研究背景和目的：腹主动脉瘤（Abdominal Aortic Aneurysm, AAA）是全球范围内除动脉粥样硬化外最常见的主动脉疾病，是导致老年人群高死亡率的主要心血管疾病之一。AAA一旦破裂出血，可迅速导致失血性休克甚至死亡。目前尚未发现有效预防AAA破裂或抑制其进展的药物，但已有研究证实持续性炎症反应是AAA重要的致病机制之一。本研究采用单细胞RNA测序（Single-cell RNA Sequencing, ScRNA-seq）技术，探究AAA的炎症特征、细胞异质性及基因网络表达差异，并发现潜在的治疗靶点；同时，通过建立动物和细胞实验模型进行验证，以期为AAA的治疗提供新的理论依据。

研究方法：本研究从公共数据库获取AAA相关的ScRNA-seq数据，采用R语言软件Seurat包进行数据清洗与分析。首先对数据进行整合、质控、标准化、降维及聚类分析；然后使用多种方法对聚类的细胞群体进行注释；最后提取巨噬细胞亚群分析基因表达差异、功能富集通路及转录因子调控模式，并筛选出药物4-辛基衣康酸盐（4-Octyl itaconate, 4-OI）。为验证4-OI的治疗效果，本研究通过血管紧张素II诱导建立小鼠AAA模型，给予外源性4-OI干预，通过血管彩超、组织病理学、分子生物学分析评估4-OI对AAA形成的抑制作用。此外，采用THP-1细胞构建巨噬细胞焦亡模型，探究4-OI对细胞焦亡通路的影响。最后，通过整合人和小鼠主动脉的组织病理学、动物和细胞实验结果，系统验证4-OI对AAA的抑制作用并阐明其潜在分子机制。

研究结果：在AAA的主动脉壁中，免疫细胞占比高达85%，其中淋巴细胞占比最高，其次是单核巨噬细胞。对巨噬细胞进行亚群分类，并对各亚群的高表达基因进行功能富集分析，结果显示这些高表达基因在TNF-Α_SIGNALING_VIA_NF-KB Pathway、IL6_JAK_STAT3_SIGNALING Pathway、NOD-like Receptor Pathway以及INFLAMMATORY_RESPONSE Pathway等通路显著富集。转录因子网络调控分析发现sMAF蛋白家族转录活性明显升高，提示NRF2是参与调控的潜在关键转录因子。4-OI是一种能够激活NRF2的天然分子。人AAA组织切片免疫组化染色结果发现，与乳内动脉相比，AAA动脉壁NLRP3以及CD68表达明显升高。在细胞实验中，我们利用LPS/Nigerin诱导THP-1细胞构建巨噬细胞焦亡模型，发现4-OI干预能够显著减轻巨噬细胞焦亡，细胞焦亡通路相关蛋白的表达均显著降低，而NRF2的表达则显著升高。使用NRF2拮抗剂ML385进行干预，我们发现ML385能够部分逆转4-OI的抗细胞焦亡作用。在动物实验中，我们发现外源性补充4-OI后，与对照组相比，4-OI组小鼠的生存率显著提高，同时主动脉成瘤率和破裂率明显降低；超声测量和病理组织切片分析发现，4-OI有效抑制了主动脉壁扩张和弹性纤维蛋白层的断裂；新鲜组织WB、qRT-PCR实验和石蜡切片免疫组化染色表明，4-OI组小鼠主动脉中焦亡通路相关蛋白（NLRP3、N-GSDMD、Caspase-1 p20、IL-18）表达明显降低，NRF2蛋白表达升高，且组织冰冻切片多重免疫荧光发现NLRP3与巨噬细胞标志物CD68存在共定位现象。

研究结论：AAA核心病理特征是持续性激活的炎症反应，巨噬细胞焦亡是导致AAA形成和进展的致病机制之一。4-OI能够抑制小鼠AAA的形成和进展，其潜在机制是激活NRF2，阻断巨噬细胞NLRP3/Caspase-1 p20/N-GSDMD信号轴，抑制细胞焦亡延缓AAA的病理进展。这一发现为AAA的治疗提供了新的理论依据和潜在干预靶点。

