CX3CR1/P2X4R signaling-mediated spinal microglial M1 polarization contributes to chronic pelvic pain in endometriosis

BACKGROUND: Deeply infiltrating endometriosis (DIE), characterized by its extrauterine invasion, is strongly associated with chronic pelvic pain (CPP). Peripheral-central sensitization synergistically contribute to the pathogenesis of CPP in endometriosis. METHODS: CPP severity was assessed in 66 endometriosis patients using visual analog scale. Mechanical and thermal pain thresholds were assessed in C57BL/6 mouse model of endometriosis via von Frey filaments and a hot plate test. Vascular injury was evaluated via immunofluorescence and transmission electron microscopy. To mimic the ischemic microenvironment of ectopic lesions, neurons were subjected to oxygen-glucose deprivation and then co-cultured with microglia to assess the effects on microglial activation. Finally, the therapeutic efficacy of pharmacological intervention was validated in a mouse model of endometriosis. RESULTS: DIE patients exhibited neuropathic CPP accompanied by increased nerve fiber infiltration, and endometriosis mouse models displayed widespread nociceptive sensitization. Ectopic lesions showed significant enrichment of inflammatory mediators and vascular injury, collectively driving peripheral sensitization. Additionally, spinal microglia exhibited M1 polarization in endometriosis mice. Mechanistically, neuronal ligands triggered microglial M1 polarization through CX3CR1 and P2X4R-mediated activation of the IκBα/NF-κB signaling pathway. Subsequently, M1 microglia released pro-inflammatory mediators (IL-1β, TNF-α and iNOS), driving central neuroinflammation and potentiating central sensitization. Pharmacological inhibition of CX3CR1 and P2X4R attenuated pain hypersensitivity, reduced spinal cytokine levels, and suppressed lesion progression in murine models. CONCLUSIONS: This study revealed a synergistic mechanism whereby lesional inflammation, vascular pathology, and spinal microglial M1 polarization collectively contribute to CPP in endometriosis. The CX3CR1/P2X4R-NFκB pathway represents a promising therapeutic target for endometriosis-associated chronic pain.