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. 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.