内质网应激调控蛛网膜下腔出血后神经元自噬的机制研究

Subarachnoid hemorrhage(SAH) is a devastating disease with high mortality and significant morbidity among patients. Early brain injury following SAH is considered as the primary cause of poor outcome after SAH and remains no effective therapy. Autophagy is a homeostatic process that involves the sequestration of cytoplasmic components in double-membraned autophagosomes. Autophagy acts as a survival mechanism under conditions of stress, maintaining cellular integrity by regenerating metabolic precursors and clearing subcellular debris. Our previous study have demonstrated that autophagy activation is associated with neuroprotection against apoptosis in a rat model of SAH. However, the exact molecular mechanism of that still remains obscure. Endoplasmic reticulum (ER) stress participates in a arrange of neurological diseases, but whether and how ER stress is involved in the pathogenesis of autophagy activation after SAH remains unknown. Our preliminary inspections showed that ER stress was strongly activated in the process of autophagy in SAH rat model, and specific ER stress inhibitor, TUDCA,could significantly suppressed the progression of autophagy and aggravated brain injury. Therefore, we hypothesize that enhanced ER stress following SAH could triggers autophagy and finally promotes cell survival. This study was undertaken to investigate the role and the mechanisms of ER stress PERK-eIF2a-ATF4 pathway in autophagy activation following SAH in both animal and cellular levels, and thus to provide a new target for SAH prevention and treatment.

蛛网膜下腔出血（SAH）是一种严重危害人民健康的疾病，它引发的早期脑损伤是其首要的致病机制，但目前仍缺乏有效的干预手段。自噬是细胞内溶酶体依赖的物质降解过程，它对维持细胞自稳态、应对内外环境刺激具有重要意义。我们前期研究发现，自噬的激活可通过抑制凋亡等途径显著缓解SAH后早期脑损伤，但其激活调控机制尚不明确。内质网应激广泛参与许多神经系统疾病的发生发展，但未有报道其与SAH后神经元自噬间的联系及机制。我们最新研究发现内质网应激在SAH后神经元自噬过程中显著激活，且内质网应激抑制剂TUDCA可显著降低SAH后神经系统自噬水平，加重早期脑损伤。因此我们将围绕内质网应激PERK-eIF2a-ATF4通路，利用SAH模型大鼠和原代神经元细胞，采用药物抑制与激活、免疫印迹、RNA干扰等技术，从体内、体外两个层面，阐明该通路对SAH后神经元自噬的作用和机制，为临床治疗SAH提供新思路和理论依据。

蛛网膜下腔出血（SAH）是一种严重危害人民健康的疾病，它引发的早期脑损伤是其首要的致病机制，但目前仍缺乏有效的干预手段。自噬是细胞内溶酶体依赖的物质降解过程，它对维持细胞自稳态、应对内外环境刺激具有重要意义。我们前期研究发现，自噬的激活可通过抑制凋亡等途径显著缓解SAH后早期脑损伤，但其激活调控机制尚不明确。内质网应激广泛参与许多神经系统疾病的发生发展，但未有报道其与SAH后神经元自噬间的联系及机制。我们最新研究发现内质网应激在SAH后神经元自噬过程中显著激活，且内质网应激抑制剂TUDCA可显著降低SAH后神经系统自噬水平，加重早期脑损伤。因此我们围绕内质网应激PERK-eIF2a-ATF4通路，利用SAH模型大鼠，采用药物抑制与激活、免疫印迹、RNA干扰等技术，探索该通路对SAH后神经元自噬的作用与具体机制。研究结果发现，SAH大鼠模型建立后，内质网应激PERK-eIF2a-ATF4通路显著激活，并于SAH后72小时到达高峰。PERK蛋白主要表达于神经元细胞中。激活的PERK蛋白可显著促进神经细胞凋亡，加重SAH大鼠神经功能障碍。应用PERK特异性抑制剂GSK2606414后，可显著提高抗凋亡因子Bcl-2的表达，降低促凋亡因子Bax, caspase-3的水平，减少神经元凋亡，改善SAH大鼠神经功能障碍。与此同时， GSK2606414可显著减轻SAH大鼠脑水肿，减少SAH大鼠脑组织中Evans blue 染料的渗出，提高血脑屏障紧密连接蛋白ZO-1、Occludin、Claudin5的表达水平，缓解SAH大鼠血脑屏障的破坏。进一步的机制研究显示，GSK2606414的神经保护作用主要通过激活Akt通路而实现，抑制Akt可显著逆转GSK2606414的神经保护作用。

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