| Multiple cell signaling pathways and biological processes are involved in cerebral ischemia/reperfusion (I/R) injury, among which, autophagy is a most confusing one. Studies suggest that autophagy is activated during ischemia and reperfusion (I/R), however there's no data to dynamically mimic autophagy level during the whole process of I/R, and it's still not known whether autophagy functions as a guardian or plays a detrimental role in I/R injury.Here we employed simulated I/R of N2a cells (mouse neuroblastoma cells) as an in vitro model of I/R injury to the neurons. LC-3II level was checked by immunoblot analysis at different time points of ischemia process, and different intervals of reperfusion. Results showed that autophagy was gradually activated as ischemia went on, and greatly elevated during the process of reperfusion after 90minutes'ischemia insult. I/R manipulation was also applied to N2a cells transfected with EGFP-LC3, punctate EGFP-LC3 was monitored, percentage of cells displaying obvious EGFP-LC3 puncta coincided with the forenamed immunoblot results. Also, we found that autophagy was induced in hippocampus and cortex of rats underwent MCAO for 90 minutes and reperfusion for a week.Beclin-1 in cytosol, p-mTOR, p-P70 S6K were also checked in ischemia/reperfusion groups by immunoblot analysis, results indicated that Beclin-1 in cytosol shared the same change pattern with LC-3II, while p-P70 S6K and p-mTOR showed delayed decrease after reperfusion. Administration of 3MA, inhibitor of class III PI3K, definitely abolished autophagy during reperfusion, while employment of rapamycin, inhibitor of mTORC1, surprisingly weakened autophagy activation during reperfusion.Analyses of mitochondria function by relative cell viability were performed in I/R model groups and 3MA-treated groups, results came that autophagy inhibition by 3MA definitely hold back the decline of mitochondria function during the process of reperfusion. |
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