Ginsenoside Rh2 mitigates doxorubicin‐induced cardiotoxicity by inhibiting apoptotic and inflammatory damage and weakening pathological remodelling in breast cancer‐bearing mice

Figures

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  FIGURE 1. Ginsenoside Rh2 synergistically sensitize the chemotherapeutic toxicity of Doxorubicin in breast cancer xenograft. (A) Schematic for experimental regime. (B) Tumour weight. *p < .05; **p < .01; ***p < .001 versus control; # p < .05; ## p < .01; ### p < .001 versus Dox

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  FIGURE 2. Histological examination of the heart. (A) The normal cardiac muscle fibres are longitudinally settled with oval nuclear and acidophilic striated sarcoplasm (red arrow). Flat fibroblasts constitute the interstitial space (yellow arrow). (B) Disorganization and separation of cardiac fibres with heightened interstitial spaces (yellow arrow). (C) Extravasation of red blood cells leading to interstitial haemorrhage (white arrows). (D) Cardiac damage was characterized by myofiber vacuolation (black arrows). (E) Focal myocardial necrosis into areas of preserved myocardium (red arrows). (F) Mononuclear cellular infiltration. Scale bars = 100 μm. (G) ELISA assays of CK‐MB and Troponin T release in cardiomyocytes. *p < .05; **p < .01; ***p < .001 versus control; # p < .05; ## p < .01; ### p < .001 versus Dox

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  FIGURE 3. The protective effect of Rh2 on the heart exhibited distinct functional properties and transcriptome profiles. (A) Heatmap of DEGs in six groups: Control, Rh2‐20 mg/kg, Rh2‐30 mg/kg, Rh2‐20 mg/kg + Dox, Rh2‐30 mg/kg + Dox, and Dox‐2 mg/kg. Gene expression is represented as Z‐score for the normalized value across rows. (B) The volcano plots of DEGs in the indicated groups

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  FIGURE 4. Functional and pathway enrichment analysis of DEGs. (A) Venn diagram of genes common or unique in Dox, Rh2‐20 mg/kg + Dox and Rh2‐30 mg/kg + Dox, as compared to Control group. (B) Network of the enrichment pathways for the common DEGs. (C) Network of the enrichment pathways for the unique DEGs

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  FIGURE 5. Rh2 alleviated cardiac apoptosis. (A) Extracts from mice heart treated with or without ginsenoside Rh2; Doxorubicin‐challenged mice, treated or not with ginsenoside Rh2, were assayed by western blotting for the indicated apoptosis marker proteins caspase 3, 7, and 9. GAPDH was used as a loading control. (B) Cleaved caspase 3 staining on the doxorubicin‐evoked heart in mice (positive staining mainly located at left ventricle and indicated with red arrows). Scale bars = 100 μm. (C) Heart tissues from mice treated with doxorubicin combined with or without ginsenoside Rh2 or vehicle were analysed by apoptosis antibody arrays. Signals from vehicle‐treated mice were used as a baseline. Colour intensities represent log2‐fold changes from baseline. Signals higher than the baseline are shown in red; signals lower than the baseline are shown in green. Shown is the average of three independent experiments. *p < .05 versus control; # p < .05 versus Dox

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  FIGURE 6. Rh2 suppressed cardiac inflammation. (A) Heart lysates were analysed by ELISA for TNF‐α, IL‐6, and IL‐1β. Quantifications were shown. (B) Fold changes of mRNA levels of adverse remodelling markers. Levels of each protein in the control group were arbitrarily set to zero. Data shown represent a log2‐fold change in expression relative to control. *p < .05 versus control; # p < .05 versus Dox

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  FIGURE 7. Rh2 Attenuated cardiac fibrosis. (A) Heart sections were stained to identify fibrosis by Masson trichrome and Sirius Red methods. Shown are representative images of interstitial and perivascular fibrosis from doxorubicin‐treated mouse hearts. Scale bars = 100 μm. (B) IHC staining of α‐SMA in doxorubicin‐administrated mouse myocardium. Red arrows indicate replacement fibrosis. Scale bars = 100 μm. (C) Downstream effectors smad2 and smad3 protein levels in heart lysates were analysed using western blot. GAPDH was used as a loading control. (D) Human cardiac fibroblasts were exposed to ginsenoside Rh2 at 2.5, 5, 10 μg/ml for 12 h, then subjected to 10 ng/ml of TGF‐β1 for extra 24 h. Protein extracts were analysed with the indicated antibodies. GAPDH was used as a loading control. (E) HUVECs were treated with 100 nM doxorubicin for 7 days, followed by 2.5, 5, 10 μg/ml of Rh2 exposure for extra 7 days. Cell extracts were incubated with endothelial markers CD31 and myofibroblast markers α‐SMA and vimentin. Beta Actin was used as a loading control. (F) Detection of CD206 expression in the vehicle and doxorubicin‐treated heart sections by immunohistochemistry analysis. Scale bars = 100…

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  FIGURE 8. Rh2 specifically induced senescence in cardiac fibroblasts differentiated myofibroblasts. (A) Heart tissues were analysed with p21 at both mRNA and protein levels. Quantifications were shown. GAPDH was used as a control. (B) Representative sections of hearts stained with p21 and p16 and quantifications. Positive cells were indicated by yellow arrows. Scale bars = 100 μm. (C) HCF and HUVEC cells were treated with 100 nM doxorubicin for 7 days, followed by treatment with Rh2 or DMSO for 7 days. Cells were then fixed, washed, and probed with the p21 antibody. The expression level of p21 was determined by fluorescence microscopy (one representative of three independent experiments is shown). Scale bars = 100 μm. (D) Protein levels of p16 and phosphorylated Rb were analysed by western blot. GAPDH was used as a loading control. (E) p21 or p53 silenced Dox‐treated HCF cells were analysed for α‐SMA by western blot. Cells were treated with or without 10 μg/ml of Rh2 for 48 h and then transfected with siRNAs for extra 48 h; quantifications of siRNA efficiency of p21 and p53 were shown. GAPDH was used as a loading control. *p < .05 versus control; # p < .05 versus Dox

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  FIGURE 9. Schematic representation of protection by Rh2 against Dox‐mediated cardiotoxicity in breast tumour‐bearing mice