NEAT1/miR-181a-5p/HMGB1 Axis Regulates Macrophage Polarization and Inflammation in Sepsis Models

Sepsis is characterized by a dysregulated host immune response and remains a leading cause of mortality worldwide. Long non-coding RNA NEAT1 has been implicated in inflammatory diseases, but its specific role in macrophage polarization during sepsis has not been fully defined. Here, we systematically examine the NEAT1/miR-181a-5p/HMGB1 axis across clinical samples, cultured macrophages, and a cecal ligation and puncture (CLP) mouse model. Quantitative PCR, western blotting, dual-luciferase reporter assays, and RNA pull-down experiments are used to confirm the competitive endogenous RNA interaction among NEAT1, miR-181a-5p, and HMGB1. Functional assays, including immunofluorescence, transwell migration, and TUNEL staining, are applied to assess macrophage polarization, migration, and apoptosis. In vivo, the CLP model combined with ELISA and histopathology validates the impact of NEAT1 knockdown on cytokine profiles and organ injury. NEAT1 and HMGB1 are upregulated, whereas miR-181a-5p is downregulated, in patients with sepsis and in lipopolysaccharide-stimulated macrophages. Silencing NEAT1 promotes M2 macrophage polarization, reduces pro-inflammatory cytokines, impairs macrophage migration, and alleviates tissue damage in septic mice via the miR-181a-5p/HMGB1 axis. To our knowledge, this is the first integrated protocol to characterize this lncRNA–microRNA–HMGB1 regulatory circuit in sepsis using harmonized clinical, in vitro, and in vivo approaches, and it provides a methodological framework for targeting NEAT1-related ceRNA networks as potential therapeutic strategies.

脓毒症（Sepsis）以宿主免疫应答失调为核心特征，仍是全球范围内致死的主要原因之一。长链非编码RNA NEAT1（long non-coding RNA, lncRNA）已被证实与炎症性疾病密切相关，但其在脓毒症进程中巨噬细胞极化过程中的具体作用尚未完全阐明。本研究借助临床样本、体外培养的巨噬细胞与盲肠结扎穿刺（cecal ligation and puncture, CLP）小鼠模型，系统探究了NEAT1/miR-181a-5p/HMGB1调控轴的作用机制。研究采用定量聚合酶链反应（quantitative PCR, qPCR）、蛋白质免疫印迹（western blotting, WB）、双荧光素酶报告基因实验以及RNA下拉实验，验证了NEAT1、miR-181a-5p与HMGB1之间的内源竞争RNA（competitive endogenous RNA, ceRNA）相互作用关系。同时通过免疫荧光、Transwell迁移实验以及末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色等功能实验，评估巨噬细胞的极化状态、迁移能力与凋亡水平。在体内层面，本研究结合CLP小鼠模型、酶联免疫吸附实验（enzyme linked immunosorbent assay, ELISA）与组织病理学分析，验证了NEAT1基因沉默对脓毒症小鼠细胞因子谱与器官损伤的影响。研究发现，临床脓毒症患者以及脂多糖（lipopolysaccharide, LPS）刺激的巨噬细胞中，NEAT1与HMGB1的表达水平显著上调，而miR-181a-5p的表达则显著下调。沉默NEAT1可通过调控miR-181a-5p/HMGB1轴，促进脓毒症小鼠体内的M2型巨噬细胞极化，降低促炎细胞因子的分泌，抑制巨噬细胞迁移，并减轻组织损伤。据我们所知，本研究首次通过整合临床、体外与体内的标准化实验方法，系统解析了脓毒症中该lncRNA-微小RNA-HMGB1调控环路，并为靶向NEAT1相关ceRNA网络的潜在治疗策略提供了方法论框架。