Attenuation of doxorubicin-induced cardiotoxicity by cryptotanshinone detected through association analysis of transcriptomic profiling and KEGG pathway

Figures

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  Figure 1. Cryptotanshinone (CPT) protected H9c2 cardiomyocytes against doxorubicin-induced damage. Chemical structure of CPT (A). Cytotoxicity of CPT in vitro (B). Effects of CPT on the viability of H9c2 cardiomyocytes induced by various concentrations of doxorubicin at the indicated time-points (C). Cell-size measurement of H9c2 cardiomyocytes (green-F-actin staining) induced by doxorubicin in the presence or absence of CPT treatment (D). The number of apoptotic H9c2 cells determined using Annexin V/ Propidium Iodide (PI) staining (E). Assessment of reactive oxygen species (ROS) by flow cytometry analysis after treatment with doxorubicin in the presence or absence of CPT (F). The JC-1 monomers and aggregates of H9c2 cells stimulated by doxorubicin with/without CPT detected by flow cytometry (G). Values are mean ± standard error of the mean; all experiments were performed in three replicates. *Significant difference (P<0.05), DOX + CPT vs DOX.

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  Figure 2. Oral administration of cryptotanshinone (CPT) ameliorated cardiac dysfunction in doxorubicin-induced injury. Evaluation of cardiac function by echocardiography with/without CPT treatment, indicated by ejection fraction (EF, %) of LV and fractional shortening (FS, %) of LV, in the doxorubicin-induced rat model (A, B). Measurement of invasive hemodynamic examinations for analyzing both LV pressure and volume (pressure-volume loop) simultaneously, also in the rats model, indicated by end-systolic pressure (Pes), end-diastolic pressure (Ped), maximal rates of the increase in ventricular pressure (dp/dtmax) and maximal rates of decline in ventricular pressure (dp/dtmin) (C). *P<0.05 vs control, #P<0.05 vs DOX.

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  Figure 3. Cryptotanshinone (CPT) gavage attenuated myocardial fibrosis and apoptosis in rats treated with doxorubicin. Hematoxylin and eosin staining for paraffin section (A). Masson’s trichrome staining for fibrosis (B). TUNEL assay for apoptotic cardiac cells (C). Values are expressed as mean ± standard error of the mean. *P<0.05 vs control, #P<0.05 vs DOX. Scale bar: 50 μm.

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  Figure 4. Cryptotanshinone (CPT) alleviated myocardial mitochondrial membrane potential (MMP) decline and oxidative stress in rats treated with doxorubicin. JC-1 fluorescent mitochondrial depolarization of LV cardiomyocytes for evaluating MMP (A). Detection of intracellular ROS by observing the fluorescence intensity of DCF in the LV of the rats (B). The levels of SOD, MDA, CAT, and GSH-Px in LV tissues of the rats induced by doxorubicin (C). Values are expressed as mean ± standard error of the mean. *P<0.05 vs control, #P<0.05 vs DOX. CAT, Catalase; GSH-Px, glutathinone peroxidase; MDA, malonydialdehyde; SOD, superoxide dismutase. Scale bar: 50 μm.

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  Figure 5. Effects of the different regulated genes and pathways of cryptotanshinone (CPT) on the heart tissues of the DOX-treated rats using transcriptomic profiling. Changed genes in the heart tissues of the two groups displayed by a heat map (D1-3 = DOX group, DC1-3 = DOX + CPT group) (A). The volcano map exhibits the differentially regulated genes by comparison of the two groups (DOX and DOX + CPT groups); the gray scatter denotes the non-significantly differentially expressed genes; both red and green scatters denote the significantly differentially expressed genes; the x-axis represents log2 fold change, and the y-axis represents the –log10 P value (B). KEGG pathway classification; the horizontal axis represents the ratio (%) of the total number of genes (differentially expressed genes) annotated with each level 2 metabolic pathway and the genes (differentiated genes) annotated to the KEGG pathway, and the vertical axis represents the level 2 pathway term; the number on the right side of the column represents the annotation of the number of differentially expressed genes in the level 2 pathway term (C).

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  Figure 6. Top 20 of pathway enrichment statistics based on the differentially expressed gene in the rat hearts after cryptotanshinone (CPT) treatment. Scatter plot of KEGG pathway enrichment statistics (the total number of differentially expressed genes) (A). Scatter plot of KEGG pathway enrichment statistics (the number of differentially expressed genes, which were downregulated (B). p53 signaling pathway in a KEGG map (C). Enrichment score is the ratio of the number of differentially expressed genes to the number of all genes in this pathway term.

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  Figure 7. Cryptotanshinone (CPT) treatment adjusted p53 signaling pathway in the rat heart induced by doxorubicin. Representative blots of 14-3-3σ, p-JNK, JNK, PI3K p85, p-AKT, AKT, and GAPDH (A). Representative blots of Bax, Bak, Bim, PUMA, Bcl-2, Bcl-xl, and GAPDH (B). Representative blots of pro-caspase-3/cleaved caspase-3, pro-caspase-9/cleaved caspase-9, and GAPDH (C). Representative blots of Foxo1, p53, PCNA, and β-tubulin (D). GAPDH, β-tubulin and PCNA were used as internal reference.

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  Figure 8. Statistical analysis of the relative protein expression of 14-3-3σ, PI3K p85, p-JNK, p-AKT, cleaved caspase-9, cleaved caspase-3, Bax, Bak, Bim, PUMA, Bcl-xl, and Bcl-2. Semi-qualification of 14-3-3σ, PI3K p85, p-JNK and p-AKT (A). Semi-qualification of Bax, Bak, Bim, PUMA, Bcl-xl, and Bcl-2 (B). Semi-qualification of cleaved caspase-9 and cleaved caspase-3 (C). Both GAPDH and PCNA were used as internal reference. *P<0.05 vs control, #P<0.05 vs DOX.

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  Figure 9. Schematic diagram of the cardioprotective effects of cryptotanshinone (CPT) in a DOX-induced myocardial damage model.