The Research Of NLRP3 Inflammasome Mediated-Pyroptosis After Cardiopulmonary Resuscitation

Background and Objective:Cardiac arrest is one of the most common critical illnesses.Cardiopulmonary resuscitation（CPR）has been proved to be an effective treatment and has been continuously improved,but the outcomes of cardiac arrest patients have not been significantly improved.Cardiac arrest survivors usually have different degrees of neurological dysfunction.Mechanism of inflammatory factor plays a cardinal role in cerebral ischemia-reperfusion（I/R）injury.However,during systemic I/R injury after CPR,the specific mechanism of inflammatory response induced by cerebral I/R injury is still unclear.Pyroptosis,one of programmed cell death patterns,has become a hot topic in recent years.The study aims to explore the role of NLRP3 Inflammasome-mediated pyroptosis and its related mechanism during cerebral I/R injury after CPR.Methods:1.C57BL/6 mice and NLRP3^-/-mice were divided into pre-recovery group and model group respectively.The mice of model group were used to make CPR model with high potassium and asphyxia.And then the survival mice after CPR was divided into four groups randomly according to the time points after CPR,such as 2h after CPR,6h after CPR,12h after CPR and 24h after CPR.The general evaluation and neurological function scores at each group were observed and recorded.The expressions of NLRP3,Caspase-1 and Gasdermin D（GSDMD）were detected in brain tissue by Western blot（WB）.The expressions and locations of NLRP3 and GSDMD in hippocampus at each group were observed by Immunofluorescence.2.U87 human glioma cells were cultured,and then transfected with NLRP3 RNAi lentiviral particles and negative control lentiviral particles respectively,resulting in constructing stable cell lines of U87 NLRP3 RNAi and U87 negative control with puromycin.What is more,normal U87 cells and U87 NLRP3 RNAi cells were dealt with oxygen-glocuse deprivation for 12h and reoxygenation for 12h.Lastly,WB was used to detect the expressions of proteins of NLRP3,Caspase-1 and GSDMD,CCK8 was used to detect the cell viability of each group,and ELISA kit was used to detect the concentration of IL-1βin cell supernatant.Results:1.Compared with C57BL/6 mice,the rate of spontaneous circulation restoration,the rate of spontaneous respiratory restoration and 2-hour survival rate after CPR in NLRP3^-/-mice were higher（P<0.05）.What is more,the time of spontaneous circulation restoration and spontaneous respiratory restoration were significantly shortened（P<0.05）.In addition,the neurological function scores in NLRP3^-/-mice at 2 h after CPR,6 h after CPR,12 h after CPR and 24 h after CPR were significantly higher than those of C57BL/6 mice respectively（P<0.05）.2.C57BL/6 mice and NLRP3^-/-mice initiated pyroptosis during cerebral I/R injury after CPR.The peaks of proteins of NLRP3,Caspase-1 and GSDMD in brain tissue of C57BL/6 mice were at 6h after CPR,and then the proteins were gradually decreased,but the expression levels at 24h after CPR did not return to the pre-recovery levels（P<0.001）.Compared with C57BL/6 mice at the same time after CPR,NLRP3^-/-mice did not express protein of NLRP3 in the brain tissue during cerebral ischemia/reperfusion injury,and the Caspase-1 protein and GSDMD protein showed significantly low expression（P<0.001）.3.The location of NLRP3 was in the cytoplasm of hippocampus,and the expression of GSDMD was located in the cell membrane of hippocampus.4.The normal U87 cells were treated with oxygenation-glucose deprivation for 12 h/reglucose and reoxygenation for 12 h,resulting in upregulation the levels of NLRP3,Caspase-1,GSDMD and IL-1β（P<0.001）.What is worse,their cell morphology were significantly changed,the ability of cell attachment and cell viability were also significantly reduced;Treating U87NLRP3 RNAi cells with oxygenation-glucose deprivation for 12h/reglucose and reoxygenation for 12h,the expression levels of NLRP3,Caspase-1 and GSDMD were significantly lower（P<0.001）,the production of IL-1βwas significantly reduced（P<0.001）.The cell morphology,the ability of cell attachment and the cell viability were all significantly improved（P<0.001）.Conclusions:（1）This study demonstrates that NLRP3 inflammasome-mediated pyroptosis aggravates cerebral I/R injury after CPR,especially in the hippocampus;（2）Silencing or knocking down NLRP3 gene can inhibit the activation of pyroptotic signal axis NLRP3/Caspase-1/GSDMD,improve cell morphology,enhance cell attachment and cell viability,reduce the production of IL-1β,and then significantly improve the rate of spontaneous circulation restoration,the rate of spontaneous respiratory restoration and 2-hour survival rate in mice after CPR,significantly shortened the time of spontaneous circulation restoration and spontaneous respiratory restoration after CPR,and can also significantly improve the neurological function scores of mice after CPR.