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·916 · 南 京 医 科 大 学 学 报 2024年7月

［Abstract］ Objective：To identity core genes of neuroinflammation mediated by microglia in sepsis ⁃ associated encephalopathy
（SAE）through bioinformatics analysis and validate them through in vitro cellular experiments. Methods：Transcriptomic datasets of
peripheral blood from sepsis patients（GSE65682）and in vitro lipopolysaccharide（LPS）⁃stimulated microglial cell activation model
（GSE103156）were obtained from the Gene Expression Omnibus（GEO）database. Weighted gene co ⁃ expression network analysis
（WGCNA）was used to screen the modules significantly related to clinical diagnosis of sepsis in the GSE65682 dataset. The
intersection between differentially expressed genes（DEG）in LPS⁃treated and untreated microglial cells from the GSE103156 dataset
and the WGCNA modules was determined. Functional enrichment analysis of DEG was performed using gene ontology（GO）and Kyoto
Encyclopedia of Genes and Genomes（KEGG）. A protein⁃protein interaction network was constructed using STRING，and core genes
were screened by Cytoscape and Lasso regression analysis. An in vitro cellular activation model of LPS⁃induced BV2 microglial was
established，and gene expression was detected using quantitative real⁃time PCR（RT⁃qPCR）. Histone deacetylase 9（HDAC9）was
overexpressed in microglia using the lentiviral vector method，and Western blot was employed to detect the inflammation related
molecule expression. Results：The WGCNA analysis identified nine modules associated with the clinical diagnosis of sepsis in the
GSE65682 dataset，comprising 332 genes. Limma analysis identified 1 272 DEGs in LPS ⁃ stimulated microglial cells from the
GSE103156 dataset. Eighteen overlapping genes were obtained，and the Lasso regression analysis further selected four hub genes：G
protein ⁃ coupled receptor 183（GPR183），HDAC9，nicotinamide adenine dinucleotide kinase（NADK），and leucine rich repeat
containing 25（LRRC25）. RT⁃qPCR confirmed downregulation of mRNA expression of Gpr183 and Hdac9 genes and upregulation of
Lrrc25 expression in the inflammatory activation model of LPS ⁃ stimulated microglial cell，with no significant change in Nadk
expression. Western blot showed that overexpression of HDAC9 promoted the expression of pro⁃inflammatory cytokines interleukin（IL）⁃
1β and inducible nitric oxide synthase（iNOS）in LPS ⁃ induced microglial cells and enhanced JAK1 ⁃ STAT3 phosphorylation.
Conclusion：This study identitfies four key genes mediating neuroinflammation in SAE through bioinformatics analysis and
preliminarily demonstrates that HDAC9 has pro ⁃ inflammatory activity in microglia，providing new insights and data for further
mechanistic research on SAE.
［Key words］ sepsis associated encephalopathy；microglia；bioinformatics analysis；differential genes；core genes
［J Nanjing Med Univ，2024，44（07）：915⁃926］

［6］
脓毒症相关性脑病（sepsis associated encepha⁃ 经营养因子，维持脑内环境稳态 。然而，全身性感
lopathy，SAE）是指由于脓毒症全身性感染及机体炎 染状态下，细菌脂多糖（lipopolysaccharides，LPS）等
症反应失调而导致的弥漫性脑功能障碍，急性期表 外 源 性 病 原 相 关 分 子 模 式（pathogen associated
现为行为异常、谵妄甚至昏迷，与高死亡率密切相 molecular pattern，PAMP）及白介素⁃1β（interleukins⁃1β，
关，慢性期则表现为持续认知功能受损，可累及超 IL⁃1β）等内源性损伤相关分子模式（damage associ⁃
50%的脓毒症幸存者，严重影响其生活质量 ［1- 2］ 。 ated molecular pattern，DAMP）通过血⁃脑屏障进入脑
SAE 症状可出现于脓毒症明确诊断之前，对于 SAE 实质，激活小胶质细胞，引发高水平神经炎症，导致神
的早期识别与积极治疗有助于提高脓毒症患者的 经元继发性损伤，是SAE的重要病理基础 ［7-8］ 。以小
生存率，改善远期预后 。由于 SAE 的病理机制还 胶质细胞介导的神经炎症为切入点，是解析SAE关
［3］
未完全阐明，临床缺乏明确的诊断及特异有效的治 键基因与发病机制的重要突破口。
疗方案，因此对于SAE的关键机制及分子靶点的深 随着基因芯片、生物信息学等技术的迅速发
［4］
入研究具有重要意义 。 展，高通量转录组数据被广泛用于研究疾病生物学
脓毒症患者全身性感染、免疫炎症反应失调、 特性。本研究通过对公共数据库中脓毒症患者血液
低灌注等因素导致脑缺血/出血损伤、血⁃脑屏障受 标本及体外LPS刺激小胶质细胞的转录组数据进行
损、神经炎症反应、神经突触受损、神经兴奋性毒性 整合分析，筛选SAE中神经炎症相关核心基因，并在
［3］
等，是 SAE 的重要致病机制 。作为脑内唯一常驻 体外小胶质细胞炎性活化模型中进行初步验证。
固有免疫细胞，小胶质细胞介导的神经炎症在 SAE
1 材料和方法
病理进程中发挥重要作用 。正常生理状态下，小
［5］
胶质细胞具有免疫防御、免疫监视等重要功能，及 1.1 材料
时清除损伤凋亡细胞，参与神经突触重塑、释放神 LPS（L2880，Sigma公司，美国）；胎牛血清（A511⁃