Inhibition of Panx1/P2X7R/NLRP3 Signaling Attenuates Bone Cancer Pain and Spinal Microglial Activation

Objectives: This study aims to investigate whether regulation of the Panx1/P2X7R/NLRP3 signaling axis in spinal microglia alleviates bone cancer pain. Methods: An in vivo bone cancer pain model was established by inoculating osteolytic tumor cells into the femoral medullary cavity of C3He/J mice. Nociceptive behaviors were evaluated using paw withdrawal mechanical thresholds and spontaneous flinch counts. The expression levels of Panx1, P2X7R, and the NLRP3 in the spinal dorsal horn (SDH) were examined. Analgesic effects of intrathecally administered 10Panx, BBG, and MCC950 were evaluated, and the intracellular localization of Panx1 was determined using immunofluorescence. In Panx1-knockdown BV2 cells, ATP-induced activation of the P2X7R-Panx1 pathway, IL-1β and IL-18 production, and ROS accumulation were assessed. Results: Tumor inoculation induced mechanical allodynia and increased spontaneous flinches. Moreover, Panx1, P2X7R, and NLRP3 were upregulated in the mouse SDH, accompanied by increased levels of caspase1, IL-1β, and IL-18. Intrathecal administration of 10Panx, BBG, and MCC950 significantly alleviated bone cancer pain related behaviors in mice. Panx1 was predominantly localized in activated microglia of the SDH following tumor inoculation. In ATP-stimulated BV2 cells, Panx1 knockdown markedly attenuated P2X7R and NLRP3 expression, reduced IL-1β and IL-18 production, and diminished intracellular ROS accumulation. Conclusions: Tumor cell inoculation promotes the release of pro-inflammatory cytokines and enhances nociceptive transmission by activating the Panx1/P2X7R/NLRP3 signaling axis in the spinal microglia, thereby contributing to the development of bone cancer pain. Targeting the Panx1/P2X7R/NLRP3 signaling axis may therefore represent a promising therapeutic strategy for treating bone cancer pain.