Carnosic acid protects normal mouse hepatocytes against H2O2-induced cytotoxicity via sirtuin 1-mediated signaling

AimCarnosic acid (CA) is well known for its antioxidant properties. The aim of this study was to examine the effects of CA on cytotoxicity under oxidative stress. MethodsPrimary hepatocytes and AML12 cells were treated with: (i) 0.1M, 1M and 10M CA; (ii) 3mM H2O2 with or without 1M CA; or (iii) 3mM H2O2 with 1M CA and 0.04M sirtuin 1 (SIRT1) inhibitor EX527 or 10M mitogen-activated protein kinase (MAPK) inhibitor U0126. Cell viability, intracellular reactive oxygen species (ROS) and lactate dehydrogenase (LDH) leakage were determined. In addition, total protein levels of cleaved caspase 3, SIRT1, phosphorylated Nrf2, 5-adenosine monophosphate-activated protein kinase (AMPK) and MAPKs were evaluated by western blot analysis and suspension array system. ResultsFirst, although 10M CA produced cytotoxicity, CA at concentrations at or below 1M did not inhibit cell viability. Second, H2O2 increased total cellular ROS and LDH leakage and decreased cell viability, whereas co-treatment with H2O2 and 1M CA significantly inhibited these effects of H2O2. Third, CA at 1M increased protein levels of SIRT1. Pretreatment with EX527 or transfection of siRNA-targeting SIRT1 weakened the protective effects of CA against H2O2-induced cell death. Fourth, H2O2 induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes. U0126 inhibited oxidative damage induced by H2O2. Co-treatment with CA inhibited ERK1/2 activation induced by H2O2. ConclusionOur data indicate that CA protects against oxidative stress-induced cytotoxicity via SIRT1 by regulating subsequent downstream factors such as ERK1/2.