Eupatilin Ameliorates Spinal Cord Injury by Inhibiting Damage-associated Microglia and Optimizing the Regenerative Microenvironment

Microglia represent critical therapeutic targets in spinal cord injury (SCI), with damage associated microglia (DAM) playing key roles in neuroinflammation and tissue repair.Through integrated in-silico analysis of scRNA-seq and microarray datasets, we identified DAM subsets specific to acute SCI characterized by hub genes Fcer1g, Grn, and Gusb. Using a C57BL/6 mouse spinal cord contusion model, we validated increased DAM accumulation post-injury and demonstrated their propensity to transition toward homeostatic microglia (MG2). Eupatilin treatment promoted DAM-toMG2 differentiation, as confirmed through bulk and single-cell RNA sequencing analyses revealing supportive gene expression changes. These findings establish DAM as functionally distinct microglial populations in acute SCI and identify Eupatilin as a therapeutic agent that facilitates beneficial microglial polarization. This work provides mechanistic insights into microglial dynamics during SCI and suggests targeted modulation of DAM-to-MG2 transitions as a promising therapeutic strategy for promoting inflammation resolution and functional recovery. Overall design: Prior to surgery, mice were fasted for 6 hours received anesthesia with a single intraperitoneal injection of 270-330 mg/kg Avertin (Sigma-Aldrich, MO, USA, T48402). The T9 lamina was surgically exposed while preserving the integrity of the dura mater, followed by a 30-second aneurysm clip compression for all injured groups. The muscle and skin were then sutured closed. Following SCI, manual bladder expression was performed 3 times daily until bladder function recovered. For the first 3 days, intrathecal injections were administered: the treatment group received Eupatilin (30 µl, 1.5 mg/mL, Bide, Shanghai, China, BD298186), while the SCI control group received an equivalent volume of saline.

小胶质细胞是脊髓损伤（spinal cord injury, SCI）的关键治疗靶点，损伤相关小胶质细胞（damage associated microglia, DAM）在神经炎症与组织修复过程中发挥核心调控作用。本研究通过对单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与微阵列数据集的整合生物信息学分析，鉴定出以Fcer1g、Grn及Gusb为核心基因的急性脊髓损伤特异性DAM亚群。随后利用C57BL/6小鼠脊髓挫伤模型，验证了损伤后DAM聚集量显著升高，并证实其具有向稳态型小胶质细胞（MG2）转化的趋势。滨蒿内酯（Eupatilin）处理可促进DAM向MG2分化，批量RNA测序与单细胞RNA测序分析均显示，该处理能诱导有益的基因表达变化。本研究确立了DAM作为急性脊髓损伤中功能独特的小胶质细胞群的地位，并确定滨蒿内酯是一种可介导有益小胶质细胞极化的治疗药物。本研究阐明了脊髓损伤过程中小胶质细胞动态变化的机制，并提出靶向调控DAM向MG2的转化作为促进炎症消退与功能恢复的潜在治疗策略。实验设计：手术前小鼠禁食6小时，通过腹腔单次注射270~330 mg/kg的Avertin（西格玛奥德里奇公司，美国密苏里州，货号T48402）进行麻醉。暴露T9节段椎板并维持硬脑膜完整性，所有损伤组均采用动脉瘤夹压迫30秒。随后缝合肌肉与皮肤。脊髓损伤后，每日手动辅助排空膀胱3次直至膀胱功能恢复。术后前3天进行鞘内注射：治疗组给予滨蒿内酯（30 μl，1.5 mg/mL，必德公司，中国上海，货号BD298186），脊髓损伤对照组给予等体积生理盐水。