Caffeic Acid Phenethyl Ester Protects Against Doxorubicin-Induced Cardiotoxicity via Inhibiting the ROS-MLKL-Mediated Cross-Talk Between Oxidative Stress and Necroptosis

Figures

• 

  Figure 1. The chemical formula of CAPE.

• 

  Figure 2. CAPE alleviated the DOX-induced myocardial injury in vivo. (A) Schematic diagram of animal experiment process. (B) ECG was used to evaluate the cardiac function of the mice in each group. (C) H&E staining was used to detect the cardiac toxicity. Scale bar: 100 μm. (D) The ELISA kits were used to detect the levels of cTn-I, CK-MB and LDH in serum. Values are represented as the x¯ ± SD (n = 10). ### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DOX-treatment group.

• 

  Figure 3. CAPE alleviated the DOX-induced myocardial injury in vitro. The (A) H9c2 and (B) AC16 cells were incubated with CAPE (0–80 µM) for 24 h. The cell viability was examined by MTT assays. (C) H9c2 cells and (D) AC16 cells were pre-treated with CAPE (0–80 µM) for 6 h, followed by 18 h of incubation with DOX (2.5 µM). The cell viability was assessed by MTT assays. Values are represented as the x¯ ± SD. ### p < 0.001 vs. control; * p < 0.05, *** p < 0.001 vs. DOX treatment group.

• 

  Figure 4. CAPE attenuated the DOX-induced oxidative stress. The H9c2 and AC16 cells were pre-treated with CAPE or NAC for 6 h, followed by 18 h of incubation with DOX (2.5 μM). The ROS levels of H9c2 (A) and AC16 (B) were assessed and quantified by flow cytometry after the DCFH-DA staining. A GSH kit was used to assess the GSH levels of the H9c2 (C) and AC16 (D). The Mito-ROS levels of the H9c2 (E) and AC16 (F) were assessed and quantified by flow cytometry after the MitoSOX Red staining. The mitochondrial damage of the H9c2 (G) and AC16 (H) were shown by TEM. Scale bar: 1 μm. Red arrows indicate damaged mitochondria. The OCRs of the H9c2 (I) and AC16 (J) were examined by an O2K cell energy metabolism analysis system. The basal respiration, ATP production, maximal respiration and spare respiratory capacity of the H9c2 (K–N) and AC16 (O–R) were quantified. Values are represented as the x¯ ± SD. ## p < 0.01, ### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DOX treatment group.

• 

  Figure 5. CAPE inhibited the DOX-induced necroptosis by inhibiting the RIPK1/RIPK3/MLKL pathway. The H9c2 (A) and AC16 (B) cells were pre-treated with various death inhibitors, namely, DSF (10 μM), Z-VAD (10 μM), DFO (10 μM), Fer-1 (10 μM), CQ (10 μM), 3-MA (10 μM), NAC (200 μM) and Nec-1 (100 μM) for 6 h, followed by a treatment with DOX for a total of 24 h. The cell viability was examined by MTT assays. The H9c2 (C,D) and AC16 (E,F) cells were pre-treated with CAPE or Nec-1 for 6 h, followed by 18 h of incubation with DOX (2.5 μM). The protein levels related to the RIPK1/RIPK3/MLKL pathway were examined by Western blotting. (G) The expression levels of RIPK1, RIPK3 and p-MLKL in the mouse hearts were examined by IHC. Scale bar: 100 μm. Values are represented as the x¯ ± SD. ### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DOX treatment group.

• 

  Figure 6. The cross-talk between oxidative stress and necroptosis played a vital role in the DOX-induced cardiotoxicity. The H9c2 and AC16 cells were pre-treated with NAC for 6 h, followed by 18 h of incubation with DOX (2.5 μM). The protein levels of the H9c2 (A) and AC16 (B) cells related to the RIPK1/RIPK3/MLKL pathway were examined by Western blotting. The H9c2 and AC16 cells were pre-treated with Nec-1 for 6 h, followed by 18 h of incubation with DOX (2.5 μM). (C,D) The ROS level was assessed and quantified by flow cytometry after the DCFH-DA staining. (E,F) The GSH levels were assessed. (G,H) The Mito-ROS level was assessed and quantified by flow cytometry after MitoSOX Red staining. The mitochondrial damage of H9c2 (I) and AC16 (J) cells were shown by TEM. Scale bar: 1 μm. (K,L) OCR was examined by an O2K cell energy metabolism analysis system. Basal respiration, ATP production, maximal respiration and spare respiratory capacity of H9c2 (M–P) and AC16 (Q–T) cells were quantified. Values are represented as the x¯ ± SD. ## p < 0.01, ### p < 0.001 vs. control; *** p < 0.001 vs. DOX treatment group.

• 

  Figure 7. CAPE regulated the cross-talk mediated by the ROS-MLKL signal pathway. The H9c2 and AC16 cells were pre-treated with CAPE for 6 h, followed by 6 h of incubation with H2O2 (400 μM). (A,B) The ROS level was assessed and quantified by flow cytometry after the DCFH-DA staining. (C,D) The protein levels related to the RIPK1/RIPK3/MLKL pathway were examined by Western blotting. The H9c2 and AC16 cells were pre-treated with CAPE for 6 h, followed by 48 h of interference with H2O2. The values are represented as the x¯ ± SD. ### p < 0.001 vs. control; *** p < 0.001 vs. H2O2 treatment group. (E,F) The protein level of MLKL was examined by Western blotting. (G,H) The ROS level was assessed and quantified by flow cytometry after DCFH-DA staining. (I,J) The GSH level was assessed. (K,L) The Mito-ROS level was assessed and quantified by flow cytometry after MitoSOX Red staining. (M,N) The OCR was examined by O2K cell energy metabolism analysis system. The basal respiration, ATP production, maximal respiration and spare respiratory capacity of the H9c2 (O–R) and AC16 (S–V) cells were quantified. Values are represented as the x¯ ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. NC; * p < 0.05, **…