Ethyl acetate extract from Platycladus orientalis leaves ameliorates diabetic cardiomyopathy via alleviating oxidative stress and suppressing myocardial fibrosis

Figures

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  FIGURE 1. Representative base peak intensity (BPI) total ion chromatogram (TIC) of EAEPOL analyzed by UPLC-QTOF-MS in negative electrospray ionization (ESI−) mode. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using a gradient elution of 0.1% formic acid in ultrapure water (A) and methanol (B) at a flow rate of 0.3 mL/min (gradient program detailed in Table 1). Mass spectrometric detection was conducted in ESI− mode over m/z 100–1,000. Key annotated peaks corresponding to the major bioactive constituents of EAEPOL are labeled with their retention times (tR, min) and accurate m/z values, including: ginkgetin (tR = 13.27 min, m/z 551.0814), myricitrin (tR = 5.13 min, m/z 463.0750), cyanin (tR = 6.09 min, m/z 447.0839), and amentoflavone (tR = 9.87 min, m/z 537.0654).

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  FIGURE 2. EAEPOL ameliorates random blood glucose in a time-dependent manner in STZ-induced diabetic cardiomyopathy (DCM) mice. (A) Longitudinal monitoring of random blood glucose levels in normal control, DCM model, and EAEPOL-treated mice over 6 weeks of intervention. Data are presented as mean ± SD (n = 10 per group). (B) Random blood glucose levels at the 6-week endpoint, with individual data points overlaid on the bar graph to show distribution. Data are presented as mean ± SD (n = 10 per group). Intergroup comparisons were performed by one-way ANOVA followed by Tukey’s HSD post hoc test. ****P < 0.0001 vs. normal control group; ####P < 0.0001 vs. DCM group.

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  FIGURE 3. EAEPOL ameliorates cardiac structural and functional abnormalities in a mouse model of diabetic cardiomyopathy (DCM). (A) Representative short-axis M-mode echocardiographic images of the left ventricle in rats from the normal control group, DCM model group, and EAEPOL-treated group. These cross-sectional views illustrate the gross morphological changes in the left ventricular chamber. (B) Representative long-axis M-mode echocardiographic tracings of the left ventricle in the same three groups. These tracings are used for quantitative assessment of cardiac functional parameters, including left ventricular ejection fraction (LVEF) and ejection fraction (EF). Note: Echocardiography was performed to evaluate left ventricular structure and function in all experimental groups. The DCM group exhibited characteristic features of DCM, including left ventricular dilatation and impaired systolic function, which were significantly attenuated by EAEPOL treatment.

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  FIGURE 4. EAEPOL attenuates myocardial fibrosis in mice with diabetic cardiomyopathy (DCM). (A) Representative images of hematoxylin-eosin (HE), Masson’s trichrome, and Picro-Sirius red staining of myocardial paraffin sections from each group of mice. Scale bar = 50 μm (magnification, ×100). Top row: H&E staining showing proliferation of cardiac fibroblasts. Middle row: Masson’s trichrome staining highlighting interstitial collagen deposition (blue). Bottom row: Picro-Sirius red staining for collagen fibers (pink). The bar graphs quantify the fibrotic area percentage from Masson’s and Picrosirius red staining, respectively. (B) Quantitative analysis of hydroxyproline content in myocardial tissues (mg/g). (C) Representative immunohistochemical staining images and quantitative analysis of Collagen I and Collagen III in cardiac tissues. Data are presented as mean ± standard deviation (SD); n = 10 biologically independent mice for (A), n = 4 biologically independent mice for (B), and n = 3 biologically independent mice for (C). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. ***P < 0.001, ****P < 0.0001 vs. normal control…

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  FIGURE 5. EAEPOL preserves endothelial integrity and inhibits myofibroblast activation in mice with diabetic cardiomyopathy (DCM). (A) Representative Western blot images (top panel) and quantitative analysis (bottom panel) of the endothelial integrity marker cluster of differentiation 31 (CD31) and the myofibroblast activation marker α-smooth muscle actin (α-SMA). (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the endothelial junction marker cadherin 5 (CDH5) and the fibrosis-related marker fibroblast-specific protein 1 (FSP1). (C) Representative immunofluorescence staining images of α-SMA (red) counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue) in cardiac tissue (scale bar = 30 μm). Data are presented as mean ± standard deviation (SD); n = 3 independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. normal control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. DCM group.

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  FIGURE 6. EAEPOL attenuates myocardial oxidative stress in mice with diabetic cardiomyopathy (DCM). (A) Myocardial levels of malondialdehyde (MDA), superoxide dismutase (SOD) activity, and total antioxidant capacity (T-AOC) in each indicated group (n = 4 per group). Data are presented as mean ± standard deviation (SD). (B) Representative Western blot images (top panel) and quantitative analysis (bottom panel) of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression in mouse myocardium (n = 3 independent experiments). Data are presented as mean ± SD. (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of HO-1 and Nrf2 mRNA expression. Data are presented as mean ± SD; n = 3 independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. **P < 0.01, ****P < 0.0001 vs. normal control group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. DCM group.