Quercetin Activates NRF2/SLC7A11/GPX4 Signaling to Restore Mitochondrial Function Inhibit Ferroptosis Thereby Alleviating Doxorubicin-Induced Cardiotoxicity

Figure 1. QUE exerts protective effects against DOX-induced myocardial injury in vivo. (A) Schematic diagram of the animal experimental protocol. (B–F) Echocardiographic parameters of left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), ejection fraction (EF), and fractional shortening (FS) in SD rats (n = 8 per group). (G,H) Masson staining for myocardial fibrosis (×400, scale bar = 50 μm) and quantitative analysis of the fibrotic area. (I) Hematoxylin–eosin (H&E) staining of myocardial histology (×400, scale bar = 50 μm). (J,K) Serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB). (L,M) Myocardial glutathione (GSH) content and glutathione peroxidase (GSH-Px) activity. (N) Transmission electron microscopy (TEM) of mitochondrial morphology in rat myocardium (scale bar = 1 μm; insets, scale bar = 200 nm). Data are expressed as the mean ± standard error from three independent experiments. Statistical significance was indicated as follows: # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DOX model group.

Figure 2. QUE alleviates DOX-induced myocardial injury, upregulates GSH and GSH-Px, and preserves mitochondrial ultrastructure in vitro. (A,B) Cell viability of H9c2 cells treated with QUE (40–160 μM) and/or DOX (2 μM) assessed by MTT assay. (C) Lactate dehydrogenase (LDH) release in H9c2 cells. (D) Hoechst 33258 staining for nuclear morphology (scale bar = 50 μm). (E) Transmission electron microscopy (TEM) of mitochondrial morphology in H9c2 cells (scale bar = 1 μm; insets, scale bar = 200 nm). (F,G) Intracellular GSH content and GSH-Px activity in H9c2 cells. Data are presented as mean ± SEM from three independent experiments. Statistical significance was indicated as follows: # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DOX model group.

Figure 3. QUE alleviates DOX-evoked oxidative stress and mitochondrial impairment in H9c2 cardiomyocytes. (A,B) Intracellular reactive oxygen species (ROS) levels detected by DCFH-DA staining and flow cytometry. (C,D) Representative DCFH-DA fluorescence images and quantitative intensity (scale bar = 100 μm). (E,F) Mitochondrial membrane potential (MMP) measured by JC-1 staining and flow cytometry. (G,H) TMRE staining for MMP (scale bar = 50 μm) and quantitative fluorescence intensity. (I) Superoxide dismutase (SOD) activity in H9c2 cells. (J,K) Mitochondrial ROS detected by MitoSOX staining (scale bar = 50 μm) and quantitative intensity. Data are presented as mean ± SEM from three independent experiments. Comparisons with the control group are denoted as # p < 0.05, ## p < 0.01, ### p < 0.001, while comparisons with the doxorubicin (DOX) model group are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4. QUE suppressed ferroptosis via the NRF2/SLC7A11/GPX4 pathway. (A) AC16 cells were analyzed by flow cytometry with DCFH-DA after 24 h of treatment. (B) Images of JC-1 stained AC16 cells, and the histogram showed JC-1 fluorescence. Scale bar = 50 μm. (C) The expression levels of NRF2, SLC7A11 and GPX 4 in AC16 cells, with GAPDH as internal reference. 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 < 0.001vs. DOX group. Original Western blot images can be found in https://zenodo.org/records/21643385 (accessed on 29 July 2026).

Figure 5. QUE activates the NRF2/SLC7A11/GPX4 antioxidant pathway and regulates iron metabolism-related proteins in DOX-injured rats and H9c2 cardiomyocytes. (A–F) Western blot detection of NRF2, SLC7A11, GPX4, FTL and FTH1 protein expression in rat myocardial tissues from different groups. Corresponding bar graphs show the relative protein expression levels. (G,H) Quantitative detection of ferrous ion (Fe2+) and malondialdehyde (MDA) contents in rat myocardial tissue. (I) Quantitative determination of intracellular ferrous ion (Fe2+) levels in H9c2 cardiomyocytes. (J–O) Western blot analysis of NRF2, SLC7A11, GPX4, FTL and FTH1 protein profiles in H9c2 cardiomyocytes among different treatment groups. Relevant bar graphs present the relative expression of these proteins. Data are expressed as the mean ± standard error from three independent experiments. Comparisons with the control group are denoted as # p < 0.05, ## p < 0.01, ### p < 0.001, while comparisons with the DOX model group are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. Original Western blot images can be found in https://zenodo.org/records/21643385 (accessed on 29 July 2026).

