ABC | Volume 112, Nº2, February 2019

Original Article Arq Bras Cardiol. 2019; 112(2):154-162 Naderi et al Garlic and exercise increase cardiac angiogenesis Figure 1 – Real-time quantitative PCR analysis of miR-126 in the heart tissue of experimental groups. The values represent means ± S.E.M for 7 animals. *p < 0.05 vs control group, $$ p < 0.01 and $$$ p < 0.001 vs diabetes group, && p < 0.01 vs Diabetes+Exercise group, and # p < 0.05 vs Diabetes+Garlic group. 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 * $$$ $$ $$$&&# MiR-126: rno-miR-191 (Fold change) Control Diabetes Diabetes+Garlic Diabetes+Exercise Diabetes+Garlic+Exercise CA, USA). The amount of PCR products was normalized with housekeeping rno-miR-191 for miR-126 and miR-210. 37 We used the 2 -(ΔΔCt) method to determine the relative quantitative levels of miR-126 and miR-210. Results were expressed as the fold-difference to the relevant controls. Immunostaining for PECAM-1/ CD31 To investigate angiogenesis in the myocardium, transversal sections of the ventricles at their midportion were immediately isolated and fixed in 10% buffered-formalin solution, dehydrated in ascending grades of alcohol and embedded in paraffin. Then, serial 3 µm-thick sections were cut from them and floated onto charged glass slides according to standard histological processing. Tissue pieces were deparaffinised in xylene and dehydrated in a graded series of ethanol. Slides were incubated sequentially in proteinase K and 0.3% hydrogen peroxide to block endogenous peroxidase activity. Sections were overlaid by primary antibody CD31 (Santa Cruz, USA) – an angiogenesis marker – and incubated at +4°C overnight. Afterwards, the sections were washed and incubated with standard avidin–biotin complex (ABC; Santa Cruz) according to the protocol. Then the slides were incubated in DAB (Diamino-benzidine, Santa Cruz) as the chromagen, and counterstained with Mayer's hematoxylin. Finally, the sections were cleared in xylene, mounted with Entellan and analyzed with a light microscope. Assessment of immunostaining To evaluate immunostaining, 3 to 5 sections of 1 mm 2 were randomly selected at a magnification of 400×, depending on the size of the sample section. Both staining intensity and number of positive cells were evaluated semi-quantitatively. Intensity scoring for CD31 staining was obtained within each area at a 400× magnification. Each endothelial cell cluster of immunoreactivity expressing CD31 and forming lumen or vessels was counted as individual microvessels. Vascular structures positive for CD31 were counted for 5 to 6 slides per animal and 10 fields per slide. To assess immunostaining, we used the granulation tissue as a positive control, and the intensity of the staining was scored as follows: 0 (<10%); 1 (10% to 25%); 2 (25% to 50%); 3 (50% to 75%) or 4 (75% to 100%). 22 Lipid profile measurement Blood samples were obtained from the inferior vena cava, then centrifuged at 3500 rpm for 10 min at 4°C, and serum was collected. Triglycerides serum level was determined by enzymatic kits (ZiestChem Diagnostic kits, Iran) using glycerol as the standard. Additionally, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels were determined based on enzymatic methods by diagnostic kits, (ZiestChem, Iran) using cholesterol as the standard. Statistical analysis All results are expressed as mean ± SEM for seven animals, and analyses were performed using SPSS statistical software version 16. All parameters were tested for normality using the theone-sample Kolmogorov-Smirnov test. Data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test. The significant level was set at p < 0.05. Results Effects of garlic and voluntary exercise on miR-126 in the myocardium As shown in Figure 1, myocardial miR-126 expression level was significantly lower (p < 0.05) in rats with diabetes than in the control group. Treatment with garlic (p < 0.001), voluntary exercise (p < 0.01), or both combined increased 156