EZETIMIBE PREVENTS ISCHEMIA/REPERFUSION-INDUCED OXIDATIVE STRESS THROUGH UP-REGULATION OF NRF2/ARE AND UPR SIGNALING PATHWAYS AND INDUCTION OF AUTOPHAGY

While reperfusion is crucial for survival after an episode of ischemia, it also causes oxidative stress and cell damages through production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Evidences indicate that nuclear factor-E2-related factor 2 (Nrf2) and unfolded protein response (UPR) pathways are protective against oxidative stress (OS) and ER stress. Ezetimibe (Eze), a cholesterol absorption inhibitor, has been shown to activate Nrf2 pathway in a p62-dependent manner. In this study, we evaluated whether Eze may affect OS as well as Nrf2 and UPR gene expression in cellular models (THP-1 monocytic cells and cardiomyocytes) of I-R. After an overnight (ON) treatment with 50μM of Eze, cells were subjected to ischemia (2 hours and 24 hours respectively) followed by reperfusion (1 hour and 24 hours respectively). I-R significantly increased ROS production and % apoptotic cells without up-regulation of Nrf2, related antioxidant response element (ARE) gene expression and pro-survival UPR activating transcription factor 6 (ATF6) gene, whereas it significantly increased the pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). Eze significantly decreased cellular ROS formation and apoptosis induced by I-R. These effects were paralleled by the up-regulation of Nrf2/ARE and ATF6 gene expression and by a down-regulation of CHOP. Concerning the mechanism of Nrf2 activation, we also found that Eze significantly increased the phosphorylation of p62 that was dependent on adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) since Compound C (CC), a pan inhibitor of AMPK, blunted the activation of p62 and hence Nrf2. Since p62 phosphorylation has been shown to be dependent also on mTORC1 kinase, we performed further experiments using Rapamycin (Rap), a mTORC1 inhibitor. Despite the inhibition of mTORC1-dependent p62 phosphorylation by Rap, Nrf2 was activated. On the contrary, the co-treatment with Eze plus Rap reduced Nrf2 nuclear translocation because of the interaction of p62 with autophagic LC3B protein, which is basically increased both by Eze and Rap treatment, leading to the activation of autophagy. Finally, we tested our I-R model in human cardiomyocytes finding that, as in THP-1 cells, Eze significantly reduced ROS formation as well as I-R-induced apoptosis increasing Nrf2 nuclear translocation. Taken together, data demonstrated the new pleiotropic effects of Eze in counteracting I-R-induced oxidative stress through Nrf2 and UPR pathway activation as well as autophagy induction.