Phosphorylated AMP-Activated Protein Kinase Is Involved In Hyperglycemia Aggravating Ischemia-reperfusion Neuron Injury

Objective Hyperglycemia aggravates ischemic/reperfused neuron injury,but the molecular mechanism remains unclear.The purpose of this study was to investigate the role of phosphorylated AMP-activated protein kinase in hyperglycemia aggravating cerebral ischemia-reperfusion injury and its possible molecular mechanism.Methods Hyperglycemia was induced by streptozotocin.The focal cerebral I /R was made by middle cerebral artery occlusion(MCAO)with suitable thread.The group is composed of normoglycemic sham group(NG sham),hyperglycemic sham group(HG sham),normoglycemic ischemia-reperfusion 24 h group(NG I/R 24 h),normoglycemic ischemia-reperfusion 72 h group(NG I/R 72 h),hyperglycemic ischemia-reperfusion 24 h group(HG I/R 24 h),hyperglycemic ischemia-reperfusion 72 h group(HG I/R 72 h).The brain tissue damage,localization of P-AMPK and expression of P-AMPK between groups were comparatively studied.Hypoxia reoxygenation model(ODR)was prepared by combined use of anoxic incubator and normoxic incubator,divided into normal glucose control group(NG-con),normal glucose hypoxia 4h reoxygenation 24 h group(NG-ODR),high glucose control group(HG-con),high glucose hypoxia 4h reoxygenation 24 h group(HG-ODR)and high glucose hypoxia 4h reoxygenation 24 h with AMPK activator group(HG-ODR-A),observe Cell morphology by inverted microscope,flow cytometry measure apoptosis,ROS determination,immunofluorescence and Western blot were used to determine the changes of caspase3,caspase9,Apaf-1,P-AMPK and other factors between the treatment groups.Results In vivo studies,blood glucose in HG group was significantly higher than thatin NG group(P < 0.05),the body mass of HG group was significantly lower than that in NG group(P < 0.05).The neurological deficit scores of HG group at each time point of ischemia-reperfusion were significantly higher than those of NG group(P < 0.05).The cerebral ischemia area of NG group was mainly distributed in the striatum area,Compared with the NG group,the HG group cerebral ischemia area increased to the cerebral cortex,and the degree of cerebral edema and the number of pyknotic neurons increased significantly.The expression of P-AMPK in the HG I/R 24 h group and the I/R 72 h group was lower than that in the NG group(P < 0.05).P-AMPK was co-expressed with neurons but not co-expressed with astrocytes.The number of P-AMPK-positive neurons in the HG group I/R24 h group and I/R 72 h group was significantly lower than that in the NG group(P < 0.05).In vitro studies,hypoxia 4h,reoxygenation 24 h + different sugar concentration treatment,it can be seen that with the increasing of sugar concentration,cell activity gradually decreased,the protein expression of caspase3 gradually increased,some cells showed shrinkage,the cell gap was widened,and more apoptotic cells were observed.moreover cell fragmentation and dissolution were observed.The expression level of P-AMPK and the cell activity in the high glucose medium treated with hypoxia 4h,reoxygenation 24 h was significantly lower than that in the normal glucose medium group,hypoxia-reoxygenation of the high glucose medium+AICAR group attenuated the inhibitory effect of high glucose on the expression of P-AMPK and the decreased effect of high glucose on the cell activity in hippocampal neurons under hypoxia-reoxygenation.the amount of ROS and the rate of apoptotic cells in the hypoxia-reoxygenation high glucose medium group was higher than that in the normal glucose medium,The hypoxia-reoxygenation+AICAR group in the high glucose medium attenuated the increase of ROS and the rate of apoptotic cells in the hypoxia-reoxygenation state of hippocampal neurons in high glucose medium.The expression levels of caspase3,caspase9 and Apaf-1 in the high glucose medium group after hypoxia for 4 h reoxygenation for 24 h were higher than that in the normal glucose medium hypoxia-reoxygenation group,The high-glucose medium hypoxia-reoxygenation+AICAR group attenuated the expression of caspase3,caspase9 and Apaf-1 in hippocampal neurons under hypoxia-reoxygenation high glucose medium.Conclusion Hyperglycemia aggravates ischemia-reperfusion neuron injury;Hyperglycemia inhibits phosphorylated AMP-activated protein kinase;Phospho-AMP-activated protein kinase participates in the reduction of hyperglycemia aggravated ischemia-reperfusion neuron injury by reducing apoptosis.