| Objective: This study was designed to investigate the protective effects of FGF-2inH2O2-induced apoptosis of H9c2cardiomyocytes, as well as the possible molecularmechanisms involved.Methods:1.The optimal concentration of H2O2to induce apoptosis was determined by Methylthiazolyl tetrazolium (MTT) assay in vitro culture of H9c2cardiomyocytes. Thepretreatment of different concentrations of FGF-2for0.5h was given followed bystimulation with H2O2for6h in H9c2cardiomyocytes, and the optimal concentrationof FGF-2against apoptosis in H9c2cardiomyocytes was ascertained by MTT assay.2.Western blot was used to detect the total and phosphorylated protein expression ofp-Akt, p-FoxO3a and Bim in H9c2cardiomyocytes treated with H2O2.3.The experiments were further divided into four groups as follows:(l) Control group:H9c2cardiomyocytes were cultured in DMEM for6h;(2) H2O2group: H9c2cardiomyocytes was stimulated with100μmol/L H2O2for6h;(3) FGF-2+H2O2group:H9c2cardiomyocytes were incubated with10ng/ml FGF-2for0.5h before addingH2O2;(4) FGF-2+H2O2+LY294002group (PI3K/Akt inhibitor group): H9c2cardiomyocytes were pretreated with FGF-2and LY294002, and then treated with100μmol/L H2O2for6h. Methyl thiazolyl tetrazolium (MTT) assay, Hoechst andTUNEL staining, and Flow cytometry were used to determine the apoptosis rate ofH9c2cardiomyocytes; Western blot were conducted to determine the expression of p-Akt, p-FoxO3a and Bim protein.Results:1.MTT assay showed that H2O2could facilitate H9c2cardiomyocytes apoptosis in adose dependent way; FGF-2can reduce H2O2induced H9c2myocardial apoptosis, theoptimal concentrations of H2O2and FGF-2were100μmol/L and10ng/ml respectively.After adding PI3K/Akt signaling pathway inhibitor LY294002, the protective effect ofFGF-2on H2O2induced H9c2myocardial apoptosis was inhibited significantly.2. Hoechst33258and TUNEL staining results showed that the apoptotic cells in H2O2group was increased significantly, compared with the control group; and comparedwith H2O2group, the apoptotic cells in FGF-2+H2O2group was significantly reduced.After incubation with PI3K/Akt inhibitor LY294002, the apoptotic cells inFGF-2+H2O2+LY294002group was increased significantly, compared withFGF-2+H2O2group.3. Flow cytometry results showed that the apoptosis rate in H2O2group was27.24%,which is significantly higher than control group (P<0.01). Compared with H2O2group,the apoptosis rate in FGF-2+H2O2group was obviously reduced by21.21%(P<0.01);after incubation with PI3K/Akt inhibitor LY294002,the apoptosis rate of FGF-2+H2O2+LY294002group significantly increased by11.68%， compared with FGF-2group (P<0.05).4. A dose-dependent decrease was found in the phosphorylation of Akt and FoxO3aprotein after treatment of H9c2cardiomyocytes with H2O2stimulation in differenttime （P﹤0.05）, while the expression of total Akt and FoxO3a protein was notchanged significantly. The expression of Bim protein was significantly increased(P<0.05). Meanwhile, the nuclear protein FoxO3a was obviously increased in atime-dependent manner after H2O2stimulation (P<0.05); On the contrary, FoxO3aprotein in cytoplasmic subfraction was evidently decreased (P<0.05).5. Compared with H2O2group，FGF-2treatment resulted in a significant increase inexpression of p-Akt and p-FoxO3a protein，and a reduction of the expression of Bimprotein (P<0.05). At the same time, the nuclear protein FoxO3a was obviouslydecreased (P<0.05), On the other hand, FoxO3a protein in cytoplasmic subfraction was significantly increased (P<0.05). When H9c2cells were pretreated with thePI3K/Akt inhibitor LY294002and FGF-2, the phosphorylation of Akt and FoxO3awas inhibited (P<0.05). Compared with the FGF-2+H2O2group, PI3K/Akt inhibitorgroup showed a significant inhibitive influence against the antiapoptotic effectinduced by FGF-2.Conclusions: These results suggest that FGF-2protects H9c2cardiomyocytes againstH2O2-induced apoptosis via activation of the PI3K/Akt/FoxO3a pathway. |
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