Abstract WP271: Epicatechin Protects Neurons from in vitro Ischemia through the Nrf2 Pathway.

Abstract

Introduction: Foods rich in polyphenols, such as flavanols, can lower the risk of ischemic heart disease and we recently demonstrated beneficial outcomes in ischemic stroke; however, the mechanism of protection has not been fully established. In this study, we assessed the hypothesis that epicatechin provides neuroprevention against oxygen glucose deprivation (OGD) in primary cortical neurons, and this protection occurs through activation of Nrf2. Epicatechin targets genes regulating expression of heme oxygenase 1 (HO1), ferritin and biliverdin, which can prevent free radical. Methods: Experiments were carried out to optimize and standardize various conditions for OGD and reoxygenation. Seven day old cultures from wildtype and Nrf2 knockout mice were pretreated with 50 and 100uM of epicatechin before 4h oxygen glucose deprivation followed by 24h reoxygenation. Neuronal survival was assessed by live calcein AM, LDH and MTT assay after 24h reoxygenation. Changes in protein expression levels were measured by Western blotting. Results: OGD of 4h followed by a reoxygenation of 24h with 4-6mg/ml glucose concentration in culture medium was found to be the optimum conditions to create ischemic stroke like situations under in vitro environment using primary postnatal neurons. Significant increase in live cells (35.7±4.3 vs 64.3±4.9 and 87.5±9.7, p<0.05 and p<0.0001) was recorded in OGD induced cultures pretreated with 50 and 100uM epicatechin respectively by calcein-AM assay and increase in viability (0.2±0.1 vs 0.3±0.1 and 0.3±0.1, p<0.0001) by MTT assay. Significant decrease in cytotoxicity (3102.0±180.4 vs 2684.0±53.5 and 1752.0±64.5, p<0.0001) was also recorded by LDH assay. Western blots from epicatechin pretreatment showed an increase in HO1 and L-ferritin protein expression. Interestingly, pretreatment of epicatechin could not protect Nrf2 knockout neurons from OGD. Conclusion: Cell viability assays suggested that pretreatment with epicatechin protect neurons against oxidative stress induced by OGD. Interestingly, the epicatechin-associated neuroprotection was mostly abolished in neurons derived from Nrf2 knockout mice. These results suggest that epicatechin exerts part of its beneficial effect through activation of Nrf2.