Tetrahedral Framework Nucleic Acids Inhibit Oxidative Stress and Cardiomyocyte Apoptosis in Doxorubicin-Induced Cardiotoxicity

Figures

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  Figure 1. Synthesis and characterization of the tFNAs. (A) Schematic diagram of tFNAs synthesis. (B) Molecular weight detection by 2% AGE of each ssDNA and tFNAs. (C) Images acquired by AFM. (D) Sizes and zeta potentials of tFNAs measured by DLS. (E) Stability evaluation of tFNAs in fetal bovine serum by 2% AGE. (F) Immunofluorescence images for cellular uptake of Cy5-labeled tFNAs in AC16 cells (Cy5: red; nucleus: blue).

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  Figure 2. tFNAs alleviated DOX-induced oxidative stress and cardiomyocyte apoptosis in vitro. (A) Representative images of intracellular ROS measurement by DCFH assay. (B) Statistical analysis of DCFH assay, n=6. (C) Representative images of intracellular ROS measurement by MITOSOX assay. (D) Statistical analysis of MITOSOX assay, n=6. (E) Representative images of intracellular ROS measurement using JC-1 assay. (F–I) MDA, NADPH, GSH, SOD contents in AC16 cells, n=6. AC16 cells were preincubated with tFNAs (250 nM) or PBS for 12 h before being treated with DOX (1μM) for 12 h. The data are presented as the mean ± SD, **p < 0.01.

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  Figure 3. Effects of tFNAs on DOX-induced fibrosis in vitro. (A) qPCR results of AC16 cells with different treatment. (B) Heatmap of the previous qPCR results. (C–E) IF staining of Collagen I, TGF-β1 and MMP9 TGF-β1 of AC16 cells with different treatment. AC16 cells were preincubated with tFNAs (250 nM) or PBS for 12 h before being treated with DOX (1μM) for 12 h. ***p < 0.001.

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  Figure 4. AKT/p53 signaling may be responsible for tFNAs-mediated protective role on cardiomyocyte apoptosis against DOX insult. (A and B). Representative images of live/dead staining of AC16 cells and quantification, n=3. (C) Representative images of TUNEL staining of AC16 cells of different groups. (D and E) Protein levels of cleaved-caspase-3, caspase-3, cleaved-PARP, PARP, phospho-AKT (Ser473), p53, and GAPDH detected by Western blot and their quantitative evaluation, n=3. AC16 cells were preincubated with tFNAs (250 nM) or PBS for 12 h before being treated with DOX (1μM) for 8/12 h. The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 5. tFNAs exerted cardioprotective effects to mice hearts in response to DOX insult. (A) Representative echocardiographic images of cardiac function. (B–D) Quantitative analysis of fraction shortening, ejection fraction and heart rate, n=6. (E) Protein levels of ANP and GAPDH detected by Western blot and their quantitative evaluation, n=3. (F) Quantitative analysis of mouse body weight, n=6. (G) Quantitative analysis of HW/TL. HW, heart weight; TL, tibia length. (H) Protein levels of collagen I and GAPDH detected by Western blot and their quantitative evaluation, n=3. (I) Representative images of Masson staining. (J) Representative TEM images of mice hearts in different groups. (K–N) MDA, NADPH, GSH, SOD contents in mice hearts, n=6. Mice were administrated with DOX (15 mg/kg, intraperitoneally) in three weekly doses, resulting in a cumulative total dose of 15 mg/kg. For those in DOX and tFNA-treated group, 100 μL of tFNA solution (250 nM) was administered via tail vein injection 12 hours prior to each DOX administration, over a 3-week period. The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 6. Schematic illustration of the antioxidant, anti-fibrosis, anti-apoptosis and cardioprotective effects of tFNAs in DOX-stimulated cardiomyocyte and mice model of DIC.