MaR1 and NGF co-treatment promotes sciatic nerve repair by modulating GSK-3β/NF-κB signaling and suppressing inflammasome-mediated pyroptosis

OBJECTIVE: This study aimed to investigate the therapeutic effects of Maresin 1 (MaR1) combined with nerve growth factor (NGF) on peripheral nerve injury (PNI) repair and to explore whether the underlying mechanism is associated with the regulation of GSK-3β/NF-κB signaling and inflammasome-mediated pyroptosis. METHODS: An in vitro PC12 cell injury model was established by H₂O₂ exposure for 4 h, followed by treatment with NGF, MaR1, or their combination. Cell viability was assessed using the CCK-8 assay, while PI staining, LDH release, and ROS fluorescence were used to evaluate cell injury, membrane integrity disruption, and oxidative stress. Western blotting was performed to detect inflammasome- and pyroptosis-related proteins, including NLRP3, cleaved caspase-1, IL-1β, IL-18, TNF-α, GSDMD-N, and full-length GSDMD (GSDMD-FL), as well as the GSDMD-N/GSDMD-FL ratio and GSK-3β/NF-κB signaling-related proteins. Immunofluorescence staining was used to examine the expression of GSK-3β, NLRP3, and p-p65. The ROS scavenger NAC was used to further assess the involvement of ROS in H₂O₂-induced inflammasome/pyroptosis-related responses, and the GSK-3β activator DIF-3 was applied for reversal experiments. In vivo, a sciatic nerve transection and microsurgical repair model was established in 6-week-old male Sprague-Dawley rats. NGF, MaR1, their combination, or saline was locally administered every other day for three injections. Functional recovery was evaluated using the sciatic functional index (SFI), gastrocnemius muscle wet weight, and muscle atrophy. Toluidine blue staining and NF200/MBP double immunofluorescence were performed to assess axonal regeneration and remyelination. Inflammasome-, pyroptosis-, and GSK-3β/NF-κB signaling-related markers were further examined in injured sciatic nerve tissues. RESULTS: In vitro, MaR1 and NGF co-treatment more effectively improved PC12 cell viability and reduced LDH release, ROS accumulation, and PI fluorescence intensity compared with either treatment alone, indicating attenuation of H₂O₂-induced cell injury and membrane integrity disruption. The combined treatment also downregulated NLRP3, cleaved caspase-1, IL-1β, IL-18, TNF-α, and GSDMD-N expression and reduced the GSDMD-N/GSDMD-FL ratio, suggesting inhibition of inflammasome-mediated pyroptosis-like responses. NAC treatment decreased ROS levels and concomitantly attenuated PI staining and pyroptosis-related protein expression, supporting the involvement of ROS accumulation in H₂O₂-induced inflammasome/pyroptosis-related injury. In addition, MaR1 and NGF co-treatment reduced the p-p65/p65 ratio, suppressed p65 activation, and modulated GSK-3β/NF-κB signaling. DIF-3 partially reversed the inhibitory effects of the combined treatment on inflammasome activation, pyroptosis-related markers, and NF-κB signaling. In vivo, MaR1 and NGF co-treatment improved SFI, alleviated gastrocnemius muscle atrophy, improved the morphology of myelinated nerve fibers, and enhanced NF200- and MBP-positive axonal regeneration and remyelination more effectively than monotherapy. The combined treatment also decreased the GSDMD-N/GSDMD-FL ratio and inflammasome-related protein expression in injured sciatic nerve tissues, accompanied by modulation of GSK-3β/NF-κB signaling. CONCLUSION: MaR1 and NGF co-treatment attenuates oxidative stress, neuroinflammation, and inflammasome-mediated pyroptosis-like responses after peripheral nerve injury, thereby promoting sciatic nerve structural repair and motor functional recovery. These protective effects may be at least partly associated with modulation of the GSK-3β/NF-κB signaling pathway.