Bml 275

SH-SY5Y neuroblastoma cells was obtained from American Type Culture Collection (ATCC, VA, USA). They were cultured at 37 °C with the high glucose DMEM medium (Gibco, Gaithersburg, USA) supplemented with 10% FBS (Gibco, Gaithersburg, USA) and 1% penicillin–streptomycin (Gibco, Gaithersburg, USA) in a humidified atmosphere of 5% CO₂. Two cell models were used in this study: oxygen and glucose deprivation/reoxygenation (OGD/R) model and tert-butyl hydroperoxide (t-BHP) induced oxidative stress model. OGD/R model was established according to Feng et al. [19] (link). Briefly, cells were washed with PBS (Sigma, St. Louis, USA), and then cultured with no glucose DMEM medium (Gibco, Gaithersburg, USA) in a hypoxia chamber (Billups-Rothenberg, Delmar, USA) perfused with 95% N₂ and 5% CO₂ for 8 h at 37 °C. After that, cells were cultured at the normal cultural conditions for another 16 h to achieve reoxygenation. t-BHP-induced oxidative stress model was performed by treating cells with 100 μM of t-BHP (Sigma, St. Louis, USA). Cells were pretreated with CSA for 24 h before they were subjected to OGD/R or t-BHP damages. The inhibitors, including SB203580 (p38 MAPK & AKT, 5 μM), SP600125 (JNK, 5 μM), U0126 (Erk1/2, 10 μM) [20] (link), BML-275 (AMPK, 5 μM) [21] (link) and brusatol (Nrf2, 0.5 μM, Sigma, St. Louis, USA) [22] (link), were added along with CSA if needed.