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Carnosic acid protects normal mouse hepatocytes against H 2 O 2 ‐induced cytotoxicity via sirtuin 1‐mediated signaling
Author(s) -
Wang Ting,
Takikawa Yasuhiro
Publication year - 2016
Publication title -
hepatology research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.123
H-Index - 75
eISSN - 1872-034X
pISSN - 1386-6346
DOI - 10.1111/hepr.12563
Subject(s) - viability assay , protein kinase a , oxidative stress , lactate dehydrogenase , chemistry , cytotoxicity , reactive oxygen species , kinase , microbiology and biotechnology , biochemistry , apoptosis , biology , enzyme , in vitro
Aim 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 H 2 O 2 with or without 1 μM CA; or (iii) 3 mM H 2 O 2 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, H 2 O 2 increased total cellular ROS and LDH leakage and decreased cell viability, whereas co‐treatment with H 2 O 2 and 1 μM CA significantly inhibited these effects of H 2 O 2 . 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 H 2 O 2 ‐induced cell death. Fourth, H 2 O 2 induced phosphorylation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes. U0126 inhibited oxidative damage induced by H 2 O 2 . Co‐treatment with CA inhibited ERK1/2 activation induced by H 2 O 2 . Conclusion Our data indicate that CA protects against oxidative stress‐induced cytotoxicity via SIRT1 by regulating subsequent downstream factors such as ERK1/2.