ABC | Volume 114, Nº1, January 2019

Original Article Alegre et al. Açaí and myocardial ischemia-reperfusion in rats Arq Bras Cardiol. 2020; 114(1):78-86 Table 1 – Morphological variables and feed intake in rats submitted to global myocardial ischemia-reperfusion C (n = 9) A (n = 10) p-value Initial BW (g) 274 ± 15 281 ± 11 0.274 Final BW (g) 468 ± 29 448 ± 40 0.225 LV weight (g) 1.03 ± 0.09 1.02 ± 0.07 0.934 RV weight (g) 0.30 ± 0.03 0.29 ± 0.03 0.431 Liver weight (g) 14.4 ± 1.6 12.9 ± 2.6 0.154 Lung weight (g) 1.62 ± 0.03 1.54 ± 0.18 0.359 Daily chow intake (g) 26.1 ± 2.2 26.6 ± 1.8 0.246 C: control group; A: açaí group; BW: body weight; LV: left ventricle; RV: right ventricle. Values are expressed as the means ± standard deviation; p-value: t test. Evaluation of energy metabolism Samples of approximately 100 mg of LV tissue were homogenized in a sodium phosphate buffer (0.1 M, pH 7.0) and centrifuged. The supernatant was used to determine protein concentration and activity of the enzymes β -hydroxyacyl coenzyme A dehydrogenase, phosphofructokinase, lactate dehydrogenase, pyruvate dehydrogenase and citrate synthase. 23 The pellet was re-suspended with sodium phosphate buffer (0.1 M) containing 250 mM sucrose and 2 mM EDTA and used to determine the activity of enzymatic complexes of the electron transport chain (complexes I, II and ATP synthase). 24 Readings were performed on a microplate reader with controlGen5 2.0 software, and all reagents were obtained from the Sigma-Aldrich laboratory (Saint Louis, USA). Western blot LV samples (80 mg) were homogenized with 1 ml of radio immune precipitation assay extraction buffer (RIPA), centrifuged, and the supernatant was collected. The protein in the samples was quantified by the Bradford method; and the samples were used to determine total and phosphorylated nuclear factor signaling pathway κ B (NF- κ B), sirtuin 1 (SIRT1), and forkhead box protein O1 (FOXO1) protein expression. To determine the nuclear factor erithroid 2 (Nrf-2), LV samples were extracted with Nuclear Extraction Buffer. 25 All samples were diluted in Laemmli buffer. Protein electrophoresis was performed at 4°C on an 8 to 10% polyacrylamide gel (Mini-Protean 3 Electrophoresis Cell System, Bio-Rad, Hercules, USA). After electrophoresis, the gels were transferred to nitrocellulose membranes (Mini Trans-Blot system, Bio-Rad, Hercules, USA) in a wet transfer system followed by blocking with 5% skim milk powder solution. The membrane was washed, and primary antibodies were added (Santa Cruz Biotechnology, Inc., Europe). After overnight incubation, the membrane was washed with basal solution, and secondary antibodies were added (Santa Cruz Biotechnology, Inc., Europe). After 2 hours, the membrane was washed again in basal solution. Immunodetectionwasperformedwith thechemiluminescence method using the SuperSignal West Pico Chemiluminescent Substrate Kit (ThermoScientific, USA). Photo documentation by ImageQuant LAS 4000 (General Eletrikcs) was used to generate images, which were analyzed in Gel-Pro 32 (Media Cybernetics, Rockville, USA). The results obtained for the target proteins were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and the same control animal was included in all electrophoreses for standardization between the experiments. Statistical analysis Values obtained are presented as the means ± standard deviation (variables with normal distribution) or median and 25 and 75% quartile (variables with non-normal distribution). Normality was verified by the Kolmogorov–Smirnov test. Comparisons between the groups were performed with a non‑pared Student’s t- test (variables with normal distribution) or Mann-Whitney test (variables with non-normal distribution). Statistical analysis was carried out by use of the SigmaStat software, considering significance level at 5% for all analyses. Results General features The animals’ initial and final body weight did not differ between groups. Additionally, the left and right ventricles, liver and lung weights were similar between groups. Daily food intake was approximately 26 g for both groups (Table 1). Infarcted myocardial area We observed no difference in the area of myocardial infarction between groups A and C, as observed in Figure 1. Oxidative stress and myocardial energy metabolism Açaí supplementation promoted lower myocardial lipid hydroperoxide concentration, and greater activity of the enzymes catalase, SOD and GSH-Px in the myocardium of these rats was observed (Table 2). Regarding myocardium energy metabolism, açaí pulp supplementation promoted lower activity of the enzymes lactate dehydrogenase and phosphofructokinase and higher activity of the enzymes β -hydroxyacyl-coenzyme A dehydrogenase, citrate synthase and pyruvate dehydrogenase. In addition, we observed higher activity of complex I, complex II and ATP synthase in the group supplemented with açaí (Table 3). 80