Carnosic acid protects normal mouse hepatocytes against H2 O2 -induced cytotoxicity via sirtuin 1-mediated signaling.

Abstract

Carnosic 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. Primary hepatocytes and AML12 cells were treated with: (i) 0.1μM, 1μM and 10μM CA; (ii) 3mM H2 O2 with or without 1μM CA; or (iii) 3mM H2 O2 with 1μM CA and 0.04μM sirtuin 1 (SIRT1) inhibitor EX527 or 10μM 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. First, although 10μM CA produced cytotoxicity, CA at concentrations at or below 1μM did not inhibit cell viability. Second, H2 O2 increased total cellular ROS and LDH leakage and decreased cell viability, whereas co-treatment with H2 O2 and 1μM CA significantly inhibited these effects of H2 O2 . Third, CA at 1μM increased protein levels of SIRT1. Pretreatment with EX527 or transfection of siRNA-targeting SIRT1 weakened the protective effects of CA against H2 O2 -induced cell death. Fourth, H2 O2 induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes. U0126 inhibited oxidative damage induced by H2 O2 . Co-treatment with CA inhibited ERK1/2 activation induced by H2 O2 . Our data indicate that CA protects against oxidative stress-induced cytotoxicity via SIRT1 by regulating subsequent downstream factors such as ERK1/2.