中文摘要
研究背景：
脑出血（intracerebral hemorrhage, ICH）是一种严重的卒中亚型，但截至目前的治疗方法有限。程序性细胞死亡（Programmed cell death, PCD）对免疫平衡至关重要，包括坏死、焦亡、凋亡、铁死亡、坏死等。然而，这些程序性细胞死亡模式在 ICH 后的优先级和程度仍有待进一步研究。我们利用单细胞转录组（Single-cell Transcriptome, ScRNA-seq）和空间转录组（Spatial Transcriptome, ST）技术分析了出血性脑卒中后大鼠脑组织超急性期（1h）、急性期（24h）和亚急性期（7d）不同程序性细胞死亡方式相关基因的变化趋势。我们发现铁死亡是 ICH 后的主要 PCD 过程，并且主要发生在成熟的少突胶质细胞里面。结合体内外实验，系统探讨了脑出血后成熟少突胶质细胞铁死亡的表型、机制及靶向干预的潜在疗效。
方法：
本研究基于单细胞转录组测序和空间转录组测序技术，运用SingleR和CellMarker工具，识别出14种细胞类型。进一步分析各细胞类型中程序性细胞死亡相关特征分数，以鉴定ICH后铁死亡相关基因的表达水平变化。此外，结合Western Blot（WB）、免疫荧光（IF）和透射电子显微镜（TEM）等技术手段，验证了Lcn2小胶质细胞亚群在ICH后通过Csf1/Csf1r信号通路介导少突胶质细胞铁死亡的潜在机制。为深入探究Lcn2在ICH后神经功能恢复中的作用，本研究构建了小胶质细胞Lcn2基因敲低模型，并通过改良Garcia评分、转棒实验和水迷宫测试等行为学评估方法，系统评价Lcn2靶向干预对脑出血后神经功能恢复的影响。
结果：
ICH后，铁死亡评分显著高于其他PCD评分，最早在ICH后1小时出现，并在24小时达到高峰。少突胶质细胞表现出明显的铁死亡基因激活，且铁死亡相关基因主要分布在海马和脉络丛。体内外实验进一步证实了ICH后少突胶质细胞中铁死亡的发生。WB和IF结果显示，Hemin处理后，Lcn2+小胶质细胞通过Csf1/Csf1r通路显著上调少突胶质细胞中的铁死亡相关蛋白ACSL4、xCT、FTH1，降低GPX4水平。TEM观察到线粒体结构的损伤，ROS水平升高。Lcn2基因敲低显著改善ICH后24小时和7天的神经功能缺损，提高运动能力和空间学习记忆能力。
结论：
本研究揭示了铁死亡是ICH后主要的PCD机制，主要发生在少突胶质细胞中，并通过Csf1/Csf1r通路介导。靶向Lcn2可显著改善ICH后的神经功能恢复，为脑出血的治疗提供了新的靶点。

Background: Intracerebral hemorrhage (ICH), a devastating subtype of stroke, remains therapeutically intractable with limited treatment options. Programmed cell death (PCD), encompassing necroptosis, pyroptosis, apoptosis, ferroptosis, and necrosis, plays a pivotal role in immune homeostasis. However, the hierarchy and spatiotemporal dynamics of these PCD modalities post-ICH remain poorly characterized. Leveraging single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST), we systematically profiled PCD-associated gene expression trajectories in rat brain tissues during hyperacute (1h), acute (24h), and subacute (7d) phases following hemorrhagic stroke. Our findings identified ferroptosis as the predominant PCD mechanism post-ICH, predominantly occurring in mature oligodendrocytes. Through integrated in vivo and in vitro experiments, we further elucidated the phenotypic features, molecular mechanisms, and therapeutic potential of targeting oligodendroglial ferroptosis post-ICH. Methods: In this study, based on single-cell transcriptome sequencing and spatial transcriptome sequencing, 14 cell types were identified using SingleR and CellMarker tools. PCD enrichment scores were calculated to determine dominant pathways. Mechanistic validation included Western blot (WB), immunofluorescence (IF), and transmission electron microscopy (TEM) to confirm Lcn2+ microglia-mediated oligodendroglial ferroptosis via the Csf1/Csf1r axis. A microglial Lcn2-knockdown model was established to assess neurofunctional recovery using modified Garcia tests, rotarod assays, and Morris water maze. Results: Ferroptosis scores significantly surpassed other PCD modalities post-ICH (peaking at 24h), with oligodendrocytes exhibiting robust activation of ferroptosis-related genes predominantly localized to the hippocampus and choroid plexus. In vivo and in vitro experiments confirmed iron accumulation (Perls staining), glutathione depletion, and lipid ROS elevation in oligodendrocytes. Hemin-treated Lcn2+ microglia upregulated ferroptotic markers (ACSL4↑, xCT↑, FTH1↑, GPX4↓) in oligodendrocytes via Csf1/Csf1r signaling, accompanied by mitochondrial ultrastructural damage (fragmented cristae, vacuolization) and elevated ROS levels. Lcn2 knockdown markedly ameliorated neurological deficits， enhanced motor coordination, and restored spatial memory at 24h and 7d post-ICH. Conclusions: This study establishes ferroptosis as the dominant PCD mechanism in oligodendrocytes post-ICH, mediated via the Csf1/Csf1r axis. Targeting Lcn2 significantly improves functional recovery, highlighting its therapeutic potential for ICH management.