Pannexin-1通道蛋白介导的自噬反应在蛛网膜下腔出血后早期脑损伤中的作用机制研究

Subarachnoid hemorrhage (SAH) is known as a cerebrovascular disease with a high rate of mortality and disability. Recent advances in SAH research suggest that autophagy plays a protective role in the period of early brain injury (EBI) after SAH. Pannexin-1 channels release ATP into the extracellular space, which may activate the AMPK/mTOR/ULK1 mediated autophagy. We raised a hypothesis that activation of pannexin-1 channels could initiate the autophagy and protect the neurons from EBI after SAH. Pannexin-1 channels gene knockdown and overexpression were achieved by adenovirus associated virus (AAV) administration. In addition, quantitative real-time polymerase chain reaction (PCR), immunofluorescence staining and western blot were performed to explore the potential interactive mechanism between pannexin-1 channels and autophagy related neuroprotective effects on EBI after SAH in vivo and vitro study. There by providing a novel therapeutic target and theoretical basis for SAH treatment.

蛛网膜下腔出血（SAH）是一类具有高致残率，高致死率的脑血管疾病。研究证实自噬反应在SAH后早期脑损伤（EBI）中发挥重要的脑保护作用，Pannexin-1通道蛋白参与细胞内ATP胞外释放，继而激活AMPK/mTOR/ULK1自噬信号通路，可能在SAH后EBI中起脑保护作用。综上我们提出假说：Pannexin-1通道蛋白的开放，激活自噬反应，保护神经元细胞，改善SAH后EBI。为了验证该假说，我们采用腺相关病毒转染技术基因敲除和过表达Pannexin-1通道蛋白，利用实时定量PCR、免疫荧光染色、western blot等实验技术在大鼠体内体外SAH模型中观察Pannexin-1通道蛋白介导的自噬反应在SAH后EBI中的脑保护作用，同时验证Pannexin-1通道蛋白激活自噬反应的信号通路机制，为治疗SAH提供新的靶点和理论基础。

蛛网膜下腔出血（SAH）后早期脑损伤的病理生理机制与患者的整体预后息息相关。前沿研究发现，自噬反应在SAH后早期脑损伤中具有积极的神经保护作用。我们研究发现，Pannexin-1通道蛋白在SAH后大量开放并向胞外释放ATP，但Pannexin-1通道蛋白的表达总量并不会发生变化。随后我们通过腺相关病毒转染技术特异性上调Pannexin-1通道蛋白的表达，进一步增加释放出胞外的ATP总量，进而顺次激活了自噬相关的AMPK/mTOR/ULK1信号通路，从而增强了SAH后自噬反应。与此同时，我们观察到在增强SAH后自噬反应后可以改善SD实验大鼠的短期与长期的神经功能障碍，减少神经元细胞的过度凋亡坏死，修复SAH后血脑屏障的破坏，最终实现了针对SAH后早期脑损伤的治疗作用。通过本课题的研究我们明确了Pannexin-1通道蛋白调控的自噬反应在SAH后起神经保护作用以及可以将Pannexin-1通道蛋白作为今后治疗SAH的有效靶点之一。