P2Y2 receptors mediate astrocytic hyperplasia by increasing hydrogen peroxide production: Effects on neural regeneration after spinal cord injury

JOURNAL/rmrep/04.03/02273995-990000000-00039/figure1/v/2026-05-22T144211Z/r/image-tiff Objectives: Reactive astrogliosis impedes neural repair after spinal cord injury, but its regulation remains incompletely understood. Our previous research revealed that the G protein-coupled receptor P2Y2 receptor (P2Y2R) facilitates motor function recovery during the chronic phase of spinal cord injury, but the interaction between the receptor and astrocytes remains unclear. This study aimed to investigate whether P2Y2R modulates astrocyte reactivity via hydrogen peroxide and to evaluate the therapeutic potential of P2Y2R antagonism in spinal cord injury. Methods: Primary astrocytes were exposed to 25 µM hydrogen peroxide with or without the P2Y2R antagonist AR-C118925XX. In vivo , a T10 incomplete spinal cord injury model was established in female C57BL/6J mice, which received AR-C118925XX (1.0 mg/kg) intraperitoneally for 10 days post-injury. Hydrogen peroxide level determination, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling apoptosis detection, immunofluorescence (for glial fibrillary acidic protein, microtubule-associated protein-2, neuron-specific nuclear protein, and myelin basic protein), western blotting, behavioral assessments (Basso Mouse Scale and footprint analysis), and transmission electron microscopy were performed. Results: In vitro , AR-C118925XX reduced hydrogen peroxide release from reactive astrocytes, decreased apoptosis, and increased microtubule-associated protein-2 immunoreactivity in cocultures. In vivo , P2Y2R expression correlated positively with glial fibrillary acidic protein expression from the acute (1 day) phase to the chronic (28 days) phase, and P2Y2R was colocalized with astrocytes and neurons. AR-C118925XX treatment improved hindlimb motor function (Basso Mouse Scale score, P < 0.01; stride length, P < 0.05), reduced glial fibrillary acidic protein expression, and attenuated glial scar formation. It also increased neuronal survival (neuron-specific nuclear protein- and microtubule-associated protein-2–positive cells), reduced Bax expression, preserved the myelin ultrastructure (lower G-ratio, P < 0.001), and improved mitochondrial morphology. Notably, the neuroprotective effects were associated with a reduction in hydrogen peroxide levels in spinal cord tissue. Conclusion: P2Y2R critically regulates astrocyte reactivity and hydrogen peroxide production after spinal cord injury. Pharmacological inhibition of P2Y2R with AR-C118925XX promotes functional recovery, enhances neuronal survival, and preserves myelin and mitochondrial integrity, partly through the modulation of hydrogen peroxide-mediated astrogliosis. These findings identify P2Y2R as a promising therapeutic target for spinal cord injury.