apoE短肽在蛛网膜下腔出血后早期脑损伤的神经保护作用及机制研究

Early brain injury (EBI) refers to immediate injury to the brain before the development of vasospasm during the first 72h after subarachnoid hemorrhage (SAH). EBI has been pointed to be the primary cause of mortality and disability in SAH patients. However, its mechanism is not completely clear and there is still not a definitive treatment to prevent EBI after SAH. Previous studies have shown that apolipoprotein E (APOE = gene, apoE = protein) polymorphisms play an important role in early responses to traumatic brain injury and COG1410, a synthetic peptide derived from the apoE receptor binding region has anti-inflammatory effects after experimental traumatic brain injury. Notably, APOEε4 allele is also a risk factor to brain function aggravation in the early stage of aneurysmal SAH, and it may contribute to EBI after SAH. Thus we hypothesize that apoE peptide could result in suppression of microglia activation, further lead to reduce inflammatory mediators, which generate a protective action in early responses after SAH. To prove the hypothesis, this study consists of three parts. 1. Experimental SAH was induced in APOE-targeted replacement mice, APOE knockout mice and wild type mice. For 72h following injury, the effects of 1mg/kg COG1410 versus saline administered intravenously starting 30 min were evaluated and daily functional assessments were made. Mice were then sacrificed at each time point (6h, 12h, 24h, 48h and 72h). The transcription and expression level of certain inflammatory factors in mice brain were examined by fluorescence quantitive RT-PCR and ELISA at different time points. Moreover, the state of activation of microglia and astroglia were determined by immunofluorescence and histochemistry in cortex and hippocampus region. And BBB permeability and brain edema were also examined. 2. To determine if apoE peptide could direct suppress inflammatory reaction in early stage after SAH instead of protect neuron directly, an anoxia and reoxygenation (A/R) model was established using cultured primary hippocampal neurons which derived from 18d embryonic mice. MTT assay method and LDH releasing rate were used to measure the cell viability, which evaluated the cellular injury. 3. To identify that apoE peptide could effect suppress microglia activation and to explore its molecular mechanism, microglia activation cell model was induced by lipopolysaccharide (LPS) in vitro. CD11b expression was observed by flow cytometry. IL-1β, TNF-α, IL-6 and IL-10 were examined by ELISA. Furthermore, the expression of TRL-4, p38 MAPK, ERK, and Akt/IκBa were determined by western blot. The current project is significant to reveal the neuroprotective effect and mechanism of apoE peptide under specific pathological conditions and offer new ideas for development of effective brain injury protection drugs.

近期研究表明，蛛网膜下腔出血（SAH）后继发的早期脑损伤（EBI）已成为其主要的致死或致残因素，但病理机制尚未阐明，治疗效果仍不理想。我们前期创伤性脑损伤的研究表明APOE亚型早期即可影响伤情转归及预后；早期应用apoE短肽（COG1410），可通过抑制炎症反应发挥神经保护作用。值得关注的是，APOE亚型与SAH后继发的EBI也存在相关性。据此我们提出，apoE短肽在SAH中早期神经保护作用的评价及其临床药物开发转化的关键是在阐明APOE亚型与EBI相关性的基础上，通过SAH在体模型及神经元离体损伤模型明确apoE短肽的神经保护作用与其抗炎症反应特性直接相关；并进一步采用小胶质细胞活化的体外模型，从表面标志物的表达、炎症因子的释放及相关信号通路的活化等三方面来证实其抗炎症反应特性及其具体通路。结合前期研究基础，本课题对于揭示apoE短肽在特定病理条件下的神经保护作用及其机制具有重要意义。

研究表明，蛛网膜下腔出血（SAH）后继发的早期脑损伤（EBI）已成为其主要的致死或致残因素。课题组前期创伤性脑损伤的研究已表明APOE亚型早期即可影响伤情转归及预后；早期应用apoE短肽（COG1410），可通过抑制炎症反应发挥神经保护作用，这为进一步研究COG1410在出血性卒中后早期脑损伤中的作用奠定了基础。本项目以载脂蛋白E为切入点，通过SAH在体模型及神经元离体损伤模型明确apoE短肽的神经保护作用与其抗炎症反应特性直接相关；并进一步采用小胶质细胞活化的体外模型，从表面标志物的表达、炎症因子的释放及相关信号通路的活化等三方面证实其抗炎症反应特性，并进一步证实其对血脑屏障及脑白质的保护作用及其机制。该项目以神经炎症反应为靶点，从小胶质细胞活化、血脑屏障破坏等多方面探讨apoE短肽在SAH后早期脑损伤中的保护作用，对于揭示apoE短肽在特定病理条件下的神经保护作用及其临床转化具有重要意义。