NLRP3炎性小体在缺血缺氧后神经细胞necroptosis信号通路中的机制研究

With the speed of population aging being accelerated, the incidence and prevalence of cerebrovascular diseases show an increased trend year by year. The present studies found that necroptosis, the manner of cell death mediated by RIP1-RIP3, is one of the mechanism of delayed ischemic brain injury, and can be used as a new target to protect the nerve which would expand the treatment time window. NLRP3 inflammasome, which is a hot spot in the inflammatory diseases, have been found to be also involved in the pathogenesis of ischemic reperfusion in recent studies, yet whether it is related to the mechanism of neurons necroptosis is unclear. Our previous study found that the expression of NLRP3 is increased in oxygen-glucose deprivation-induced necroptosis of cortical neurons.On the basis of previous studies, this subject will focuse on the mechanism of necroptosis after oxygen- glucose deprivation from the cells and animal levels, and further explore the relationship between cell death mediated by NLRP3 and necroptosis through biochemistry, histochemistry, molecular biology methods, then explore whether oxidative stress related compound INrf2/Nrf2 is involved in its regulation, in order to provide a theoretical basis for the study of mechanism of necroptosis after cerebrovascular diseases and targets to protect damaged nerves.

随着人口老龄化速度的加快，脑血管病发病率和患病率呈逐年增高趋势。目前研究发现RIP1-RIP3介导的死亡方式necroptosis是迟发的缺血性脑损伤机制之一，可以作为扩大治疗时间窗的神经保护新靶标。NLRP3炎性体是炎性疾病的研究热点，新近研究发现它也参与了缺血再灌注的发病机制，而其是否与神经元细胞necroptosis的发生机制相关尚不明确。我们的前期研究发现缺糖缺氧诱导的大鼠皮质神经元细胞necroptosis中NLRP3表达增加。在既往研究基础上，本课题将从细胞水平、动物水平对缺糖缺氧后necroptosis 的发生机制进行研究，用生物化学、组织化学、分子生物学等方法探讨由NLRP3介导的细胞死亡与necroptosis的关系，及氧化应激相关的INrf2/ Nrf2复合物是否参与其调节。从而为脑血管病后细胞necroptosis死亡机制以及神经损伤保护靶点的研究提供理论基础。

目前脑血管病发病率和患病率呈逐年增高趋势。急性期的有效处理可以显著降低死亡率和致残率，Necroptosis是迟发的缺血性脑损伤机制之一，NLRP3炎性体是炎性疾病的研究热点，新近研究发现它也参与了缺血再灌注的发病机制，而其是否与神经元细胞necroptosis的发生机制相关尚不明确。本实验应用广谱caspase抑制剂Q-Vd-Oph，缺糖缺氧诱导原代皮质神经元及大脑中动脉缺血诱导皮质神经元necroptosis，结果提示该死亡方式与NLRP3炎性体表达存在相关性。原代皮质神经元细胞通过应用ROS激活剂H2O2和抑制剂NAC，激光共聚焦观察ROS水平变化与TXNIP、NLRP3炎性体表达的相关性，结果显示ROS促进了TXNIP与NLRP3的结合，并导致坏死过程中NLRP3炎症激活。通过Nrf2基因敲减，western blot检测，HO-1的表达量呈下降趋势，而TXNIP的表达量呈上升趋势；扩增Nrf2后，HO-1、TXNIP蛋白相反变化趋势；随后应用了HO-1的激活剂Hemin及抑制剂Znpp，应用免疫沉淀检测发现Nrf2/HO-1通过调控TXNIP与NLRP3的结合，从而调控NLRP3炎性体在necroptosis信号通路中的表达，且该调节Nrf2/HO-1是通过调控ROS水平的变化实现的。此外我们在大鼠动物在体水平也进一步验证了上游蛋白Nrf2可以负调节ROS水平，从而负向调控NLRP3炎性体活性。综上，本课题从细胞及在体水平验证了Nrf2/HO-1 通过调控TXNIP 与NLRP3 的结合，从而调控NLRP3炎性体在necroptosis 信号通路中的表达。因此NLRP3炎性体可以作为急性脑血管病扩大治疗时间窗的新靶标。