| Brain injury was the major cause of deaths after cardiac arrest(CA)/cardiopulmonary resuscitation(CPR).Hypothermia had a protective effect on brain injury after CPR,but the mechanism was unclear.Nuclear factor erythroid 2-related factor 2(Nrf2)was an important endogenous antioxidant factor,and it was found that Nrf2 may be involved in the process of brain protection by hypothermia.Recent studies had found that besides Kelch-like ECH associating protein 1(Keap1),Akt/(glycogen synthase kinase-3 beta)GSK-3 beta also downregulated Nrf2 expression,but whether Akt/GSK-3 beta/Nrf2 pathway was involved in brain protection by hypothermia,there was no literature report.In order to clarify the exact relationship between Nrf2 and brain protection by hypothermia,and explore whether Akt/GSK-3 beta pathway regulated the expression of Nrf2 and influenced neurological function after CPR,we performed our study in vivo and vitro.Firstly,we observed the effect of hypothermia on the expression of Nrf2 in model of CA/CPR in hippocampus in rats.Secondly,we detected the expression of GSK-3 beta and HO-1,which were upstream and downstream signaling molecules of Nrf2 respectively,and examined intracellular reactive oxygen species(ROS)and cell apoptosis in the model of ischemia/reperfusion injury in primary hippocampal neurons.Then we detected the indicators above again after pretreatment with shNrf2 and pathway inhibitor.The aim of the study was to find whether Akt/GSK-3 beta/Nrf2 pathway was involved in the protective effect for ischemia/reperfusion hippocampal neurons by hypothermia.Part ⅠEffects of hypothermia on expression of Nrf2 in hippocampus after cardiopulmonary resuscitation in ratsObjective:The present study was undertaken to observe the effect of hypothermia on the expression of Nrf2 in the model of CA/CPR in hippocampus in rats,and explore the relationship between the level of Nrf2 and neurological function.Methods : Fourty-six male SD rats were randomized into sham group(n=6),normothermia CA/CPR group(NT group,n=20)and hypothermia CA/CPR group(HT group,n=20).NT group and HT group were futher divided into two subgroups according to observation time respectively(NT-24,HT-24,NT-72 and HT-72).The rats were experienced regular operations,such as weighing,anesthesia(1% pentobarbital sodium,50mg/kg),fixed,shaving,endotracheal intubation,and femoral artery vein catheterization.The heart rate(HR)and mean arterial pressure(MAP)were monitored by multi-channel physiological signal acquisition processing system and the arterial blood gas was measured in baseline.The model of CA/CPR was performed by clamping the endotracheal tube.Manual chest compression and mechanical ventilation were started until systolic blood pressure(SBP)droped to 25 mmHg and lasted 5 minutes.Epinephrine(10ug/kg)was injected after CPR for 1 minute.Regular CA/CPR parameters,rectum temperature,HR and MAP were continuously recorded for 4 hours.The control group was sacrificed after regular operations were completed,and the rats which were successful resuscitation were sacrificed at 24 h or 72 h according to groups.Changes in the histopathology and scanning electron microscope of hippocampus specimens were evaluated.Malondialdehyde(MDA)content and Superoxide Dismutase(SOD)activity were assayed using a spectrophotometer.The expressions of caspase-3 and Nrf2 were measured by the method of western blot.Finally,neural deficit scores(NDS)was observed at 24 h and 72 h.Results:1.No statistical difference in body weight,HR,MAP,rectum temperature,pH,PaO2,PaCO2 and lactic acid was observed in baseline between groups,no statistical difference in the rate of successful resuscitation and the rate of survival from ROSC to observation time was observed,and no statistical difference in HR and MAP during the whole observing times was observed.2.Rectum temperature in HT group was significantly decreased compared to NT group.3.The morphology,ultramicro damages of nucleus and mitochondria in hippocampus in HT and NT group were evident compared to sham group,but the degree of damage was educed in HT-24 group compared to NT-24 group.4.MDA content and