Alpha-lipoic acid pre- and post-treatments provide protection against in vitro ischemia-reperfusion injury in cerebral endothelial cells via Akt/mTOR signaling

Alpha-lipoic acid (ALA) is an endogenous short-chain fatty acid that has beneficial protective effects against various vascular diseases. In this study, we sought to determine whether ALA could induce pre- or post-treatment protective effects against simulated ischemia and reperfusion-induced cerebral endothelial cell (CEC) injury by activating the Akt/mTOR pathway. CECs are currently considered to be an important target for ischemia therapy. Mouse brain endothelial cells (bEnd.3) and primary cultures of CECs were subjected to 6h of oxygen glucose deprivation (OGD) followed by 4h of simulated reperfusion, either alone or together with ALA administration before (pre-treatment) or immediately after (post-treatment) OGD. We found that pre-treatment administration of ALA reduced the OGD and simulated reperfusion-induced lactate dehydrogenase (LDH) release in bEnd.3 cells in a dose-dependent manner and that 1 mM ALA pre- and post-treatments provided protection in both bEnd.3 cells and primary cultures of CECs. However, rapamycin, an mTOR inhibitor, was able to thoroughly abolish the protective effects of ALA. Western blotting showed that the ALA pre- and post-treatments up-regulated the phosphorylation of Akt, mTOR, S6K and 4E-BP1 in both bEnd.3 cells and primary cultures. However, after pre-treatment with rapamycin, the level of Akt phosphorylation was decreased in primary cultures of CECs but could still be restored by ALA, whereas the levels of mTOR, S6K and 4E-BP1 phosphorylation were significantly decreased and could not be restored. These results suggest that ALA pre- and post-treatments provide protective effects against simulated ischemia and reperfusion-induced CEC injury by promoting the Akt/mTOR pathway and that mTOR is required for ALA protection. (C) 2012 Elsevier B.V. All rights reserved.