Hyperoside Alleviates Myocardial Fibrosis by Reducing Oxidative Stress via the GATA4/HIF-1α Pathway in Mice

Figures

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  Figure 1. Molecular docking and molecular dynamics simulation of Hyp-GATA4 complex. (A) Three-dimensional docking conformation of Hyp-GATA4 complex. (B) Two-dimensional interaction diagram of Hyp-GATA4 complex. (C) RMSD distribution of Hyp relative to GATA4 skeleton. The dark blue line represents the RMSD trajectory smoothed by Gaussian filtering (σ = 2), the red dotted line represents the average RMSD, and the light red region shows a fluctuation range of ±0.01 nm. (D) Solvent accessible surface area (SASA) distribution map of Hyp in GATA4 complex. The meanings of dark blue lines, red dashed lines and light red areas are the same as those in (C). (E) Three-dimensional free energy landscape of Hyp-GATA4 complex. (F) Two-dimensional free energy surface of Hyp-GATA4 complex.

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  Figure 2. Molecular docking and molecular dynamics simulation of Hyp-HIF-1α complex. (A) Three-dimensional docking conformation of Hyp-HIF-1α complex. (B) Two-dimensional interaction diagram of Hyp-HIF-1α complex. (C) RMSD distribution of Hyp relative to HIF-1α skeleton. The dark blue line represents the RMSD trajectory smoothed by Gaussian filtering (σ = 2), the red dotted line represents the average RMSD, and the light red region shows a fluctuation range of ±0.01 nm. (D) Solvent accessible surface area (SASA) distribution map of Hyp in HIF-1α complex. The meanings of dark blue lines, red dashed lines and light red areas are the same as those in (C). (E) Three-dimensional free energy landscape of Hyp-HIF-1α complex. (F) Two-dimensional free energy surface of Hyp-HIF-1α complex.

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  Figure 3. Effects of different doses of Hyp on MF in mice. (A) Representative diagram of echocardiography in mice. (B–E) EF, FS, LVIDd and LVIDs were measured by echocardiography in mice (x¯ ± s, n = 6). (F) Cardiac index (x¯ ± s, n = 6). (G,I) H&E staining representative figure and H&E score of mouse heart tissue (Data are presented as median (IQR), n = 3). Black arrows indicate inflammatory cell infiltration (200×: 100 μm; 400×: 50 μm). (H,J) Sirius Red staining of mouse heart tissue and myocardial collagen volume fraction (x¯ ± s, n = 3). Black arrows indicate collagen deposition (200×: 100 μm; 400×: 50 μm). (K–N) The levels of Col I, Col III, α-SMA and TGF-β mRNA in the heart tissue of mice were measured (x¯ ± s, n = 3). (O,P) The mRNA levels of GATA4 and HIF-1α in mouse heart tissue (x¯ ± s, n = 3). (Q–S) The content of Col I, Col III and α-SMA protein in serum of mice with central muscle fibrosis (x¯ ± s, n = 6). (T–W) Serum oxidative stress-related indicators HIF-1α, HO-1, ROS, SOD protein content in mice (x¯ ± s, n = 6), * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05.

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  Figure 4. GATA4 affects the improvement of Hyp on cardiac injury in MF mice. (A) Representative diagram of echocardiography in mice. (B–E) EF, FS, LVIDd and LVIDs were detected by echocardiography in mice (x¯ ± s, n = 6). (F–J) The contents of cTnT, cTn1, CK-MB, LDH 1 and LDH 2 in the serum of mice were measured (x¯ ± s, n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05.

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  Figure 5. GATA4 affects the improvement of Hyp on cardiac injury in MF mice. (A,B) cTnT immunofluorescence representative map and cTnT positive cell count in mouse heart tissue (x¯ ± s, n = 3), (200×: 100 μm; 400×: 50 μm). (C–G) The mRNA levels of cTnT, Col I, Col III, TGF-β, and α-SMA in mouse heart tissue (x¯ ± s, n = 6). (H,I) The representative figure and average optical density of Col I immunohistochemical staining in mouse heart tissue (x¯ ± s, n = 3). (J,K) The representative figure and average optical density of Col III immunohistochemical staining in mouse heart tissue. (L,M) The WB representation of α-SMA in mouse heart tissue and the relative expression of α-SMA protein (x¯ ± s, n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05.

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  Figure 6. Hyp improves MF in mice through GATA4/HIF-1α pathway. (A,B) GATA4 immunofluorescence staining representative map and fluorescence quantification of mouse heart tissue (x¯ ± s, n = 3) (200×: 100 μm; 400×: 50 μm). (C,D) The HIF-1α immunofluorescence staining representative map and fluorescence quantification of mouse heart tissue (x¯ ± s, n = 3) (200×: 100 μm; 400×: 50 μm). (E) The representative figures of GATA4, HIF-1α and HO-1 WB in mouse heart tissue. (F–H) The relative expression levels of GATA4, HIF-1α and HO-1 proteins (x¯ ± s, n = 3). (I,J) The mRNA levels of GATA4 and HIF-1α in mouse heart tissue (x¯ ± s, n = 3). (K) The content of HIF-1α protein in serum of mice (x¯ ± s, n = 3). (L,M) ROS flow cytometry representative figure and protein content in mouse cardiomyocytes (x¯ ± s, n = 3). (N,O) The levels of ROS and HO-1 mRNA in mouse heart tissue (x¯ ± s, n = 3). (P,Q) The content of ROS and HO-1 protein in serum of mice (x¯ ± s, n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05.

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  Figure 7. Hyp improves oxidative stress in MF through the GATA4/HIF-1α pathway. MF can cause pathological damage to the heart muscle and subsequently affect cardiac function. Hyp can exert a protective effect on the heart by targeting cardiac muscle cells and regulating the GATA4/HIF-1α pathway to inhibit oxidative stress and reduce collagen deposition. Eventually, this can alleviate pathological damage to the heart and improve MF.

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  Figure 8. Flow chart of the experiment.