Tamarixetin suppresses neuronal ferroptosis in ischemic stroke rats by targeting and facilitating nuclear factor erythroid-2-related factor 2 expression

Objective: Neuronal ferroptosis has emerged as a promising therapeutic target for ischemic stroke. Tamarixetin, a natural dietary flavonoid, exerts protective effects against ischemic stroke by modulating neuroinflammatory responses and mitigating oxidative stress. However, its potential role in regulating neuronal ferroptosis remains unclear. Methods: A rat model of middle cerebral artery occlusion and reperfusion and an erastin-treated SH-SY5Y cell model were used for in vivo and in vitro experiments, respectively. The neurological function of the rats was evaluated using a series of behavioral tests, the Garcia scoring system, and 2,3,5-triphenyltetrazolium chloride staining. Neuronal damage was detected via immunofluorescence staining and TdT-mediated dUTP nick-end labeling. Commercial kits, western blotting, and coimmunoprecipitation were used to analyze neuronal ferroptosis and the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. The direct target protein of tamarixetin was examined using the cellular thermal shift assay, drug affinity-responsive target stability assay, surface plasmon resonance, and molecular docking. Cellular Nrf2 was knocked down using small interfering RNA. Results: Tamarixetin mitigated the neurological dysfunctions of MCAO/R rats, including motor dysfunction, limb coordination impairment, neurological deficit, cerebral infarction, and reduced neuronal loss. Furthermore, it alleviated neuronal ferroptosis in vivo and in vitro by lowering the levels of iron ions, reactive oxygen species, malondialdehyde and Acyl-CoA synthetase long-chain family member 4 and upregulating the expressions of superoxide dismutase, glutathione, glutathione peroxidase 4, heme oxygenase-1 and solute carrier family 7 member 11. Tamarixetin activated the Nrf2 signaling pathway by suppressing Keap1 protein expression, weakening Keap1 and Nrf2 combination, upregulating Nrf2 protein expression and nuclear translocation, and promoting antioxidant response element activity. Nrf2 is the direct binding protein of tamarixetin. It specifically interacts with amino acid residues at arginine 72, arginine 515, and lysine 518. The effects of tamarixetin on Nrf2 signaling pathway activation and neuronal ferroptosis inhibition were abrogated in Nrf2 knockdown cells challenged with erastin. Conclusions: Our findings not only identify tamarixetin as a novel ferroptosis inhibitor but also elucidate its mechanism of action via direct binding and Nrf2 pathway activation, providing a promising therapeutic candidate for ischemic stroke. Graphical abstract: http://links.lww.com/AHM/A203