Cyanidin ameliorates cisplatin-induced cardiotoxicity via inhibition of ROS-mediated apoptosis

Figures

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  Figure 1.. Effect of cisplatin on H9c2 cells viability and protective effects of cyanidin. (A) H9c2 cells were incubated with different concentrations of cisplatin (0–40 µM) for 24 h, cells viability were detected by MTT assay. (B) H9c2 cells were incubated with cisplatin for 0, 6, 24 and 48 h, cells viability were detected by MTT assay. (C) Cells were pre-treated with cyanidin (40–80 µM) for 3 h, then exposed to cisplatin (40 µM) for 24 h. Data represent means ± SD, *P<0.05, **P<0.01 compared to control group, #P<0.05, ##P<0.01 compared with cisplatin group.

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  Figure 2.. Cyanidin reduces cisplatin-induced H9c2 cell apoptosis. (A) Apoptotic cells were detected by TUNEL after incubated with cisplatin (40 µM) alone or co-treated with cyanidin (80 µM) for 24 h. (B) Representation images of the effects of cyanidin pretreatment on cisplatin-induced apoptotic cell distribution in H9c2 cells. (C) The apoptosis rate was expressed as the ratio of TUNEL-positive cardiomyocytes number to total number of cardiomyocytes. (D) The statistical analysis of apoptotic cells. The apoptotic cells show hypodiploid DNA contents (Q3 area), the data were expressed in the histogram and analyzed by Modfit Software *P<0.05 compared with control group, #P<0.05 compared with cisplatin group.

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  Figure 3.. Cyanidin attenuates cisplatin-induced accumulation of ROS and ΔΨm depolarization in H9c2 cells. (A) Representative images of ROS assay. (B) Quantitative analysis of ROS assay. (C) Representative images of JC-1 staining. (D) Quantitative analysis of JC-1 staining. (a) control; (b) cisplatin (40 µM); (c) cisplatin (40 µM) + cyanidin (40 µM); (d) cisplatin (40 µM) + cyanidin (80 µM); (e) cyanidin (40 µM); (f) cyanidin (80 µM). Data represent means ± SD, *P<0.05, **P<0.01 compared to control group, #P<0.05, ##P<0.01 compared with cisplatin (40 µM) group.

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  Figure 4.. Cyanidin alleviates cisplatin-induced activation of caspases in H9c2 cells. (A-C) Caspase 9, caspase 8, and caspase 3 were detected by spectrophotometry. (D) After cells pretreatment, cleaved caspase 3 and caspase 3 proteins were extracted and detected by western blotting. All experiments were repeated twice with similar results and the data is normalized after grayscale scanning. Data represent means ± SD, *P<0.05 compared to control group, #P<0.05, compared with cisplatin group.

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  Figure 5.. Cyanidin promotes the translocation of Bax and the expression of anti-apoptotic protein in H9c2 cells. (A) H9c2 cells were pre-treated with cisplatin (40 µM) for 24 h in the presence or absence of cyanidin (80 µM), then Bax and cytochrome c located in the cytoplasm and mitochondria were extracted for immunblotting. (B) Relative quantification of Bax in cytoplasm and mitochondrial. (C) Relative quantification of cytochrome c in cytoplasm and mitochondrial. (D) H9c2 cells were pre-treated same as (A and B) and the quantitative expression of Bak and Bcl-2 were detected by western blotting. (E) The ratio of Bak/β-actin. (F) The ratio of Bcl-2/β-actin. Data represent means ± SD, *P<0.05, **P<0.01 compared with control group, #P<0.05, ##P<0.01 compared with cisplatin group.

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  Figure 6.. Cyanidin suppresses cisplatin-induced ERK activation. (A) H9c2 cells were pre-treated with cisplatin at varying doses for 24 h, and immunblotting was performed to detected ERK and ERK phosphorylation. (B) H9c2 cells were incubated with cisplatin (20 µM, 40 µM) for 24 h in the presence or absence of cyanidin (80 µM) and U0126 (10 µM), then tested by western blotting. (C) The ratio of pERK to ERK was expressed as a histogram. (D) The ratio of pERK/ERK in different groups. Data represent means ± SD, *P<0.05, **P<0.01 compared with control group, #P<0.05 compared with cisplatin (40 µM) group.