Ginsenoside Rb3 Promotes Opa1‐Mediated Regenerative Neurogenesis via Activating the Ido1 Pathway in Ischemic Stroke

ABSTRACT The activation of neural stem cells (NSCs) residing in the subventricular zone (SVZ) and dentate gyrus (DG) has been shown to promote the restoration of damaged brain tissues. Ginsenoside Rb3 (Rb3) is a bioactive substance known for its pharmacological properties in treating neurological disorders. This study investigated the effects of Rb3 on neural regeneration following ischaemic stroke (IS) and the underlying mechanisms involved. Male C57BL/6 mice were utilized and were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Post‐ischemia, Rb3 was administered through intraperitoneal (i.p.) injection for either 7 or 28 days. The promotion of Rb3 on regenerative neurogenesis was detected by immunofluorescence staining. NSCs were pretreated with different concentrations of Rb3 for 24 h before oxygen–glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, immunofluorescence staining and flow cytometry were used to detect the migration and proliferation of Rb3 in OGD/R‐induced NSCs. Furthermore, Adeno‐associated virus (AAV) transduction experiments, siRNA transfection experiments, gene knockout experiments, targeted metabolomics analysis, molecular dynamics simulation, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assays were used to explore the promotion and mechanism of Rb3 on regenerative neurogenesis following IS. Rb3 promoted Opa1‐mediated NSCs migration and proliferation. Knockdown of Opa1 blunted the above‐promoting effects of Rb3 in both the brains of ischemia–reperfusion (I/R)‐treated mice and OGD/R‐treated NSCs. Mechanistically, targeted metabolomics, molecular dynamics, molecular docking, CETAS, and DARTS experiments showed that Rb3 promoted Opa1‐mediated neural regeneration required the activation of Ido1 and that Ido1 served as a direct target of Rb3 to repair I/R injury. Moreover, studies in siRNA‐mediated knockdown and KO mice revealed that inhibition of Ido1 attenuated the enhancing effect of Rb3 on mitochondrial fusion. Our study provides novel evidence that Rb3 promotes neurogenesis through an Ido1/Opa1‐mediated pathway involving the interaction between Rb3 and Ido1, leading to improved long‐term neurological function. These results indicate that Rb3 or other mitochondrial fusion promoters could be a potential neurorestorative strategy for regenerative neurogenesis following IS.