Calycosin inhibits doxorubicin-induced mitochondrial oxidative stress and ferroptosis by activating the NRF2 pathway

fig.1 Calycosin significantly ameliorated cardiac dysfunction in DIC mice. (A) Echocardiographic assessment (n = 6). (B) Quantitative analysis of cardiac function parameters (n = 6). (C) Serum biomarkers of myocardial injury (n = 4). (D) Representative histopathological images (H&E staining; scale bar=100 μm). fig.2 Calycosin alleviated DOX-mediated mitochondrial dysfunction in vivo. (A) TEM analysis showing calycosin treatment preserved mitochondrial architecture. (B-C) Cardiac oxidative stress parameters (lipid peroxidation marker MDA and antioxidant enzyme SOD; n = 3). (D) Representative immunofluorescence images of DNA damage evaluation via 8-OHdG (scale bar=20 μm). fig.3 Calycosin stimulated the NRF2/NRF1-TFAM signaling pathway in mice model. (A) The expression of NRF2 visualized by immunofluorescence microscopy (scale bar=20 μm). (B-C) Cardiac expression levels of NRF2, NRF1, TFAM, NDUFB8, COX1 and GPX4 (n = 6). fig.4 Calycosin rescued mitochondrial respiration chain in NRF2-dependent way. (A) Dose-response of calycosin on H9c2 viability (n = 6). (B) Rescue of DOX-impaired cell viability by calycosin cotreatment (n = 6). (C) NRF2 nuclear localization (n = 3). (D-E) Western blot analysis of NRF2, NRF1, TFAM, COX1, NDUFB8, SDHB and ATP5A (n = 3). fig.5 Calycosin alleviated ferroptosis by triggering the NRF2 signaling pathway in H9c2 cells. (A-B) Intracellular Fe²⁺ levels visualized by FerroOrange staining in H9c2 cardiomyocytes (scale bar = 20 μm). (C) Western blot analysis of iron regulatory proteins FPN and FTMT . fig.6 Calycosin protected mitochondrial respiration chain in NRF1-dependent way. (A-B) The expression of NRF2, NRF1 after interference with NRF2 siRNA, NRF1 siRNA. (C-D) Western blot of NRF1, TFAM, COX1, NDUFB8 after interference with NRF1 siRNA (n = 3 per group). fig.7 Calycosin alleviated mitochondrial DNA damage, oxidative stress and membrane potential in H9c2 cells through activation of the NRF2–NRF1 pathway. (A) Immunofluorescence detection of oxidative DNA damage (8-OHdG). (B) Mitochondria (MitoTracker Green, green) and mitochondrial ROS (MitoSOX Red, red) in live cells (n = 3). (C) Variations in mitochondrial membrane potential following treatment (Scale bar = 50 μm). (D) Quantification of the ratio of mitochondria to mitochondrial ROS in live cells. (E) Quantitative analysis of the ratio of JC-1 monomer to JC-1 aggreates. fig.8 Calycosin protected against DIC by activation of NRF2. (A) Representative M-mode echocardiographic images (n = 4). (B) Quantitative analysis of cardiac function parameters (n = 4). (C) Serum biomarkers of myocardial injury (CK-MB and LDH, n = 4). (D) MDA and SOD levels in cardiac tissues of mice (n = 3). (E) Hematoxylin and eosin staining . fig.9 Calycosin regulated mitochondrial proteins and ferroptosis-associated proteins by activating NRF2. (A-B) Western blot analysis of NRF2, NRF1, TFAM, COX1, and NDUFB8 expression levels. (C-D) FPN and FTMT expression levels (n = 3).