Icariin Protects H9c2 Rat Cardiomyoblasts from Doxorubicin-Induced Cardiotoxicity: Role of Caveolin-1 Upregulation and Enhanced Autophagic Response

Figures

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  Figure 1. The effect of Doxorubicin treatment on H9c2 cell viability. H9c2 cells were treated with Doxorubicin (Doxo) 1 μM at three-time points (18 h, 24 h and 48 h). Treatment with Doxo induces a time dependent reduction in cell viability. Data are presented as mean ± SEM. ** p < 0.01 vs. Ctrl (24 h); *** p < 0.001 vs. Ctrl (48 h); Mann Whitney test (n = 8). Data is from three different independent experiments.

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  Figure 2. The effect of Icariin on Doxo-treated H9c2 cell viability. (A). Incubation of H9c2 cells with Icariin (Ica) alone, at increasing concentrations, did not result in a reduction of cell viability compared to the Ctrl group. (B). Pretreatment with increasing concentrations of Ica for 3 h protects cardiomyoblasts from Doxo induced toxicity. Data are expressed as mean ± SEM *** p < 0.001 vs. Ctrl; °°° p < 0.001 vs. Ica 1 μM; ^^ p < 0.01 vs. Ica 5 μM; ## p < 0.01 vs. Doxo; ### p < 0.001 vs. Doxo; Mann Whitney test (n = 8). Data is from three different independent experiments.

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  Figure 3. Ica reduces intracellular reactive oxygen species overproduction. (A). Reactive oxygen species (ROS) overproduction was determined by immunofluorescence assay. Oxidative stress was quantified by counting ∼1000 myoblasts/slide, for a minimum of three slides per sample, and the ROS positive fraction was expressed as a percentage of total myoblasts. (B). Representative confocal images of H9c2 cells treated as described above. Green staining showed fluorescent probe 2’,7’—dichlorodihydrofluoresceindiacetate (DCFH-DA), blue staining showed DAPI (4’,6-diamidino-2-phenylindole), used for nucleic acid staining. Cell images were collected using a 40× confocal microscope objective. Data are expressed as mean ± SEM, ** p < 0.01 vs. Ctrl; °° p < 0.01 vs. Ica 1 μM; ^ p < 0.05 vs. Ica 5 μM; # p < 0.05 vs. Doxo; ## p < 0.01 vs. Doxo; Mann Whitney test (n = 3). Data is for three independent experiments.

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  Figure 4. The effect of Ica on mitochondrial superoxide overproduction. (A). Superoxide (O2−) levels were determined by immunofluorescence assay. Fluorescence was quantified using ImageJ® software by converting the intensity to a greyscale value based on the RGB colour model. For each treatment, 30–40 cells were processed. Significant differences in mean fluorescence were found between all treated cells. (B). Representative confocal images of H9c2 cells treated as described above. Red staining showed the MitoSOX fluorescent probe used to identify mitochondrial O2− overproduction, blue staining showed DAPI (4’,6-diamidino-2-phenylindole), used for nucleic acid staining. Cell images were collected using a 40× confocal microscope objective. Data are expressed as mean ± SEM., *** p < 0.001 vs. Ctrl; °°° p < 0.001 vs. Ica 1 μM; ^^^ p < 0.001 vs. Ica 5 μM; # p < 0.05 vs. Doxo; ### p < 0.001 vs. Doxo; Kruskal-Wallis test and Dunn’s multiple comparisons test (n= 3). Data is for three independent experiments.

