| Background: Cardiac arrest is one of the leading causes of death and disability worldwide.Post-cardiac arrest syndrome,which consists of brain injury,myocardial dysfunction,and systemic ischemia/reperfusion,has a poor prognosis for patients with successful resuscitation.Global cerebral ischemia/reperfusion injury,triggering neuronal cell death,leading to irreversible brain injury.At present,there is no effective drug intervention to improve the neuronal cell death and neurological dysfunction in patients with brain injury after cardiac arrest.Neuronal excitotoxicity is a well-recognized mechanism of ischemic brain injury.Several drugs have been designed for excitotoxicity to prevent neuronal overactivity,but they have failed at the clinical.The GABAB receptor is responsible for coordinating the slow inhibitory neurotransmission of the central nervous system.The GABAB receptor is involved in many neurological disorders and is considered an attractive therapeutic molecular target.The neuroprotective effect of baclofen,a GABAB receptor-specific agonist,has been demonstrated in animal and neuronal culture models.However,few studies have explored the role of GABAB receptors in brain injury after cardiac arrest.In this study,we hypothesized that activation of GABAB receptors improves neuronal death and neurological dysfunction after cardiac arrest.We verified the effect of GABAB receptor activation on behavior and neuronal cell survival through a rat model of asphyxial cardiac arrest and an hypoxia-hypoglycemia/reoxygenation(OGD/R)cell model,and further experiments to verify the specific mechanism of GABAB receptor effect on neuronal cell death.This study will provide a theoretical basis for the mechanism of brain injury after cardiac arrest and targeted therapy with drugs.Methods: 1.Rats with a weight of 300–350 g.They were divided into five groups at random: sham group(sham operation group),CA group(cardiac arrest group),BCA5 group(cardiac arrest +5mg/kg baclofen group),BCA10 group(cardiac arrest +10mg/kg baclofen group),and BCA20 group(cardiac arrest +20mg/kg baclofen group).All groups except sham group were treated with cardiac arrest caused by asphyxia for 10 minutes.After resuscitation,rats in the BCA5 group,the BCA10 group and the BCA20 group were treated with intraperitoneal injection of baclofen at 5mg/kg,10mg/kg and 20mg/kg respectively.After 24 hours,autonomic movement ability and neurological function were assessed by open field test and modified neurological deficit score(m NSS).The PC12 cells were administered with 10μM,25μM,50μM,100μM and 200μM baclofen.The effect of baclofen on the activity of normal cells was observed by CCK-8 kit.The cells in the OGD/R model were treated with baclofen at the dose of 50μM,100μM and 200μM respectively,to observe the effect of baclofen on the cell activity in the OGD/R model.After 24 hours of cardiac arrest,the expressions of GABAB receptors in the hippocampus and cortex of rats in sham group,CA group and BCA group(cardiac arrest +20mg/kg baclofen group)were detected by western blotting and q RT-PCR.TUNEL staining was used to detect apoptosis in the hippocampus and cortex,while Nissl staining was used to observe cell damage in the hippocampus and cortex.2.The rats were divided into five groups at random: sham group(sham operation group),CA-12 h group,CA-24 h group,CA-48 h group,and CA-72 h group.The expression levels of NLRP3 and GSDMD at different time points in the brain were detected by western blotting.PC12 cells were randomly divided into 4 groups: control group,OGD/R 6h group(6 h after hypoxia-hypoglycemia /reoxygenation group),OGD/R 12 h group(12 h after hypoxia-hypoglycemia /reoxygenation group)and OGD/R 24 h group(24 h after hypoxia-hypoglycemia /reoxygenation group).The expressions of NLRP3,caspase-1 and GSDMD at different time points in the cell model were detected by western blotting.After 24 hours of resuscitation from cardiac arrest,the expressions of pyroptosis related molecules caspase-1 and GSDMD in the hippocampus and cortex of rats in sham group,CA group and BCA group(cardiac arrest +20mg/kg baclofen group)were detected by western blotting,q RT-PCR and immunofluorescence staining.Western blotting,q RT-PCR,and immunofluorescence staining were used to detect the expressions of GABAB receptor,caspase-1,and GSDMD in PC12 cells after 6 hours of OGD/R in the control group,OGD/R group(6 hours after hypoxia-hypoglycemia/reoxygenation)and OB group(6 hours after hypoxia-hypoglycemia /reoxygenation+baclofen group).3.The rats were divided into three groups at random: sham group(sham operation group),CA group(cardiac arrest group),and BCA group(cardiac arrest +20mg/kg baclofen group).Western blotting,q RT-PCR,and immunofluorescence staining were used to detect the expression and location of DDX3 X and NLRP3 in the hippocampus and cortex of rats 24 hours after cardiac arrest.PC12 cells were randomly divided into 3 groups: control group,OGD/R group(6 h after hypoxia-hypoglycemia /reoxygenation)and OB group(6 h after hypoxia-hypoglycemia /reoxygenation+baclofen group).After 6 hours of OGD/R,the expressions of DDX3 X and NLRP3 in PC12 cells were detected by western blotting,q RT-PCR and immunofluorescence staining.4.PC12 cells were randomly divided into 5 groups: control group,OGD/R group(6 h after hypoxia-hypoglycemia/reoxygenation group),OGD/R+Baclofen group(6 h after hypoxia-hypoglycemia/reoxygenation+baclofen group),OGD/R+nigericin group(6 h after hypoxia-hypoglycemia/reoxygenation+nigericin group),OGD/R+Baclofen +nigericin group(6 h after hypoxia-hypoglycemia /reoxygenation +baclofen+nigericin group).CCK-8 and LDH assays were used to determine the viability of PC12 cells in each group after 6 hours of OGD/R,and western blotting was used to assess the expressions of NLRP3,caspase-1,and GSDMD in