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
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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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