Gnetupendin A attenuates ischemic stroke injury by modulating oxidative stress through AMPK/SIRT1 pathway.

Oxidative stress represents a critical pathological mechanism underlying cerebral ischemia-reperfusion injury (CIRI) progression. This study investigated the therapeutic potential of gnetupendin A (GA) against CIRI by targeting oxidative stress. In middle cerebral artery occlusion/reperfusion (MCAO/R) rats, GA treatment significantly ameliorated neurological deficits, reduced infarct volume and cerebral edema, alongside preserved neuronal integrity. GA exhibited potent antioxidant properties by reducing in vivo and in vitro levels of superoxide anion, reactive oxygen species (ROS), and lipid peroxides. Furthermore, GA reversed the excessive activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 2 by inhibiting the translocation of p47phox and p67phox and the expression of gp91phox. GA also preserved mitochondrial homeostasis by enhancing membrane potential, attenuating cytochrome C (cyt c) leakage, and suppressing mitochondrial ROS release. Mechanistically, GA enhanced the interaction between AMP-activated protein kinase (AMPK) α1 and silent information regulator of transcription 1 (SIRT1), facilitated by an elevated NAD+/NADH ratio and subsequent deacetylation of liver kinase B1 (LKB1). Molecular docking, drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) experiments confirmed a direct and robust binding affinity of GA for both AMPK and SIRT1. Further evidence indicated that GA mitigates CIRI-induced NOX2 activation and mitochondrial dysfunction via an AMPK/SIRT1 interdependent mechanism, thereby alleviating oxidative stress. Collectively, these findings reveal a novel mechanism for GA-mediated neuroprotection in CIRI and support its development as a promising antioxidant therapeutic agent.