title: Carnosic acid protects normal mouse hepatocytes against H2 O2 -induced cytotoxicity via sirtuin 1-mediated signaling.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Primary hepatocytes and AML12 cells were treated with: (i) 0.1 μM, 1 μM and 10 μM CA; (ii) 3 mM H2 O2 with or without 1 μM CA; or (iii) 3 mM H2 O2 with 1 μM CA and 0.04 μM sirtuin 1 (SIRT1) inhibitor EX527 or 10 μM 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.
  
  
  RESULTS
  First, although 10 μM CA produced cytotoxicity, CA at concentrations at or below 1 μM 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 1 μM CA significantly inhibited these effects of H2 O2 . Third, CA at 1 μM 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 .
  
  
  CONCLUSIONS
  Our data indicate that CA protects against oxidative stress-induced cytotoxicity via SIRT1 by regulating subsequent downstream factors such as ERK1/2.
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, Iwate Medical University, Morioka, Iwate, Japan.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, Iwate Medical University, Morioka, Iwate, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takikawa
keywords: carnosic acid; extracellular signal-regulated kinase 1/2; oxidative stress; sirtuin 1