ENOPH1-ADI1双重调控多胺和MT1-MMP促进缺血早期血脑屏障损伤的分子机制

hemorrhagic transformation（HT） is the most dangerous complication in tPA thrombolytic therapy for ischemic stroke, which occurs as a consequence of endothelial cell apoptosis of the blood brain barrier (BBB) during thrombolytic reperfusion. ENOPH1 has been increasingly recognized to be implicated in regulating stress via serving as the key transmitter for receiving and transmitting cell stress signals. Our previous study data shown that cerebral ischemia (3h) induced ENOPH1 upregulation and knockdown of ENOPH1 ameliorated BBB injury, and ENOPH1 promotes ADI1 translocation (ADI1 is a downstream molecule of ENOPH1). It is known that ADI1 interacts with MT1-MMP . We hypothesize that early ENOPH1 in cerebral ischemia may be a "trigger point" that triggers the process of BBB endothelial cell apoptosis and BBB damage. We have proposed the following in vivo and in vitro experiments to test our hypothesis: 1) to analyze the relationship between the destruction of the blood-brain barrier and the formation of polyamines related in the activation of ENOPH1; 2) to explore the ADI1 translocation mediated by ENOPH1 was engaged in integrity and stability of BBB basement membrane through modulating the activity of MT1-MMP ; 3) to confirm the effect of ENOPH1 on the expression of tight junction proteins in endothelial cells induced by early ischemia. This study major focused on investigating the molecular events of early BBB injury in ischemia and providing a new target for the early warning of cerebral hemorrhage after thrombolysis.

溶栓治疗脑卒中易导致出血性转化 (HT)，缺血早期血脑屏障（BBB）损伤与HT直接相关。烯醇磷酸酶1（ENOPH1）是参与多胺代谢的一种酶，多胺代谢与脑缺血损伤密切相关，但机制尚不清楚。我们前期研究发现：脑缺血早期诱导ENOPH1高表达, ENOPH1可促进脑微血管内皮细胞凋亡并介导ADI1的转位（ADI1是ENOPH1的下游分子），据文献报道ADI1可抑制MT1-MMP活性。我们推测：ENOPH1-ADI1通过双重调控多胺合成和MT1-MMP的活性促进缺血早期BBB损伤。本项目拟采用卒中模型，结合CRISPR-Cas9、质谱等技术，分析ENOPH1促进多胺的合成与血脑屏障破坏之间的相关性；明确ENOPH1介导ADI1转位通过调控MT1-MMP影响BBB基底膜稳定性；观察缺血早期ENOPH1对促进溶栓再灌后脑出血发生的影响。本研究以缺血早期BBB损伤为着眼点，为HT早期预警提供理论依据。

脑卒中又称为中风，是由于脑血管突然梗塞或者破裂，导致局部脑组织缺血缺氧性病变坏死，其中缺血性脑卒中占80%以上。脑卒中为我国居民第一位的死亡和残疾原因，且呈持续上升趋势。临床上缺血性脑卒中的主要治疗手段是溶栓取栓，疏通血管，使缺血区域再灌注。再灌注虽可使受损脑组织重新获得供血供氧，但也会造成急性血管损伤，破坏血脑屏障(BBB)的结构与功能，并增加出血转化的风险。以往关注较多的是再灌注后血脑屏障的破坏，然而急性缺血性脑卒中早期血脑屏障功能的分子调控机制却不清楚。针对这些待解决的关键科学问题，本项目主要围绕ENOPH1-ADI1-MT1-MMP轴在缺血早期BBB损伤的作用机制展开研究，同时探讨了紧密连接蛋白Occludin的降解在脑缺血早期BBB内皮细胞对于再灌注损伤的作用，并研究了PAQR3基因缺血神经元凋亡过程中的作用机理，开展了脑缺血早期BBB微血管内皮细胞外泌体miRNA和外泌体膜表面蛋白的表达谱研究，以及研究Co-Fe3O4纳米酶和uPA-CDs两种纳米生物材料在缺血性卒中BBB及神经损伤中的作用。本项目取得以下研究发现：1） 发现ENOPH1通过干预ADI1-MT1-MMP信号通路加剧缺血早期BBB功能障碍；2）发现脑缺血后内皮细胞Occludin降解诱导凋亡导致内皮细胞更易受再灌注损伤；3）发现敲除PAQR3基因可能通过PI3K/AKT信号通路发挥神经保护作用；4）获得了脑缺血超早期BBB内皮细胞外泌体miRNA和表面蛋白谱的特征图谱；5）并在动物脑缺血模型中应用Co-Fe3O4纳米酶和uPA-CDs纳米杂化材料发现前者具有神经保护作用，而后者拥有BBB示踪功能。本项目执行期内发表SCI文章7篇，申请和授权发明专利5项、培养硕士研究生3名。本项目增加了我们对急性缺血性脑卒中BBB的分子调节机制及神经功能保护的理解，为脑缺血损伤的临床治疗提供了新的思路、靶点和方法。