The Effect And Mechanism Of P2X7 Receptor Activation On Oxidative Stress After Intracerebral Hemorrhage In Mice

Background: Secondary injury is an important pathological mechanism of neurological deterioration after intracerebral hemorrhage(ICH).There is still no safe and effective method to intervene the secondary injury after ICH.It is particularly important to clarify the pathological mechanism of secondary injury after ICH.ATP is released by gentle mechanical stimulation from most,if not all,cell types,as well as from dead or dying cells.Oxidative stress caused by abnormally accumulated extracellular ATP-activated P2X7 receptor plays an important role in the development of central nervous system degenerative diseases.At present,little is known about the role of P2X7 receptor in oxidative stress after ICH.This study aimed to investigate whether P2X7 receptor mediated oxidative stress after ICH and its possible underlying mechanism.Methods: The ICH model was induced by the stereotactic injection of type VII collagenase in C57BL/6 mice,this research consists of three parts.In the first part,the expression of P2X7 receptor in the tissue surrounding the hematoma at different time points after ICH was detected by western blot,and the localization of P2X7 receptor was determined by immunofluorescence staining.Treatment with the P2X7 receptor inhibitor A438079 by intraperitoneal injection or agonist Bz ATP by intraventricular injection was used to investigate the role of P2X7 R in ICH.P2X7 receptor expression was assessed by Western blot;the levels of MDA,SOD,GSH,and GSH/GSSH in the tissue around the hematoma were measured to reflect the degree of oxidative stress at 24 h after ICH;TUNEL staining was performed to detect cell apoptosis in the tissue surrounding the hematoma at 24 h after ICH;brain water content measured by wet/dry method was used to evaluate the brain edema at 24 and 72 h after ICH;neurological impairment was assessed by modified Garcia score,corner test,and forelimb placement test at 24 and 72 h after ICH.In the second part,mice with ICH were treated with P2X7 receptor agonist Bz ATP or inhibitor A438079.Western blot was conducted to detect the expression of NOX2 in the tissue surrounding the hematoma at24 h after ICH.Subsequently,mice with ICH were injected intraperitoneally with NOX2 inhibitor GSK2795039.The levels of MDA,SOD,GSH,and GSH/GSSH in the tissue surrounding the hematoma were measured to reflect the degree of oxidative stress at 24 h after ICH;brain water content was calculated by wet/dry method to evaluate the brain edema at 24 and 72 h after ICH;neurological impairment was assessed by modified Garcia score,corner test,and forelimb placement test at 24 and 72 h after ICH.In the third part,mice were treated with P2X7 receptor agonist Bz ATP or inhibitor A438079.ERK1/2,JNK,p38,NF-κB and their corresponding phosphorylation levels were detected by Western blot and immunofluorescence staining.Mice were further treated with ERK1/2 activation inhibitor U0126 or NF-κB transcription activity inhibitor JSH-23 by lateral ventricle injection,and the expression of NOX2 was detected by western blot at 24 h after ICH;the MDA,SOD,GSH,and GSH/ GSSH levels in the tissue surrounding the hematoma were measured to reflect the degree of oxidative stress at 24 h after ICH;TUNEL staining was performed to detect cell apoptosis in the tissue surrounding the hematoma at 24 h after ICH.Results: In the first part,we found that the expression of P2X7 receptor increased as early as6 h,peaked at 24 h,and then gradually decreased after ICH in mice.Immunofluorescence staining showed that the positive rate of P2X7 receptor on neurons were significantly higher than that on astrocytes and microglia.The western blot result indicated that the P2X7 receptor expression was further increased with Bz ATP treatment at 24 h after ICH,while the P2X7 receptor inhibitor A438079 treatment significantly decreased the expression of P2X7 receptor.Inhibition of P2X7 receptor remarkably decreased the MDA level and increased SOD activity,GSH level,and GSH/GSSH in the tissue surrounding the hematoma at 24 h after ICH;meanwhile,treatment with A438079 also remarkably alleviated cell apoptosis,brain edema,and neurological damage after ICH.In the second part,we found that the expression of NOX2 in the tissue surrounding the hematoma was further up-regulated with P2X7 receptor agonist Bz ATP treatment at 24 h after ICH,while treatment with P2X7 receptor inhibitor A438079 significantly reduced the expression of NOX2.We also showed that inhibition of NOX2 by GSK2795039 remarkably alleviated the oxidative stress,brain edema and neurological damage caused by P2X7 receptor activation after ICH.In the third part,we found that P2X7 receptor agonist Bz ATP treatment further increased the phosphorylation level of ERK1/2 and NF-κB in the tissue surrounding the hematoma at 24 h after ICH,while treatment with P2X7 receptor inhibitor A438079 significantly decreased the ERK1/2 and NF-κB Phosphorylation levels.The western blot result showed that the NOX2 expression was remarkably downregulated at 24 h after ICH with U0126 treatment which inhibited the activation of ERK1/2;consistently,inhibition of ERK1/2 activation also significantly decreased oxidative stress and cell apoptosis induced by P2X7 receptor activation at 24 h after ICH.Simultaneously,antagonist of NF-κB transcriptional activity by JSH-23 also remarkably decreased the NOX2 expression,oxidative stress and cell apoptosis induced by P2X7 receptor activation after ICH.Conclusions: Our study suggests that the P2X7 receptor activation aggravates NOX2-induced oxidative stress via ERK1/2 and NF-κB pathways following ICH in mice.At the same time,the novel NOX2 inhibitor GSK2795039 may have a great prospect for the treatment of secondary injury after ICH.