Gnetupendin A protects against ischemic stroke through activating the PI3K/AKT/mTOR-dependent autophagy pathway

Autophagy has been recently emerged as a prominent factor in the pathogenesis of ischemic stroke (IS) and is increasingly being considered as a potential therapeutic target for IS. Gnetum parvifolium has been identified as a potential therapeutic agent for inflammatory diseases such as rheumatism and traumatic injuries. However, the pharmacological effects of Gnetupindin A (GA), a stilbene compound isolated from Gnetum parvifolium, have not been fully elucidated until now.

Here we identified the therapeutic potential of GA for IS, deeply exploring the possible mechanisms related to its regulation of autophagy.

The mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R) and the oxygen-glucose deprivation reperfusion (OGD/R)-exposed cells served as models to study the protection of GA against IS. The adeno-associated virus (AAV) encoding shAtg5, in conjunction with autophagy inhibitor 3-Methyladenine (3-MA) were utilized to explore the role of GA in regulating autophagy following IS. Molecular docking, CETSA, and DARTS were used to identify the specific therapeutic target of GA. PI3K inhibitor LY294002 was employed to test the participation of PI3K in GA-mediated autophagy and neuroprotective effects following IS.

Our findings revealed that treatment with GA significantly alleviated the brain infract volume, edema, improved neurological deficits and attenuated apoptosis. Mechanistically, we found that GA promoted autophagic flow both in vivo and in vitro after IS. Notably, neural-targeted knockdown of Atg5 abolished the neuroprotective effects mediated by GA. Inhibition of autophagy using 3-MA blocked the attenuation on apoptosis induced by GA. Moreover, molecular docking, CETSA, and DARTS analysis demonstrated that GA specifically targeted PI3K and further inhibited the activation of PI3K/AKT/mTOR signaling pathway. LY294002, which inhibits PI3K, reversed GA-induced autophagy and neuroprotective effects on OGD/R-treated cells.

We demonstrated, for the first time, that GA protects against IS through promoting the PI3K/AKT/mTOR-dependent autophagy pathway. Our findings provide a novel mechanistic insight into the anti-IS effect of GA in regulating autophagy.