Exosome-mediated delivery of 3-n-butylphthalide rescues microglial energy crisis and ameliorates neuroinflammation in ischemic stroke

Stroke remains the second leading cause of death and the primary cause of long-term disability worldwide, with ischemic stroke accounting for the majority of cases. Ischemia triggers robust microglial activation, yet the precise regulatory mechanisms underlying microglial functional reprogramming remain incompletely understood. Here, we demonstrate that excessive mitophagy drives metabolic energy failure in microglia following cerebral ischemia, resulting in impaired phagocytosis and exacerbated neuroinflammation. Analysis of single-cell RNA-sequencing data from mouse brains in the sham, transient middle cerebral artery occlusion (tMCAO, mMCAO), and permanent middle cerebral artery occlusion (pMCAO, sMCAO) groups revealed that mitophagy was markedly activated in microglia under sustained ischemia and was associated with impaired phagocytic and cytoskeletal pathways. In vitro oxygen-glucose deprivation (OGD) assays showed that phagocytosis of apoptotic neurons by microglia induced upregulation of Drp1, triggering excessive mitochondrial fission and mitophagy, which caused ATP depletion and reduced clearance capacity. The mitophagy inhibitor 3-methyladenine alleviated inflammatory responses but failed to restore mitochondrial quality. In contrast, 3-n-butylphthalide (NBP) stabilized mitochondrial membrane potential, restored ATP production, and improved microglial phagocytic defects and inflammation. To achieve targeted delivery, we constructed BV2 microglia-derived exosomes encapsulating NBP (BV2 exo @ NBP), which efficiently enhanced drug accumulation in ischemic lesions and significantly improved neurological outcomes in stroked mice. These results identify excessive mitophagy as a core mechanism underlying microglial energy crisis after cerebral ischemia and provide a mitochondria-targeted therapeutic strategy for ischemic stroke. Importantly, the neuroprotective efficacy, mitochondrial restoration, and anti-inflammatory effects of BM@NEB were fully recapitulated in 18-month-old aged mice, a clinically relevant model that more closely reflects the stroke patient population, supporting the translational potential of this exosome-based therapeutic strategy.