Sheng-Mai-Yin inhibits doxorubicin-induced ferroptosis and cardiotoxicity through regulation of Hmox1

Figures

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  Figure 1. The HPLC analysis of SMY and its main components, including 5-HMF (1), lobetyolin (2), Schisandrin B (3), schizandrin (4). (A) Standards. (B) Samples.

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  Figure 2. SMY prevented DOX-induced cardiomyopathy. (A, B) Cardiac sections were prepared from control mice and mice treated with DOX. SMY-L or SMY-H or mitoTEMPO were treated. The heart tissues were stained with hematoxylin and eosin (H&E, Top) or Sirius red (Bottom). (C) Cardiac collagen quantification (% of collagen of area) in heart tissue. (D) The heart/body weight ratio was calculated in control mice and mice treated with DOX with or without SMY-L or SMY-H or mitoTEMPO (n = 9 mice per group). The results were presented as mean ± SEM. ##means compared with control group, P < 0.01; *means compared with DOX group, P < 0.05, **means compared with DOX group, P < 0.01.

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  Figure 3. SMY inhibited iron overload, lipid peroxidation, and serum enzymology. (A, B) Serum (A), cardiac mitochondrial, and cytosolic (B) nonheme iron were detected by an iron assay kit (#ab83366, Abcam). (C, D) Serum (C) and cardiac (D) MDA contents were measured by lipid peroxidation MDA assay kit (Beyotime, S0131S) in control mice and mice treated with DOX with or without SMY or mitoTEMPO. (E, F) Serum LDH (E) and CK-MB (F) levels were examined by commercial assay kits (Beyotime, C0016 and ZCIBIO, ZC-38269) according to the manufacturer’s instructions. The results were presented as mean ± SEM. ##means compared with control group, P < 0.01; *means compared with DOX group, P < 0.05, **means compared with DOX group, P < 0.01.

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  Figure 4. SMY scavenged ROS generation and alleviated mitochondria dysfunction. (A) Images of DHE staining of ROS. (B) Histogram showed the DHE+ cells of the area in different groups. (C) Representative images of JC-1 staining in different groups. Red fluorescence represented aggregation of JC-1, and green fluorescence represented monomeric JC-1. (D) Histogram of aggregate JC-1/monomeric JC-1. The JC-1 probe was applied to measure ΔΨm, which was expressed as the ratio of red (the aggregate form of JC-1, which denotes intact ΔΨm) to green (a monomeric form of JC-1, which signifies dissipation of ΔΨm). The results were presented as mean ± SEM. ##means compared with control group, P < 0.01; *means compared with DOX group, P < 0.05, **means compared with DOX group, P < 0.01.

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  Figure 5. The effect of SMY on molecular markers of ferroptosis. (A, B) Ptgs2 mRNA (A) and Hmox1 mRNA (B) levels were measured by qPCR. (C) Quantification of the intensity of immunofluorescence of Hmox1. (D) Representative images of immunofluorescence of Hmox1. The green fluorescence represented Hmox1 and the blue fluorescence indicated cell nucleus. (E) Representative blot images of FTH1, TFR1, GPX4, and Hmox1 protein levels. (F) Analysis of FTH1 protein levels by column graph, adjusted by GAPDH. (G) Analysis of TFR1 protein levels by column graph, adjusted by GAPDH. (H) Analysis of GPX4 protein levels by column graph, adjusted by GAPDH. (I) Analysis of Hmox1 protein levels by column graph, adjusted by GAPDH. The results were presented as mean ± SEM. ##means compared with control group, P < 0.01; *means compared with DOX group, P < 0.05, **means compared with DOX group, P < 0.01.

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  Figure 6. Overexpression of Hmox1 counteracted the effect of SMY on ferroptosis. (A) The heart/body weight ratio was calculated. (B, C) Cardiac mitochondria and cytosolic nonheme iron (B) and serum nonheme iron (C) were detected by an iron assay kit (#ab83366, Abcam) in mice treated with DOX with or without SMY or Hmox1-OE. (D, E) Western blot analysis of Hmox1 and GAPDH protein levels in control mice, AAV-control mice, and AAC-Hmox1 mice. (F–J) Western blot analysis of FTH1, TFR1, GPX4, and Hmox1 protein contents. The results were presented as mean ± SEM. *means compared with the other group, P < 0.05, **means compared with the other group, P < 0.01.

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  Figure 7. Overexpression of Hmox1 counteracted the effect of SMY on DIC. (A) Hematoxylin and eosin (H&E) staining of heart tissues. (B) Sirius red staining of mice treated with DOX with or without SMY or Hmox1-OE. (C) Cardiac collagen quantification (% of collagen of area) of heart tissues. (D) Representative images of JC-1 of heart tissues. JC-1 aggregation is represented by red fluorescence and monomeric JC-1 formation by green fluorescence. (E) Histogram of aggregate JC-1/ monomeric JC-1 in mice treated with DOX with or without SMY or Hmox1-OE. The results were presented as mean ± SEM. *means compared with the other group, P < 0.05, **means compared with the other group, P < 0.01.