基于脑微血管内皮质膜微囊、细胞缝隙连接和血管基膜的调控，探讨生脉散制剂固摄rtPA引起的血脑屏障损伤的作用机理

Ischemic stroke is a major disease with high mortality and morbidity, for which recombinant tissue type plasminogen activator (rtPA) is the choice of treatment. However, administration of rtPA later than 4.5 hours after onset of stoke frequently results in impairment of blood brain barrier, leading to angioedema and hemorrhage, which remains a challenge for clinicians. Effusion and hemorrhage belong to Qi-deficiency and non-astringent, to deal with which ShengMaiSan, a compound Chinese medicine with potential of tonifying Qi and astringent, is effective. The primary study of the applicant revealed that ShengMaiSan is able to lessen the cerebral microvascular effusion and hemorrhage caused by rtPA thrombolysis in mice with carotid thrombi that formed 4.5 hours before. The underlying mechanism is unclear, however. The present project, using C57BL/6 mice subjected to 4.5 hours carotid thrombosis as a model, evaluates the attenuating effect of ShengMaiSan on rtPA thrombolysis-caused blood brain barrier injury in terms of thrombosis dynamics, cerebral blood flow, cerebral venular albumin leakage, hemorrhage, and cerebral infarction, and explores the effect and mechanism of ShengMaiSan in tonifying Qi and attenuating blood brain barrier injury by assessment of cerebral microvascular endothelial cells caveolae, tight junction proteins, basement membrane proteins collage type IV and laminin, mitochondria respiratory chain, energy metabolism, oxidative stress, and F-actin.

缺血性中风是发病率高致死率高的病种，重组组织型纤溶酶原激活剂（rtPA）是首选的溶栓药，但是，在缺血性中风发病4.5小时后使用rtPA，常损伤血脑屏障，引发脑血管性水肿和出血，是尚未解决的临床问题。脑血管性水肿和出血属于气虚不固摄范畴，具有补气固摄作用的生脉散是治疗气不固摄相关疾病的有效复方。申请者前期研究证实了生脉散制剂可以减轻小鼠颈总动脉血栓形成4.5小时后rtPA溶栓引起的脑血管性水肿和出血，但是，其作用机理尚不清晰。本课题用C57BL/6小鼠颈总动脉血栓形成4.5小时的模型，通过血栓动态、脑血流量、脑细静脉血浆白蛋白渗出、出血和脑梗死，评价生脉散制剂对rtPA溶栓引起的血脑屏障损伤的改善作用；通过脑微血管内皮细胞质膜微囊、缝隙连接蛋白、血管基底膜的IV型胶原蛋白和层粘连蛋白，以及线粒体呼吸链、能量代谢、氧化应激、细胞骨架等，探讨生脉散制剂补气固摄作用及其减轻血脑屏障损伤的机理。

脑卒中已成为我国居民首位致死原因，其中缺血性卒中占 60%~ 80%。组织型纤溶酶原激活剂 (tissue plasminogen activator，tPA) 是目前唯一被批准用于治疗急性缺血性脑卒中的药物。而临床上使用tPA静脉溶栓治疗可能继发脑血管水肿和脑出血等严重并发症。注射用益气复脉 (冻干) (YiQiFuMai, YQFM) 是2006年批准上市的 (国药准字Z20060463)，质量可控的生脉散 (红参、五味子、麦冬) 注射用冻干粉针剂，具有补气生津、固摄生脉的作用。本项目采用氯化铁化学刺激法制备缺血性卒中的小鼠模型，和缺氧再复氧的细胞模型，观察了益气复脉静脉滴注给药，对tPA诱导的血脑屏障破坏的改善作用。结果表明，益气复脉抑制了tPA所加重的脑梗死、神经行为学缺损、伊文思蓝渗出、白蛋白漏出、脑水肿和脑出血；降低了tPA所致的质膜微囊相关蛋白 Caveolin-1 的高表达和 Src 蛋白的磷酸化水平升高;逆转了tPA引起的能量代谢相关蛋白的减少；抑制了tPA所致的紧密连接蛋白 zonula occludens-1、junctional adhesion molecule 1、Occludin、Claudin-5，黏附连接蛋白 VE-cadherin和基底膜主要成分CollagenIV、Laminin 的低表达和排列不连续；部分逆转了tPA所致 MMP-9的高表达，脑细静脉黏附白细胞的增多和浸润白细胞数的增多。益气复脉能抑制卒中后tPA所加重的血脑屏障损伤，改善脑水肿和脑出血，有望作为一种提高tPA安全性的辅助药物应用于临床。