PC12 cells in each group.5.PC12 cells were randomly divided into 5 groups: control group,OGD/R group(6 h after hypoxia-hypoglycemia/reoxygenation group),OGD/R+Baclofen group(6 h after hypoxia-hypoglycemia/reoxygenation+baclofen group),OGD/R+OE-DDX3 X group(6 h after hypoxia-hypoglycemia/reoxygenation+overexpressed DDX3 X group),OGD/R+Baclofen+OE-DDX3 X group(6 h after hypoxia-hypoglycemia/ reoxygenation +baclofen+DDX3X group).After 6 hours of OGD/R,the expressions of DDX3 X and NLRP3 in PC12 cells of each group were detected by western blotting.Results: 1.The total distance traveled by rats in the CA group was substantially less than that of the sham group(p < 0.01).The total distance traveled in the BCA20 group was substantially higher(p < 0.01)than in the CA group.The m NSS in the CA group was considerably higher(p < 0.01)than in the sham group.The m NSS of the BCA20 group was substantially lower(p < 0.01)than that of the CA group.CCK-8 results showed that cell viability was significantly increased at concentrations of 100μM and 200μM(p < 0.01).Compared with the OGD/R group,the cell viability after OGD/R treated with 200μM baclofen was significantly increased(p < 0.01).When compared to the sham group,the results of Western blotting and q RT-PCR revealed that the expression level of GABAB receptor in hippocampus and cortex of CA group was significantly decreased,while the expression level was significantly increased after administration of baclofen(p < 0.01).TUNEL results showed that compared with sham group,hippocampal and cortical cell apoptosis was significantly increased in CA group.After the addition of baclofen in the BCA group,compared with the CA group,the apoptosis was significantly reduced(p < 0.01).The sham group’s hippocampal and cortical cells displayed complete structure,homogeneous staining,complete tissue structure,and a distinct nucleus at the cell’s center,according to Nissl staining data.In contrast,the CA group exhibited disorganized tissue structures,vacuole damage,and deep staining.However,treatment with baclofen reduced this pathological damage.2.Western blotting results in animal model showed that the protein levels of NLRP3 and GSDMD in CA-12 h,CA-24 h,CA-48 h and CA-72 h were significantly increased compared with that in sham group,but the increases of NLRP3 and GSDMD in CA-24 h were the most significant compared with that in sham group(p < 0.01).The protein levels of NLRP3 and caspase-1 after 6 hours after OGD/R were considerably higher than in the control group,according to Western blotting(p < 0.01),and decreased with the time.GSDMD protein levels at 6 and 24 hours were significantly higher than those in the control group(p < 0.01).The protein levels of caspase-1 and GSDMD in the hippocampus and cortex of the CA group were considerably higher in comparison to the sham group,according to Western blotting,but these increases were significantly reduced by baclofen(p < 0.01).Similar results were found with q RT-PCR and immunofluorescence.The results of Western blotting,q RT-PCR,and immunofluorescence in the cell model revealed that the OGD/R group had considerably lower GABAB receptor expression than the control group.The expression of GABAB receptor in the OB group was substantially higher(p < 0.01)than in the OGD/R group.The protein levels of caspase-1 and GSDMD in the OGD/R group were considerably greater than those in the control group,according to Western blotting data.Caspases-1 and GSDMD protein levels in the OB group were substantially lower(p < 0.01)than in the OGD/R group.Caspases-1 expression was considerably elevated in the OGD/R group when compared to the control group(p < 0.01),while caspase-1 expression was dramatically lowered in the OB group when compared to the OGD/R group(p < 0.01).3.The expression levels of DDX3 X and NLRP3 in the hippocampus and cortex of the CA group were considerably higher(p < 0.01)than in the sham group,according to Western blotting and q RT-PCR results of animal models.The expressions of DDX3 X and NLRP3 in the hippocampus and cortex of the BCA group were considerably lower(p < 0.01)than in the CA group.Immunofluorescence revealed that in the CA group,NLRP3 expression in the hippocampus and cortical neurons was considerably higher(p < 0.01)than in the sham group.NLRP3 expression in the BCA group was substantially lower(p < 0.01)than in the CA group.The expression levels of DDX3 X and NLRP3 in the OGD/R group were considerably higher(p < 0.01)than in the control group,according to Western blotting,q RT-PCR,and immunofluorescence results of the cell model.The expression levels of DDX3 X and NLRP3 in the OB group were considerably lower(p < 0.01)than in the OGD/R group.4.CCK-8,LDH and western blotting results show that compared with the OGD/R group,only nigericin-activated NLRP3 aggravates cell death,which is related to the increased expressions of NLRP3,caspase-1 and GSDMD.Coadministration of baclofen and nigericin not only eliminated the protective effects of baclofen,but also counteracted the detrimental effects of nigericin on the pharmacological activation of NLRP3.5.The expression levels of DDX3 X and NLRP3 in the OGD/R+OE-DDX3 X group were substantially higher in the OGD/R+OE-DDX3 X group compared to the OGD/R group(p < 0.01).The expression levels of DDX3 X and NLRP3 in the OGD/R+Baclofen+OE-DDX3 X group were considerably higher than in the OGD/R+Baclofen group(p < 0.01).Conclusions:(1)Activation of GABAB receptor improves neuronal damage and neurological dysfunction after cardiac arrest.(2)Neuronal pyroptosis exists after cardiac arrest;Activation of GABAB receptor reduces the expression of neuronal pyroptosis-related molecules.(3)Activated GABAB receptor regulate neuronal pyroptosis through the DDX3X/NLRP3 pathway. |
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