siNgR1调控Notch通路干预巨噬细胞极化方向对心跳骤停后神经功能损伤的保护作用及机制

Survival rates for cardiac arrest (CA) patients, both in and out of hospital, are poor. Furthermore, survival without severe neurological impairment has not improved over the past few decades. Macrophages at the site of injury were traditionally suggested to have a negative role in secondary tissue degeneration, via the secretion of proinflammatory cytokines, as well as increased production of reactive oxygen and nitrogen species. Novel data show that only M1 macrophages are neurotoxic and M2 macrophages promote a regenerative growth response in adult sensory axons, even in the context of inhibitory substrates that dominate sites of CNS injury. However, the mechanism of the shift in macrophage polarization is unknown. Our team had shown Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage-associated genes. In addition, we had proved sNgR1-Fc can stimulate axonal regeneration via Notch signaling pathway in vitro, with promoted neuronal regeneration and functional recovery. Thus, we hypothesize that blocking NgR1 after CA, using siNgR1, might cause the change of phenotype and activities of macrophage in CNS via Notch pathway, limiting inflammation, preventing secondary degeneration, and promoting function repair. Understanding how to harness these macrophages holds great potential as a therapeutic approach to the degenerating CNS after CA.

心跳骤停(CA)的救治成功率始终偏低，严重的神经功能损伤是导致心脏复跳患者最终死亡的首位原因。既往观点认为巨噬细胞在CNS损伤过程中介导炎症反应、参与神经元二次损伤，目前研究证实是由M1型巨噬细胞导致，而M2型巨噬细胞具有趋化、吞噬神经元髓鞘碎片，发挥促进神经元生长和修复的作用，但调节巨噬细胞极化分型的具体机制不详。课题组前期研究发现拮抗髓鞘抑制蛋白受体NgR1能够调控Notch通路、调控组织局部的炎症反应程度并最终发挥抑制神经细胞凋亡促进轴突再生的作用；另一方面的研究则发现Notch途径可以调节巨噬细胞分型：在缺失Notch途径的巨噬细胞难以产生特定类型的M1细胞，而表现出低的炎性表型。本项目拟进一步探索siNgR1是否通过Notch通路调节巨噬细胞功能和极化分型- - 活化M2型巨噬细胞，发挥迅速清除髓鞘碎片、分泌营养因子和减轻局部过度炎症反应作用，探寻改善CA后神经功能的新方法。

心跳骤停(CA)的救治成功率始终偏低，严重的神经功能损伤是导致心脏复跳患者最终死亡的首位原因。既往观点认为巨噬细胞在CNS损伤过程中介导炎症反应、参与神经元二次损伤，目前研究证实是由M1型巨噬细胞导致，而M2型巨噬细胞具有趋化、吞噬神经元髓鞘碎片，发挥促进神经元生长和修复的作用，但调节巨噬细胞极化分型的具体机制不详。本课题组通过成功制备大鼠心肺复苏模型，通过构建热图谱对CA后CNS损伤导致的巨噬细胞极化分型以及重要分泌因子表达情况进行可视化分析，从而确定巨噬细胞与CNS 损伤之间存在直接关联，CA 造成对CNS损伤过程中存在 M1/M2巨噬细胞极化分型改变。采集CA后脑组织海马部位标本，并对该区域巨噬细胞 M1/M2相关蛋白表达情况以及分泌细胞因子的测定，以及 Notch 通路中关键因子，如 ATF、c-JUN、JNK 以及RHOA等的检测，验证巨噬细胞极化与Notch通路相关；采用iPS-MSCs干预后，损伤部位的海马组织 M1/M2极化情况及 Notch 通路相关因子显著下降。通过构建糖氧剥夺模型的体外试验也得到相似结果,证实 M1/M2 巨噬细胞极化参与了CA后的 CNS 损伤，这一过程通过Notch通路完成，并且采用 iPS-MSCs 干预，可以逆转CNS 的损伤，改善 CA 后脑损伤预后。