Ergothioneine promotes functional recovery after spinal cord injury by stabilizing VPS35 to suppress mitochondrial damage-driven neuroinflammation and oxidative stress

Background Spinal cord injury (SCI) triggers a complex secondary injury cascade involving neuroinflammation, oxidative stress, and mitochondrial dysfunction. These interconnected pathological processes exacerbate neuronal damage and hinder functional recovery. However, effective therapeutic strategies targeting these interrelated pathways remain limited. Ergothioneine (EGT), a naturally occurring antioxidant with potent cytoprotective properties and a favorable safety profile, has emerged as a promising candidate for SCI intervention, yet its mechanisms of action in this context remain unclear.Methods A mouse model of contusive SCI was established to evaluate the neuroprotective potential of ergothioneine. Mice received a moderate contusion injury at the T10 spinal segment by using a weight-drop impactor (5 g × 5 cm). Animals were randomly divided into four groups (n=6 per group): Sham, SCI+vehicle, SCI+50 mg/kg EGT, and SCI+100 mg/kg EGT, with EGT administered intraperitoneally for 7 consecutive days starting at 6 h post-injury. Functional recovery was assessed using behavioral tests, histological analyses, and electrophysiological recordings. Proteomic profiling, limited proteolysis-mass spectrometry (LiP-MS), molecular docking, and molecular dynamics simulations were employed to identify direct molecular targets of ergothioneine. In vitro studies using LPS-stimulated BV-2 microglia were conducted to investigate its effects on mitochondrial function, oxidative stress, and inflammatory responses. VPS35 knockdown experiments were performed to validate its mechanistic involvement.Results Ergothioneine treatment, particularly at 100 mg/kg, significantly improved locomotor recovery, preserved tissue integrity, and enhanced electrophysiological function in SCI mice. It markedly suppressed microglia-mediated neuroinflammation and oxidative stress, as indicated by reduced pro-inflammatory cytokine expression and decreased reactive oxygen species (ROS) production. Mechanistically, VPS35 was identified as a direct binding target of ergothioneine. Ergothioneine stabilized VPS35 expression, maintained mitochondrial membrane potential, reduced mitochondrial ROS generation, and alleviated mitochondrial dysfunction in activated microglia. Notably, VPS35 knockdown significantly attenuated these protective effects, demonstrating that ergothioneine exerts its actions in a VPS35-dependent manner.Conclusion Our study demonstrates that EGT confers neuroprotection after SCI by targeting VPS35, stabilizing mitochondria, and suppressing microglia-driven neuroinflammation and oxidative damage. These findings nominate EGT as a promising, well-tolerated therapeutic candidate for translational SCI treatment.