Huoxin pill prevents acute myocardial ischaemia injury via inhibition of Wnt/β‑catenin signaling

Figures

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  FIGURE 1. HXP improves cardiac function following ischaemic injury. (A‐B) Ejection fraction (EF%) and fractional shortening (FS%) parameters in mice administrated with PBS, low‐dose HXP‐L (3 mg kg d−1) or high‐dose HXP‐H (9 mg kg d−1) at 4 weeks post‐MI. n = 5 or more in each group, * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Representative M‐mode echocardiographic images showing LV end‐systolic and end‐diastolic dimensions in mice administrated with PBS, low‐dose HXP‐L (3 mg kg d−1) or high‐dose HXP‐H (9 mg kg d−1) at 4 weeks post‐MI. (D‐G) Echocardiographic measurements of heart rate (D), LV end‐diastolic wall thickness in diastole at the infarct border zone (E), and remote zone (F), and cardiac function parameters (G) in sham, PBS, low‐dose HXP‐L (3 mg kg d−1), or high‐dose HXP‐H (9 mg kg d−1) mice at 4 weeks post‐MI. Data are presented as mean ± sem. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., no significance. n = 5 or more in each group. LVID;d—Left Ventricular Internal Dimension; diastolic; LVID;s—Left Ventricular Internal Dimension; systolic

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  FIGURE 2. HXP prevents MI‐induced cardiac remodelling. (A) Masson's trichrome staining of hearts at 4 weeks post‐MI in mice administrated with PBS, low‐dose HXP‐L (3 mg kg d−1), or high‐dose HXP‐H (9 mg kg d−1) showing 5 different levels (L1‐L5) of heart transverse cross sections from the site of ligation towards the apex. Muscle fibres are stained red, and collagen‐rich fibrotic regions are stained blue. n = 9 or more for each group. Scale bar, 1 mm. (B) Quantification of infarct size in cardiac sections. Infarct size (%) = Mean endocardial infarct arc lengths at L3‐L4 divided by mean left ventricular endocardial arc lengths at L3‐L4 × 100. (C‐G) Relative mRNA expression levels of cardiac fibrosis genes (collagen‐I and collagen‐III) and heart failure genes (ANP, BNP, skeletal α‐actin) in the infarct regions of mice treatment with PBS, low‐dose HXP‐L (3 mg kg d−1) or high‐dose HXP‐H (9 mg kg d−1) at 2 weeks post‐MI. Data are presented as mean ± sem, n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.01. n = 5 or more for each group

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  FIGURE 3. HXP attenuates ischaemia‐induced DNA damage in vitro and in vivo. (A) Representative immunoblots (left) and quantification (right) showing γ‐H2AX expression in ischaemic hearts from mice treated with PBS, low‐dose HXP‐L (3 mg kg d−1) or high‐dose HXP‐H (9 mg kg d−1) at 1‐week post‐MI. GAPDH was used as the loading control. Data are presented as mean ± sem, ** p < 0.01, *** p < 0.001. n = 3 for each group. (B) Representative immunofluorescence staining (left) and quantification (right) of γ‐H2AX foci (red), cardiac muscle marker Troponin T (green) and DAPI (blue) in the infarcted LV myocardium in SHAM‐operated mice and those treated with PBS, low‐dose HXP‐L (3 mg kg d−1) or high‐dose HXP‐H (9 mg kg d−1) at 1‐week post‐MI. Data are presented as mean ± sem, * p < 0.05, ** p < 0.01. n = 4 for each group. Scale bar, 50 μm. (C) Representative immunoblot (left) and quantification (right) showing expression of γ‐H2AX following pretreatment with 1 µg ml HXP for 2 hours and treatment with 100 µM H2O2 for 30 minutes in AC16 cardiomyocytes. GAPDH was used as the loading control. Data are presented as mean ± sem, * p < 0.05, *** p < 0.001 versus control group. n = 3 for each group

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  FIGURE 4. HXP is a potent inhibitor of Wnt/β‑catenin activation‐induced cardiac injury and DNA damage. (A‐C) Reporter gene assay using TOPflash analysis. Wnt3a (A), LiCl addition (B), or full‐length LRP6 (C) were transfected with TOPflash in HEK293 cells for 48 h. HXP or LiCl were directly added to the transfected cells at varying concentrations, as indicated. RLU, relative light units. Values are presented as the mean ± sem. n.s., no significance, * p < 0.05, *** p < 0.001 versus control group. n = 3 for each experiment. (D) Representative immunoblot (left) and quantification (right) showing expressions of γ‐H2AX and β‐catenin following siRNA‐mediated knockdown of β‐catenin for 48 hours, subsequent pretreatment with 1 µg ml−1 HXP for 2 hours and treatment with 100 µM H2O2 for 30 minutes. GAPDH was used as the loading control. Data are presented as mean ± sem, * p < 0.05, ** p < 0.01. n = 3 for each. (E, F) Representative immunoblot showing the expression of cytoplasmic phosphorylated‐β‐catenin (E) and nuclear β‐catenin (F) in the infarct region of mice treated with PBS, low‐dose HXP‐L (3 mg kg d−1), or high‐dose HXP‐H (9 mg kg d−1) at 1 h and 2 h post‐MI. GAPDH was used as the cytoplasmic…

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  FIGURE 5. HXP attenuates H2O2‐induced DNA damage via inhibition of Wnt/β‐catenin pathway. (A) Representative immunoblot (top) and quantification (bottom) showing the expression of nuclear β‐catenin following LiCl stimulation at different time intervals in AC16 cardiomyocytes. TBP was used as the loading control. n.s., no significance, * p < 0.05, ** p < 0.01 versus control group. n = 3 for each experiment. (B) Representative immunoblot (top) and quantification (bottom) showing the expression of nuclear β‐catenin following LiCl stimulation at different concentrations in AC16 cardiomyocytes. TBP was used as the loading control. n.s., no significance, * p < 0.05, ** p < 0.01 versus control group. n = 3 for each experiment. (C) Representative immunoblot (left) and quantification (right) showing the expression of γ‐H2AX and β‐catenin following pretreatment with 1 µg ml−1 HXP for 2 hours, 40 mM LiCl for 4 hours, and subsequent treatment with 100 µM H2O2 for 30 minutes. GAPDH was used as the loading control. Data are presented as mean ± sem, * p < 0.05. n = 3 for each