Hyssopus cuspidatus Boriss. Attenuates Myocardial Ischemia/Reperfusion Injury by Modulating the Keap1-Nrf2-GPX4 Axis: Identification of 4,5-Dicaffeoylquinic Acid as a Keap1-Binding Constituent

Objective: This study aimed to elucidate the molecular mechanisms and potential active constituents of SXCF (the active fraction of Hyssopus cuspidatus Boriss.) against myocardial ischemia/reperfusion injury (MIRI). Results: SXCF pretreatment significantly reduced myocardial infarct size, ameliorated histopathological damage, and suppressed oxidative stress and inflammation in I/R mice. Network pharmacology integrated with lipidomics suggested that SXCF primarily targets lipid peroxidation. SPR further demonstrated that 4,5-dicaffeoylquinic acid, a candidate active constituent of SXCF, directly binds to Keap1 with high affinity (KD = 1.86 μM), which may interfere with Keap1-mediated Nrf2 regulation and facilitate Nrf2 nuclear translocation. This was accompanied by the upregulation of GPX4 and SLC7A11, downregulation of ACSL4, and reduced 4 HNE production, suggesting the involvement of the Keap1-Nrf2-GPX4 axis and effective inhibition of lipid peroxidation. Methods: UPLC HRMS was used to characterize the chemical composition of SXCF and identify its absorbed blood constituents. A mouse myocardial ischemia/reperfusion (I/R) model was established to evaluate the cardioprotective effects of SXCF. Network pharmacology combined with targeted lipid mediator profiling was applied to predict and verify its regulatory effects on cardiac lipid metabolism. Molecular docking and surface plasmon resonance (SPR) were utilized to identify direct binding targets, while immunofluorescence, Western blot, and qRT PCR were performed to validate key molecules of the Keap1-Nrf2-GPX4 axis. Conclusions: SXCF alleviates MIRI by suppressing lipid peroxidation. Its candidate active constituent, 4,5-dicaffeoylquinic acid, directly binds to Keap1 and may interfere with Keap1-mediated regulation of Nrf2, potentially engaging the Keap1-Nrf2-GPX4 axis to attenuate ferroptosis-associated lipid peroxidation. This study provides a potential therapeutic strategy and material basis for the treatment of MIRI.