Flavonoids from Hippophae rhamnoides Linn. Revert Doxorubicin-Induced Cardiotoxicity through Inhibition of Mitochondrial Dysfunction in H9c2 Cardiomyoblasts In Vitro

Figures

• 

  Figure 1. Chemical structures of three flavonoids (F-A, F-B, and F-C) from Hippophae rhamnoides L.

• 

  Figure 2. The effect of three flavonoids on Dox-induced cytotoxicity in H9c2 cells. Cell viability of H9c2 cells after exposure to different concentrations of F-A (A), F-B (B), and F-C (C) without Dox and the protective effect of F-A (D), F-B (E), and F-C (F) on Dox-induced cytotoxicity in H9c2 cells. Results are mean ± SEM from three independent experiments. ## p < 0.01 vs. control, ** p < 0.01 vs. Dox treated group. ns, not significant.

• 

  Figure 3. Flavonoids from Hippophae rhamnoides Linn affect the protein expression level of cleaved caspase-3 (A,B) and p-JNK (C,D) in Dox-induced H9c2 cells. Cells were pretreated with same concentrations of F-A, F-B, and F-C (10 µM) for 1 h, then exposed to Dox for 24 h. Relative folds of cleaved caspase-3, p-JNK. (B) The relative expression level of cleaved caspase-3 in Dox group significantly increased compared to the NC group (p < 0.01). There was no significant activation of cleaved caspase-3 in the F-A, F-B, and F-C group without Dox. But F-A, F-B, and F-C can significantly inhibit the Dox-induced activation of caspase-3 in comparison to the Dox model group (p < 0.01). (D) H9c2 cells treated with 2.5 μM Dox rapidly increased the relative expression level of phosphorylation of JNK (p < 0.01). Compared to the NC group, F-A, F-B, and F-C with the absence of Dox has no significant increase in the expression level of p-JNK. The expression levels of p-JNK compared to the Dox group was significantly suppressed by the administration of F-A, F-B, and F-C in the presence of 2.5 µM Dox (p < 0.01). Results are mean ± SEM from three independent experiments. ## p < 0.01 vs. control, * p < 0.05, **…

• 

  Figure 4. The effects of three flavonoids on p-ERK1/2 (A,B), p-P38 (C,D), and p-Akt (E,F) protein expression level of Dox-treated H9c2 cells. Cells were pretreated with same concentrations of F-A, F-B, and F-C (10 µM) for 1 h, then exposed to Dox for 24 h. Relative folds of p-Akt, p-ERK1/2, and p-P38. (B,D,F) The relative expression level of p-P38, p-ERK, and p-Akt in Dox group significantly increased compared to the NC group (p < 0.01). There was no significant activation of p-P38, p-ERK, and p-Akt in the F-A, F-B, and F-C group without Dox. However, F-A, F-B, and F-C had no significantly protective effect to reduce protein phosphorylation. Results are mean ± SEM from three independent experiments. ## p < 0.01 vs. control, ** p < 0.01 vs. Dox group. ns, not significant.

• 

  Figure 5. The effects of three flavonoids on intracellular ROS level (A) and ATP content (B) under Dox treatment in H9c2 cells. Cells were pretreated with same concentrations of F-A, F-B, and F-C (10 µM) for 1 h, then exposed to Dox for 24 h. CCCP (carbonyl cyanide 3-chlorophenylhydrazone) as a negative contrast group. (A) The DCF fluorescence intensity in Dox group significantly increased compared to the NC group (p < 0.01). There was no marked change of DCF fluorescence intensity in the F-A, F-B, and F-C group without Dox. But F-A, F-B, and F-C can significantly inhibit the Dox-induced elevation of ROS level in comparison to the Dox model group (p < 0.01). (B) H9c2 cells treated with 2.5 μM Dox rapidly reduced the content of ATP. Compared to the NC group, F-A, F-B, and F-C with the absence of Dox has no significant decrease (p > 0.05) the content of ATP. The content of ATP compared to the Dox group was significantly increased by the administration of F-A, F-B, and F-C in the presence of 2.5 µM Dox. Results are mean ± SEM from three independent experiments. ## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. Dox group. ns, not significant.

• 

  Figure 6. The effects of three flavonoids on mitochondrial fusion proteins Mfn-1 (A,B) and Mfn-2 (C,D) during Dox-induced cardiotoxicity. Cells were pretreated with same concentrations of F-A, F-B, and F-C (10 µM) for 1 h, then exposed to Dox for 24 h. Relative folds of Mfn1 (C) and Mfn2 (D). (B,D) The relative expression level of Mfn1and Mfn2 in Dox group significantly decreased compared to the NC group (p < 0.01). There was no significant change of Mfn1and Mfn2 in the F-A, F-B, and F-C group without Dox. But F-A, F-B, and F-C can significantly enhance the Dox-induced reduction of Mfn1 and Mfn2 in comparison to the Dox model group (p < 0.01). Results are mean ± SEM from three independent experiments. ## p < 0.01 vs. control, ** p < 0.01 vs. Dox group. ns, not significant.

• 

  Figure 7. The effects of three flavonoids on mitochondrial ultrastructure during Dox-induced cardiotoxicity by SEM (5000×). Cells were pretreated with same concentrations of F-A, F-B, and F-C (10 µM) for 1 h, then exposed to Dox for 24 h. (A) NC Group; (B) 2.5 μM Dox Group; (C) F-A Group; (D) F-B Group; (E) F-C Group; (F) F-A + 2.5 μM Dox Group; (G) F-B + 2.5 μM Dox Group; and (H) F-B + 2.5 μM Dox Group.

• 

  Figure 8. The effects of three flavonoids on Sab, p-Src, and p-IRE1α protein expression level of Dox-treated H9c2 cells. Cells were pretreated with same concentrations of F-A, F-B, and F-C (10 µM) for 1 h, then exposed to Dox for 24 h. Relative folds of Sab (A,B), p-Src (A,D), and p-IRE1α (A,C). (B) The relative expression level of p-IREα in Dox group significantly increased compared to the NC group (p < 0.01). There was no significant activation of p-IREα in the F-A, F-B, and F-C group without Dox. F-A, F-B, and F-C had no significantly protective effect to reduce Dox-induced p-IREα protein phosphorylation. (C) The relative expression level of Sab in Dox group significantly decreased compared to the NC group (p < 0.01). There was no significant change of Sab in the F-A, F-B, and F-C group without Dox. But F-A and F-C can significantly enhance the Dox-induced reduction of Sab in comparison to the Dox model group (p < 0.05). (D) The relative expression level of p-Src in Dox group significantly decreased compared to the NC group (p < 0.01). There was no significant change of p-Src in the F-A, F-B, and F-C group without Dox. But F-B and F-C can significantly enhance the Dox-induced reduction…

• 

  Figure 9. Molecular docking to predict the binding of three flavonoids to JNK-1 via AutoDock Vina, and PyMOL. Demonstration of the predicted binding conformation and corresponding interaction amino acid residues of F-A (A), F-B (B), and F-C (C) docking into JNK-1.

• 

  Figure 10. Possible signaling mechanism pathways.