Background and Objectives: Abdominal aortic aneurysm (AAA) is the second most common aortic disease globally after atherosclerosis and one of the leading cardiovascular diseases contributing to high mortality rates in the elderly. Once AAA ruptures and bleeds, it can rapidly lead to hemorrhagic shock or even death. Currently, no effective drugs have been identified to prevent AAA rupture or inhibit its progression, but researches have confirmed that persistent inflammatory response is one of the key pathogenic mechanisms of AAA. The present study uses single-cell RNA sequencing (ScRNA-seq) technology to explore the inflammatory characteristics, cellular heterogeneity, and gene network expression differences in AAA compared to normal aortic wall, and to identify potential therapeutic targets. Additionally, animal and cell models will be established for validation, aiming to provide a new theoretical basis for the treatment of AAA.

Methods: This study obtained AAA-related ScRNA-seq data from public databases and performed data cleaning and analysis using the Seurat package in the R software. The analysis was first integrated, quality controlled, standardized, and reduced dimensions of the data, followed by clustering. Subsequently, various cell annotation methods were applied to precisely annotate the cell populations. Finally, macrophage subgroups were extracted for gene expression differential analysis, functional enrichment pathway analysis and gene regulatory network (GRN) analysis. 4-Octyl itaconate (4-OI) was found to be the potential molecule to inhibit AAA progression. To validate the therapeutic effect of 4-OI, an Ang II induced mouse AAA model was established, and exogenous 4-OI intervention was given. The effect of 4-OI on AAA formation was evaluated by vascular ultrasound, histopathology, and molecular biology analysis. In addition, THP-1 cells were used to construct a macrophage pyroptosis model to investigate the effect of 4-OI on the pyroptosis pathway. Finally, human and mouse aortic tissue pathology, animal, and cell experimental results were integrated to systematically validate the inhibitory effect of 4-OI on AAA and elucidate its potential molecular mechanism.

Results: Immune cells account for as much as 85% of AAA wall, with lymphocytes being the most abundant, followed by monocyte macrophages. Subgroup classification of macrophages and functional enrichment analysis of highly expressed genes in each subgroup revealed significant enrichment in pathways such as TNF-α signaling via NF-κB, IL6-JAK-STAT3 signaling, NOD-like receptor pathway, and inflammatory response. GRN analysis showed a marked increase in the transcriptional activity of the sMAF protein family, suggesting that NRF2 is a potential key transcription factor involved in regulation. 4-OI is a natural molecule that can activate NRF2. Immunohistochemistry of human AAA tissue slices revealed significantly elevated expression of NLRP3 and CD68 in the abdominal aorta compared to the mammary artery. In cell experiments, LPS/Nigerin was used to induce THP-1 cells to construct a macrophage pyroptosis model, 4-OI intervention significantly reduced macrophage pyroptosis, and pyroptosis-related proteins expression level was significantly lower, while NRF2 expression level was significantly higher. The use of the NRF2 antagonist ML385 partially reversed the anti-pyroptosis effect of 4-OI. In animal experiments, after exogenous supplementation with 4-OI, the survival rate of mice in the 4-OI group was significantly higher compared to the control group, and the incidence and rupture rate of aortic aneurysms were significantly lower. Ultrasound measurements and histological analysis revealed that 4-OI effectively inhibited aortic wall dilation and the rupture of elastic fiber layers. WB, qRT-PCR, and immunohistochemical staining showed a significant reduction in the expression of pyroptosis-related proteins (NLRP3, N-GSDMD, Caspase-1 p20, IL-18) in the 4-OI group, with NRF2 expression increased. Multi-immunofluorescence staining of tissue frozen sections showed colocalization of NLRP3 and the macrophage marker CD68.

Conclusion: The core pathological feature of AAA is the persistent activation of inflammatory responses, and macrophage pyroptosis is one of the pathogenic mechanisms leading to AAA formation and progression. 4-OI can inhibit AAA formation and progression in mice, and its potential mechanism is the activation of NRF2, blocking the macrophage NLRP3/Caspase-1 p20/N-GSDMD signaling axis, and inhibiting pyroptosis to delay the pathological progression of AAA. This finding provides a new theoretical basis and potential intervention targets for the treatment of AAA.