Protective effect of urotensin II receptor antagonist urantide and exercise training on doxorubicin-induced cardiotoxicity

Figures

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  Figure 1. The study flow diagram for animal experiments. (A) The treatment groups were injected Urantide at 10, 20, and 30 μg/kg/day for 7 days consecutively. (B) The treatment groups were injected Urantide and (or) p38 MAPK-IN-1. (C) The treatment groups were injected Urantide and (or) exercise.

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  Figure 2. Urantide improves DOX-induced cardiac damage in vivo. (A) Representative images of global appearance of the heart in different groups. Results of LVEF (B) and LVFS (C) in the five groups (n = 6 per group). Enzyme‐linked immunosorbent assay was used on serum from rats to determine the expression of LDH (D) and CK-MB (E). (F) Average body weight change in 5 wk in rats from five groups (n = 6 per group). *P < 0.05, vs. the control; #P < 0.05 vs. the DOX group.

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  Figure 3. Urantide improves DOX-induced cardiac damage, myocardial fibrosis and cardiac myocyte hypertrophy in vivo. (A) HE staining of the heart tissue sections from Wistar rats treated. Bar = 200 μm. (B) Masson staining showing collagen content in the three Urantide treatment groups was decreased. (C) WGA staining of left ventricular tissue from rats for detect cardiac myocyte hypertrophy. Bar = 400 μm. (D) Transmission electron microscopy showing betterment of mitochondrial ultrastructure in Urantide group compared to DOX‐treated rats. Bar = 0.5 μm. (n = 6).

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  Figure 4. Urantide attenuates DOX-induced apoptosis in vivo. (A) Staining cells with red indicated TUNEL‐positive cells, DAPI staining (blue) indicated nucleus. Bar = 100 μm. Percentages of TUNEL‐positive cells of total cells were shown. (B) Protein expression of Bcl‐2 and Bax in the different treatment groups. (n = 6). *P < 0.05, vs. the control; #P < 0.05 vs. the DOX group.

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  Figure 5. The attenuation of DOX-induced myocardial toxicity by Urantide was dependent on the downregulation of p38 in the MAPK pathway in vivo. (A) The expression level of the p38 MAPK pathway was downregulated in Urantide-treated. (B) CK-MB and LDH of the inhibitor group was decreased compared to Urantide‐treated rats. (C) Myocardial fiber appearance was more intense of the inhibitor group in HE staining. (D) Masson staining showed that the collagen content in the inhibitor group was significantly lower. (E) Transmission electron microscopy showing the beneficial of inhibitor. (F) Representative images of TUNEL staining of the heart tissues. (G) The levels of anti-apoptotic proteins were increased by the inhibitor. (n = 6). *P < 0.05, vs. the control; #P < 0.05 vs. the DOX group; $P < 0.05 vs. the Urantide group.

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  Figure 6. Urantide attenuates DOX-induced apoptosis in vitro. (A) Effects of Urantide (10 nmol/L, 100 nmol/L, 1000 nmol/L for 24 h) on DOX (5 μmol/L for 24 h)‐induced dissipation of mitochondrial membrane potential measured in H9C2 cells loaded with JC‐1 using fluorescence microscopy. (B) Representative images of TUNEL staining of H9C2 cells. (C) The relative expression of Bcl‐2 and Bax in H9C2 cells with or without Urantide treatment under DOX stimulation. *P < 0.05, vs. the control; #P < 0.05 vs. the DOX.

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  Figure 7. The protective effect of Urantide on DOX-induced H9C2 cells in vitro depended on the downregulation of the p38 MAPK pathway. (A) The expression level of the p38 MAPK pathway was downregulated in H9C2 cells with Urantide-treated compared to DOX group. (B) The expression level of the p38 MAPK pathway of the inhibitor group and Urantide group was decreased compared to DOX‐treated. (C) Determination of mitochondrial membrane potential by JC-1 method, the inhibitor group resulted in orange fluorescence. (D) Staining cells with red indicated TUNEL‐positive cells were obviously decreased in the inhibitor group and Urantide group. (E) The relative expression of Bcl‐2 and Bax in five groups. *P < 0.05, vs. the control; #P < 0.05 vs. the DOX.

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  Figure 8. The protective effect of Urantide on DOX-induced primary cardiomyocytes depend on the down-regulation of the p38 MAPK pathway. (A,C) The expression level of the p38 MAPK pathway was downregulated in primary cardiomyocytes with Urantide or inhibitor-treated compared to DOX group. (B,D) The relative expression of Bcl‐2 and Bax in five groups. *P < 0.05, vs. the control; #P < 0.05 vs. the DOX.

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  Figure 9. Urantide and (or) exercise alleviate DOX‐induced cardiac damage and apoptosis. (A) Myocardial fibers were arranged more neatly in the three treatment groups in HE staining. (B) Masson staining showed that the collagen content in the Urantide and (or) exercise group were significantly lower. Enzyme‐linked immunosorbent assay was used on serum from rats to determine the expression of LDH (C) and CK-MB (D). (E) DOX increased the Bax/Bcl-2 ratio, which was decreased by Urantide and (or) exercise treatments. (F) TUNEL-positive cells in the treatment group was significantly lower than in the DOX group. (G) The expression level of the p38 MAPK pathway was significantly downregulated by Urantide and (or) exercise. (H) The CCK8 was performed in breast cancer cells in vitro. *P < 0.05, vs. the control; #P < 0.05 vs. the DOX. NSP > 0.05 vs. the DOX.

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  Figure 10. The expression level of the p38 MAPK pathway was upregulated in rats exposed to DOX, while Urantide and exercise were downregulated p38 MAPK and attenuated cardiac damage.