ANALYSIS OF THE PPARD GENE EXPRESSION LEVEL CHANGES IN FOOTBALL PLAYERS IN RESPONSE TO THE TRAINING CYCLE
Domańska-Senderowska D, Snochowska A, Szmigielska P, Jastrzębski Z, Jegier A, Kiszałkiewicz J, Dróbka K, Jastrzębska J, Pastuszak-Lewandoska D, Cięszczyk P, Maciejewska-Skrendo A, Zmijewski P, Brzeziańska-Lasota E
*Corresponding Author: Piotr Zmijewski, Ph.D., Faculty of Medicine, University of Information Technology and Management in Rzeszow, Rzeszow, Poland. Tel: +48-22-384-08-12. Fax: +48-22-835-09-77. E-mail: zmijewski@op.pl
page: 19

MATERIAL AND METHODS

The study was approved by the Medical University of Lodz Ethics Committee (RNN /157/16/KE). All participants gave full written informed consent prior to commencement of the study. Twenty-two young male football players (17.5±0.7 years, 178±0.7 cm, 68.05±9.18 kg) participated in the study. Before the experiment, all the players took part in the 2 months of preliminary training. This experiment took place during 2 months training cycle (from middle of April to middle of June 2016). All the players were subjected to the same football training that consisted of strength, speed and endurance exercises. The typical weekly training load during the experiment involved different training drills in the work week (two mornings and five afternoons during the week) and the competition game on Saturday. Training drills included: interval run, small-sided games and plyometric, speed, technical, coordination, tactical and aerobic exercises. Small-sided games were carried out on the field (44 × 33 m) on Tuesdays with 120 square meters per football player. The subjects played four games, 4 min. each with 3 min. active break that consisted of walking and muscle relaxing exercises. The intensity of the training was imposed by the heart rate (HR) that was equal or higher than anaerobic threshold (AnT) value but did not exceeded 90.0% HRmax value. Individual maximal intensity run and run at lactate threshold of the player was determined on a synthetic field at the beginning of an experiment. The test protocol included 3.5-5.0 min. running stages separated by a 1 min. rest, during which a capillary blood sample was taken from the fingertip. The initial speed was set at 2.8 m/s and increased by 0.4 m/s after each stage until exhaustion [20]. Collection of Biological Material. Blood samples were collected before (T1) and 12 hours after training (T2). Before blood collection, the players had been resting in the supine position for 10 min. Blood was aspirated into 5 mL EDTA -containing tubes. For lymphocyte isolation, a density gradient cell separation solution Histopaque®-1077 (Sigma-Aldrich Co., St. Louis, MO, USA) was used. Blood needed for determination of lipid profiles was collected into the serum separator tubes. Gene Expression Analyses. RNA isolation was performed using the mirVana™ miRNA Isolation Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. The quality and quantity of isolated RNA was spectrophotometrically assessed (Eppendorf BioPhotometrTM Plus; Eppendorf, Hamburg, Germany). The purity of total RNA (ratio of 16S to 18S fraction) was determined by automated electrophoresis using the RNA Nano Chips LabChipplates in Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Complementary DNA (cDNA) was transcribed from 100 ng of total RNA, using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) in a total volume of 20 μL, according to manufacturer’s protocol. The relative expression analysis was performed in 7900HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan probes for the study gene PPARD (Hs00987008_m1) and ACTB gene (Hs99999903_m1) used as an endogenous control. The PCR mixture contained cDNA (1-100 ng), 20 × TaqManR Gene Expression Assay, 2 × KAPA PROBE Master Mix (2 ×) ABI PRISM® Kit (Kapa Biosystems, Wilmington, MA, USA), and RN ase-free water in a total volume of 20 μL. The expression levels relative quantification (RQ) values of the studied gene were calculated using the ΔΔ CT method, with the adjustment to the β-actin expression level and in relation to the expression level of calibrator, for which RQ value was equal to 1. Lipid Profile Analyses. The concentration of chosen plasma lipids was determined using a high performance laboratory analyzer (Olympus AU680; Beckman Coulter, Atlanta, GA, USA). The analytical enzymatic methods used in our study were: method with esterase and cholesterol oxidase for total cholesterol, enzymatic method with esterase and cholesterol oxidase after prior forming an immunological complex with other lipoproteins for highdensity lipoprotein (HDL) cholesterol and the enzymatic method with phosphoglycerol oxidase (determination of H2O2 using peroxidase) for triglycerides (TGs). The lowdensity lipoprotein (LDL) cholesterol concentration was calculated. Body Fat Analyses. The body FAT data, absolute FAT (kg) and relative FAT (%), were determined using an electrical bioimpedance method with mode for athletes (Tanita MC-980 MA, Abdominal Fat Analyzer AB-140; Tanita, Tokyo, Japan). The football players were tested in the morning (in a fasting state), 24 hours after training. Statistical Analyses. A Shapiro-Wilk test was carried out to assess the normal distribution. The Wilcoxon signed rank test was used to compare the levels of relative expression values (RQs) in both time points. Spearman’s rank correlation coefficient was used to assess the correlation between gene relative expression level and cholesterol concentration or fat mass in both time points. Outcomes of p <0.05 were considered to be statistically significant. Calculations were based on the Statistica for Windows, version) 13.0 program.



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