Calycosin Safeguards Mitochondrial Genomic Stability and Iron Homeostasis via the NRF2/NRF1-TFAM Axis to Mitigate Doxorubicin-Induced Cardiotoxicity

Figures

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  Figure 1. Calycosin improves cardiac dysfunction in a DIC mouse model. (A) Echocardiographic evaluation (n = 6 mice per group). (B) Quantitative analysis of cardiac function parameters (n = 6 mice per group). (C) Measurement of serum biomarkers of myocardial injury (n = 4 mice per group). (D) Representative histological images obtained by H&E staining (scale bar = 100 μm). (E) Viability of H9c2 cells treated with different concentrations of CAL (n = 6 independent biological experiments). (F) Effects of CAL and DOX on H9c2 cell viability (n = 6 independent biological experiments). (G) Viability of 4T1 breast cancer cells following DOX treatment with or without CAL (n = 6 independent biological experiments). Data represent mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

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  Figure 2. Transcriptomic analysis of mouse hearts revealed that calycosin attenuated DIC by restoring mitochondrial energy metabolism and suppressing ferroptosis. (A) A three-dimensional PCA plot illustrating the clustering patterns among the Control group (C), the DOX-treated model group (M), and the DOX + CAL group (CAL), (n = 3 mice per group). The same abbreviations are used throughout Figure 2. (B) Bar chart showing the number of differentially expressed genes (DEGs) in the M-vs-C and CAL-vs-M comparisons. (C) GO enrichment analysis of downregulated genes in the M-vs-C comparison. (D) GO enrichment analysis of upregulated genes in the CAL-vs-M comparison. (E) KEGG pathway enrichment analysis of upregulated genes in the M-vs-C comparison. (F) KEGG pathway enrichment analysis of downregulated genes in the CAL-vs-M comparison. Terms highlighted in red represent the primary focus of this study.

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  Figure 3. Calycosin ameliorates DOX-mediated mitochondrial dysfunction in vivo. (A) TEM analysis showing calycosin treatment preserved mitochondrial architecture (n = 3 mice per group). (B) Quantitative analysis of mitochondrial cristae volume density in TEM images (n = 3 mice per group). (C,D) Cardiac oxidative stress parameters (lipid peroxidation marker MDA and antioxidant enzyme SOD; n = 3 mice per group). (E) Quantitative colocalization analysis of 8-OHdG and Tom20 using Pearson’s correlation coefficient (n = 3 mice per group). (F) Representative immunofluorescence images illustrating DNA damage evaluated by 8-OHdG staining (scale bar = 20 μm; n = 3 mice per group). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with DOX group.

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  Figure 4. Molecular docking and molecular dynamics simulation analysis of the calycosin-NRF1 complex. (A) Schematic illustration of the docking conformations of calycosin within the binding pockets of NRF1 and NRF2. (B) Binding energy profile of calycosin with NRF1. (C) RMSD analysis of the calycosin-NRF1 complex. (D) RMSF profile of NRF1. (E) Rg of the NRF1 backbone. (F) SASA analysis. (G) Hydrogen bond interactions within the complex. (H) Hydrogen bond interactions within the complex. (I) FEL of the calycosin-NRF1 complex in 3D.

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  Figure 5. Calycosin activates the NRF2/NRF1-TFAM signaling axis and promotes NRF2 nuclear translocation. (A) Representative Western blot and quantitative analysis of NRF2 and NRF1 protein expression in cardiac tissues (n = 6 mice per group). (B–D) Immunofluorescence analysis of NRF2 localization in cardiac tissue sections (n = 3 mice per group) and H9c2 cardiomyocytes (scale bar = 20 μm; n = 3 independent biological experiments). (E,F) Western blot analysis of TFAM and mitochondrial respiratory chain subunits (NDUFB8 and COX1; n = 6 mice per group). ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. DOX group; ### p < 0.001 vs. the DOX + CAL group.

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  Figure 6. Calycosin preserves mitochondrial biogenesis and suppresses ferroptosis via the NRF2-mediated NRF1/TFAM axis. (A,B) Assessment of mitochondrial biogenesis. NRF2 knockdown abolished the protective effects of CAL on the protein levels of NRF1, TFAM, COX1, and NDUFB8, while no significant changes were observed in SDHB or ATP5A (n = 3 independent biological experiments). (C,D) NRF2 knockdown abolished CAL-mediated ferroptosis suppression (n = 3 independent biological experiments). (E) NRF2 knockdown abolished CAL-mediated iron regulation. Following siRNA-mediated silencing of NRF2, CAL failed to upregulate the expression of FTMT and FPN (n = 3 independent biological experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DOX group; # p < 0.05 vs. DOX + CAL group, ns: not significant.

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  Figure 7. NRF1 functions as the essential downstream effector linking NRF2 signaling to mitochondrial genomic integrity and function. (A–D) Effects of NRF1 silencing on CAL-mediated mitochondrial protection. siRNA-mediated knockdown of NRF1 did not alter NRF2 protein levels, but completely abrogated CAL-induced upregulation of TFAM, COX1, and NDUFB8 (n = 3 independent biological experiments). (E,F) NRF1 knockdown abolishes CAL-mediated preservation of mitochondrial genome integrity and function. Loss of NRF1 abrogated CAL’s protective effect against DOX-induced mtDNA damage, as reflected by elevated 8-OHdG levels and resulted in a reduction in mitochondrial membrane potential (Δψm; n = 3 independent biological experiments). ns, not significant; * p < 0.05, *** p < 0.001, and **** p < 0.0001 vs. DOX group.

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  Figure 8. Pharmacological inhibition of NRF2 abolishes CAL-mediated cardioprotection, mitochondrial recovery, and iron homeostasis in vivo. (A,B) Echocardiographic assessment of cardiac function. CAL treatment significantly improved DOX-impaired Ejection Fraction (EF) and Fractional Shortening (FS), whereas co-administration with the NRF2 inhibitor ML385 completely negated this functional recovery (n = 6 mice per group). (C) Serum levels of cardiac injury markers. CAL significantly reduced DOX-induced elevations in CK-MB and LDH, effects that were effectively abrogated by ML385 co-treatment (n = 4 mice per group). (D) Representative H&E staining of cardiac tissue sections. CAL ameliorated DOX-induced myofibril disarray and inflammatory cell infiltration; however, this structural preservation was largely abolished upon NRF2 inhibition. (E) Cardiac oxidative stress parameters. CAL suppressed DOX-induced MDA elevation and restored SOD activity, both of which were reversed by ML385 co-administration (n = 3 mice per group). (F,G) Western blot analysis of NRF2/NRF1-TFAM axis and iron metabolism proteins. ML385-mediated inhibition of NRF2 abolished CAL-induced upregulation of NRF1, TFAM, COX1,…

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  Figure 9. Schematic diagram illustrating that calycosin exerts cardioprotective effects via the NRF1/NRF2-TFAM signaling axis. Calycosin restores both NRF2 and NRF1 expression and promotes NRF2 activation. Activated NRF2 contributes to mitochondrial iron homeostasis through regulation of FTMT and FPN and further supports downstream NRF1–TFAM signaling. In parallel, calycosin-mediated restoration of NRF1 promotes TFAM and mitochondrial respiratory-chain components, including COX1 and NDUFB8, thereby contributing to the maintenance of mitochondrial genomic integrity and function. Through NRF2-mediated regulation of mitochondrial iron homeostasis and NRF1–TFAM-dependent maintenance of mitochondrial function, calycosin attenuates DOX-induced oxidative stress, ferroptosis, and myocardial injury.