Cardiac protection of wogonin in mice with pulmonary fibrosis by regulating Sirt1/ γ-H2AX pathway

Figures

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  FIGURE 1. Wogonin alleviates bleomycin-induced mouse pulmonary fibrosis. (A) Timeline of wogonin administration. (B) Representative images of Masson staining with the lung sections, and (C) Fibrosis scores based on stained lung sections. N = 4 mice/group. (D) Immunohistochemical analysis of α-SMA with the lung tissues, and relative quantification of α-SMA at the right. Scale bars, 500 µm (black bars), 100 µm (red bars). N = 4/group, *p < 0.05, **p < 0.01, compared with control animals; ##p < 0.01, compared with BLM group, one-way ANOVA with a post-hoc Tukey’s test. All data are presented as the mean ± SEM. BLM, bleomycin; Wog, wogonin.

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  FIGURE 2. Wogonin protects against cardiac dysfunction in mice with pulmonary fibrosis. (A) Representative M-mode echocardiographic imagings. (B) Ejection fraction; (C) Fractional shortening; (D) Heart rates; (E, F) Left ventricle internal diameter at end-systole (LVIDs) and at end-diastole (LVIDd); (G) Heart weight-to-body weight ratio (HW/BW). N = 6-7 mice/group. **p < 0.01, compared with control animals; #p < 0.05, ##p < 0.01, compared with BLM group, one-way ANOVA with a post hoc Tukey’s test. All data are mean ± SEM. BLM, bleomycin; Wog, wogonin.

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  FIGURE 3. Wogonin suppresses cardiac fibrosis. (A) Representative images with Masson staining and Sirius red staining. Scale bars, 50 µm. (B) Relative quantification of collagen deposition assayed by Masson staining, n = 4/group; (C) The protein levels of α-SMA by Western blots. Mean data at the right, GAPDH as an internal control; n = 3/group; *p < 0.05, **p < 0.01 compared with controls; ##p < 0.01, compared with BLM, one-way ANOVA with a post-hoc Tukey’s test. All data are presented as the mean ± SEM. BLM, bleomycin; Wog, wogonin.

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  FIGURE 4. Wogonin mitigates myocardial DNA damage and apoptosis. (A) Immunohistochemical analysis of γ-H2AX in the hearts. Scale bars, 50 µm (black bars), 10 µm (red bars); (B) Relative quantification of γ-H2AX staining; (C) Apoptosis evaluated by TUNEL staining in heart sections. Scale bar: 100 μm; (D) Quantitation of TUNEL positive staining; (E) Protein levels of cleaved-caspase 3 and γ-H2AX in heart tissues by Westerns; (F, G) Mean data of protein immunoblots; n = 3–4/group; **p < 0.01 compared with controls; #p < 0.05, ##p < 0.01 compared with BLM groups, one-way ANOVA with a post-hoc Tukey’s test. All data are presented as the mean ± SEM. BLM, bleomycin; Wog, wogonin.

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  FIGURE 5. Wogonin attenuates DNA damage in H9C2 cardiomyocytes. (A, B) Immunofluorescence staining of γ-H2AX in H9C2 cells induced by CoCl2 and H2O2 respectively. Scale bar, 50μm; (C, E) Representative images of neutral comet assay in CoCl2 or H2O2-stimulated H9C2 cells with or without wogonin treatment. Scale bar, 50 μm. (D, F) Quantification of comet tail moment (a.u., arbitrary units) shown at the right respectively. At least 50 cells/group. *p < 0.05, **p < 0.01, compared with controls; #p < 0.05, ##p < 0.01, compared with respective CoCl2 or H2O2 group. 2-way ANOVA with a post-hoc Tukey’s test. All data are presented as the mean ± SEM.

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  FIGURE 6. Wogonin diminishes DNA damage via upregulation of Sirt1. (A) Western blot analysis of Sirt1 proteins in H9C2 cells following the treatment with different concentrations of wogonin (0–20 μM) for 12 h. Mean data shown at the right. N = 3/group; **p < 0.01 compared to non-treatment; (B) Protein levels of γ-H2AX and Sirt1 in H2O2-stimulated H9C2 cells with or without wogonin treatment. Mean data at the right. N = 3/group; *p < 0.05, **p < 0.01, compared with controls; #p < 0.05, ##p < 0.01, compared with H2O2 group; (C) Immunohistochemical analysis of Sirt1 in the mouse heart sections. Scale bars, 50 µm (black bars), 10 µm (red bars). Relative quantification of Sirt1 shown at the right. N = 4/group; (D) Western blot analysis of Sirt1 proteins in mouse heart tissues. Mean data at the right, n = 3/group. **p < 0.01, compared with control animals; #p < 0.05, ##p < 0.01, compared with BLM group, one-way or two-way ANOVA with a post-hoc Tukey’s test. All data are presented as the mean ± SEM. BLM, bleomycin; Wog, wogonin.

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  FIGURE 7. Sirt1 is a direct target of wogonin. (A) Chemical structure of wogonin and biotin-labeled wogonin (Bio-Wog); (B) Schematic showing steps for biotin-affinity pull-down assay; (C) Physical association of Sirt1 and wogonin evaluated by pull-down assay. Biotinylated wogonin (Bio-Wog) was used to pull-down Sirt1 with H9C2 lysates. Biotin alone as a control, total lysates as input; (D) Distribution of molecular electrostatic potential presented on Sirt1 (PDB code: 4ZZH); Blue and red color are the positive and negative potential, respectively. 3D molecular surface representation of the superimposition of wogonin (green sticks) against Sirt1; (E) The amino acid residues of Sirt1 with potential polar bonds with wogonin and the distance between each residue and wogonin; (F) 3D cartoon depiction of molecular interaction of docking complex between Sirt1 (yellow) and wogonin (green). The docking investigation revealed the presence of hydrogen bonds (blue dotted lines) and π-π stacking (green dotted lines).