ABC | Volume 113, Nº6, December 2019

Original Article Barbosa et al Nrf2, NF- κ B and PPAR β / δ in CAD patients Arq Bras Cardiol. 2019; 113(6):1121-1127 and is inhibited by its cytosolic repressor protein, Kelch‑like ECH-associated protein 1 (Keap1), which through the action of certain substances, including the ROS, which alters the conformation, decouples Nrf2 and thereby facilitates accumulation and nuclear translocation of Nrf2. In the nucleus, Nrf2 binds to regulatory sequences called AREs acting on genes that encode antioxidant and phase II detoxifying enzymes, including NADPH: quinone oxidoreductase 1 (NQO1). 15 The role of Nrf2 in reducing inflammation is related to the ability to antagonize NF- κ B indirectly by removing ROS. In addition, antioxidant enzymes appear to act directly on the reduction of inflammatory mediators. 15 Besides Nrf2, another target that has attracted interest and attention from research is the peroxisome proliferator- activated receptor- β / δ (PPAR β / δ ). However, the biological functions of PPAR β / δ and its effectiveness as a therapeutic target in the treatment of hypertension and CVD have not been elucidated. 16 PPAR β / δ  is the predominant subtype in the heart and several lines of evidence suggest a cardioprotective function of PPAR β / δ . 17 Preclinical studies suggest that PPAR β / δ activation promotes antihypertensive effects in established animal models 18 and the pharmacological activation of PPAR β / δ prevents endothelial dysfunction and downregulates inflammatory responses. 19,20 Furthermore, PPAR β / δ suppresses the activities of several transcription factors, including the NF‑ κ B. 21 Based on the fact that there are no studies about gene expression of Nrf2, NF- κ B and PPAR β / δ and its profile in CAD patients, the objective of this study was to evaluate the transcription factors NF- κ B and Nrf2 and PPAR β / δ mRNA expression in patients with CAD. Methods Subjects Forty-seven patients were enrolled in this study through a convenience sample where patients composed each group according to the presence or absence of CAD. Thirty-five patients (17 men and 18 women, mean age 62.4 ± 7.5 years, BMI 28.9 ± 4.9 kg/m 2 ) with CAD and/or abnormal findings of myocardial perfusion scintigraphy comprised the CAD group and twelve patients (5 men and 7 women, mean age 63.5 ± 11.5 years, 26.5 ± 6.2 kg/m 2 ) without CAD comprised the group without CAD. Eligible patients were older than 18 and attended the Nuclear Medicine Section at Hospital Universitário Antônio Pedro (Niterói, Rio de Janeiro, Brazil) to undergo myocardial scintigraphy. Patients with infection, cancer, chronic kidney disease (estimated glomerular filtration rate <60 mL/min), acquired immune deficiency syndrome (AIDS) and autoimmune disease were excluded. The control group consisted of hypertensive, dyslipidemic and/or diabetic patients not diagnosed with CAD, from the same hospital. Anthropometric Measures Anthropometric measurements were made by a trained staff member using standard techniques. Body mass index was calculated as weight in kilograms divided by height in square meters. 22 Blood pressure assessment BP was measured by the indirect method using auscultatory technique with sphygmomanometer and appropriate cuff in accordance with the dimensions of the patient's arm. Aneroid arterial pressure device – AD-2 was used on caster (pedestal), brand UNITEC Hospitalar (INMETRO ML 095 2007/ANVISA 10432300016). To assess BP, the procedure was initially explained to the patient who was resting for more than five minutes. The patient was sitting, feet resting on the floor, back resting on the chair, arm at heart level (mid-point of the sternum), supported, free of clothing, with the palm of the hand facing upwards and the elbow slightly flexed. HA was defined when systolic BP (SBP) values were greater or equal to 140 mmHg. 23 Analytic procedures and sample processing Blood was collected from each participant in the morning, after 12-hour overnight fasting, using a tube containing EDTA anticoagulant (1.0 mg/mL). Plasma was centrifugated and separated (15 min, 3000× g , 4°C) and stored at −80°C until analysis. Peripheral blood mononuclear cells (PBMCs) were collected, blood samples with EDTA were diluted in PBS and cells were separated in 5 mL Histopaque (Sigma-Aldrich) by centrifugation at 1800 g for 30 min. PBMCs were collected and washed twice with cold PBS and re-suspended and stored (−80°C) with 1 mL of Recovery TM cell culture freezing medium (Thermo Fisher Scientific) for RNA isolation. Biochemical and inflammation parameters Total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride, glucose and ultra-sensitive C-reactive protein levels were determined using Bioclin® automatic biochemical analyzer kits (Bioclin BS-120 Chemistry Analyzer). LDL-c was calculated using the Friedewald et al. equation. 24 Real-time quantitative PCR analysis Nrf2, NF- κ B, NQO1 and PPAR β / δ mRNA expressions were evaluated using real-time quantitative PCR (qPCR) from PBMCs according to Cardozo et al. 25 TaqMan ® Gene Expression Assays (Applied Biosystems) were used to detect Nrf2 (Hs00975961_g1), NF- κ B (Hs00765730_m1), NQO1 (Hs00168547_m1), PPAR β / δ (Hs00975961_g1) mRNA and the control gene GAPDH (Hs02758991_g1). Statistical analysis Shapiro-Wilk test was applied to test sample distribution. Results were expressed as mean ± SD (age, BMI, SBP, lipidic profile, glucose, Nrf2, NF-B, NQO1, PPAR β / δ ), median (interquartile range) (CRP) or percentage (hypertension, dyslipidemia, diabetes), as applicable. Unpaired Student’s t -test was used to compare the variables and groups with normal distribution and the Mann-Whitney-Wilcoxon test was used for nonparametric data. Correlations between variables were assessed by Pearson’s or Spearman coefficient correlation according to the distribution of the 1122