NLRP3-inflammasome介导的细胞焦亡在脑出血神经元继发性损伤的作用和机制研究

Secondary injury of neurons is an important part of brain injury in intracerebral hemorrhage, in which hemin induced neuronal death is involved, but the regulatory mechanism of hemin induced neuron cell death remains to be clarified. We previously found that hemin could induce neuron cell death through a necroptosis pathway rather than Caspase-3-mediated apoptosis. Our preliminary experiments further showed that hemin also could induce the expression of NLRP3 inflammasome in neurons, and inhibit the expression of NLRP3-Caspase1 mediated neuron pyroptosis could alleviate neuron cell death by blocking necrosome activation, we thus speculate that this process is regulated by the "NLRP3-IRAK1-TRAF6-TAK1" axis. In this study, we will clarify the mechanism of NLRP3/capase1 mediated pyroptosis in modulating necroptosis activation by using biochemical molecular biology, pharmacology and gene silencing methods at the levels of clinical specimens, animal model and in vitro cultures. Our results may shed light on the molecular network between inflammatory response and necroptosis, this study will provide new theoretical basis and therapeutic targets for clinical prevention and treatment of intracerebral hemorrhage.

神经元继发性损伤是脑出血后引起脑组织损伤的重要环节，血红素所诱导的神经元死亡参与其中，但该细胞死亡方式的调控机制有待阐明。我们前期发表的文章表明血红素诱导神经元死亡的方式是通过程序性坏死而不依赖于Caspase3介导的细胞凋亡。在此基础上我们进一步预实验发现，血红素可诱导神经元NLRP3炎症体、IRAK1、TAK1及坏死相关蛋白表达上调，阻断NLRP3-Capase1依赖的细胞焦亡可阻抑神经元程序性坏死。因此，我们推测：血红素可能通过激活NLRP3-IRAK1-TRAF6-TAK1轴，导致继发性脑损伤。本项目拟在临床标本、动物水平及体外细胞三个层次通过生化分子生物学、药理学和基因干预等方法深入研究NLRP3-Capase1依赖的细胞焦亡对神经元程序性坏死的调节机制，研究结果将可能揭示炎症反应-程序性坏死互相调控的分子网络，从而为脑出血的临床诊治提供新的理论依据和治疗靶点。

脑出血后神经元继发性损伤严重影响患者不良预后，我们前期工作发现程序性坏死参与血红素诱导的神经元死亡。本项目进一步进行扩展，NOD样受体作为胞内危险模式相关分子受体，在临床标本结合体内外实验中发现NLRP3依赖的炎症反应同样参与神经元损伤。进一步通过机制研究发现:1 NLRP3 依赖的细胞焦亡和necrosome 依赖的程序性坏死互为调控关系2. NLRP3通过与IL1R1偶联，激活下游IRAk1 和IRAK4，IRAK1/4 与RIPK1/3 直接互作，调控necrosome的活化，另一方面IRAk1 通过TRAF6—TAK1 调控NFκB的激活，调控炎症反应。基于以上探索，我们证实了NLRP3在脑出血时作为细胞内感受器能感受危险模式相关分子刺激活化，并作为细胞内外信息传导的枢纽，通过下游信号传导通路调控程序性坏死的激活，可能成为调控脑出血神经元细胞死亡的重要靶点之一。