Apigenin Suppresses NLRP3 Inflammasome Activation and Pyroptosis and Promotes Functional Recovery by Promoting Mitophagy in Experimental Spinal Cord Injured Rats

Zuomeng Wu,1,2,* Yunxiao Fang,1,2,* Yixiang Dong,1,2 Yue Qin,1,2 Ao Liu,1,2 Tianyu Han,1,2 Peiwen Song,1,2 Cailiang Shen1– 3 1Department of Orthopedics (Spinal Surgery), The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, People’s Republic of China; 2Laboratory of Spinal and Spinal Cord Injury Regeneration and Repair, The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, People’s Republic of China; 3Anhui Province Research Center for the Clinical Application of Digital Medical Technology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230022, People’s Republic of China*These authors contributed equally to this workCorrespondence: Cailiang Shen, Email [email protected]: Secondary damage following spinal cord injury (SCI) is closely associated with pyroptosis and mitochondrial dysfunction. Apigenin (API), a natural flavonoid, possesses notable anti-inflammatory and antioxidant properties. However, whether API can inhibit microglial pyroptosis via the mitophagy pathway, thereby exerting neuroprotective effects, remains unclear. This study aimed to elucidate the mechanism by which API mitigates post-SCI inflammatory responses through modulation of the mitophagy-NLRP3 axis.Methods: Neurological recovery was assessed using the Basso, Beattie, and Bresnahan scale, neuroelectrophysiological recordings, and histological analyses. The effects of API on NLRP3 inflammasome activation, reactive oxygen species (ROS) generation, and mitochondrial membrane potential were assessed using ELISA, quantitative PCR, immunofluorescence, and JC-1 staining.Results: API significantly improved locomotor function in SCI rats, reduced scar formation, and promoted axonal regeneration. Mechanistically, API downregulated NLRP3/ gasdermin D expression in microglia, reduced the release of inflammatory factors, and enhanced mitophagy. Notably, the protective effects of API were reversed by Mdivi-1 and mimicked by Urolithin A, confirming that mitophagy is the primary mechanism mediating API’s anti-pyroptotic effects.Conclusion: API attenuates microglial pyroptosis and facilitates SCI repair by enhancing mitophagy-mediated clearance of damaged mitochondria and suppressing activation of the ROS/NLRP3 inflammasome pathway in rats. These findings provide important preclinical evidence supporting the development of multi-target neuroprotective strategies derived from natural compounds.Plain Language Summary: API effectively suppresses NLRP3 inflammasome activation and mitigates microglial pyroptosis.API enhances mitophagy in microglia following SCI.API inhibits pyroptosis through mitophagy-mediated regulation of NLRP3 activation.API significantly promotes locomotor recovery and neural regeneration in spinal cord-injured rats. Keywords: spinal cord injury, apigenin, mitophagy, pyroptosis