Experimental protocol

All procedures involving animals were in compliance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals [19], and ethical approval was granted by the Ethics Committee of São Paulo State University (1393/2021) approved on 28 September 2021.

Groups characterization

The sample size of the present study was calculated based on the work on myocardial collagens I and III of obese rats fed a saturated high-fat diet [20]. Thus, a sample of 24 animals was estimated with a sampling power of 90%, a two-tailed alpha of 0.05, and a sample loss of approximately 25%. Male Wistar rats (±187 g, n = 24) were kept in an environmentally controlled room (22 °C ± 3 °C; 12 h light-dark cycle and relative humidity of 60 ± 5%) and randomly distributed into 2 experimental groups by 20 weeks. During this period, the animals received ad libitum water and a control diet (C, n = 8) or a high sugar-fat diet (HSF, n = 16) containing 25% sucrose. The diets were prepared in our laboratory as previously published [21]. After this period, echocardiography was performed on the animals, and a 95% confidence interval for cardiac remodeling was constructed based on morphological, systolic, and diastolic variables in the HSF and C groups. This criterion is important because animals subjected to different dietary models do not always exhibit the expected response. This can lead to misclassification of animals and, consequently, false conclusions [22]. Therefore, the HSF animals that did not develop cardiac remodeling were excluded from the experiment, and two animals in the control group died during the experiment due to gavage. After, the animals were redivided into 3 groups to begin treatment with BLE by gavage daily (50 mg/kg) for 10 weeks: control diet + placebo (C, n = 6), HSF diet + placebo (HSF, n = 6), HSF + BLE (n = 6). The nutritional composition of both diets is presented in Table 1.

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Table 1. Diet composition and nutritional values.

https://doi.org/10.1371/journal.pone.0334015.t001

At the end of 30 weeks, the animals were fasted for 8 h and then anesthetized with thiopental (120 mg/Kg/i.p.) and euthanized by decapitation after verification of the absence of palpebral, foot, interdigital and caudal reflexes. Blood samples were collected, and the plasma was separated by centrifugation (800 × g at 4 °C for 10 min) for metabolic analyses. The adipose tissue was isolated, dissected, and weighed for nutritional parameter assessment and cardiac tissue was also collected from each animal for further analysis.

BLE

Bergamot leaves were harvested in a farm located in the Reggio Calabria region, Italy, and the dry extract was obtained at H&AD (Herbal & Antioxidant Derivatives S.r.l.) plant located in Localit`a Chiusi, 89032 Bianco (RC), Italy (www.head-sa.com). The processes involved in the extraction and administration were described and published by our research group [23]. The dosage was defined according to published fruit juice data [24].

Nutritional and metabolic parameters

The nutritional parameters included the following parameters: chow fed, water intake, caloric intake, final body weight (FBW) and adiposity index. Caloric intake was determined by multiplying the energy value of each diet (g × Kcal) by the daily food consumption. For the HSF group, caloric intake also considered the calories from water (0.25 × 4 × mL consumed). After euthanasia, the fat deposits (visceral (VAT), epididymal (EAT) and retroperitoneal (RAT)) were used to calculate the adiposity index by the following formula [25]:

Triglycerides levels were measured using specific kits (BIOCLIN^®, Belo Horizonte, MG, Brazil) and analyzed by a colorimetric-enzymatic method in an automatic enzymatic analyzer system (Chemistry Analyzer BS-200, Mindray Medical International Limited, Shenzhen, China). The homeostatic model of insulin resistance (HOMA-IR) was used as an insulin resistance index, calculated according to the formula [26]:

Glucose levels were measured in a blood drop using a handheld glucometer (Accu-Chek Performa, Roche Diagnostics Brazil Limited, São Paulo, Brazil), while insulin levels were assessed by an enzyme-linked immunosorbent assay (ELISA) method using commercial kits (Linco Research Inc., R&D Systems, Millipore and B-Brigde International Inc.) and the reading was performed in a Spectramax 190 microplate spectrophotometer (Molecular Devices^®, Sunnyvale, CA, USA).

Systolic blood pressure (SBP)

Evaluation was assessed in conscious rats by the non-invasive tail-cuff method with a NarcoBioSystems® Electro-Sphygmomanometer (International Biomedical, Austin, TX, USA). The animals were kept in a wooden box (50 × 40 cm) between 38 and 40 °C for 4–5 min to stimulate arterial vasodilation [27]. After this procedure, a cuff with a pneumatic pulse sensor was attached to the tail of each animal. The cuff was inflated to 200 mmHg pressure and subsequently deflated. The blood pressure values were recorded on a Gould RS 3200 polygraph (Gould Instrumental, Valley View, OH, USA). The average of three pressure readings was recorded for each animal.

Echocardiographic study

Doppler echocardiographic evaluation was performed by a single examiner at the 20th and 30th weeks. Animals were anesthetized with ketamine (50 mg/kg, i.p.) and xylazine hydrochloride (1 mg/kg, i.p.). After trichotomy of the anterior chest region, the animals were placed in slight left lateral decubitus for the exam. The equipment used was model Vivid S6 (General Electric Medical Systems, Tirat Carmel, Israel) with a multifrequency ultrasonic transducer 5.0–11.5 MHz. To implement structural measurements of the heart, the images were obtained in one-dimensional mode (M-mode) guided by the images in two-dimensional mode with the transducer in the parasternal position, minor axis [23].

