LCN2通过24p3R/ERK信号调节中性粒细胞募集在脑梗死静脉溶栓后出血转化中的作用研究

Hemorrhagic transformation (HT) is the most serious complication of cerebral infarction following the use of intravenous recombinant tissue plasminogen activator (rtPA) thrombolytic therapy. One prominent feature of the development of HT after rtPA treatment is neutrophil recruitment into ischemic brain in the early stages. As an innate immunity molecule, Lipocalin-2 (LCN2) has been implicated as an important factor mediating neutrophil functions and the process of neutrophil migration. However, the role of LCN2 in neutrophil recruitment in ischemic stroke and the relationship between LCN2 and HT remain unknown. Our previous study indicated that delayed rtPA treatment at 4 hours after thromboembolic stroke (inducing HT) promotes the expression of LCN2 in neurons, astrocyte, microglia and endothelial cells, with concomitant increase in brain neutrophil recruitment. Moreover, suppression of neutrophils attenuates blood–brain barrier disruption and HT after delayed rtPA treatment in thromboembolic stroke rats. We therefore conducted a systematic study to verify our hypothesis that LCN2-mediated neutrophil recruitment contributes to HT after thrombolysis in acute cerebral infarction in thromboembolic focal cerebral ischemia rats and in vitro models of neutrophil migration. Moreover, we also explore whether LCN2 and its downstream components can be used as important prognostic markers for the HT. This study is to systematically study what it is in central nervous system that triggers and regulates neutrophil recruitment after rtPA treatment in stroke, thus providing novel ways for prediction and treatment of HT in patients with stroke treated with rtPA.

出血转化（HT）是急性脑梗死静脉溶栓治疗中最严重的并发症，其主要病理特征之一是中性粒细胞募集引起的神经血管单元损伤。近期研究表明，固有免疫分子Lipocalin-2（LCN2）能够调节中性粒细胞功能及其迁移过程。但LCN2在脑梗死后中性粒细胞募集过程中的作用及其与溶栓后HT的关系尚不清楚。我们前期研究显示，与对照组相比，HT组大鼠脑缺血区LCN2表达明显增高，并且中性粒细胞募集也显著增加；抑制中性粒细胞明显减少脑梗死溶栓后血脑屏障破坏和HT。基于此，我们将采用大鼠脑栓塞溶栓后HT模型、中性粒细胞体外迁移模型，运用分子生物学、组织病理学等实验方法，研究LCN2能否介导中性粒细胞募集参与脑梗死溶栓后HT并探讨其分子机制；以及结合临床研究探讨LCN2及下游组分能否作为溶栓后HT的预测指标。本项目期望为发现急性脑梗死静脉溶栓后HT新的预测及治疗方法提供理论依据。

出血转化（HT）是急性脑梗死静脉溶栓及血管内介入再通治疗后最严重的并发症，其主要病理机制之一是缺血再灌注损伤。近期研究表明，炎症因子脂质运载蛋白Lipocalin-2(LCN2)和中性粒细胞在脑缺血再灌注损伤中起着重要作用。但LCN2在脑梗死后中性粒细胞募集过程中的作用及其与缺血再灌注损伤的关系尚不清楚。本项目旨在探究使用抗LCN2抗体(LCN2mAb)对脑缺血再灌注损伤后神经功能的保护作用以及对中性粒细胞表达的影响，并进一步阐明LCN2mAb的保护作用和中性粒细胞极化之间的关系。我们以小鼠大脑中动脉阻塞(MCAO)模型模拟脑缺血再灌注损伤。缺血前分别予以LCN2mAb（侧脑室给药）、Anti-Ly6G（腹腔给药）及LCN2mAb+Anti-Ly6G联合给药。体外利用HL-60为细胞模型。结果显示，脑缺血再灌注损伤后，LCN2、中性粒细胞Ly6G在急性期表达量明显升高(P<0.05)，24h达到高峰，而后逐渐降低。LCN2mAb预处理小鼠脑梗死体积减小(P<0.05)；Evans blue结果、Occludin蛋白的表达均显示血脑屏障损伤减轻(P<0.05)；Longa评分、Open field、步态等行为学结果显示神经功能得到改善(P<0.05)；中性粒细胞Ly6G的表达在蛋白水平和流式检测中无明显差异(P>0.05)；但N2型中性粒细胞表达量明显增高(P<0.05)；脑损伤区域炎症因子在转录水平CD206表达明显增加(P<0.05)，TNF-α表达明显减少(P<0.05)，而IL-1β、IL-10未见明显变化(P>0.05)。与LCN2mAb+Anti-Ly6G联合治疗组相比，LCN2mAb处理的小鼠脑梗死体积、血脑屏障损伤减少；神经功能得到改善(P<0.05)。使用LCN2mAb预处理N1-HL-60细胞，在蛋白水平和流式检测中，N2-HL-60细胞均明显增加(P<0.05)。最后，在临床研究中我们不仅发现中性粒细胞与出血转化及不良预后相关，我们还发现嗜酸性粒细胞与卒中相关肺炎、出血转化、不良预后独立相关。总之，LCN2和中性粒细胞都参与了MCAO后缺血再灌注损伤，且时间上具有相关性；LCN2mAb对急性缺血性卒中有神经保护作用；LCN2mAb可能通过调节中性粒细胞向N2型中性粒细胞的极化改善缺血性卒中的结果，这为临床治疗提供了新思路。