Activation of PI3K/Akt mediates the protective effect of diallyl trisulfide on doxorubicin induced cardiac apoptosis

Fig. 1. Dox induces the apoptosis of cardiac cells. Cells were treated with Dox at concentrations of 0.5, 1, and 1.5 μM for 24 h. The expression of PI3K/Akt, Bcl-2, and caspase-3 were evaluated by western blot analysis. β-actin was used as a loading control. Values shown are means ± SD. Quantification of the results is shown (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 versus control cells.

Fig. 2. DATS exhibited a dose-dependent reduction in the doxorubicin-induced generation of ROS. H9c2 cells were initially exposed to 1 μM Dox for a duration of 2 h, followed by treatment with DATS at different concentrations (1, 5, and 10 μM) for an additional 22 h. Cellular ROS was detected using the dichlorofluorescein (DCF) assay for 15 min and examined by flow cytometry. Values shown are means ± SD. Quantification of the results is shown (n = 3); **p < 0.01, ***p < 0.001 versus Dox-treated cells; ###p < 0.001 versus control.

Fig. 3. DATS increases the survival of cardiac cells. Cells were treated with DATS at the indicated concentrations (1, 5, and 10 μM) for 24 h. The expression of apoptotic and survival proteins was examined by western blot analysis. β-actin was used as a loading control. Values shown are means ± SD. Quantification of the results is shown (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 versus control cells.

Fig. 4. DATS inhibits apoptosis by decreasing the mitochondrial pathway in cardiac cells following exposure to Dox. H9c2 cells were initially exposed to 1 μM Dox for a duration of 2 h, followed by treatment with DATS at different concentrations (1, 5, and 10 μM) for an additional 22 h. Cells only treated with 10 μM of DATS for 24 h were used as a positive control. (A) Cell viability was examined using an MTT assay. (B) Protein expression was evaluated by western blot analysis. β-actin was used as a loading control. Values shown are means ± SD. Quantification of the results is shown (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 versus Dox-treated cells; ##p < 0.01, ###p < 0.001 versus control.

Fig. 5. DATS suppresses Dox-induced apoptosis through the activation of the PI3K/Akt signaling pathway. H9c2 cells were initially exposed to 1 μM Dox for a duration of 2 h, followed by treatment with 10 μM DATS and combined with LY294002 (a PI3K/Akt inhibitor) or PI3K siRNA for an additional 22 h. Protein expression was evaluated by western blot analysis. β-actin was used as a loading control. Values shown are means ± SD. Quantification of the results is shown (n = 3); **p < 0.01, ***p < 0.001 versus Dox-treated cells; ###p < 0.001 versus control; &&p < 0.01, &&&p < 0.001 versus Dox + DATS-treated cells.

Fig. 6. Mediation of PI3K/Akt signaling in the antiapoptotic effect of DATS on the Dox-induced apoptosis of cardiac cells. Cells were treated as described in Fig. 5. Apoptosis of cells was examined by TUNEL assay; green representing apoptosis and blue (DAPI) representing cell nuclei.