Mitochondrial Inhibitor Rotenone Triggers and Enhances Neuronal Ferroptosis Following Intracerebral Hemorrhage

Ferroptosis, a form of regulatory non-apoptotic cell death driven by iron-dependent lipid peroxidation, accounts for more than 80% of the total types of neuronal death in the acute phase of intracerebral hemorrhage (ICH). Mitochondria have essential roles in energy production, macromolecule synthesis, cellular metabolism, and cell death regulation. However, its role in ferroptosis remains unclear and somewhat controversial, especially in ICH. This study aimed to investigate whether damaged mitochondria could trigger and enhance neuronal ferroptosis in ICH. The isobaric tag for relative and absolute quantitation proteomics on human ICH samples suggested that ICH caused significant damage to the mitochondria, which presented ferroptosis-like morphology under electron microscopy. Subsequently, use of the mitochondrial special inhibitor Rotenone (Rot) to induce mitochondrial damage showed that it has significant dose-dependent toxicity on primary neurons. Single Rot administration markedly inhibited neuronal viability, promoted iron accumulation, increased malondialdehyde (MDA) contents, decreased total superoxide dismutase (SOD) activity, and downregulated ferroptosis-related proteins RPL8, COX-2, xCT, ASCL4, and GPX4 in primary neurons. Moreover, Rot enhanced these changes via hemin and autologous blood administration in primary neurons and mice, mimicking the in vitro and in vivo ICH models, respectively. Furthermore, Rot exacerbated the ICH-induced hemorrhagic volumes, brain edema, and neurological deficits in mice. Together, our data revealed that ICH induced significant mitochondrial dysfunction and that mitochondrial inhibitor Rot can trigger and enhance neuronal ferroptosis.

铁死亡（Ferroptosis）是一种由铁依赖性脂质过氧化驱动的调节性非凋亡细胞死亡形式，在脑出血（intracerebral hemorrhage, ICH）急性期的神经元死亡总类型中占比超过80%。线粒体在能量产生、大分子合成、细胞代谢及细胞死亡调控中发挥关键作用。然而其在铁死亡中的作用仍不明确且存在一定争议，在ICH场景中尤为突出。本研究旨在探究受损线粒体是否可触发并加剧ICH中的神经元铁死亡。
对人类ICH样本开展的同量异位素相对与绝对定量标记蛋白组学分析显示，ICH可对线粒体造成显著损伤，后者在电子显微镜下呈现出铁死亡样形态特征。随后，使用线粒体特异性抑制剂鱼藤酮（Rotenone, Rot）诱导线粒体损伤的实验表明，该抑制剂对原代神经元具有显著的剂量依赖性毒性。单次给予鱼藤酮可显著抑制原代神经元的细胞活性，促进细胞内铁蓄积，升高丙二醛（malondialdehyde, MDA）含量，降低总超氧化物歧化酶（superoxide dismutase, SOD）活性，并下调铁死亡相关蛋白RPL8、COX-2、xCT、ASCL4及GPX4的表达水平。
此外，在分别模拟体外和体内ICH模型的原代神经元及小鼠实验中，经血红素（hemin）和自体血处理后，鱼藤酮可进一步加剧上述异常变化。进一步实验证实，鱼藤酮可加重小鼠ICH诱导的出血体积、脑水肿及神经功能缺损。
综上，本研究数据表明，ICH可引发显著的线粒体功能障碍，且线粒体抑制剂鱼藤酮能够触发并加剧神经元铁死亡。