Atractylenolide III alleviates cerebral ischemia-reperfusion injury by targeting Keap1 to activate the Nrf2/HO-1 pathway.

BACKGROUND: Atractylenolide III (ATL III), a natural sesquiterpene lactone, exhibits neuroprotective properties against cerebral ischemia-reperfusion injury (CIRI), but its underlying mechanism remains unclear.

PURPOSE: To identify the pharmacological target and elucidate the mechanism of ATL III in CIRI protection.

METHODS: Middle cerebral artery occlusion/reperfusion (MCAO/R) in mice and oxygen-glucose deprivation/reperfusion (OGD/R) in HT22 cells were employed to evaluate the protective effects of ATL III on neurological function, oxidative stress, and apoptosis. Network pharmacology, molecular docking, surface plasmon resonance, and molecular dynamics were used to identify its direct target. Molecular mechanisms were validated using Western blot, immunofluorescence, and a pharmacological inhibitor both in vitro and in vivo.

RESULTS: ATL III (10, 20, 40 mg/kg) significantly reduced infarct volume in MCAO/R mice from 42 % to 33 %, 27 %, and 20 %, and decreased neurological scores from 13 to 9, 7, and 5. Histology revealed alleviated hippocampal neuronal damage. In HT22 cells, ATL III decreased OGD/R-induced apoptosis, lowered ROS levels from 374 % to 202 %, increased SOD and GSH, and reduced MDA levels. Keap1 was identified as a direct target; ATL III disrupted the Keap1-Nrf2 interaction, reduced Keap1 protein levels, increased Nrf2 expression and nuclear translocation, and upregulated HO-1 expression. Nrf2 inhibition with ML385 abolished these antioxidative and neuroprotective effects in both models.

CONCLUSION: ATL III protects against CIRI by directly targeting Keap1 and activating the Nrf2/HO-1 pathway. Given its favorable safety profile and blood-brain barrier (BBB) permeability, ATL III holds promising translational potential as a plant-derived prophylactic agent for ischemic stroke.