Cathepsin_B在脑梗死后出血转化中的作用及其炎症调控通路的分子机制研究

Haemorrhagic transformation (HT) occurs in about 1/3 of patients with ischaemic stroke, which is associated with higher incidence of mortality and morbidity; however, currently there are no effective strategies for its prevention or treatment. Cathepsin B is an intracellular cysteine enzyme, which degrades basal lamina of Blood-Brain Barrier (BBB) ex vivo but its mechanisms are unknown. Cathepsin B co-exists with Caspase-1 in brain tissues of stroke animal models and up-regulates IL-1β expression in vitro, of which the latter destroys BBB in stroke models. Based on these findings, current project will explore the effect and molecular mechanisms of Cathepsin B in HT in vivo: 1) explore the effect of Cathepsin B on extravascular matrix protein HSPG-2 and HT, by inhibiting Cathepsin B expression through stereostatic ventricle injection of neutrophilic adeno-associated virus-transfected shRNA; 2) explore the expression and sequential activation of Cathepsin B/Caspase-1/IL-1β pathway in a rat middle cerebral artery occlusion (MCAO) model; 3) preliminarily explore associations between plasma levels of Cathepsin B/Caspase-1/IL-1β and occurrence of HT in stroke patients. By clarifying effects of Cathepsin B in HT and its molecular mechanisms, this study may identify potential targets to prevent and/or treat HT.

出血转化致残率高、病死率高，但机制不明。体外研究发现Cathepsin B可降解正常脑组织血管外基质并可在脑梗模型上调Caspase-1和IL-1β，且IL-1β介导的炎症反应可破坏梗死区血脑屏障。由此我们推测Cathepsin B可通过其炎症调控通路导致出血转化，但目前尚无相关研究。本课题拟探索Cathepsin B在出血转化中的作用及其分子机制：1）通过大鼠出血转化模型抑制Cathepsin B基因，探索其对血管外基质蛋白HSPG-2的作用，并从分子和组织水平探索其对出血转化的影响；2）向大鼠MCAO模型脑室定向转染shRNA验证Cathepsin B/Caspase-1/IL-1β序贯激活通路；3）测定脑梗患者外周血Cathepsin B、Caspase-1和IL-1β血浆浓度，分析其与出血转化的关系。本课题所阐明的Cathepsin B通路分子机制将为出血转化防治靶点提供研究依据。

脑梗死后脑实质出血（出血转化）是急性脑梗死自然病程的一部分，也是溶栓取栓等再灌注疗法的常见并发症，其不仅与脑梗死不良预后相关，也是限制再灌注疗法使用的重要原因。目前对于出血转化尚缺乏有效的防治措施，亟需研究其发生发展机制，为临床防治提供干预靶点。本项目通过大鼠模型发现出血转化大鼠梗死脑组织Cathepsin B表达增加而血脑屏障组成蛋白Occludin表达降低，通过脑室立体定位特异性抑制Cathepsin B可降低梗死脑组织Cathepsin B（p=0.03）和Caspase-1（p=0.004）表达且增加血脑屏障组成蛋白Occludin表达（p=0.01），提示Cathepsin B通过降解Occludin破坏血脑屏障。脑梗死患者临床队列中，入院NIHSS评分高（中位数 13 vs. 6分, p=0.001）、合并瓣膜性心脏病（OR=2.35, 95% CI 1.11-5.00）、接受急诊血管内治疗（OR=4.44, 95% CI 2.60-7.57）的患者更易发生出血转化；与非出血转化患者相比，出血转化患者的3月不良预后发生率更高（p<0.001）；脑梗死患者外周血Cathespin B、Caspase-1和IL-1β血浆浓度在出血转化组和非出血转化组之间无显著差异（p>0.05）。本研究结果提示梗死脑组织中Cathepsin B通过降解血脑屏障组成蛋白破坏血脑屏障完整性，且与Caspase-1相关，Cathepsin B可能是防治脑梗死后血脑屏障破坏的潜在干预靶点。但上述机制在出血转化发生中的作用尚未在临床队列证实，提示可能存在Cathepsin B以外的其他机制导致出血转化，有待进一步研究；另一方面，Cathepsin B是否参与脑水肿等其他血脑屏障破坏相关并发症有待后续研究阐明。