Ligustrazine Alleviates Nitroglycerin‐Induced Migraine by Regulating Mitochondrial Energy Metabolism Through the Concurrent Regulation of CypD, ANT, and PAR2

ABSTRACT To investigate the protective effects of ligustrazine (Lig) against nitroglycerin (NTG)‐induced migraine and explore its underlying mechanisms, with a particular focus on mitochondrial energy metabolism and the coordinated regulation of protease‐activated receptor 2 (PAR2), cyclophilin D (CypD), and adenine nucleotide translocator (ANT). An NTG‐induced rat migraine model and NTG‐challenged PC12 cells were utilized. Migraine‐like behaviors, cell viability, oxidative stress, and mitochondrial function parameters (reactive oxygen species [ROS], nitric oxide [NO], mitochondrial membrane potential [MMP], ATP, and lactic acid) were evaluated. The expression of PAR2, CypD, and ANT was analyzed via Western blotting. Erastin was applied to verify the specific mechanisms. Furthermore, immunoprecipitation‐mass spectrometry (IP‐MS) was performed to identify CypD‐interacting proteins. Validating IP‐MS results through protein docking. Lig significantly alleviated NTG‐induced migraine‐like behaviors in rats and improved the viability of NTG‐challenged PC12 cells. Biochemically, Lig mitigated oxidative stress and mitochondrial dysfunction by reducing ROS, NO, and lactic acid levels while restoring MMP and ATP production. Mechanistically, Lig effectively suppressed the NTG‐induced upregulation of PAR2, CypD, and ANT. The application of Erastin abolished Lig's protective efficacy, confirming its dependence on mitochondrial energy metabolism pathways. Additionally, IP‐MS successfully identified 46 CypD‐interacting proteins, expanding the mechanistic understanding of migraine pathogenesis. The protein docking results also support the reliability of the IP‐MS results. Lig effectively alleviates NTG‐induced migraine by restoring mitochondrial energy metabolism, in close association with the coordinated modulation of PAR2, CypD, and ANT expression. These findings highlight Lig as a promising therapeutic candidate and offer potential molecular targets for clinical migraine management.