脑缺血早期occludin降解促血脑屏障内皮细胞凋亡的分子机制

Intracerebral hemorrhage is the most dangerous complication intPA thrombolytic therapy for ischemicstroke, which occurs as a consequence ofendothelial cell apoptosis of the blood brain barrier (BBB) during thrombolytic reperfusion. However, what drives the evolution of endothelial cell injury toward apoptosis remains unclear. Wehave obtained preliminary datathatcerebral ischemia (2 h) inducedBBB disruption, tight junction protein occludindegradation and a concurrent increase of blood levels of cleaved occludin fragments.More recently, we found thatoccludin degradation concurrently occurred with the reduction of anti-apoptotic molecule c-Yes and the activation of caspase-8. However, it remains unknown about the roles of these changes and whether they are secondary to occludin degradation. In addition to its primary role as a BBB structural protein, occludin has been increasingly recognized to be implicated in regulating apoptosis via serving as the key transmitter for receiving and transmitting cell survival and death signals. Therefore, we hypothesized thatoccludin degradation in the early phase of cerebral ischemia is the “trigger” to initiate the evolution of endothelial injury toward apoptosisduring thrombolytic reperfusion. We have proposed the followingin vivo and in vitro experiments to test our hypothesis: 1) to correlate pre-thrombolysis blood occludinlevels to post-thrombolysis hemorrhagic volume in the ischemic brain; 2) to analyze the effects of inhibition of occludin degradation by mutating its cleavage site; 3) to determine whether occludindegradation leads to the release of cleaved fragments into the extracellular space which activates the extrinsic apoptotic pathway(Fas-FADD-caspase-8)to induce caspase-3 activation and apoptosis; 4) to determine whether activated caspase-3 will in turn degrade occludin to form a vicious cycle between occludin degradation and caspase-3 activation to exacerbate endothelial cell injury. The completion of this project is expected to not only help to develop new blood biomarker for the evaluation of the risk of hemorrhagic complication associated with tPA thrombolysis, but also provide new targets for therapeutic intervention for ischemic stroke.

脑出血是tPA溶栓治疗脑卒中最危险的并发症，直接原因为溶栓后血脑屏障（BBB）内皮细胞凋亡。我们前期发现：脑缺血2小时可引起BBB损伤、紧密连接蛋白occludin（ODN）降解及外周血降解片段升高；ODN降解伴抗凋亡分子c-Yes降低和caspase-8激活，但是否存有因果关系不明。文献报道ODN蛋白可接收和传递细胞应激信号，为调节凋亡的重要传感器。我们推测：脑缺血早期ODN的降解可能是启动受累BBB内皮细胞在溶栓再灌过程中向凋亡演变的“触发器”。本项目拟采用脑缺血动物和细胞模型，观察tPA溶栓前外周血ODN片段水平和溶栓后脑出血的关系；观察突变ODN降解位点对c-Yes和细胞凋亡的影响；检测和清除培养上清中ODN降解片段，分析该片段是否激活外源死亡受体途径、诱导凋亡以及活化的caspase-3降解ODN而形成恶性循环。本研究将为早期预测和防治脑出血提供可靠的生物标记物和药物作用新靶点。

本项目聚焦于缺血早期血脑屏障（BBB）损伤的分子机制及BBB保护展开研究工作，重点分析脑缺血早期occludin的降解这一事件在溶栓再灌过程中的作用，阐明脑缺血早期BBB损伤与tPA溶栓后脑出血转化相关的分子机制等。通过建立大鼠大脑中动脉闭塞（MCAO）模型和体外糖氧剥夺（OGD）模型模拟脑缺血，采用siRNA干扰、免疫共沉淀、慢病毒过表达等多种实验手段，获得了以下研究发现：（1）Occludin蛋白降解与BBB通透性呈正相关；（2）Occludin的降解与脑组织中MMP-2的释放增加有关；（3）血液中的occludin水平与卒中早期BBB损伤程度密切相关，血液中紧密连接蛋白occludin可以作为评估缺血性卒中早期BBB损害的一个指标；（4）Occludin的下调增加了脑微血管内皮细胞对细胞凋亡的敏感性；（5）ENOPH1的激活可促进ROS生成和细胞凋亡相关蛋白的表达，加重OGD诱导的内皮细胞衰竭和BBB破坏；（6）HIF-1α抑制剂YC-1的预处理能够显著抑制2小时大脑中动脉梗塞引起的血脑屏障损伤，并且伴随着Occludin的降解和血管内皮生长因子（VEGF）的mRNA上调，阻断β2-AR介导的HIF-1α上调可改善脑缺血后BBB的破坏；褪黑素通过激活AMPK和抑制gp91phox表达上调减少LPS诱导的BBB损伤；（7）氟代柠檬酸通过下调GFAP和Cx43表达抑制星形胶质细胞活化，从而明显改善卒中所导致的记忆障碍，这种保护作用还伴随着缺血性卒中大鼠海马区域CRTC1、HMGB1和SYP表达水平的增加。以上实验发现为缺血性脑卒中早期BBB破坏的分子机制和神经血管单元修复提出实验基础和理论依据，并可能对减少溶栓相关的脑出血并发症提供分子靶点，从早期BBB损伤角度，occludin可作为溶栓后所致脑出血的风险评估潜在的生物标记物。