环境的影响尚不明确。本项目应用Nrf2基因敲除小鼠TLR4基因敲除小鼠复制心肺复苏模型，应用SiRNA技术分别沉默表达p47,TLR4,Nrf2,Keap1建立缺氧/复氧巨噬细胞模型，观察炎症/抗炎，氧化/抗氧化，免疫功能，比较上述三条信号通路的激活，以及Nrf2介导的抗氧化信号通路调控氧化应激和炎症的可能性。期望解析 keap1/Nrf2/HO-1在心脏骤停后综合征从SIRS- Sepsis-MODS进行性发展中的调控机制，为临床在心肺复苏后重建免疫稳态提供新的治疗思路。

In the previous study from our group has shown that NADPH oxidase complex- dependent reactive oxygen species (ROS) play crucial roles in the systemic inflammatory response syndrome after cardiac arrest. Ox-LDL/LOX-1 and NADPH oxidase / ROS form a positive loop that continuously induces the inflammatory response. Most of important, this systemic ischemia- reperfusion injury activates both the NADPH oxidase-dependent reactive oxygen species (ROS) and the Keap1/Nrf2/H0- 1 antioxidant (innate immunity) signaling pathway. We speculate that activation of Nrf2 is one of the regulatory mechanisms of the inflammatory response that helps to limit inflammation. To date, it is unclear how to cross-talk among NADPH oxidase / ROS signaling pathway, Nrf2/ARE/H0-1 signaling pathway, and TLR4/MyD88 / NF-κB signaling pathway during the pathogenesis of PCAS(post cardiac arrest syndrom) . The interaction among these three signaling pathways effect on immunization homoeostasis is either unknown. To address them, we tend to investigate the effects of short interfering RNA (siRNA)-mediated Keap1, or Nrf2, or p47phox, or TLR4 knockdown on inflammation, oxidant, apoptosis, and immune function in the anoxia-reoxygenation cell model of RAW 264.7 murine macrophages. Further, we will observe the incidence of SIRS, MODS, mortality, dysfunction of vital organs after ROSC in a cardiac arrest model of Nrf2-/ - mice(on the background of C57BL/6). In this study, we focus on the upstream signaling events responsible for the activation of Nrf2/H0-1 signaling pathway and its positive or negative feedback to the NADPH oxidase / ROS signaling pathways, and the TLR4 / MyD88 / NF-κB signaling pathway. Together, this research will demonstrate deeply the regulatory mechanism of Keap1/Nrf2/H0-1 signaling pathways in cardiac arrest syndrome from SIRS - Sepsis- MODS, and provide new ideas and strategies for rebuilding the immune homeostasis after cardiopulmonary resuscitation.

本课题组建立标准化的小鼠心脏骤停心肺复苏模型，完成氧化/抗氧化, 炎症/抗炎症，免疫稳态以及相关信号通路的研究；完成小鼠单核巨噬细胞在缺氧/复氧条件下，炎症因子表达，线粒体自噬，细胞焦虑性死亡对免疫内稳态影响的研究。以细胞膜上的Toll受体为切入点，探讨了TLR4/MyD88/IRAK/NF-κB 炎症反应信号通路与Keap1/ Nrf2/ HO-1 抗氧化信号通路二者对免疫内稳态的影响；以位于胞质内的NLRP3模式识别受体为切入点，探讨了mtROS/ NLRP3 inflammasome / Caspase-1 / mtROS与细胞自噬之间的调控机制对免疫内稳态的影响。 本课题组已发表的论著和研究证实NADPH氧化酶/ROS 与 oxLDL/LOX-1信号通路形成一个正向反馈系统，不断放大氧化应激信号和包括LOX-1在内的多种炎症信号分子，形成瀑布样炎症反应。心肺复苏初期激活NADPH氧化酶依赖性ROS的同时，也激活体内最重要的抗氧化信号通路Nrf2/ARE/H0-1。激活Nrf2是机体炎症反应的监管机制。本课题组已完成的论著和研究证实，系统性缺血再灌注损伤线粒体，释放大量线粒体活性氧 (mtROS)，启动二个路径，①线粒体损伤→启动线粒体自噬→修复损伤；②线粒体损伤→激动NLRP3 inflammasome炎症复合体→触发Caspase-1依赖性线粒体损伤→阻止线粒体自噬→释放更多DAMPs→加重细胞焦亡（pyroptosis）。线粒体自噬和NLRP3炎症小体之间存在着负调节关系，我们已完成的研究证实免疫功能正常的C57BL/6小鼠在心肺复苏早期及时启动线粒体自噬，所以抑制NLRP3炎症小体持续高表达。NOD/SCID小鼠在心肺复苏早期没有正确启动线粒体自噬，致使线粒体ROS→NLRP3炎症小体→Caspase1→线粒体ROS这一信号环路持续扩增应激信号，持续扩大炎症反应，导致Caspase 1依赖性细胞焦亡。 本课题组由急诊医学，重症医学，麻醉医学领域的科研技术人员组成，是一个在心肺脑复苏领域长期从事临床和科研工作的学术团队。通过本项目的实施深入剖析了心脏停搏后综合征（PCAS）从心脏骤停后全身炎症反应综合征（CA-SIRS）到心脏骤停后多脏器功能衰竭（CA-MODS）进行性发展的机制，为心肺复苏后重建免疫稳态积极寻找新的治疗靶点和策略。