P2Y2 receptors mediate astrocytic hyperplasia by increasing H 2 O 2 production to affect neural regeneration after spinal cord injury

The reactivity of astrocytes is a key factor impeding nerve repair and regeneration after spinal cord injury (SCI). However, there is heterogeneity in the role of reactive astrocyte. Our previous research revealed that the G protein-coupled receptor P2Y2 receptor (P2Y2R) facilitates motor function recovery during the chronic phase of SCI, but the interaction between this receptor and astrocytes remains unclear. We established an in vitro model of responsiveness of primary astrocytes by 25 μM H₂O₂, and observed the effects of AR-C118925XX on astrocyte reactivity and H ₂ O ₂ levels. In vivo, the C57BL/6J mice with T10 incomplete transection as SCI model were used. The recovery of neurological and motor functions after SCI was evaluated by BBB score and footprint analysis. Molecular and histological methods were used to detect astrogliosis and neural repair. The level of H ₂ O ₂ were determined using hydrogen peroxide assay kits. In vitro, we found that (AR-C118925XX) P2Y2 receptor inhibitor reduced the concentration of hydrogen peroxide (H ₂ O ₂ ) released by reactive astrocytes. By TUNEL and immunofluorescence (IF) staining, we found that cells treated with the inhibitor exhibited lower apoptosis rate and higher mean immunofluorescence intensity of MAP2 ⁺ . To gain a better understanding of this interaction, we conducted animal experimentation that revealed a strong correlation between P2Y2R and GFAP expression (glial fibrillary acidic protein) from the acute phase (1 day after SCI) to the chronic phase (28 days). In addition, co-localization of the P2Y2R with astrocyte and neuron was found. It was shown that inhibition of P2Y2R expression inhibited astrocyte scar formation, significantly improved neuronal survival and regeneration, and attenuated axonal demyelination and mitochondrial damage. Intriguingly, the neuroprotective effect of the P2Y2R inhibitor may be realized by reducing the level of H ₂ O ₂ released by astrocytes after SCI. Our findings emphasize that P2Y2R is a key molecule in regulating H ₂ O ₂ levels, promoting neuronal survival and regeneration, and inhibiting the reactivity of astrocytes in the chronic phase of SCI. These findings provide new insights and potential therapeutic strategies for the treatment of SCI.