Pinnatifolin from Syringa pinnatifolia protects against myocardial ischemic injury in mice by targeting GPX4 to suppress ferroptosis

BACKGROUND The peeled stem of Syringa pinnatifolia Hemsl. (SP) has traditionally been used to treat ischemic heart diseases in Inner Mongolia region, China. Through a modified bioactivity-guided fractionation, our group previously identified I3 as the equivalent and pharmacological fraction of total SP extract; however, its specific constituents and mechanisms remain unknown. PURPOSE The study aims to identify the anti-acute myocardial ischemia (AMI) constituents of SP and elucidate the molecular target and mechanisms of the pharmacological bioactive constituents. METHODS The fraction I3 was separated by using ODS and silica gel column chromatography, and five monoeoxylignans (1-5) were isolated. The anti-AMI effects of fractions and a single molecule pinnatifolin (3) were evaluated in mice. Ferroptosis and mitochondrial functions were assessed by biochemical assays, fluorescence imaging, and western blotting techniques. Potential molecular targets were screened by proteomic profiling and further validated by molecular docking, cellular thermal shift assay, and SPR. RESULTS Fraction P retained the equivalent anti-AMI effect comparable to that of I3. P suppressed ferroptosis, and preserved mitochondrial function, accompanied by activation of the AMPK/PGC-1α pathway. P2 was demonstrated as a bioactive subfraction, from which five monoeoxylignans were isolated. Among them, pinnatifolin improved cardiac function and attenuated ferroptotic injury. GPX4 was proved as a potential molecular target underlying the cardioprotective effect of pinnatifolin. CONCLUSIONS A major lignan pinnatifolin exerts anti-AMI effects equivalent to its parent fraction P2 and P of SP.P exerts dual cardioprotective effects via AMPK/PGC-1α activation and ferroptosis suppression, while pinnatilolin acts through GPX4-dependent ferroptosis inhibition.