Kaempferol protects against doxorubicin-induced myocardial damage by inhibiting mitochondrial ROS-dependent ferroptosis

Figures

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  Figure 1.. KP alleviated DOX-induced myocardial damage (A) Cell viability assay of H9c2 cardiomyocytes when treated with 0, 10, 20, 40, 80, 160, 320 µM of KP for 24-h, n = 3. (B) H9c2 cells were cultured with 2 µM DOX in combination with KP at various concentrations (0, 10, 20, 40, 80, 160, 320 µM) for 24-h. Cell viability was measured by MTT method, n = 3. (C–F) Effect of KP administration on DOX-induced myocardial injury and oxidative stress damage markers LDH, SOD, GSH, and GSH-px activity in H9c2 cardiomyocytes, n = 3. Data were expressed as mean ± SEM of three independent replicates; #P < 0.05, ##P < 0.01, ###P <0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. DOX group.

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  Figure 2.. KP protected DOX-induced myocardial damage by counteracting ferroptosis. (A,B) The level of Fe2+ in H9c2 cardiomyocytes after 24-h treatment of DOX (2μM) in combination with Fer-1 (1μM) or KP after 24-h treatment of DOX in combination with Fer-1, n = 3. (C,D) H9c2 cells were analyzed by flow cytometry with DCFH-DA after 24-h treatment of DOX (2μM) in combination with Fer-1, n = 2. (E–H) Representative protein blotting bands of GPX4, SLC7A11 and ACSL4 in H9c2 cells with GAPDH as internal reference after 24-h treatment of DOX in combination with Fer-1, n = 3. Data were expressed as mean ± SEM; #P < 0.05, ###P <0.001 vs. control group; *P < 0.05, ***P < 0.001 vs. DOX group.

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  Figure 3.. KP rescued disruption of iron homeostasis and lipid peroxidation induced by Erastin. (A) H9c2 cells were analyzed by flow cytometry with DCFH-DA after 24-h treatment of Erastin (32μM) in combination with Fer-1 or KP, n = 2. (B) Images of MitoSOX stained H9c2 cells, and the histogram showed MitoSOX fluorescence. Scale bar=50 μm, n = 3. (C) Images of C11-BODIPY 581/591 stained H9c2 cells. Histogram presented fluorescence intensity of oxidized BODIPY (Oxidized/Reduced). Histogram presented fluorescence intensity of oxidized BODIPY. Scale bar=50 μm, n = 3. (D–F) Effect of KP and Fer-1 administration on Erastin-induced oxidative stress damage markers GSH, GSH-px activity and the level of Fe2+, n = 3. Data were expressed as mean ± SEM; ###P <0.001 vs. control group; **P < 0.01, ***P < 0.001 vs. DOX group.

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  Figure 4.. KP attenuated mitochondrial damage in Erastin-induced ferroptosis. (A) H9c2 cells were stained with JC-1 and analyzed by flow cytometry after 24-h treatment of Erastin in combination with Fer-1 or KP. Two independent experiments were performed, n = 2. (B) Images of TMRE stained H9c2 cells, and the histogram showed TMRE fluorescence. Scale bar=50 μm, n = 3. (C) Images of Mito-Ferro Green staining stained H9c2 cells, and the histogram showed Mito-Ferro Green fluorescence, detecting mitochondrial Fe2+ level. Scale bar=50 μm, n = 3. Data were expressed as mean ± SEM; ###P <0.001 vs. control group; **P < 0.01, ***P < 0.001 vs. DOX group.

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  Figure 5.. KP mitigated DOX-induced cardiomyocyte ferroptosis via suppressing mitochondrial ROS-dependent lipid peroxidation. (A,B) Images and level of DCFH-DA. and the histogram showed DCFH-DA fluorescence. Scale bar=100μm, n = 2. (C) Images of MitoSOX stained H9c2 cells, and the histogram showed MitoSOX fluorescence. Scale bar=50 μm, n = 3. (D) Images of C11-BODIPY 581/591 stained H9c2 cells. Histogram presented fluorescence intensity of oxidized BODIPY (Oxidized/Reduced) Scale bar=50 μm, n = 3. Data were expressed as mean ± SEM of three independent replicates; ##P < 0.01, ###P<0.001 vs. control group; **P < 0.01, ***P < 0.001 vs. DOX group.