SOD activity in HT and NT group were significantly increased compared to sham group,and MDA content was decreased and SOD activity was increased in HT-24 group compared to NT-24 group.5.Caspase-3 expressions in HT and NT group were significantly increased compared to sham group,but caspase-3 expressions was decreased in HT-24 group compared to NT-24 group.6.The total and nuclear Nrf2 expressions in HT and NT group were significantly increased compared to sham group,whereas the cytoplasic Nrf2 expressions were significantly decreased.The total and nuclear Nrf2 expressions were increased and cytoplasic Nrf2 expressions was decreased in HT-24 group compared to NT-24 group.7.NDS in HT and NT group were significantly decreased compared to sham group,and significantly decreased in NT-24 group compared to HT-24 group.Conclusions:Results demonstrated that Nrf2 expression was significantly increased in hippocampus after CPR in rats,and oxidative stress injury,apoptosis and neurological function were significantly aggravated.Whereas hypothermia further increased Nrf2 expression and nuclear translocation,and relieved oxidative stress injury,apoptosis and neurological function.Part ⅡRoles of Akt/GSK-3 beta/Nrf2 pathway in hypothermia protective effect for ischemia/reperfusion hippocampal neuronsObjective:Through observation of the effect of hypothermia on ischemia/reperfusion hippocampal neurons,to explore whether Akt/GSK-3 beta/Nrf2 pathway was involved in hypothermia protective effect for ischemia/reperfusion hippocampal neurons.Methods:Primary hippocampal neurons are isolated from rat embryos,then cultured,and microtubule associated protein 2(MAP 2)was detected via immunohistochemical staining method to identify neurons.The model of ischemia/reperfusion hippocampal neurons was performed by the method of hypoxia/reoxygenation.The hippocampal neurons were divided into four groups,the control group,the hypoxia group,the hypothermia group and the hypoxia/hypothermia group.GSK-3 beta,Nrf2 and HO-1 expressions were measured via quantitative real-time PCR and western blot methods.The level of ROS was detected by fluorescent dyes and laser confocal microscope.Neuron apoptosis was observed by AnnexinV/PI method.Furthermore,before the model of ischemia/reperfusion hippocampal neurons was established,hippocampal neurons were pretreatmented with shNrf2 and wortmannin respectively,and then the expression of GSK-3 beta,Nrf2 and HO-1,the level of ROS and neuron apoptosis were observed again.Results:1.We selected rat hippocampal neurons which were cultured for 7 days for further experiments through MAP 2 immunohistochemical staining method.2.The expression of total and nuclear Nrf2 in the hypoxia/hypothermia group was significantly increased compared to the hypoxia roup,and the tendency of cytoplasic Nrf2 was opposite.The expression of HO-1 and p-GSK-3 beta in the hypoxia/hypothermia group was significantly increased compared to the hypoxia roup.We found that the level of ROS and neuron apoptosis were significantly alleviated in the hypoxia/hypothermia group compared with the hypoxia roup.3.After hippocampal neurons were pretreatmented with shNrf2,no statistical difference in the expression of Nrf2 and HO-1was observed between groups.We found that expression of p-GSK-3 beta were were significantly increased in the hypoxia/hypothermia group compared with the hypoxia group.No statistical difference in the level of ROS and neuron apoptosis was observed between groups.4.After hippocampal neurons were pretreatmented with wortmannin,no statistical difference in the expression of Nrf2,HO-1 and p-GSK-3 beta was observed between groups,and no statistical difference in the level of ROS and neuron apoptosis was observed between groups.Conclusions:Hypothermia promoted Akt phosphorylation in ischemia/reperfusion hippocampal neurons,accelerated the GSK-3 beta phosphorylation and made its inactivation,attenuated its negative effect on Nrf2,induced the expression of HO-1,and suppressed oxidative stress injury and apoptosis,thus finally protected neural function. |
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