Hydroxysafflor Yellow A Protects HT22 Cells Against Ferroptosis-Associated Injury with Keap1 as a Candidate Interaction Partner

Hydroxysafflor yellow A (HSYA), a bioactive constituent of Carthamus tinctorius L., has antioxidant and cytoprotective properties. Whether Keap1 is a candidate interaction partner of HSYA in RSL3-challenged hippocampal-derived cells remains unresolved. We examined mouse HT22 cells exposed to RSL3 using viability, mitochondrial, iron, oxidative stress, lipid peroxidation, and protein assays. HSYA pretreatment followed by continued co-incubation with RSL3 preserved viability and mitochondrial ultrastructure and attenuated labile Fe2+ accumulation, reactive oxygen species, and lipid peroxidation. These changes were accompanied by increased SLC7A11 and GPX4 abundance, reduced ACSL4, and attenuation of the RSL3-induced increase in HO-1. HSYA treatment was also associated with increased nuclear Nrf2 and the GAPDH-normalized p-GSK-3β (Ser9) signal. Docking, DARTS, lysate-based CETSA, and low-amplitude SPR responses provided complementary, exploratory evidence consistent with an HSYA–Keap1 interaction. These findings support the protective effects of HSYA against ferroptosis-associated injury and identify Keap1 as a candidate interaction partner. Integrating cellular protection with exploratory interaction evidence advances the pharmacological characterization of HSYA and provides a testable molecular hypothesis for further investigating its neuroprotective potential.