溶酶体内铁超载介导SAH后神经元氧化应激损伤的机制研究

The product iron of blood clot degradation following subarachnoid hemorrhage (SAH) is a major cause of neuronal damage induced by oxidative stress. However, its exact mechanism is unknown. Preliminary studies conducted by the applicant's group confirmed that the iron mediating oxidative stress damage mainly located in lysosomes. Therefore, we propose that the lysosomal iron overload following SAH catalyzes a Fenton reaction to produce hydroxyl radicals, increase permeability of lysosomal membrane, and release cathepsin; and this series of cascade reactions initiate apoptosis, thus leading to neuronal damage. The project intended to establish in vitro and in vivo SAH models to investigate the relationship between lysosomal iron overload, hydroxyl radicals, lysosomal membrane permeability, lysosomal cathepsin release, and apoptosis at cellular and molecular levels. We applied a lysosomotropic iron chelator and cathepsin inhibitor, respectively, to observe their protective effect on neurons after SAH. The results demonstrated that lysosomal iron overload resulted in increased lysosomal membrane permeability, thus triggering neuronal apoptosis. The finding of this project will not only academically clarify the mechanism of neuronal damage caused by oxidative stress following SAH, but also provide a strong academic foundation for making a breakthrough in clinical treatment.

蛛网膜下腔出血（SAH）后血凝块降解产物铁是引起神经元氧化应激损伤的重要原因，但具体机制不明。申请者课题组前期研究证实介导细胞氧化应激损伤的铁主要位于溶酶体内，因此，我们提出：SAH后溶酶体内铁超载通过催化Fenton反应，产生羟自由基，引起溶酶体膜通透性增加，释放组织蛋白酶，这一系列级联反应启动细胞凋亡，导致神经元损伤。本项目拟建立离体和在体SAH模型，在整体、细胞和分子水平研究溶酶体内铁超载与羟自由基、溶酶体膜通透性、溶酶体组织蛋白酶释放、细胞凋亡的关系，并分别应用亲溶酶体的铁螯合剂LAP和组织蛋白酶抑制剂，观察其对SAH后神经元的保护作用，证实溶酶体内铁超载导致溶酶体膜通透性增加，进而引发神经元凋亡。本项目的研究成果将不仅在学术上阐明SAH后氧化应激性神经元损伤的机制，也为临床治疗学的突破提供有益的学术基础。

观察大鼠蛛网膜下腔出血（SAH，Subarachnoid Hemorrhage）早期两种主要的溶酶体组织蛋白酶Cathepsin B/D 与凋亡蛋白caspase-3的时相性变化，探讨溶酶体组织蛋白酶与早期脑损伤的关系，并分析去铁敏（DFX）对其的干预及对早期脑损伤各项指标的影响，为研究蛛血后早期脑损伤的发病机制提供理论依据。我们结果提示溶酶体可能参与了SAH后早期脑损伤的过程，SAH后溶酶体膜可能被破坏，导致溶酶体组织蛋白酶（Cathepsin B/D）的释放并诱发凋亡途径。铁鳌合剂DFX能抑制溶酶体组织蛋白酶的表达，抑制细胞凋亡，并能显著减轻脑水肿、改善血脑屏障，改善神经认知功能，从而减轻SAH后早期脑损伤。稳定溶酶体膜，减少Cathepsin B/D的释放可能为SAH后早期脑损伤提供新的治疗途径。硫辛酸胺 (α-lipoic acid-plus LAP) 是LA（α-lipoic acid）胺的衍生物，能通过螯合溶酶体内铁来保护神经元免受氧化应激损伤，然而其在蛛网膜下腔出血（subarachnoid hemorrhage SAH）的神经保护作用机制尚未明了。因此，我们拟利用LA，LAP来干预SAH模型大鼠，通过分析脑组织内的溶酶体组织蛋白酶B/D（cathepsin B/D），凋亡蛋白（caspase-3），促凋亡蛋白（bax），铁蛋白（ferritin），血红素加氧酶（HO-1）的表达情况以及其他相关早期脑损伤（early brain injury EBI）指标来评估LA，LAP在SAH后EBI中神经保护作用的差异。LA，LAP在实验性SAH后EBI中均可发挥神经保护作用。但LAP抗氧化应激能力明显强于LA，可能与其靶向溶酶体同时作用于溶酶体内铁密切相关。