DiOHF Protects Against Doxorubicin-Induced Cardiotoxicity Through ERK1 Signaling Pathway

Figures

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  Figure 1. The protective effects of DiOHF on H9C2 cells against the DOX-induced cytotoxicity (A) DOX led to the reduction of cell viability in a dose-dependent manner (n = 3). (B) DiOHF inhibited the DOX-induced reduction of cell viability (n = 3). (C) Effects of different concentrations of DiOHF on cell viability (n = 3). (D) The H9C2 cells were treated with vehicle or DOX (1 µM for 24 h), and DiOHF was administrated at different time points (–2, 0, 2, 4, 8, and 12 h). Cell viability was determined using CCK-8 kit (n = 3). (E) Pre-treatment with DiOHF for 2 h reduced the DOX-induced ROS production (n = 3). (F) Post-treatment with DiOHF for 2 h also decreased the DOX-induced ROS production (n = 3). Data were analyzed by ANOVA and expressed as the mean ± SEM. *P < 0.05, compared with control. # P < 0.05, compared with DOX.

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  Figure 2. DiOHF prevented and reversed the DOX-induced apoptosis. (A) H9C2 cells were pre-treated with DiOHF for 2 h followed by the treatment with DOX for 24 h. Apoptosis was assessed by flow cytometry. (B) H9C2 cells were treated with DOX for 2 h followed by the treatment with DOX + DiOHF for 22 h. Apoptosis was detected by flow cytometry. (C and D) Quantitative data of the apoptosis. (E and G) Representative Western blots. (F and H) Western blot analysis for the apoptosis-associated proteins (cleaved-caspase-3, Bcl-2, and Bid). GAPDH served as the loading control. The data were analyzed by ANOVA and expressed as mean ± SEM. *P < 0.05, compared with the control; # P < 0.05, compared with DOX.

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  Figure 3. DiOHF prevented and rescued the DOX-induced mitochondrial dysfunction in H9C2 cells (A and B). Effects of pre- and post-treatment of DiOHF (10 μM) on the loss of MMP in H9C2 cells treated with DOX (1 μM for 24 h; n = 3). JC-1 was observed as green monomers in the cytosol or as red aggregates in intact mitochondria. The change from red to green fluorescence indicated a collapse of mitochondria with intact membrane potential. The micrographs were recorded under confocal microscopy (Zeiss) at 40× magnification (C and D; n = 3), and the red JC-1 fluorescence intensity was quantified (E and F) by Image Pro Plus. *P < 0.05, compared with control. #P < 0.05, compared with DOX.

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  Figure 4. ERK1/2 participated in the protective effects of DiOHF. (A) DiOHF induced ERK1/2 activation in a time-dependent manner (n = 3). The H9C2 cells were treated with vehicle or DOX (1 µM) in the presence or absence of DiOHF (10 µM for 2 h prior to DOX exposure and 10 µM after 2 h of DOX exposure, respectively) for 24 h. The protein levels of phospho-ERK1/2 (p-ERK1/2) and ERK1/2 were measured by Western blot (C and E; n = 3). (B, D, and F; n = 3) Quantitative results of ERK1/2 and p-ERK1/2. The data were analyzed by ANOVA and expressed as the mean ± SEM. *P < 0.05, compared with control. # P < 0.05, compared with DOX.

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  Figure 5. Knock-down of ERK1 or ERK2. (A) ERK1 or ERK2 silencing at the mRNA level (n = 3). (B) ERK1 or ERK2 silencing at the protein level (n = 3). (C) Quantitative results of ERK1 or ERK2 proteins (n = 3). The data were analyzed by ANOVA and expressed as the mean ± SEM. *P < 0.05, compared with Scr-siRNA.

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  Figure 6. ERK1 siRNA abolished the cardioprotective effects of DiOHF against DOX-induced damage in H9C2 cells. Effects of ERK1 or ERK2 silencing on cell viability (A; n = 3), MMP (B; n = 3), apoptosis rate (C; n = 3), and apoptosis-associated proteins (cleaved-caspase-3, Bcl-2, and Bid) (D and E). (D and E) Results of western blot and quantitative results of the apoptosis-associated proteins. The data were analyzed by ANOVA and expressed as the mean ± SEM. *P < 0.05, compared with control. # P < 0.05, compared with DOX.

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  Figure 7. Protective effects of DiOHF against DOX-induced death, loss of body weight, cardiac dysfunction, and fibrosis in mice (A) Kaplan-Meier survival analysis in mice after vehicle or DOX administration with or without DiOHF pretreatment (p > 0.05; n = 30 for each group). (B) Serum CK-MB levels measured in all groups of mice. (C) Echocardiographic images acquired on the 7th day after DOX treatment in all the mice. (D) Representative Masson’s trichrome-stained photomicrographs exhibiting cross-sectional area of cardiomyocyte and myocardial fibrosis. (E) Quantitative analysis of the fibrotic areas (Masson’s trichrome-stained area in light blue normalized to the total myocardial area) obtained from 30–50 randomly chosen fields. (F) The levels of blood ALT (n = 7) and Urea (n = 9) in mice. *P<0.05, compared with control. #P<0.05, compared with DOX.