Bifidobacterium adolescentis attenuates cardiac remodeling in HFD/STZ-induced obesity-associated diabetic cardiomyopathy

Figures

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  Fig. 1. Schematic overview of the experimental design and cardioprotective effects of Bifidobacterium adolescentis in HFD/STZ-induced obesity-associated diabetic cardiomyopathy. (A) High-fat diet (HFD) combined with streptozotocin (STZ) injection induced obesity and diabetes, leading to cardiac remodeling characterized by hypertrophy, fibrosis, upregulation of stress- and fibrosis-related markers (ANP, BNP, TGF-β, NF-κB, GATA4, Myc), and downregulation of contractility-associated genes (α-MHC, SERCA2a, NPR1, KCNQ1), along with stereological alterations in cardiac structure. (B) Oral supplementation with Bifidobacterium adolescentis (weeks 8–16) was administered as an intervention. (C) Probiotic treatment restored cardiac structure and volume, reduced stress and fibrosis markers, and improved expression of contractility-related genes, as confirmed by stereological and transcriptomic analyses.

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  Fig. 2. Overview of stereological methods applied to cardiac tissue. (a) Left ventricular volume was determined using the Scherle immersion method. (b) Isotropic uniform random (IUR) sections of the left ventricle were generated according to the orientator method, in which the ventricle was placed on an equally divided circle and cut in random directions (illustrated here at positions 7, 6, and 1 on the cosine-weighted circle). (c) Ventricular slabs were collected, circular samples were punched out, and both slabs and samples were embedded in paraffin blocks and sectioned using a microtome. Stained sections were mounted on slides for microscopic evaluation. (d) Volume densities of myocardium, endocardium, and vessels were estimated on H&E-stained sections using the point-counting method.

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  Fig. 3. Comparison of body weight and cardiac index among the experimental groups. (a) Body weight and (b) cardiac index (heart weight/body weight × 1000) were measured in Control, HFD (after STZ injection), HFD-B. adolescentis (8W), and HFD-B. adolescentis (16W) groups. Each point represents an individual animal (n = 8 per group), and data are shown as the mean. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. Significance symbols: *p < 0.05, **p < 0.01, ***p < 0.001 versus Control; #p < 0.05, ##p < 0.01, ###p < 0.00,1 ####p < 0.0001 versus HFD.

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  Fig. 4. Stereological analysis of cardiac weight and volume in experimental groups. (a) Heart weight in control, HFD following STZ administration, and Bifidobacterium adolescentis-treated rats. (b) Left ventricular volume estimated by unbiased stereological techniques. Data are presented as mean (n = 5 rats/group). Statistical significance is indicated as follows: *p < 0.05 vs. Control; #p < 0.05 vs. HFD; &p < 0.05 HFD-B.a(8W) vs. HFD-B.a (16W). HFD high-fat diet, B.a Bifidobacterium adolescentis, 8W/16W 8 or 16 weeks of treatment.

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  Fig. 5. Stereological quantification of cardiac tissue components. (a) Myocardial volume in the left ventricle. (b) Endocardial volume assessed by systematic uniform random sampling. (c) Intramyocardial vascular volume, reflecting changes in cardiac microvasculature across groups. Data are presented as mean (n = 5 rats/group). Symbols denote statistical significance: *p < 0.05 vs. Control; #p < 0.05 vs. HFD; &p < 0.05 HFD-B.a(8W) vs. HFD-B.a(16W). HFD high-fat diet, B.a Bifidobacterium adolescentis, 8W/16W 8 or 16 weeks of treatment.

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  Fig. 6. Relative mRNA expression levels of hypertrophic, fibrotic, and inflammatory markers in cardiac tissue. Expression of ANP (a), BNP (b), TGF-β (c), GATA4 (d), Myc (e), and Nfkb (f) was assessed by qRT-PCR. Gene expression levels were normalized to β-actin and are presented as fold change relative to the control group. Data are shown as mean ± SD (n = 8 rats/group). HFD high-fat diet, B.a Bifidobacterium adolescentis, 8W/16W 8 or 16 weeks of treatment.

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  Fig. 7. Relative mRNA expression of cardiac function-related genes. Expression of (a) α-myosin heavy chain (MEF2D); (b) sarcoplasmic reticulum Ca2⁺ ATPase (SERCA2a); (c) natriuretic peptide receptor 1 (NPR1); (d) potassium voltage-gated channel subfamily Q member 1 (KCNQ1) was quantified by qRT-PCR. Data are presented as mean (n = 8 rats/group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. HFD high-fat diet, B.a Bifidobacterium adolescentis, 8W/16W 8 or 16 weeks of probiotic treatment.