脑缺血早期MMP-2快速分泌阻断内皮细胞与星型胶质细胞“互作”促进血脑屏障损伤的机制研究

We have shown that the rapid secretion of MMP-2 contributes to early blood-brain barrier (BBB) damage in acute ischemic stroke. However, the mechanism has not been elucidated. It is known that the endothelial cells (ECs) interact with astrocytes through the shared Shh pathway to maintain the integrity of the BBB via promoting the expression of junctional proteins and maintaining the anti-inflammatory properties of BBB endothelial cells. However, it remains unknown whether and how the above cross-talk between astrocytes and endothelial cells is affected during ischemic BBB injury in the early stage of ischemic stroke. Our recent co-culture experiments showed that the supernatants of OGD-treated (or ischemic) ECs could significantly down-regulate the expression of Scube2 in astrocytes and weaken the activation of Shh pathway in the ECs by the astrocytes that were pre-incubated with the conditioned media of ischemic ECs. Based on the fact that Scube2 is a key protein involved in the release of Shh and can be degraded by MMP-2, we hypothesize that the rapid secretion of MMP-2 induced by cerebral ischemia may inhibit the release of Shh from astrocytes by degrading Scube2, thus cutting off the cross-talk between astrocytes and ECs to promote the occurrence of BBB damage. To test our central hypothesis, we will apply in vivo and in vitro ischemic stroke models, co-culture model, co-immunostaining and siRNA silence to demonstrate 1) whether and how MMP-2 interacts with Scube2/Shh to affect the cross-talk between ECs and astrocytes in vivo and in vitro and 2) whether the above changes contribute to early ischemic BBB damage via downregulating the expression of junctional proteins and inhibiting the expression of chemotactic factors and cell adhesion molecules to reduce the infiltration of inflammatory lymphocytes to ischemic brain tissue. The completion of this study will not only expand our understanding the mechanisms underlying early ischemic BBB damage but will also provide new therapeutic targets for BBB protection in the early stage of stroke.

我们已经证明了MMP-2的快速分泌参与了卒中早期血脑屏障（BBB）的损伤，但机制尚未阐明。已知内皮细胞（EC）与星形胶质细胞通过共享Shh通路发生“互作”，促进连接蛋白表达和维持内皮的抗炎特性，共同维护BBB完整。那么，在卒中早期BBB的损伤中，上述互作是否被破坏，机制是什么？我们近期体外共培养实验发现，缺血EC的培养上清可显著下调星形胶质细胞Scube2的表达,同时削弱后者对ECShh通路的激活。因Scube2参与Shh的释放，又可被MMP-2降解，据此我们推测，缺血早期MMP-2的快速分泌可通过降解Scube2抑制Shh释放，进而切断星形胶质细胞与EC的互作，促进BBB损伤的发生。本项目拟采用体内外卒中模型，细胞共培养、免疫荧光双标和siRNA等方法，阐明MMP-2调控Scube2/Shh在脑缺血早期BBB损伤中的关键作用及分子机制，为寻找卒中早期BBB保护治疗靶点提供科学依据。

本项目围绕缺血早期BBB损伤及缺血再灌注神经损伤的分子机制展开，主要探讨了脑缺血早期MMP-2的快速分泌在阻断星形胶质细胞与内皮细胞“互作”促进BBB损伤中的重要作用，阐明MMP-2分泌通过靶向干预Scube2/Shh通路破坏星形胶质细胞与内皮细胞“互作”而造成BBB功能失调。 此外，本研究围绕脑缺血早期BBB损伤机制进行了研究内容的拓展，通过以上研究取得如下发现：1）缺血早期BBB三种主要细胞均会分泌MMP-2，其中星型胶质细胞分泌量最高，Scube2与MMP-2存在直接相互作用，且缺血早期导致小鼠MCAO脑缺血侧的Scube2明显降低，体内敲除星形胶质细胞中的Scube2会加剧其下游Shh通路蛋白表达的下降，同时也加剧了BBB的损伤和炎症水平的上调，体外过表达星形胶质细胞Scube2可抑制OGD/R导致的星形胶质细胞及血管内皮细胞Shh通路的下调，逆转内皮细胞屏障和抗炎功能的下降，体外抑制MMP-2的分泌可抑制OGD/R处理后星形胶质细胞及内皮细胞Scube2的表达及Shh通路的下调，逆转内皮细胞TJ蛋白的下调。2）研究发现闭锁蛋白的降解会加速脑微血管内皮细胞对于缺血应激性损伤的敏感性；3）探讨了PAQR3基因在急性脑缺血诱导的神经元凋亡过程中的作用机制，表明PAQR3基因敲除通过激活PI3K/AKT通路能够减轻OGD/R诱导的细胞凋亡；4）研究团队发现体外缺血促进了脑微血管内皮细胞中外泌体的释放以及外泌体miRNA和表面蛋白的特异性变化，这些胞外miRNAs和表面蛋白之间有明显的相关性；5）项目组新合成一种来源于野菊花的天然产物蒙花苷衍生物BLR，发现BLR对脑缺血后神经损伤具有显著保护作用，且这种作用依赖于CSPG4依赖的信号通路的激活，KDELR介导的翻译后修饰和CSPG4从ERGIC到细胞膜的泡状转运是BLR诱导的神经保护的一个重要途径；6）研究发现小胶质细胞Swell1 通道开放可激活SGK1介导的 FOXO3a/CREB 以及 WNK1 介导的 SPAK/OSR1-CCCs 信号通路，促进缺血损伤后小胶质细胞的存活和M2型极化，从而减轻炎症反应和缺血性脑损伤。本项目增加了我们对卒中早期BBB和脑缺血损伤的分子调节机制的理解，为BBB功能改善和脑缺血损伤的临床治疗提供了新思路、新靶点和新方法。