Figure 6. QUE inhibits ferroptosis in H9c2 cardiomyocytes induced by erastin or DOX via the ACSL4/SLC7A11/GPX4 signaling pathway. (A,B) Mito-Ferro Green staining was used to observe mitochondrial ferrous ion (Fe2+) levels in H9c2 cells, with representative images and quantitative results provided. Scale bar: 50 μm. (C,D) C11-BODIPY 581/591 staining was applied to assess lipid peroxidation in H9c2 cells. Representative fluorescent images and the corresponding ratio of oxidized to reduced BODIPY fluorescence intensity are shown. Scale bar: 50 μm. (E–H) A Western blot assay was performed to detect ACSL4, SLC7A11 and GPX4 protein expression in H9c2 cells from different treatment groups, with relative expression quantified by bar graphs. (I–L) The expression levels of NRF2, SLC7A11 and GPX4 in H9c2 cells of each group were examined via Western blot, and relative protein abundance was statistically analyzed. Data are expressed as the mean ± standard error from three independent experiments. Comparisons with the control group are denoted as # p < 0.05, ## p < 0.01, ### p < 0.001, while comparisons versus the DOX model group or erastin group are marked as * p < 0.05, ** p < 0.01, *** p < 0.001.…

Figure 7. QUE alleviates erastin- or DOX-induced ferroptosis in H9c2 cardiomyocytes. (A,B) Mito-Ferro Green fluorescence staining was performed to detect mitochondrial Fe2+ levels in H9c2 cells, with representative images and quantitative analysis provided. Scale bar: 50 μm. (C,D) C11-BODIPY 581/591 fluorescence staining was used to evaluate lipid peroxidation levels in H9c2 cells; representative images and corresponding quantitative data are shown. The bar graph depicts the ratio of oxidized to reduced BODIPY fluorescence intensity. Scale bar = 50 μm. (E,F) Mitochondrial reactive oxygen species (ROS) levels in H9c2 cardiomyocytes were detected and quantitatively analyzed by flow cytometry. (G) Quantitative analysis was conducted to assess QUE’s protective effect on the erastin-induced reduction of Fe2+ levels. (H) The effect of different treatment groups on superoxide dismutase (SOD) levels—a key indicator of oxidative stress—in H9c2 cardiomyocytes was examined. Data are expressed as the mean ± standard error from three independent experiments. Comparisons with the control group are denoted as # p < 0.05, ## p < 0.01, ### p < 0.001, while comparisons versus the DOX model group or erastin…

Figure 8. Inhibitory effects of QUE on Erastin- and DOX-induced ferroptosis and mitochondrial damage in H9c2 cardiomyocytes. (A,B) After 24 h of drug intervention, H9c2 cells were stained with DCFH-DA and examined by flow cytometry. (C,D) H9c2 cells were stained with TMRE, with representative fluorescent images and fluorescence intensity quantification listed. Scale bar: 50 μm. (E,F) JC-1 staining combined with flow cytometry was used for detection and quantitative analysis. All assays were performed with two independent repeats. (G,H) Mito-Ferro Green staining was used to monitor mitochondrial Fe2+ changes in H9c2 cells, along with representative images and statistical results. Scale bar: 50 μm. (I,J) C11-BODIPY 581/591 staining was adopted to evaluate cellular lipid peroxidation. Histograms were used to calculate the fluorescence intensity ratio of oxidized to reduced BODIPY. Scale bar: 50 μm. Data are expressed as the mean ± standard error from three independent experiments. Comparisons with the control group are denoted as # p < 0.05, ## p < 0.01, ### p < 0.001, while comparisons versus the DOX model group or erastin group are marked as * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 9. QUE mitigates DOX-induced ferroptosis and mitochondrial dysfunction in H9c2 cardiomyocytes through the NRF2/SLC7A11/GPX4 pathway. (A–D) Western blot detected NRF2, SLC7A11, and GPX4 protein expression in H9c2 cells from various groups (DOX, TBHQ, QUE), with quantitative analyses. (E,F) Western blot verified NRF2 protein silencing efficiency in H9c2 cardiomyocytes; bar graphs show relative protein expression. (G–J) Western blot analyzed NRF2, SLC7A11, and GPX4 expression in H9c2 cells (including the siNRF2 group); bar graphs indicate relative levels. (K–N) TMRE fluorescence staining assessed mitochondrial membrane potential in H9c2 cells treated with DOX, tert-butylhydroquinone, siNRF2, and QUE, with representative images and fluorescence intensity quantification. Scale bar: 50 μm. Original Western blot images can be found in https://zenodo.org/records/21643385 (accessed on 29 July 2026). (O) Images show QUE effects on NRF2 expression in DOX-stimulated rat tissues. Scale bar: 100 μm. (P) Images show QUE effects on NRF2 expression in DOX-stimulated H9c2 cells. Scale bar: 50 μm. Data are expressed as the mean ± standard error from three independent experiments. Statistical…

Figure 10. QUE regulates the ferroptosis signaling pathway to mitigate DOX-induced myocardial injury, with its potential mechanism clarified.