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  Figure 5. The effect of Ica on mitochondrial permeability transition pore opening in Doxo-treated H9c2 cells. (A). Mitochondrial permeability transition pore (mPTP) opening was determined by immunofluorescence assay. Fluorescence was quantified using ImageJ® software by converting the intensity to a greyscale value based on the RGB color model. For each treatment, 30–40 cells were processed. Significant differences in mean fluorescence were found between all treated cells. (B). Confocal representative images of H9c2 cells treated as described above. Green staining showed calcein, blue staining showed Hoechst 33342 used for nucleic acid staining, red staining showed MitoTracker Red CMXRos used for mitochondria staining. Cell images were collected using a 40× confocal microscope objective. Data are expressed as mean ± SEM, *** p < 0.001 vs. Ctrl; °°° p < 0.001 vs. Ica 1 μM ^^^ p < 0.001 vs. Ica 5 μM; ### p < 0.001 vs. Doxo; Kruskal-Wallis test and Dunn’s multiple comparisons test (n = 3). Data is from three independent experiments.

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  Figure 6. Cytofluorimetric evaluation of the effect of Ica against Doxo-induced apoptotic cell death. (A). H9c2 cells treated for 3 h with both Ica 1 μM and 5 μM showed no significant apoptotic or necrotic cell death. (B). Treatment with Doxo 1 μM for 24 h resulted in a detrimental increase in apoptotic cell death when compared to Ctrl cells. Conversely, pretreatment of H9c2 cells with Ica 1 μM and 5 μM significantly prevented apoptosis. (C) Representative cytofluorimetric dot plot. Data are expressed as means ± SEM.; ** p < 0.01 vs. Ctrl, ## p < 0.01 vs. Doxo; Mann Whitney test (n = 3). Data is for three independent experiments.

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  Figure 7. Ica inhibits Caveolin-1 expression levels in Doxo-treated H9c2 cells. (A) Doxo treatment significantly upregulated Caveolin-1 (Cav-1) expression levels compared to Ctrl cells. Pretreatment of Ica cells at both concentrations used significantly inhibited the expression of the molecular marker. (B) Representative images of western blot analysis. Three independent Western Blots were quantified by densitometry. The protein expression of Cav-1 was normalized with respect to the corresponding actin signals of the appropriate samples and expressed as a percentage. Data are expressed as mean ± S.E.M. *** p < 0.001 vs. Ctrl; # p < 0.05 vs. Doxo; ### p < 0.01 vs. Doxo; Mann Whitney test (n = 3).

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  Figure 8. The inhibitory effect of Ica on phosphodiesterase-5a activity. (A). Treatment with Ica 1 µM resulted in a reduction of phosphodiesterase-5a (PDE5a) activity, while sildenafil and Ica 5 µM significantly reduced PDE5a activity. (B). On H9c2 cells, the increased activity of PDE5a, determined by Doxo, was significantly reduced by ICA1 µM and by ICA5 µM. Data are expressed as mean ± S.E.M. ** p < 0.01 vs. Ctrl; *** p < 0.001 vs. Positive Ctrl; **** p < 0.0001 vs. Positive Ctrl; °° p < 0.01 vs. Ica 1 μM; ^^ p < 0.01 vs. Ica 5 μM; # p < 0.05 vs. Doxo; Tukey’s multiple comparisons test and Mann Whitney test (n = 3). Data is for three independent experiments.

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  Figure 9. The effect of Ica on the pro-autophagic markers Beclin-1 and LC3. Treatment with Doxo alone significantly increased the expression levels of Beclin-1 (A) and LC3II/LC3I ratio (C) in H9c2 cells. Pretreatment with Ica at both concentrations used inhibited the proautophagic pathway when compared to H9c2 cells treated with Doxo alone. (B,D) Representative images of western blot analysis. Three independent Western Blots were quantified by densitometry. The protein expression of Beclin-1 and LC3II/LC3I were normalized with respect to the corresponding tubulin or actin signals of the appropriate samples and expressed as a percentage. Data are expressed as mean ± S.E.M. ** p < 0.01 vs. Ctrl; ° p < 0,05 vs. Ica 1uM; ^ p < 0.05 vs. Ica 5 μM; # p < 0.05 vs. Doxo; ## p < 0.01 vs. Doxo; Mann Whitney test (n = 3).