P2RY6 activation aggravates NLRP3-dependent microglial pyroptosis via down-regulation of the PI3K/AKT pathway in a mouse model of intracerebral hemorrhage

AbstractBackground Pro-inflammatory signals generated after intracerebral hemorrhage (ICH) trigger a form of regulated cell death known as pyroptosis in microglia. Although the p2Y purinoceptor 6 (P2RY6) plays a significant role in control of inflammatory reactions in central nervous system diseases, its roles in the development of neuroinflammation and microglial pyroptosis following ICH remain unclear. Methods Type IV collagenase was injected to induce ICH. Mice were then treated with MRS2578 and LY294002 to inhibit P2RY6 and phosphatidylinositol 3-kinase (PI3K), respectively. Bio-conductivity analysis was performed to examine PI3K/AKT pathway involvement. Immunostaining and quantitative polymerase chain reaction (qPCR) analyses were conducted to examine pyroptosis following P2RY6 inhibitor treatment. Western blot and immunostaining were performed to clarify the specific mechanisms underlying microglia pyroptosis and white matter injury (WMI). A modified Garcia test, corner turning test, and forelimb placement test were used to assess neurobehavioral function. Results Increases in the expression of P2RY6 and pyroptosis-related proteins were observed after ICH, peaking 24 h post ICH. P2RY6 was only expressed on microglia. Administration of MRS2578, a specific inhibitor of P2RY6, attenuated short-term neurobehavioral deficits and WMI while decreasing both microglia pyroptosis and cerebral edema. These changes were attended by decreases in pyroptosis-relatived proteins and pro-inflammatory cytokines. Bioinformatic analysis revealed an association between the PI3K/AKT pathway and P2RY6-mediated neuroinflammation. The effects of MRS2578 were partially reversed by treatment with LY294002, a specific PI3K inhibitor. Conclusion P2RY6 activation induces microglial pyroptosis, deficits in neurological function and WMI, partially via the P2RY6/PI3K/AKT/NLRP3 pathway. Consequently, targeting P2RY6 might be a promising approach to the therapy of ICH.