Electroacupuncture relieves neuropathic pain via modulation of the ferroptosis pathway through GPX4

Objectives: Neuropathic pain (NP) is a chronic condition with complex mechanisms and limited treatments. While electroacupuncture (EA) shows promising analgesic effects, its molecular mechanisms remain unclear. This study investigated whether EA modulates ferroptosis to alleviate NP via glutathione peroxidase 4 (GPX4). Methods: A chronic constriction injury (CCI) rat model was employed. EA was administered at ST36 and GB34 for 14 consecutive days. Pain hypersensitivity, neuronal injury, glial activation, ferroptosis-related markers, mitochondrial morphology and the effect of co-administration of Ferrostatin-1 (Fer-1) were assessed. Results: EA alleviated CCI-induced pain hypersensitivity, restoring mechanical withdrawal threshold (MWT) to 77.2% and paw withdrawal latency (PWL) to 85.4% of sham group levels ( p < 0.01). Immunofluorescence analysis revealed that EA increased neuronal nuclei (NeuN)-positive cell counts by 41.5% ( p < 0.001) while reducing glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule (Iba)-1 expression by 48.2% and 54.7%, respectively ( p < 0.001). EA restored GPX4 expression (2.3-fold, p < 0.01), increased glutathione (GSH) levels, and reduced acyl-coenzyme A (CoA) synthetase long-chain family member 4 (ACSL4), malondialdehyde (MDA) and iron accumulation. Furthermore, EA reversed mitochondrial shrinkage and cristae disruption. Immunofluorescence confirmed increased GPX4 co-localization in spinal neurons. Notably, co-administration with Fer-1 further enhanced neuroprotection and pain relief, suggesting additive effects. Conclusion: These findings suggest that EA relieves NP by modulating GPX4-mediated ferroptosis, inhibiting lipid peroxidation and preserving mitochondrial integrity. The enhanced effect with EA and Fer-1 in combination further supports ferroptosis modulation as a potential mechanism underlying EA-induced analgesia.