title: [Resveratrol attenuates oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes via inactivating GSK-3β].
abstract: OBJECTIVE
  To investigate the underlying mechanism of the protective effects of resveratrol on oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes.
  
  
  METHODS
  H9c2 cells, a permanent cell line derived from embryonic rat cardiac tissue, and then randomly divided into control group [PBS, cells exposed to H2O2 (600 µmol/L) for 20 min to induce mitochondrial oxidant damage], resveratrol group (0.01, 0.1, 1, 5, 10 and 20 µmol/L for 20 min at 20 min before exposing to H2O2), resveratrol plus inhibitor group (1 µmol/L KT5823 for 10 min at 10 min before 5 µmol/L resveratrol treatment) and inhibitor group (1 µmol/L KT5823 for 10 min). Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mitochondrial permeability transition pore (mPTP) opening was evaluated by measuring the decrease of TMRE fluorescence intensity. Immunofluorescence assay was used to observe GSK-3β phosphorylation. The phosphorylation of GSK-3β and VASP were determined by Western blot. To detect intracellular NO, cells were loaded with DAF-FM DA (specific fluorescent dye of NO) and imaged with confocal microscopy.
  
  
  RESULTS
  Compared to the control group, resveratrol (0.01-5 µmol/L) attenuated H2O2-induced mitochondrial damage reflected by attenuating the H2O2-induced TMRE fluorescence intensity decrease in a dose-dependent manner and the efficacy of 10 and 20 µmol/L resveratrol was significantly lower than that of 5 µmol/L resveratrol. Resveratrol also significantly upregulated the protein expression of VASP and increased GSK-3β Ser(9) phosphorylation, which could lead the inactivation of GSK-3β. These effects of resveratrol could be significantly abolished by protein kinase G inhibitor KT5823, while KT5823 alone did not affect GSK-3β and VASP phosphorylation. Confocal microscopy showed that DAF-FM (specific NO indicator) was similar between resveratrol and control group, suggesting that resveratrol did not produce NO.
  
  
  CONCLUSIONS
  Resveratrol could attenuate oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes by inactivating GSK-3β via cGMP/PKG signaling pathway independent of NO-related mechanism.
authors.affiliation: Department of Neurology, Affiliated Hospital of Hebei United University, Tangshan, China.
authors.forename: Yong-gui
authors.initials: YG
authors.lastname: He
authors.forename: Yu-jie
authors.initials: YJ
authors.lastname: Sun
authors.forename: Yu-xi
authors.initials: YX
authors.lastname: Xie
authors.forename: Huan
authors.initials: H
authors.lastname: Zheng
authors.forename: Yi-dong
authors.initials: YD
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Guo
authors.forename: Jin-kun
authors.initials: JK
authors.lastname: Xi
mesh_terms: D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006861:Hydrogen Peroxide / Q000378:metabolism; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects; D016877:Oxidants / Q000378:metabolism; D051381:Rats; D000077185:Resveratrol; D015398:Signal Transduction / Q000187:drug effects; D013267:Stilbenes / Q000494:pharmacology*
chemical_list: D002227:Carbazoles; D016877:Oxidants; D013267:Stilbenes; C073601:KT 5823; D006861:Hydrogen Peroxide; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D017869:Cyclic GMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D006152:Cyclic GMP; D000077185:Resveratrol