Mechanism of Astragaloside IV Against Cerebral Ischemia–Reperfusion Injury: Inhibiting Neuronal Apoptosis via the CytC/Apaf-1 Mitochondrial Pathway

Background: Neuronal apoptosis is the core pathological mechanism of cerebral ischemic–reperfusion injury (CIRI); although Astragaloside IV (AS-IV) has demonstrated neuroprotective activity against CIRI, its specific molecular mechanisms underlying the regulation of this apoptosis-related pathway remain to be systematically elucidated. Methods: We establish an in vivo model of middle cerebral artery occlusion/reperfusion (MCAO/R) in rats and an in vitro model of oxygen–glucose deprivation/reperfusion (OGD/R) in PC12 cells. Six core apoptotic proteins, including CytC, Apaf-1, BAX, Bcl-2, Caspase3, and Caspase9, were detected using neurological function scoring, TTC/HE/Nissl staining, TUNEL staining, Western blot, and immunofluorescence techniques. Molecular docking and molecular dynamics simulation were utilized to analyze the binding affinity between AS-IV and the aforementioned apoptotic proteins. Results: Molecular docking and dynamics simulation demonstrated AS-IV stably binds six core apoptotic proteins, and comparative analysis with target-specific reference ligands identified Apaf-1 as its primary target with the most favorable binding properties. In rat MCAO/R models, AS-IV alleviated neurological deficits, reduced cerebral infarct volume and improved brain pathological damage; in PC12 cell OGD/R models, it decreased neuronal apoptosis. Western blot and immunofluorescence confirmed AS-IV downregulated pro-apoptotic proteins (cytoplasmic CytC, Apaf-1, BAX, cleaved-Caspase9/3) and upregulated anti-apoptotic Bcl-2. Conclusions: This study clarifies the anti-apoptotic molecular mechanism of AS-IV, it alleviates CIRI by targeting the CytC/Apaf-1 mitochondrial apoptotic pathway.