基于BBB新理论探讨Wnt通路在脑出血后的脑保护机制

Intracranial hemorrhage is a devastatingly tragic stroke subtype with a significant high mortality and morbidity rate. There are still lack of effective treatments for the brain edema and neuronal apoptosis caused by intracranial hemorrhage currently. Recent evidence revealed that molecular pathogenesis of blood-brain barrier (BBB) breakdown was the change of neurovascular unit (NVU). Present study is to investigate the expressions and significance of Wnt signaling pathway (including: Wnt-1, β-catenin, GSK-3β) after intracranial hemorrhage in the rat; meanwhile, vasogenic edema and BBB disruption as well as associated proteins such as (TJPC-3, Careolin-1) in experimental intracranial hemorrhage are evaluated. These results will interpret the molecular pathogenesis of brain injury caused by NVU after intracranial hemorrhage. . The second step of our research is to construct a lentiviral vector and select the effective sequence against Dickkopf-1 (Dkk-1) successfully. Because latest literature demonstrated that Dkk-1 played an important role in the negative modulation on Wnt pathway, which contributed to the neuronal death in models of brain ischemia and neurodegenerative disease. Present study is to determine the therapeutic outcome after the small interfering Dkk-1 (siDkk-1) injection into the lateral ventricle of rat. The neuronal death, brain edema, BBB breakdown, behavioral test and associated proteins will be detected after administration of siDkk-1 in the model of intracranial hemorrhage. These outcomes may provide a novel treatment for intracranial hemorrhage through interfering Wnt signaling pathway.

脑出血是严重危害人类健康的脑血管病之一，脑出血后继发脑水肿及神经元凋亡目前缺乏有效的治疗手段。本研究基于最新BBB破坏的基础是神经血管单元（ NVU）的变化，通过制作稳定大鼠脑出血模型，观察脑出血后Wnt通路分子包括Wnt-1、β-catenin、GSK-3β的诱导表达及意义；检测血管源性脑水肿、BBB破坏及相关蛋白如紧密连接闭合蛋白3 （TJPC-3）及Caveolin-1的表达变化，从而判断NVU的组织结构改变与脑出血后继发损伤发生的分子机制。然后根据Dkk-1作为负性调节Wnt信号通路的关键因子，设计并构建出大鼠Dkk-1的siRNA基因慢病毒载体，经体外鉴定干扰效率后筛选出一个有效的干扰片段，将其注射到大鼠脑室内治疗脑出血，探讨siDkk-1对脑出血后神经元损害、脑水肿及BBB破坏及相关蛋白表达的影响，阐明脑出血后通过激活Wnt经典通路发挥脑保护，最终为脑出血的治疗提供又一新途径。

脑出血是严重危害人类健康的常见脑血管病之一，脑出血后血脑屏障（BBB）的破坏及其引发的继发脑水肿是造成患者颅内压增高乃至死亡的重要因素，目前缺乏有效的治疗手段。本研究通过制作大鼠稳定脑出血模型，观察大鼠脑出血后血肿周围脑组织及血浆内Dkk-1的变化，检测Wnt通路分子包括Wnt-1、β-catenin、GSK-3β的在脑出血后的诱导表达；同时检测血管源性脑水肿、BBB破坏及相关蛋白如紧密连接闭合蛋白3 （TJPC-3）的表达变化，从而探讨脑出血后继发损伤发生的分子机制。然后根据Dkk-1作为负性调节Wnt信号通路的关键因子，设计并构建出大鼠Dkk-1的siRNA基因慢病毒载体，经体外鉴定干扰效率后筛选出一个有效的干扰片段，实验用量为2×107个传导单元，将其注射到大鼠脑室内治疗脑出血。结果显示脑出血后血肿周围脑组织及血浆内Dkk-1在24h和72h较对照组显著升高，Wnt-1基因表达水平较sham组显著降低(p <0.001 )，GSK-3βmＲNA 水平均显著升高（p <0.05），而经siDkk-1干预后Wnt-1和GSK-3βmＲNA水平均显著逆转，大鼠脑出血24h和72hβ-连环蛋白（β-catenin）表达较sham组显著升高，经siDkk-1干预后24h和72h，β-catenin的表达水平较假干预组显著升高(p< 0.05). 大鼠脑出血24h和72h术侧基底节区脑组织含水量及EB含量均显著增加（p< 0.01），脑组织紧密连接闭合蛋白3 （TJPC-3）的表达下降，而经siDkk-1干预后较ICH+vehicle组脑含水量及EB含量均显著下降（p< 0.01），同时伴透射电镜（TEM）示BBB组织结构改善及行为学评分的提高。研究结果表明：脑出血可诱导Dkk-1的过度表达，通过RNA干扰Dkk-1进而激活Wnt经典通路发挥脑出血后的脑保护作用，为脑出血的临床治疗提供又一新途径。