The Role And Mechanism Of P2Y6 Receptor In Microglial Pyroptosis And Neuroinflammation After Intracerebral Hemorrhage

Background:Intracerebral hemorrhage(ICH)is a type of stroke subtype that leads to high mortality and disability rates.Neuroinflammation is closely related to secondary brain damage following ICH,and the P2Y6 receptor plays an important role in regulating neuroinflammation.However,the role of P2Y6 in neuroinflammation after ICH is not yet clear.Objective:To investigate the role and mechanism of P2Y6 in microglial pyroptosis and neuroinflammation in mice after ICH.Methods:We used an Ⅳ collagenase injection into the striatum of mice to induce an ICH model in this study.We detected the trend changes of P2Y6 at different time points using Western Blot(WB)and clarified the localization of P2Y6 in microglia,neurons,and astrocytes using immunofluorescence double-labeling.The cell pyroptosis and neuroinflammation were detected by WB,real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),and immunofluorescence staining(IF)after inhibiting P2Y6.In addition,in vivo experiments were performed to evaluate the short-term neurological behavioral function of mice in different intervention groups using the modified Garcia test,forelimb placement test,and corner turn test.In the in vitro experiments,BV2 cells were stimulated with LPS to simulate the ICH inflammatory environment,and WB was used to detect the expression of P2Y6 at different time points.Results:The expression of P2Y6 gradually increased after ICH in mice,reaching a peak at 24 hours and then gradually decreasing,but still higher than that in the sham group.P2Y6 was only expressed in microglia,not in neurons or astrocytes.In LPSstimulated BV2 microglia,the expression trend of P2Y6 was consistent with that in the in vivo experiment.Intraperitoneal injection of 150μg/kg P2Y6-specific inhibitor MRS2578 after ICH improved short-term neurological behavioral defects and brain edema in mice,while reducing the transcription and protein expression levels of NLRP3,ASC,Caspase-1,GSDMD(Gasdermin D),MPO,IL-1β,and IL-18.In terms of mechanism exploration,after intraperitoneal injection of MRS2578,PI3K-specific inhibitor LY294002 was injected into the mouse’s ventricle.It was found that LY294002 reversed the anti-pyroptosis and anti-inflammatory effects of MRS2578 on microglia after ICH.In the in vivo experiments,WB was used to detect the expression of pyroptosis-related proteins and inflammatory factors in the sham group,ICH group,ICH+MRS2578 group,and ICH+MRS2578+LY294002 group.It was found that the expression of pyroptosis-related proteins and inflammatory factors in the ICH+MRS2578+LY294002 group was higher than that in the ICH+MRS2578 group.Conclusion:The P2Y6 receptor can promote microglial pyroptosis,neurofunctional deficits,and brain edema in mice after ICH through the PI3K/AKT/NLRP3 signaling pathway,thereby exacerbating secondary brain damage.Inhibiting P2Y6 can alleviate neuroinflammatory reactions after ICH through the PI3K/AKT pathway.Therefore,targeting the P2Y6 receptor may become a new approach for treating secondary brain damage after ICH.