Morroniside Attenuates Doxorubicin‐Induced Cardiotoxicity by Activating the PI3K/AKT/Nrf2/HO‐1 Pathway to Inhibit Ferroptosis and Oxidative Stress

Figures

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  Figure 1. Bioinformatics integration between Mor and DIC. (A) The chemical structure of Mor. (B) The common targets found in both the therapy (Mor) and the disease (DIC). (C) Identified targets and their protein interaction network. (D) Analysis of GO enrichment using genes with differential expression. (E) Using differentially expressed genes for KEGG pathway analysis.

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  Figure 2. In vitro experiments showed that Mor diminished the ferroptosis and oxidative stress resulting from Dox. (A) Cytotoxicity was evaluated via LDH assay in all experimental groups, n = 6. (B–F) Iron content, MDA, GSH, SOD, and CAT served as markers of ferroptosis and oxidative stress in all experimental groups, n = 6. (G) Western blotting was employed across all groups to determine the levels of ferroptosis and oxidative stress. (H–K) The levels of Nrf2, PTGS2, HO‐1, and GPX4 were assessed following western blot analysis, n = 6. (L) Immunofluorescence staining was used to detect PTGS2 and HO‐1 in all experimental groups, 100x, n = 3. (M) In the context of oxidative stress staining, DHE is marked by red and ROS by green, 100x, n = 3. Data are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 3. In vitro, the ferroptosis inducer significantly weakened the protective effect of Mor in DIC. (A) Western blotting was employed across all groups to determine the levels of ferroptosis. (B, C) The levels of PTGS2 and GPX4 were assessed following western blot analysis, n = 6. (D–G) Iron content, MDA, GSH, and SOD served as markers of ferroptosis and oxidative stress in all experimental groups, n = 6. (H) Immunofluorescence staining was used to detect PTGS2 in all experimental groups, 100x, n = 3. (I) Cytotoxicity was evaluated via LDH assay in all experimental groups, n = 6. Data are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 4. In vitro, the inhibition of ferroptosis, oxidative stress, and DIC by Mor depends on the activation of Nrf2. (A) Western blotting was employed across all groups to determine the levels of ferroptosis and oxidative stress. (B–E) The levels of Nrf2, PTGS2, HO‐1, and GPX4 were assessed following western blot analysis, n = 6. (F–J) Iron content, MDA, GSH, SOD, and CAT served as markers of ferroptosis and oxidative stress in all experimental groups, n = 6. (K, L) Cytotoxicity was evaluated via cell viability and LDH assay in all experimental groups, n = 6. (M) In the context of oxidative stress staining, DHE is marked by red and ROS by green, 100x, n = 3. Data are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 5. In vitro studies showed that Mor triggered the PI3K/AKT signaling pathway in DIC, while LY294002 inhibited this activation. (A) The 3D‐molecular docking of PI3K (Blue) and Raf‐1 (Green). The residues involved in hydrogen bonding are visible in a zoomed‐in view of the binding pocket. (B–E) RMSD, RMSF, Rg, and SASA values of the protein‐ligand complexes over time. (F) Western blotting was employed across all groups to determine the levels of PI3K and AKT. (G, H) The levels of PI3K and AKT were assessed following western blot analysis, n = 6. (I) Western blotting was employed across all groups to determine the levels of PI3K and AKT. (J, K) The levels of PI3K and AKT were assessed following western blot analysis, n = 6. Data are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 6. In vitro, Mor activates Nrf2/HO‐1 to alleviate ferroptosis, oxidative stress, and DIC, which depend on the activation of the PI3K/AKT pathway. (A, B) Cytotoxicity was evaluated via cell viability and LDH assay in all experimental groups, n = 6. (C) Western blotting was employed across all groups to determine the levels of ferroptosis and oxidative stress. (D–G) The levels of Nrf2, PTGS2, HO‐1, and GPX4 were assessed following western blot analysis, n = 6. (H–L) Iron content, MDA, GSH, SOD, and CAT served as markers of ferroptosis and oxidative stress in all experimental groups, n = 6. (M) In the context of oxidative stress staining, DHE is marked by red and ROS by green, 100x, n = 3. Data are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 7. In the DIC model in vivo, Mor also had a protective effect. (A) Measurements of survival rates were taken for every group. (B) Representative echocardiographic images from each experimental group, n = 6. (C, D) LVEF and LVFS, n = 6. (E) Representative images of H&E staining, 200x, n = 3. (F) The concentration of LDH in mouse serum, n = 6. (G) Western blotting was employed across all groups to determine the levels of ferroptosis and oxidative stress. (H–K) The levels of Nrf2, PTGS2, HO‐1, and GPX4 were assessed following western blot analysis, n = 6. (L–P) Iron content, MDA, GSH, SOD, and CAT served as markers of ferroptosis and oxidative stress in all experimental groups, n = 6. Data are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.