内质网应激-自噬途径在脑出血后继发性脑损伤中的调控作用及机制研究

Inflammatory response and apoptosis are important factors of secondary brain injury after intracerebral hemorrhage (ICH) .Endoplasmic reticulum stress, autophagy are closely related to the inflammatory reaction and apoptosis, and so far there is no study described the role of endoplasmic reticulum stress and autophagy in secondary brain injury after ICH.Our preliminary experimental results show that endoplasmic reticulum stress was enhanced continuously after ICH, autophagy was enhanced early while autophagy flow was blocked lately after ICH. But the specific mechanism remains to be further explored.To this end, we propose a hypothesis: continued endoplasmic reticulum stress may result in autophagy flow blocking by calcium overload after ICH, inducing neuronal apoptosis and inflammatory reaction in secondary brain injury. To test this hypothesis, We apply oxidized hemoglobin treated the original generation of neurons co-culture ICH model in vitro, rats ICH model in vivo and patients with clinical tissue specimens, and use methods of electron microscopy, western blot, plasmid transfectio and RNA interference. From multi-level of cellular, animals and clinical medicine, we explore the role of endoplasmic reticulum stress and Autophagy pathway in the regulation function of secondary brain injury after ICH and investigate the mechanism of which continued endoplasmic reticulum stress result in autophagy flow blocking by calcium overload after ICH. This study from the new point of view in endoplasmic reticulum stress and autophagy will provide new ideas for improving the prognosis of ICH.

炎症反应和细胞凋亡是脑出血（ICH）后继发性脑损伤的重要因素。内质网应激、自噬与炎症反应和细胞凋亡密切相关，迄今尚未见内质网应激-自噬在ICH后脑损伤中的作用研究。我们的预实验结果提示ICH后内质网应激持续增强，自噬早期增强，晚期自噬流阻断，但具体作用机制不明。为此，我们提出假说：ICH后持续内质网应激可能通过钙超载阻断自噬流，从而诱导神经元的凋亡和炎症反应导致脑损伤。为了验证该假说，我们应用氧合血红蛋白处理原代培养神经元细胞体外ICH模型、大鼠ICH模型和ICH患者脑组织标本，采用电镜，western blot，质粒转染、RNA干扰等手段，从细胞、动物和临床等多方面探讨内质网应激-自噬在ICH后继发性脑损伤中的调控作用，明确ICH后持续内质网应激通过钙超载导致自噬流阻断产生脑损伤的机制。本研究从内质网应激-自噬这个新视点揭示ICH后继发性脑损伤的发生机制，将为改善ICH预后提供新思路。

目的:研究和探讨自噬和内质网应激在ICH后大鼠继发性脑损伤中的作用及发生机制。方法:采用自噬诱导剂(雷帕霉素)和抑制剂(3-methyladenine)以及内质网应激激活剂(tunicamycin, TM)和抑制剂(tauroursodeoxycholic acid, TUDCA)。自噬体-溶酶体融合抑制剂Bafilomycin A1被用来评估自噬流。结果:脑出血后一周自噬及内质网应激增强。在ICH后6h，自噬可能过度，而自噬流可能在72h破坏，在ICH后7d恢复完整。在脑出血后6小时，TM诱导内质网应激可增强自噬，导致caspase12介导的细胞凋亡和神经元变性，而自噬诱导又进一步加重了细胞凋亡和神经元变性。脑出血7 d后，TUDCA对ER的应激抑制仍可抑制ICH诱导的SBI。自噬诱导增强了TUDCA的作用。结论:脑出血后6小时，过度自噬可能参与内质网应激所致的脑损伤；在ICH后7 d，自噬可能通过清除早期自噬流损伤产生的细胞垃圾，增强内质网应激抑制剂的保护作用。