Therapeutic time window of Sodium of Danshensu on cerebral ischemia and its mechanism of inhibiting oxidative stress and ferroptosis through Nrf2 pathway

Sodium of Danshensu (SDSS), extract of salvia miltiorrhiza root, has been shown to have neuroprotective effects on ischemic stroke (IS) in our previous studies. However, its therapeutic time window and mechanism of action remain unclear. Ferroptosis exerts a crucial feature in the development and progression of IS. Nuclear factor-E2-related factor 2 (Nrf2) can positively regulate the transcription of Recombinant Solute Carrier Family 7, member 11 (SLC7A11) and glutathione peroxidase (GPX4) genes that combat lipid peroxidation in ferroptosis. The current study aimed to assess therapeutic time window of SDSS and the pharmacological mechanism involved in Nrf2-mediated oxidative stress and ferroptosis. Mice with transient middle cerebral artery occlusion (MCAO) and HT22 cells with oxygen-glucose deprivation/reoxygenation (OGD/R) were induced to simulate IS. Mice were administered SDSS at 1, 3, 6 or 9 h after MCAO to determine the therapeutic time window of SDSS. MicroRNA-seq was conducted to analyze differentially expressed genes in both the MCAO and the SDSS treatment group. The interaction between SDSS and Nrf2 was also investigated using molecular docking, molecular dynamics (MD) simulations, and surface plasmon resonance (SPR) experiments. Furthermore, the neuroprotection of SDSS was investigated in Nrf2-deficient mice to assess the activation mechanism of the Nrf2/GPX4 axis by SDSS. The biomarkers (Fe2 + content, ROS, MDA, GSH, GSH/GSSG), mitochondrial structure, these proteins (Nrf2, SLC7A11, GPX4, FTH1, HO-1, ACSL4 and TFRC) expression were detected by commercial kits, transmission electron microscope (TEM) and Western blotting, respectively. The therapeutic time window of SDSS should be within 6 hours after MCAO, beyond which SDSS cannot play a therapeutic role. SDSS played a neuroprotective affection in mice and HT22 cells by restraining ROS, MDA and Fe2+ content, elevating GSH level and GSH/GSSG ratio. At the molecular mechanism, SDSS can bind to Nrf2, improve Nrf2 activity and nuclear expression, further enhance SLC7A11, GPX4, FTH1, HO-1 expression and reduce ACSL4 and TFRC expression. However, the neuroprotective effects of SDSS and its effect on ferroptosis-related proteins were partially reversed in Nrf2-deficient mice. The therapeutic time window of SDSS for ischemic stroke is relatively wide. The administration of SDSS can potentially mitigate brain damage through the inhibition of oxidative damage and ferroptosis, which is partly regulated by the Nrf2/GPX4 axis. Therefore, SDSS is a promising candidate for the treatment of ischemic stroke.