Effects Of AMPK-mTOR Signaling Pathways On Autophagy In The Role Of Focal Cerebral Ischemia Injury In Mice

Aim:To observe the relevance between trend variability of AMP-activated potein kinase(AMPK) and mammalian target of rapamycin(m TOR) and the effect on expression of autophagy related protein LC3 and Beclin-1 in cerebral hemisphere cortex after permanent focal cerebral ischemia injury in mice. Investigating the effect and mechanisms on ischemic brain injury induced by inhibiting AMPK activity.Methods:Adult male healthy Balb/c mice were randomly divided into sham group, vehicle group and drug intervention group. For the sham group and vehicle group, permanent middle cerebral artery occlusion(p MCAO) model was established in mice with an intraluminal silicon-coated filament. AMPK inhibitor Compound C(20mg/kg) was injected intraperitoneal in drug intervention group while vehicle(normal saline and DMSO) was injected intraperitonaeal in sham and vehicle group immediately after the onset of ischemia. At the time point of 3h, 6h, 12 h and 24 h after the occlusion, the infarction side cortices were collected and AMPK, p-AMPK, m TOR, p-m TOR, LC3 and Beclin-1 levels were detected by the methods of Western blot and immunohistochemistry. The effect of inhibiting AMPK activity on ischemic brain injury was analyzed by scoring neurological deficits, assessing brain infarction volume, determining the brain water content and quantifing the amount of nissl bodies in the brain infarcts.Results:Western blot and immunohistochemistry results showed that the expression level of p-AMPK was low in cerebral hemisphere in sham-operated group, but significantly increased and reached peak at 3h after p MCAO in cerebral ischemia hemisphere, at6 h time point the expression level was decreasing but still high, there were significant differences compared with the sham-operated group(p<0.01), at 12 h and 24 h time point the expression levels decreased continuously. The expression trend of LC3 and Beclin-1 kept the same with p-AMPK after the occlusion, the variability trendbetween p-AMPK and p-m TOR levels was inverse. After treated with Compound C,the expression level of p-AMPK/LC3/Beclin-1 was significantly decreased, while the p-m TOR level increased significantly(p<0.05). The deficiency of nissl bodies was reduced compared with the vehicle group each time after the occlusion. Neurological deficits, infract areas and brain water content were also significantly relieved at 24 h time point after the occlusion(p<0.05).Conclusion:1. Autophagy was activated in cortical tissue after ischemic brain injury in mice.2. AMPK-m TOR signaling pathways were actived and the expression was antagonistic in cortical tissue after ischemic brain injury in mice. 3. Inhibiting AMPK activity can inhibit autophagy. 4. Inhibiting AMPK activity had a protective effect on neuronal injury induced by cerebral ischemia in mice. Activing m TOR signaling pathways and reducing autophagic neuronal injury may relative on the mechanisms.