Previous studies had showed that Apelin 13 could protect against apoptosis induced by ischemic/reperfusion (I/R). However, the mechanisms whereby Apelin 13 protected brain I/R remained to be elucidated. The present study was designed to determine whether Apelin 13 provided protection through AMPK/GSK-3β/Nrf2 pathway. In vivo, the I/R model was induced and Apelin 13 was given intracerebroventricularly 15 min before reperfusion. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. For in vitro study, PC12 cells were used. To clarify the mechanisms, proteases inhibitors or siRNA were used. Protein levels were investigated by western blotting. The results showed that Apelin 13 treatment significantly reduced infarct size, improved neurological outcomes, decreased brain edema, and inhibited cell apoptosis, oxidative stress, and neuroinflammation after I/R. Apelin 13 significantly increased the expression of Nrf2 and the phosphorylation levels of AMPK and GSK-3β. Furthermore, in cultured PC12 cells, the same protective effects were also observed. Silencing Nrf2 gene with its siRNA abolished the Apelin 13’s prevention of I/R-induced PC12 cell injury, oxidative stress, and inflammation. Inhibition of AMPK by its siRNA decreased the level of Apelin 13-induced Nrf2 expression and diminished the protective effects of Apelin 13. The interplay relationship between GSK-3β and Nrf2 was also verified with relative overexpression. Using selective inhibitors, we further identified the upstream of AMPK/GSK-3β/Nrf2 is AR/Gα/PLC/IP3/CaMKK. In conclusion, the previous results showed that Apelin 13 protected against I/R-induced ROS-mediated inflammation and oxidative stress through activating the AMPK/GSK-3β pathway by AR/Gα/PLC/IP3/CaMKK signaling, and further upregulated the expression of Nrf2-regulated antioxidant enzymes.

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 (Fe^2 + 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.

Quercetin, a naturally occurring flavonoid, is mainly extracted from tea, onions, and apples. It has the underlying neuroprotective effect on experimental ischemic stroke. A systematic review and meta‐analysis were used to assess quercetin’s efficacy and possible mechanisms in treating focal cerebral ischemia. Compared with the control group, twelve studies reported a remarkable function of quercetin in improving the neurological function score (NFS) (P < 0.05), and twelve studies reported a significant effect on reducing infarct volume (P < 0.05). Moreover, two and three studies showed that quercetin could alleviate blood‐brain barrier (BBB) permeability and brain water content, respectively. The mechanisms of quercetin against focal cerebral ischemia are diverse, involving antioxidation, antiapoptotic, anti‐inflammation, and calcium overload reduction. On the whole, the present study suggested that quercetin can exert a protective effect on experimental ischemic stroke. Although the effect size may be overestimated because of the quality of studies and possible publication bias, these results indicated that quercetin might be a promising neuroprotective agent for human ischemic stroke. This study is registered with PROSPERO, number CRD 42021275656.

The present study aims to evaluate the impact of Astilbin (AST) on cortical neuron survival in vitro under conditions of oxygen-glucose deprivation and reoxygenation (OGD/R) and determine the role of NF-E2-related factor 2 (Nrf2) in this process. Primary neurons were pre-treated with various concentrations of AST for 8 h before OGD induction. Cell viability and lactate dehydrogenase (LDH) leakage were assessed to determine the optimal concentration. Biomarkers related to oxidative stress, antioxidant enzyme activities, and apoptosis were evaluated at 24 h post-OGD/R. To investigate the involvement of Nrf2 in AST-mediated neuroprotection, we conducted molecular docking and microscale thermophoresis analyses, as well as examined the expression levels of Nrf2 and its regulatory genes including heme oxygenase-1(HO-1), (NAD(P)H: quinone oxidoreductase 1 (NQO-1), and peroxiredoxin 1 (Prdx1). Additionally, lentivirus-mediated knockdown of Nrf2 and overexpression of Nrf2 with L-sulforaphane (SFN) were performed, followed by an assessment of cell viability, oxidative stress, antioxidant enzyme activities and apoptosis. Pre-treatment with AST reduced oxidative stress levels while increasing antioxidant enzyme activities and mitigating neuronal apoptosis. After OGD/R exposure, AST upregulated nuclear Nrf2 expression and increased the expression of HO-1, NQO-1 and Prdx1 in the cytoplasm. However, the knockdown of Nrf2 abolished the antioxidative and protective effects exerted by AST treatment. Conversely, combining AST with the Nrf2 agonist SFN demonstrated an enhancement in the protective effects provided by AST. These results demonstrate that Nrf2-dependent antioxidant responses contribute to AST-induced tolerance against neuronal injury caused by OGD/R injury. Overall findings support the ability of AST to protect primary neurons from OGD/R-induced damage through activation of Nrf2-dependent antioxidant responses.