Role Of AMPK-mediated Glucose Metabolism In Mitochondrial Apoptosis After Neuronal Ischemic Injury

Background and objection: Ischemic stroke is one of the major causes of mortality and morbidity worldwide The purpose of this study was to investigate the effects of AMP-activated protein kinase (AMPK) activity on GLUT1 expression and mitochondrial apoptosis in neuron ischemic injury,and to explore the protective effect of inhibition of AMPK activity on neuron ischemic injury.Methods: The model of transient middle cerebral artery occlusion (t MCAO)was established in vivo. The C57BL/6 male mice were randomly divided into sham operation group,t MCAO group and Compound C group.The Compound C treatment group was injected intraperitoneally with Compound C (25mg/kg 1 hour before operation, sham operation group and t MCAO group were injected with the same amount of saline water. Twenty-four hours after ischemia, mice were euthanised and the cerebral infarction volume was measured by TTC staining. The expression of p-AMPK, GLUT1 and Caspase-3 was detected by Western blot,immunohistochemistry and immunofluorescence.HT22 cells were used to establish the model of ischemic injury induced by glutamate in vitro,and the cells were divided into control group, GLUT group and GLUT+CC group.The cell survival rate,the expression of p-AMPK,GLUT1 and Caspase-3 were detected by Calcein-AM/PI staining,Western blot,PCR and immunofluorescence.Glucose uptake and cell oxygen consumption were measured by 2-NBDG and seahorse,respectively.The level of ROS production in HT22 cells was measured by H2 DCFDA probe method. The change of mitochondrial membrane potential (MMP) was detected by TMRE staining. What is more, apoptotic cells were detected by TUNEL staining.Results: 1. In t MCAO model, p-AMPK expression was significantly up-regulated.Inhibition of AMPK activation could reduce the cerebral infarction volume,neuronal necrosis and the expression of GLUT1 and Cleaved Caspase-3.2. The p-AMPK expression was also significantly up-regulated in the f glutamate-induced ischemic injury in HT22 cells. What is more, inhibition of AMPK activity by Compound C could rescue the HT22 cells from glutamate-induced injury. Western blot, Immunochemical staining and q PCR demonstrated that inhibition of AMPK activation suppressed the expression of GLUT1. Meanwhile, glucose uptake, oxygen consumption, reactive oxygen species and mitochondrial membrane potential loss were also inhibited by Compound C.Conclusion: Inhibition of AMPK activity can protect neurons from ischemia-reperfusion injury by inhibiting GLUT1 expression, reducing cellular energy metabolism and oxidative stress response.