神经酰胺对脑缺血/复灌过程中自噬的调控作用及机制研究

Cerebral ischemia is a complex pathological process deficient in potent therapeutic strategy. Our previous research demonstrated that autophagy protected against focal brain ischemia/reperfusion (I/R) in mice, but it is still unclear which induced autophagy in this pathological process. It has become increasingly clear that ceramide, a membrane sphingolipid and a key mediator of cell stress responses, could play a critical role in autophagy induction in various in vitro models. Our preliminary results showed that I/R resulted in endogenous ceramide accumulation. Prevention the ceramide generation by N-SMase inhibitor could hamper I/R-evoked autophagy and aggravate the neuronal injury, while exogenous ceramide conduced to autophagy activation and neuroprotection. Furthermore, the preliminary data also suggested that ceramide may mediate the selective removal of damaged mitochondria via Parkin-dependent mitophagy to alleviate the I/R injury. In this proposal, we aimed to confirm and analyze the association of ceramide with autophagy during I/R, and further clarify the underlying mechanism. These investigations will help to understand the key factor for autophagy induction during I/R, and also provide significant experimental evidences in taking ceramide as a novel therapeutic target in cerebral ischemia.

缺血性脑损伤病理机制复杂，治疗困难。申请人所在课题组前期研究表明脑缺血/复灌诱导的自噬具有重要的神经保护作用，然而该过程中自噬的调控因素尚不明确。近期研究表明，细胞膜鞘磷脂成分神经酰胺可能参与自噬诱导过程。申请人预实验研究发现，脑缺血/复灌过程伴随大量神经酰胺产生，抑制其生成可降低该过程所诱导的自噬水平，并加重神经组织/细胞损伤；而外源性给予神经酰胺则可显著上调自噬，并减轻损伤。此外，还初步发现，神经酰胺可能通过调控Parkin介导的线粒体自噬，清除损伤线粒体，进而发挥神经保护作用。本研究拟进一步利用Atg5，Parkin基因敲除动物，结合药理学、RNA干扰等手段，从整体、细胞和分子生物学等多个层面明确神经酰胺在脑缺血/复灌过程中对自噬的调控作用，并初步阐明其相关分子机制。本项目将为揭示脑缺血/复灌诱发自噬的关键因素提供重要线索，也为开发神经酰胺相关药物治疗缺血性脑卒中提供理论基础。

缺血性脑损伤病理机制复杂，其中线粒体自噬在该疾病中发挥了重要作用。线粒体自噬发生的机制尚不清除，如何通过调控线粒体自噬进而保护缺血脑组织也有待明确。本项目探索了缺血脑组织中神经酰胺的水平，发现随缺血时间延长，其水平增加，阻断神经酰胺生成抑制线粒体自噬发生。进一步发现神经酰胺的堆积与缺血后的组织酸化密切相关。利用体内、外缺血性脑损伤模型，发现了脑缺血后的酸化处理过程具有神经保护作用。酸后处理增强了线粒体自噬，而线粒体自噬抑制取消了上述神经保护作用。进一步机制研究发现，Parkin基因敲除动物中酸后处理诱导的线粒体自噬及神经保护作用均消失，提示Parkin依赖的线粒体自噬在其中发挥了重要的作用。通过利用缺血不同时间复灌的方式，研究发现Parkin的上述作用与延长缺血后的复灌时间窗密切相关。上述研究发现了Parkin依赖的线粒体自噬在脑缺血过程中的重要神经保护作用，该线粒体自噬的发生可能与神经酰胺的生成有关。