NLRP3介导中性粒细胞募集参与脑梗死溶栓后出血转化的机制研究

Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved thrombolytic agent for the early treatment of ischemic stroke. Hemorrhagic transformation (HT) is a serious complication of cerebral infarction following the use of thrombolytic therapy. One prominent feature of the development of HT after rtPA treatment is neutrophil recruitment into ischemic brain in the early stages. However, the mechanism by which tissue damage triggers and regulates neutrophil recruitment after rtPA treatment in stroke remains unknown. As intracellular pattern recognition receptors, Nod-like receptor protein 3 (NLRP3) has been implicated as an important factor mediating neutrophil recruitment. Our previous study indicated that delayed rtPA treatment at 4 hours after thromboembolic stroke (inducing HT) promotes the expression of NLRP3 in neurons, microglia and endothelial cells, with concomitant increase in brain neutrophil recruitment. We therefore conducted a systematic study to clarify the mechanisms by which NLRP3 mediated neutrophil recruitment enhances delayed rtPA-induced hemorrhagic transformation in thromboembolic focal cerebral ischemia rats (mimic the clinical human HT). Finally, we also explore whether NLRP3 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 way for prediction and treatment of HT in patients with stroke treated with rtPA.

重组组织型纤溶酶原激活剂（rtPA）静脉溶栓是急性脑梗死的首选治疗，但是溶栓后出血转化（HT）是其最严重的并发症。脑梗死溶栓后HT的主要病理特征是中性粒细胞募集，然而其启动及调节机制尚不明确。NLRP3（Nod-like receptor protein 3）作为胞质内模式识别受体，在调节中性粒细胞募集中发挥重要作用。我们前期研究发现，与对照组相比，溶栓HT组大鼠脑缺血区NLRP3表达明显增高，并且中性粒细胞募集也显著增加。基于此，我们提出假说，NLRP3介导中性粒细胞募集参与脑梗死溶栓后HT。本项目将采用大鼠脑栓塞rtPA溶栓HT模型，运用分子生物学、组织病理学等实验方法，验证上述假说并探讨其机制；此外，结合临床研究探讨NLRP3及下游组分能否作为rtPA溶栓HT的预测指标。本项目期望为发现溶栓HT新的预测及治疗方法提供理论依据。

重组组织型纤溶酶原激活剂（rtPA）静脉溶栓是急性脑梗死的首选治疗，但是溶栓后出血转化（HT）是其最严重的并发症。脑梗死溶栓后HT的主要病理特征有中性粒细胞募集，然而其启动及调节机制仍不明确。NLRP3（Nod-like receptor protein 3）作为胞质内模式识别受体，在调节中性粒细胞募集中发挥重要作用。我们运用分子生物学、组织病理学等实验方法研究发现，在脑缺血4小时后延迟rtPA处理可促进神经元、小胶质细胞和内皮细胞NLRP3的表达，BBB成分的降解和HT。敲除NLRP3可显著降低NLRP3的表达、血脑屏障的破坏和HT。它还能改善神经功能，减少中性粒细胞募集。兔抗大鼠中性粒细胞血清和NLRP3 shRNA的使用能降低rtPA治疗后的出血评分和出血量。抗大鼠中性粒细胞血清联合NLRP3 shRNA与单独使用兔抗大鼠中性粒细胞血清相比，对HT的保护作用没有进一步增强。此外，临床研究证明动态的中性粒细胞与淋巴细胞比值（NLR）与脑卒中患者溶栓后出血转化间存在相关性。本项目为溶栓后HT的预测及治疗方法提供了新的理论依据。