The Role Of P2Y1 Receptor In Neuroinflammation After Intracerebral Hemorrhage In Mice And Its Related Mechanisms

BACKGROUND:Intracerebral hemorrhage(ICH)is a common highly disabling and fatal cerebrovascular disease.Surgical removal of the hematoma can reduce the mass effect of the hematoma and reduce intracranial pressure,but does not resolve the secondary brain injury.Secondary brain injury after intracerebral hemorrhage involves inflammation,oxidative stress,and apoptosis.Neuroinflammation plays a key role,characterized by inflammatory cell activation and release of inflammatory factors.Pyroptosis,a highly pro-inflammatory form of cell death,is characterized by activation of GSDMD and the formation of pores in the cell membrane to allow cell leakage and release of inflammatory factors,which has been shown to promote the progression of neuroinflammation after intracerebral hemorrhage,Inhibition of pyroptosis can alleviate secondary brain injury after intracerebral hemorrhage.P2Y1 receptor is a purinergic receptor widely distributed in brain tissue,which can be activated by ATP/ADP released by damaged brain cells.It plays a role in disease and can regulate the release of inflammatory factors.However,whether P2Y1 plays a role in brain injury secondary to intracerebral hemorrhage and regulates neuroinflammation by mediating pyroptosis has not yet been reported.OBJECT:In this experiment,a collagenase-injected mouse model of cerebral hemorrhage was established:(1)to explore the changes of P2Y1 receptor protein levels and cell types after intracerebral hemorrhage;(2)to administer the P2Y1 agonist MRS2365 and inhibitor MRS21 79 through the lateral ventricle to understand the role of P2Y1 receptor in the The role in nerve injury after intracerebral hemorrhage;(3)P2Y1 agonist MRS2365 and inhibitor MRS2179 were administered through the lateral ventricle to clarify whether P2Y1 receptor regulates the NLRP3 inflammasome-dependent pyroptosis pathway after intracerebral hemorrhage.METHODS:The first part of the experiment:The experimental mice were divided into 6 groups(Sham group,ICH12h group,ICHld group,ICH3d group,ICH5d group and ICH7d group),and the expression of P2Y1 receptor after intracerebral hemorrhage was detected by Western Blot;The cellular localization of P2Y1 receptor in Sham group and ICH3d group was observed by immunofluorescence double-labeling technique;The second part of the experiment:The experimental mice were divided into 4 groups(Sham group,ICH group,ICH+MRS2365 group,ICH+MRS2179 group).and through neurobehavioral examination(modified neurological deficit score mNSS),cerebral edema(dry and wet weight method),apoptosis Tunel staining and pro-inflammatory and anti-inflammatory factors(interleukin-6,leukocyte Interleukin-10)assay(Western Blot)to study the role of P2Y1 receptors in nerve injury after intracerebral hemorrhage.The third part of the experiment:The experimental mice were divided into 4 groups(Sham group,ICH group,ICH+MRS2365 group,and ICH+MRS2179 group).Western blotting was used to detect NLRP3-dependent pyroptosis pathway-related proteins after cerebral hemorrhage.Expression changes of(NLRP3,pro-Caspase-1,Caspase-1 p20,pro-GSDMD,GSDMD-N,ASC,pro-IL-1β,mature IL-1β and mature IL-18)and intracerebroventricular administration of MRS2365 and MRS2179 Afterwards,it has an effect on the expression of NLRP3-dependent pyroptosis pathway-related proteins,so as to investigate whether P2Y1 receptors are involved in the regulation of NLRP3-dependent pyroptosis pathway after ICH.RESULTS:(1)The expression of P2Y1 receptor increased after intracerebral hemorrhage and was the highest on the 3rd day;the expression of P2Y1 receptor was in neurons and astrocytes in the Sham group,and in neurons,astrocytes and microglia in the ICH3d group;(2)In the case of cerebral hemorrhage,the agonist MRS2365 up-regulates the expression of P2Y1 receptor,while the inhibitor MRA2179 reduces its expression;and after cerebral hemorrhage,the experimental mice have nerve damage,which is manifested by increased mNSS score,increased cerebral water content,and apoptosis.The number of nerve cells increased and the release of pro-inflammatory factors increased;the above nerve damage was aggravated after MRS2365 activated P2Y1 receptor;the above nerve injury was alleviated after MRS2179 inhibited P2Y1 receptor;(3)After intracerebral hemorrhage,the NLRP3-dependent pyroptosis pathway is activated,and the expression of pathway-related proteins increases;MRS2365 activates the P2Y1 receptor and further activates the NLRP3-dependent pyroptosis pathway,and the expression of pathway-related proteins further increases;MRS2179 inhibits the P2Y1 receptor and causes the opposite to the above.As a result,the NLRP3-dependent pyroptosis pathway was inhibited,and the expression of pathway-related proteins was down-regulated.CONCLUSIONS:The expression of P2Y1 receptors is significantly up-regulated after ICH,in addition to astrocytes,neurons and microglia;P2Y1 receptors have a significant role in promoting neuroinjury after ICH;P2Y1 receptors are up-regulated by NLRP3-dependent pyroptosis pathway promotes neuroinflammation after intracerebral hemorrhage.