Puerarin alleviates cerebral ischemia/reperfusion injury-induced oxidative stress via the Neurl1/JAG1/Notch1 signaling pathway

Background Cerebral ischemia/reperfusion injury (CIRI) remains a major challenge in stroke therapy, with limited effective neuroprotective interventions. Puerarin (Pue), a major bioactive isoflavone from Pueraria lobata, exhibits promising neuroprotective effects, yet its precise molecular targets and mechanisms remain unclear. Purpose To elucidate the molecular mechanisms of Pue’s neuroprotection against CIRI, including its direct protein target and downstream signaling pathways. Methods Both in vivo and in vitro models were employed, including transient middle cerebral artery occlusion (tMCAO) in rats and oxygen-glucose deprivation/reperfusion (OGD/R) in HT22 cells. Neuroprotective effects were assessed by infarct volume, neurological scores, histopathology, oxidative stress markers (SOD, GSH, MDA), reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP). An integrated transcriptomic-computational screening, molecular docking, and molecular dynamics (MD) simulations were used to identify Neuralized E3 ubiquitin protein ligase 1 (Neurl1) as a potential molecular target of Pue. Functional validation of candidate genes was conducted via genetic knockdown and pharmacological modulation (JAG1 peptide, DAPT). Results Pue dose-dependently reduced cerebral infarct volume, attenuated neurological deficits and histopathological damage, and alleviated oxidative stress in tMCAO rats. In OGD/R HT22 cells, Pue suppressed cytosolic and mitochondrial ROS accumulation and preserved mitochondrial membrane potential (MMP). Neuralized E3 ubiquitin protein ligase 1 (Neurl1) was identified as a molecular target of Pue. Mechanistically, Pue restored CIRI-induced suppression of Neurl1 protein expression in both tMCAO rats and OGD/R HT22 cells, accompanied by downregulation of Jagged1 (JAG1) and inhibition of Notch1 signaling. Neurl1 knockdown partially counteracted protective effects of Pue against oxidative stress and mitochondrial dysfunction. In vivo , co-administration of a Notch1 activator (JAG1 peptide) partially reversed the neuroprotective effects of Pue, whereas Notch1 inhibition with DAPT showed neuroprotective effects comparable to those of Pue. Conclusion Pue exerts neuroprotective effects against CIRI by restoring Neurl1 expression, which is associated with modulation of the JAG1/Notch1 signaling pathway and attenuation of oxidative stress. These findings identify the Neurl1/JAG1/Notch1 axis as a potential therapeutic target for CIRI.