冷诱导RNA结合蛋白介导氧化应激促进脑缺血再灌注损伤的机制研究

The Mechanism of CIRP Mediating Oxidative Stress to Promote Cerebral Ischemia-Reperfusion Injury

摘要: 目的：探究冷诱导RNA结合蛋白(cold-inducible RNA-binding protein, CIRP)是否能通过TLR4/MyD88途径介导氧化应激促进脑缺血再灌注损伤的发生发展过程，目前尚无报道。本研究将为脑缺血再灌注损伤的治疗提供新的靶点，亦为临床急性缺血性脑卒中(acute ischemic stroke, AIS)患者术后缺血再灌注损伤的治疗应用提供了理论依据。方法：首先通过检测AIS患者术后外周血、构建HMC3细胞缺氧/复氧模型，观察缺血再灌注损伤对CIRP表达水平的影响；其次通过培养SH-SY5Y细胞，使用人重组CIRP蛋白(Recombinant human CIRP protein, rhCIRP)及C23干预，观察CIRP的表达及氧化应激水平。最后利用慢病毒敲减HMC3细胞的CIRP基因，再次构建细胞缺氧/复氧模型，探究CIRP通过TLR4/MyD88途径激活NADPH氧化酶，释放ROS上调Drp-1水平，进而促进线粒体分裂的分子机制。结果：AIS患者脑缺血再灌注后血清CIRP表达较术前显著升高。在人小胶质细胞HMC3缺氧/复氧模型中，细胞内CIRP表达水平显著升高且8小时后CIRP表达水平达到巅峰，并分泌到细胞外，激活炎症因子TNF-α、IL-6表达。rhCIRP可以作用于SH-SY5Y细胞促进TLR4、GP91、P47、DRP-1、MFN-2、Cleaved-caspase-3、TNF-α、IL-6的表达，介导氧化应激。而使用C23和敲减CIRP后则可以抑制上述蛋白和炎症因子的表达，保护组织细胞。结论：缺血再灌注损伤使小胶质细胞释放大量CIRP，分泌到细胞外，TLR-4/MyD88通路介导氧化应激，引起神经细胞缺血再灌注损伤。

Abstract: Objective: To explore whether CIRP can promote the occurrence and development of cerebral ischemia-reperfusion injury through TLR4/MyD88 pathway through oxidative stress, there is no report at present. This study will provide a new target for cerebral ischemia-reperfusion injury, and also provide a theoretical basis for the treatment and application of ischemia-reperfusion injury after acute ischemic stroke surgery. Methods: The effect of ischemia reperfusion injury on the expression of CIRP was observed by detecting the peripheral blood of patients with acute ischemic stroke after operation and constructing the hypoxia/reoxygenation model of HMC3 cells. Secondly, SH-SY5Y was cultured, and human recombinant CIRP protein (rhCIRP) and CIRP inhibitor (C23) were used to observe the level of CIRP and oxidative stress. Finally, the CIRP gene of HMC3 cells was knocked down by lentivirus, and the cell hypoxia/reoxygenation model was constructed again to explore the molecular mechanism of CIRP activating NADPH oxidase through TLR4/MyD88 pathway, releasing ROS to up-regulate the level of Drp-1, and promoting mitochondrial division. Results: The expression of serum CIRP in AIS patients after cerebral ischemia reperfusion was significantly higher than that before operation. In the hypoxia/reoxygenation model of human microglial cell HMC3, the expression level of CIRP in the cell increased significantly and reached the peak after 8 hours, and secreted to the outside of the cell to activate the inflammatory factor TNF-α, IL-6 expression. rhCIRP can be used as SH-SY5Y cells to promote TLR4, GP91, P47, DRP-1, MFN-2, Cleved-caspase-3, TNF-α, IL-6 expression, and mediate oxidative stress. C23 and knockdown of CIRP can inhibit the expression of the above proteins and inflammatory factors, and protect the tissue cells. Conclusion: Ischemia reperfusion injury causes microglia to release a large amount of CIRP, which is secreted into the extracellular space. The TLR-4/MyD88 pathway mediates oxidative stress, leading to neuronal ischemia-reperfusion injury.