Paeoniflorin Alleviates Oxygen–Glucose Deprivation/Reoxygenation Injury by Mediating Crosstalk Between Neurons and Endothelial Cells Through the VEGF/PI3K-AKT/mTOR Pathway

Background/Objectives: Cerebral ischemia–reperfusion injury (CIRI) poses therapeutic challenges because of oxidative stress, blood–brain barrier disruption, and neuronal apoptosis, limiting current treatments. Paeoniflorin (PF) from Paeonia lactiflora has neuroprotective potential, but its multi-target mechanisms remain unclear. This study investigated the role and mechanisms of PF in CIRI, focusing on neuron–endothelial crosstalk. Methods: Oxygen–glucose deprivation/reoxygenation (OGD/R) models were established using SH-SY5Y (human neuroblastoma) and HCMEC/D3 cells (human cerebral microvascular endothelial). Network pharmacology was used to predict potential PF targets and pathways. RNA sequencing, molecular docking, and molecular dynamics simulation were performed to screen and evaluate PF binding characteristics with key targets, and MTT, flow cytometry, Western blotting, and co-cultures were employed to detect paracrine interactions. Results: Network pharmacology and transcriptomics identified VEGF/PI3K-AKT/mTOR pathway enrichment. Molecular docking confirmed stable PF binding to VEGF-A (−8.4 kcal/mol), AKT (−5.5 kcal/mol), and mTOR (−9.6 kcal/mol). PF (10–80 μM) showed no cytotoxicity and reduced OGD/R injury in a concentration-dependent manner (maximal at 40 μM). PF activated VEGF/PI3K-AKT/mTOR signaling, reducing apoptosis by 57% (SH-SY5Y) and 33% (HCMEC/D3); PI3K inhibitor LY294002 abolished these effects. PF-treated HCMEC/D3-conditioned media enhanced OGD/R neuronal viability, verifying paracrine crosstalk. Conclusions: PF alleviated CIRI by directly protecting neurons and indirectly modulating neuron–endothelial crosstalk through VEGF/PI3K-AKT/mTOR activation, supporting its multi-target therapeutic potential.