GPIb通过Epac/Rap1通路调控血脑屏障加重脑缺血再灌注损伤的分子机制

Cerebral ischemia reperfusion (CI/R) can damage vascular endothelial cells, activate the central role of platelet membrane glycoprotein GPIb, induce thrombo-inflammation, and destroy blood-brain barrier (BBB). Epac (exchange factor directly activated by cyclic AMP-activated guanine nucleotide) belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream the Raslike small GTPase Rap1. Hypoxia–reoxygenation (H/R) induced inactivation of Epac pathway in cultured vascular endothelial cells accompanied by hyperpermeability and loss of barrier function. However, whether Epac plays a role in CI/R injury is not well known. We found that blocking GPIb could improve the thrombo-inflammatory lesions, enhance BBB function, alleviate CI/R injury and reduce intracranial hemorrhage, but the mechanism has not been clarified. Preliminary experiments showed that Epac in CI/R mice decreased and inhibited Rap1 production, suggesting that cAMP-mediated Epac/Rap1 pathway might be involved in the pathogenesis of CI/R, blocking GPIb regulation of Epac/Rap1 pathway to protect BBB might be a new way to prevent CI/R injury and reduce intracerebral hemorrhage. To prove this hypothesis, we will establish MCAO model in mice and H/R model in cerebral vascular endothelial cell. Meanwhile, immunofluorescence, laser confocal, siRNA and other technologies and pharmacological tools will also be used. This study will clarify the potential molecular mechanism of the role of GPIb through Epac/Rap1 pathway in regulation BBB, aggravating CI/R injury, may provide further experimental basis for new treatment strategy of stroke by targeting blockade of GPIb.

脑缺血再灌注(CI/R)损伤内皮细胞，启动血小板膜糖蛋白GPIb为核心的血栓炎性病变，破坏血脑屏障(BBB)。Epac是cAMP直接激活的新型效应分子，缺氧致内皮屏障渗漏时伴随Epac通路活性下调，但它与CI/R损伤关系鲜见报道。我们发现，阻断GPIb可改善MCAO小鼠血栓炎性病变，增强BBB功能，减少颅内出血，其机制尚未阐明。预实验显示CI/R小鼠Epac下调并抑制Rap1，提示Epac/Rap1通路可能参与CI/R发病，阻断GPIb通过调控Epac通路保护BBB可能是防治CI/R损伤并降低出血转归的新思路。为验证此假说，本项目拟建立小鼠MCAO和脑血管内皮细胞缺氧模型，应用免疫荧光、激光共聚焦、siRNA等技术及药理学工具药，研究GPIb调控脑缺血损伤致BBB渗漏的分子机制，为靶向阻断GPIb治疗脑卒中的新策略提供进一步的实验依据。

脑缺血再灌注(CI/R)损伤内皮细胞，启动血小板膜糖蛋白GPIb为核心的血栓炎性病变，破坏血脑屏障(BBB)。Epac是cAMP直接激活的效应分子，缺氧致内皮屏障渗漏时伴随Epac通路活性下调。我们推测，GPIb可能激活Epac/Rap1通路参与CI/R损伤及防治。为了证实以上假设，我们开展了以下研究：1、小鼠H/R损伤对脑微血管内皮屏障影响及GPIb激活影响。氧糖剥夺及复灌处理激活血小板，GPIb和P-selection表达增加；缺氧或用血小板激活剂花生四烯酸(AA) 激活血小板，鼠脑微血管屏障损伤加重，内皮细胞电阻抗TER值下降。2、CI/R损伤时Epac/Rap1通路变化。CI/R损伤后BBB破坏和脑功能受损，cAMP介导Epac/Rap1信号通路抑制。荧光示踪剂研究CI/R损伤后BBB功能随时间延长而加重；缺血脑组织TJs蛋白Occludin、Claudin-5和ZO-1mRNA及蛋白表达降低，肌球蛋白p-MLC/MLC上升，细胞骨架蛋白F-actin聚合增多；神经功能和运动协调能力受损，Garcia和Bederson神经评分升高，斜板和握力能力下降；脑组织cAMP水平降低，PKA活性明显增强，Epac1表达下调，并抑制Rap1-GTP、Rac1-GTP表达。3、CI/R损伤时Epac/Rap1通路参与对脑血管内皮屏障破坏。Epac激活剂8-CPT可改善CI/R诱导的BBB破坏，减轻缺血再灌损伤，保护脑功能。Epac抑制剂ESI-09、GGTI298和NSC23766作用相反，加重屏障破坏和脑损伤。4、GPIb通过Epac/Rap1通路调控血脑屏障通透性、加重CI/R损伤。GPIb拮抗剂（Anfibatide，ANF）与8-CPT作用相似，保护BBB，增加TJ表达，降低p-MLC/MLC表达，抑制F-actin细胞骨架重排，减轻脑部损伤，上调缺血皮层Epac1通路蛋白表达。而Epac特异性拮抗剂ESI -09可取消ANF的保护作用，加重损伤。研究结果显示Epac/Rap1通路参与CI/R血脑屏障的完整性和脑功能维持，GPIb调控脑缺血损伤致BBB渗漏是GPIb调控脑缺血损伤致BBB渗漏是经由抑制Epac/Rap1通路介导的，为靶向阻断GPIb治疗脑卒中的新策略提供了进一步的实验依据，同时Epac/Rap1通路也成为防治CI/R保护血脑屏障损伤的潜在治疗靶点。