Inhibition of ferroptosis after intracerebral hemorrhage

脑出血后铁下垂的抑制 自发性脑出血（ICH）是发病率和死亡率最高的卒中亚型。 因为在ICH期间大量血液被释放到细胞外空间中， 血红蛋白/游离血红素对脑恢复非常重要。血红素被血红素加氧酶降解成铁， 胆绿素和一氧化碳。重要的是，铁在大脑中的积累有助于继发性脑损伤。 脑出血后的损伤事实上，铁毒性有助于胶原酶诱导的小鼠出血性脑损伤， 用铁螯合剂减少铁积累可改善神经元存活。最近，一种铁- 在癌细胞和器官型海马切片中鉴定出非凋亡性细胞死亡的依赖形式 谷氨酸暴露后培养。它是由小分子或抑制谷胱甘肽的条件触发的 生物合成或谷胱甘肽过氧化物酶4（GPX 4）活性。这种受调控的细胞死亡的特征是广泛的 铁依赖性脂质过氧化，可被亲脂性抗氧化剂ferrostatin-1或livestatin抑制， 1.铁凋亡细胞死亡也已被证明发生在脑细胞中。迄今为止，已在小鼠中报道了 帕金森病和脑出血模型。无论是ferrostain-1还是livestain-1对铁凋亡的抑制作用降低 ICH损伤和改善老年动物的功能结局仍然未知。因此，这项研究是 预期用于表征老年动物ICH背景下的铁凋亡细胞死亡。我们的长期目标是限制 ICH损伤和改善功能结局。这一提议的科学目的是检验以下假设： 通过ferrostain-1或library stain-1抑制铁凋亡可保护ICH脑并改善功能结果。我们 将进一步确定GPX 4活性的增强是否能抑制铁凋亡并提供神经保护 ICH之后。在初步研究中，铁凋亡细胞死亡，其特征是萎缩的线粒体， 在小鼠ICH脑中的透射电子显微镜。此外，ICH后用ferrostatin-1治疗的小鼠 比用载体治疗的小鼠有更好的神经学结果。第二种独立的铁凋亡抑制剂， 利司他汀-1也表现出对ICH损伤的显著保护作用。总之，这些新的观察强烈地 支持抑制铁凋亡可能减少老年动物脑出血损伤的前提。第一个具体目标 将确定是否抑制铁依赖性脂质过氧化改善组织学和功能 两种性别老年小鼠ICH后的结局。第二个具体目标将决定是否过度表达 GPX 4的表达对ICH后的脑保护作用。这项研究将提供新的证据表明，铁凋亡细胞死亡，在 除了其他受调节的细胞死亡途径外，在患有ICH的老年动物的脑中也很突出。它还将 深入了解GPX 4活性增加预防ICH后铁凋亡的分子机制。 拟议项目的工作可以为ICH患者提供新药/治疗方法。成功验证 老年动物ICH模型中的脂质过氧化抑制剂将为以下方面提供理论依据和概念验证 未来的临床前和临床试验。

Inhibition of ferroptosis after intracerebral hemorrhage Spontaneous intracerebral hemorrhage (ICH) is the stroke subtype with the highest mortality and morbidity. Because large amounts of blood are released into the extracellular space during ICH, the metabolism of hemoglobin/free heme is very important for brain recovery. Heme is degraded by heme oxygenase into iron, biliverdin, and carbon monoxide. Importantly, iron accumulation within the brain contributes to secondary brain injury after ICH. Indeed, iron toxicity contributes to collagenase-induced hemorrhagic brain injury in mice, and reducing iron accumulation with iron chelators can improve neuronal survival. Recently, ferroptosis, an iron- dependent form of non-apoptotic cell death, was identified in cancer cells and in organotypic hippocampal slice cultures after glutamate exposure. It is triggered by small molecules or by conditions that inhibit glutathione biosynthesis or glutathione peroxidase 4 (GPX4) activity. This regulated cell death is characterized by extensive iron-dependent lipid peroxidation, which can be suppressed by lipophilic antioxidant ferrostatin-1 or liproxstatin- 1. Ferroptotic cell death also has been shown to occur in brain cells. To date, it has been reported in mouse models of Parkinson disease and ICH. Whether inhibition of ferroptosis by ferrostain-1 or liproxstain-1 reduces ICH injury and improves functional outcomes in aged animals remains unknown. Therefore, this research is intended to characterize ferroptotic cell death in the context of ICH in aged animals. Our long-term goal is to limit ICH injury and improve functional outcomes. The scientific objective of this proposal is to test the hypothesis that inhibition of ferroptosis by ferrostain-1 or liproxstain-1 protects ICH brain and improves functional outcomes. We will further determine whether augmentation of GPX4 activity inhibits ferroptosis and provides cerebroprotection after ICH. In pilot studies, ferroptotoic cell death, characterized by shrunken mitochondria, was detected by transmission electron microscopy in the mouse ICH brain. Additionally, mice treated with ferrostatin-1 after ICH had better neurologic outcomes than mice treated with vehicle. A second, independent, ferroptosis inhibitor, liproxstatin-1, also exhibited marked protection against ICH injury. Together, these novel observations strongly support the premise that inhibition of ferroptosis might reduce ICH injury in aged animals. The first specific aim will determine whether suppression of iron-dependent lipid peroxidation improves histologic and functional outcomes after ICH in aged mice of both sexes. The second specific aim will determine whether overexpression of GPX4 is cerebroprotective after ICH. This study will provide novel evidence that ferroptotic cell death, in addition to other regulated cell death pathways, is prominent in the brains of aged animals with ICH. It will also provide insight into the molecular mechanism by which increased GPX4 activity prevents post-ICH ferroptosis. Work on the proposed project could render new drugs/treatments for patients with ICH. Successful validation of lipid peroxidation inhibitors in the ICH models of aged animals will provide the rationale and proof-of-concept for future preclinical and clinical trials.