Left ventricular (LV) evaluation was performed by positioning the cursor M-mode just below the mitral valve plane at the level of the papillary muscles. The following cardiac structures were used to analyze cardiac morphology: left ventricular diastolic diameter (LVDD), posterior wall diastolic thickness (PWDT), interventricular septum diastolic thickness (IVSDT), left ventricular mass index (LVM index) and relative thickness of the left ventricular (LVRT). The systolic LV function was assessed by the following parameters: ejection fraction (EF) and posterior wall shortening velocity (PWSV). The LV diastolic function was evaluated using the following indices: isovolumic relaxation time (IVRT), E wave deceleration time (EWDT) and E/E′ ratio.

Cardiac inflammatory markers

Cardiac tissue samples were homogenized and centrifuged as already described by our research group [2,3]. The levels of TNF-α and IL-6 were evaluated using the ELISA method using commercial kits from R&D System, Minneapolis, USA. The results were corrected by the protein amount.

Myocardial MMP-2 activity

The activity was determined by the zymography technique as described by Tyagi et al. [28]. In brief, samples were diluted in extraction buffer with 50 mM of Tris pH 7.4, 0.2 M of sodium chloride (NaCl), 0.1% of Triton X, and 10 mM of calcium chloride (CaCl₂). Protein in samples was quantified by the Bradford method [29]. Samples with 20 µg of protein were then diluted in application buffer with 0.5 M of Tris pH 6.8, 50% of glycerol, and 0.05% of bromophenol blue, and loaded into wells of 8% of sodium dodecyl sulfate (SDS) polyacrylamide containing 1% gelatin. Electrophoresis was run in a Bio-Rad apparatus at 80 V for 2 h. Gel was removed, washed with 2.5% of Triton X-100, and washed with 50 mM of Tris pH 8.4. Gel was then incubated at 37 °C overnight in activation solution with 50 mM of Tris pH 8.4, 5 mM of CaCl₂, and zinc chloride (ZnCl₂). Staining was performed for 2 h with 0.5% of comassie blue and destaining in 30% of methanol and 10% of acetic acid at room temperature on a rotatory shaker. To identify the gel activity of MMP-2, we used recombinant mouse/rat MMP-2 standard (R&D Systems) as a positive control. The gels were photographed by Gel Logic 6000 Pro (Carestream Health Inc.) and the intensity of gelatinolytic action (clear bands) was analyzed by GelPro 3.1 [30].

Myocardial type III collagen protein expression

LV samples were homogenized in RIPA buffer with protease and phosphatase cocktail inhibitors, and centrifuged for 20 min at 4 °C, for the supernatant collection. Protein concentration was determined by the Bradford method [29] and samples were diluted in Laemmli buffer and loaded (50 μg of protein) into a 10% SDS–polyacrylamide gel. Electrophoresis was performed in a Mini-Protean 3 Eletrophoresis Cell system (Bio-Rad, Hercules, USA), at 4 °C, at 50 V for 30 min and then at 120 V for 2 h. After that, the gels were transferred to a nitrocellulose membrane using Trans-Blot Turbo-Transfer System (BioRad) in a semi-wet transfer for 30 min at 25 V and 1.0 A, using transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol and 0.1% SDS). Next, the membranes were blocked in 5% bovine serum albumin solution at room temperature for 2 h in order to prevent unspecific bindings with antibodies and washed with basal solution (Tris 1 M, NaCl 2.5 M and Tween 20), pH 8.0. Incubation with primary antibodies was performed overnight at 4 °C in Tris-buffered saline solution containing Tween 20 (TBS-T) and 3% bovine serum albumin [5]. Antibody dilutions were 1:1000 for anti-collagen III (rabbit monoclonal antibody, ABCAM ab184993) and 1:1000 for anti-beta actin (rabbit polyclonal antibody, ABCAM ab8227). After incubating overnight at 4 °C in TBS-T containing 1% bovine serum albumin with the Abcam secondary antibodies (dilutions 1:5000 for goat anti-rabbit IgG H&L (HRP) (ABCAM ab205718)), proteins were revealed using the chemiluminescence method according to the manufacturer’s instructions (ECL SuperSignal^® West Pico Chemiluminescent Substrate, Thermo Scientific). Band intensities were evaluated by ImageQuant TL 1D Version 8.1 (GE Healthcare Life Sciences). β-actin protein was used as an endogenous control.

Statistical analysis

Data are presented as means ± standard deviation or medians (interquartile range). Differences among the groups were determined by one-way ANOVA followed by post hoc Tukey’s test for parametric data or Kruskal-Wallis followed by post hoc Tukey’s test for non-parametric data. Statistical analyses were performed using Sigma Stat for Windows Version 3.5. (Systat Software, Inc., San Jose, CA, USA). A p value < 0.05 was considered as statistically significant.