Mechanism For GSK-3β Inactivation In Resveratrol-induced Protection Against Oxidant-induced Mitochondrial Damage In Cardiac Cell

Objective:1 To investigate whether resveratrol protect heart through inhibited mPTP opening.2 To investigate whether the inhibition of mPTP opening by resveratrol inhibits GSK-3βactivity.3 Further more, the molecular mechanism was studied, by which resveratrol inhibits GSK-3βactivity in this study.Methods:Rat heart tissue-derived H9c2 cells were purchased from ATCC. And cardiac H9c2 cells were exposed to H₂O₂ (600μM) for 20 min to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mPTP opening was evaluated by the decrease of TMRE fluorescence intensity. Immunofluorescence assay was used to test GSK-3β(Ser⁹) phosphorylation. Meanwhile the phosphorylation of GSK-3β(Ser⁹) and VASP (Ser²³⁹) were determined with Western blot. To detect intracellular NO, cells were loaded with DAF-FM DA (specific fluorescent dye of NO) and imaged with confocal microscopy.Results:1 Cardiac H9c2 cells treated with different dose of resveratrol (0.01μM-20μM) and 5.0μM resveratrol (81.9±1.1%) showed a significant prevention of the decrease in TMRE fluorescence intensity compared to the H₂O₂ group (38.6±1.9%), indicating that resveratrol(5.0μM) can prevent oxidant-induced mitochondrial damage. 2 Immunofluorescence assay and Western blot results showed that, resveratrol showed a significant increase in phosphorylation of GSK-3β(Ser⁹) compared with that of control group. These data indicated that GSK-3βcoudld mediate the cardioprotective effect of resveratrol.3 Confocal imaging study for the effect of resveratrol on the TMRE fluorescence was reversed by KT5823 (1μM),the inhibitor of PKG , suggesting that cGMP-PKG accounted for the protective effect of resveratrol. Western blot results showed that, resveratrol significance increased the phosphorylation of GSK-3β(Ser⁹) and VASP(Ser²³⁹).That was abolished by KT5823 (1μM), further supporting that the cGMP-PKG signaling pathway was responsible for inactivation of GSK-3βby resveratrol.5 Confocal microscopy showed that resveratrol (5.0μM) could not increase DAF-FM (specific NO indicator) fluorescence intensity compared with that of control, suggesting that resveratrol did not produce NO.Conclusions:These data suggest that, resveratrol prevents the mPTP opening by indicating GSK-3βthrough the cGMP/PKG signaling pathway, NO may not be involved in the action of resveratrol.