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  Figure 6.. KP reversed DOX-induced mitochondrial damage and ferroptosis through Fe²+ homeostasis regulation. (A) Transmission electron microscope images of H9c2 cells. Enlarged inserts at the down side of each image showed typical morphology of mitochondria. Each pair of images had a scale indicated in the lower right corner of each image, while the lower left corner showed the magnification. The lower image was an enlarged version of the area within the yellow box. The red arrows represented the mitochondrial membrane, and the blue arrows indicated the mitochondrial cristae, n = 3. (B) H9c2 cells were stained with JC-1 and analyzed by flow cytometry. Two independent experiments were performed, n = 2. (C) Images of TMRE stained H9c2 cells, and the histogram showed TMRE fluorescence Scale bar=50 μm, n = 3. (D) Images of Mito-Ferro Green staining stained H9c2 cells, and the histogram showed Mito-Ferro Green fluorescence, detecting mitochondrial Fe2+ level. Scale bar=50 μm, n = 3.Data were expressed as mean ± SEM of three independent replicates; ##P < 0.01, ###P < 0.001 vs. control group; **P < 0.01, ***P < 0.001vs. DOX group.

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  Figure 7.. KP restored mitochondrial function and attenuated DOX-induced ferroptosis via ROS scavenging and Fe²+ homeostasis regulation. (A) Images of TMRE stained H9c2 cells after 24-h treatment of DOX in combination with or without mitoTEMPO (8μM) or KP, Scale bar=50 μm, n = 3. (B) Images of MitoSOX stained H9c2 cells, and the histogram showed MitoSOX fluorescence. Scale bar=50 μm, n = 3. (C) Images of C11-BODIPY 581/591 stained H9c2 cells. Histogram presented fluorescence intensity of oxidized BODIPY (Oxidized/Reduced) as referred in, Scale bar=50 μm, n = 3. (D) H9c2 cells were analyzed by flow cytometry with DCFH-DA after 24-h treatment of DOX in combination with mitoTEMPO, PEG-SOD or KP. (E) Effect of mitoTEMPO, PEG-SOD or KP administration on DOX-induced oxidative stress damage markers SOD activity, n = 3. Data were expressed as mean ± SEM of three independent replicates; ###P <0.001 vs. control group; ***P < 0.001 vs. DOX group.

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  Figure 8.. KP attenuated DOX cardiotoxicity by activating NRF2/SLC7A11/GPX4 pathway to suppress mitochondrial ROS-driven ferroptosis. (A,B) The expression levels of NRF2, ACSL4, SLC7A11 and GPX 4 in H9c2 cells with GAPDH as internal reference, n = 3. (B) The expression levels of NRF2, ACSL4, SLC7A11, FTL, FTH1 and GPX 4 in H9c2 cells with GAPDH as internal reference, n = 3. Data were expressed as mean ± SEM of three independent replicates; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. DOX group.

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  Figure 9.. KP attenuated DOX cardiotoxicity by activating NRF2/SLC7A11/GPX4 pathway to suppress mitochondrial ROS-driven ferroptosis. (A–H) The expression levels of NRF2, SLC7A11 and GPX 4 in H9c2 cells with GAPDH as internal reference, n = 3. (I) Images of TMRE stained H9c2 cells after 24-h treatment of DOX in combination with or without TBHQ (2.5μM) or KP, Scale bar=50 μm, n=3. (J) Representative image of the effect of KP on NRF2 expression in DOX-stimulated cells. Scale bar = 50 μm, n=3. Data were expressed as mean ± SEM of three independent replicates; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. DOX group.

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  Figure 10.. KP protected human AC16 cardiomyocytes from DOX-Induced ferroptosis via the NRF2/SLC7A11/GPX4 pathway (A) Cell viability assay of AC16 cardiomyocytes when treated with 0, 10, 20, 40, 80, 160, 320 µM of KP for 24-h, n=3. (B) AC16 cells were cultured with 2 µM DOX in combination with KP at various concentrations (0, 10, 20, 40, 80, 160, 320 µM) for 24-h. Cell viability was measured by MTT method, n=3. (C) The expression levels of NRF2, SLC7A11 and GPX 4 in H9c2 cells with GAPDH as internal reference, n=3. Data were expressed as mean ± SEM of three independent replicates; ##P < 0.01, ###P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. DOX group.