Hydroxysafflor Yellow A Alleviates Ischemic Stroke Through Modulation of GPX4-Associated Ferroptosis: Computational Simulation and Animal Validation.

Abstract

Background: Ferroptosis has been recognized as an important pathological mechanism underlying ischemic stroke (IS). However, the precise mechanisms underlying the neuroprotective effects of Hydroxysafflor yellow A (HSYA) in IS remain unclear.

Objective: Therefore, this study aimed to investigate the protective effects of HSYA against IS and explore the underlying ferroptosis-related mechanisms.

Methods: An integrated strategy combining network pharmacology, molecular docking, molecular dynamics simulations, and a middle cerebral artery occlusion (MCAO) mouse model was employed to elucidate the neuroprotective mechanisms of HSYA. Neurological deficit scores, infarct volume, histopathology, oxidative stress, and ferroptosis-related molecules were evaluated. For in vivo validation, mice were randomly assigned to the sham, MCAO, Ras-selective lethal (RSL), HSYA, and RSL + HSYA groups (n = 12 per group). Ras-selective lethal 3 (RSL3) was used as a glutathione peroxidase 4 (GPX4) inhibitor in the RSL and RSL + HSYA groups.

Results: Network pharmacology identified GPX4 and Ferritin Heavy Chain 1 (FTH1) as potential ferroptosis-related targets of HSYA in IS. Docking analysis showed favorable binding affinities of HSYA toward GPX4 (-9.3 kcal/mol) and FTH1 (-9.1 kcal/mol), and molecular dynamics simulations further supported the stability of the HSYA-GPX4 and HSYA-FTH1 complexes. In vivo experiments showed that HSYA significantly improved neurological deficit score, reduced infarct volume, and alleviated histopathological damage in MCAO mice (p < 0.01 or p < 0.05). Moreover, HSYA significantly decreased malondialdehyde (MDA) and reactive oxygen species (ROS) levels while increasing superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities (p < 0.01 or p < 0.05). HSYA significantly upregulated the mRNA and protein levels of GPX4, FTH1, and mitochondrial ferritin (FTMT) (p < 0.01 or p < 0.05). Notably, HSYA significantly increased the relative protein expression of GPX4 compared with the MCAO group (p < 0.01). Furthermore, RSL3 partially attenuated the protective effects of HSYA on cerebral injury, oxidative stress, and the expression of GPX4, FTH1, and FTMT (p < 0.01 or p < 0.05). Collectively, the present study demonstrates that HSYA confers neuroprotection against IS, at least in part, through regulation of GPX4-associated ferroptosis pathways.

Conclusions: HSYA provides significant neuroprotection against IS. These effects may be mediated, at least in part, through GPX4-associated ferroptosis pathways and the maintenance of iron homeostasis, thereby advancing understanding of its antioxidant mechanisms and supporting its therapeutic potential for IS.