There were two different groups in the study. The athletic group included 43 male basketball, volleyball, and handball players (mean age: 20.65 ± 2.46 years, height: 189.8 ± 7.22 cm, bodyweight: 87.32 ± 14.1 kg), and the sedentary group included 43 healthy men (mean age: 22.49 ± 3.51 years, height: 178.63 ± 7.46 cm, bodyweight: 76.77 ± 11.63 kg). The athletic group consisted of individuals aged 18–35 years who had been training regularly for at least 3 months. They had been doing sport for a mean 9.87 ± 3.71 years. The sedentary group consisted of healthy individuals with similar physical characteristics to the athletes and the same age range, but who had not peformed regular exercise for at least 3 months.
The criteria for inclusion in the study were that the participants were not obese [body mass index (BMI) < 30 kg/m2] and did not use tobacco-related products, alcohol, drugs, and antioxidants. All athletes were instructed to stop training 2 days before the measurements. All participants were instructed to continue their usual nutrition habits and to attend for measurements with four hours of fasting.
The research was approved by Ege University Faculty of Medicine Clinical Research Ethics Committee (Approval number: 18 − 7.1/49).
All participants were informed about the study and written informed consent was obtained before taking the measurements.
The height and body weight measurements of the participants were performed using a portable height measuring device and digital scales (Seca, Germany). BMI was calculated using the following formula: BMI = Weight (kg) / height (m2). All participants performed a standard warm-up procedure. The procedure consisted of the first 4 speed levels of the Yo-Yo Intermittent Recovery Test level 1 (Yo-Yo IR1), dynamic stretching, basic running, and change of direction exercises.
The Yo-Yo IR1 was applied to all participants as an acute maximal exercise after standard warming up. It is a standard test that includes running periods (20 x 2 = 40 m) with gradually increasing speeds. Active rest is performed by walking or jogging at a distance of 10 m (2x5) for 10 seconds after every 40 m run. The Yo-Yo IR1 lasts 10–20 minutes depending on the ability level of the participants [23]. The test was continued until the participants were exhausted. The distance that they could not continue and their heart rate during this time were recorded. If the participants could not complete the 40 m distance despite the auditory signal and this situation occurred twice consecutively, the test was ended for these participants by the researchers.
A blood sample was taken 20–25 minutes before and 10 minutes after the Yo-Yo IR1. MMP9, TIMP1, MMP9/TIMP1 ratio, blood lipids and lipoproteins [total cholesterol (TC), high and low-density lipoprotein cholesterol (HDL-C and LDL-C)] and indicators of muscle damage [creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT)] were determined from postprandial venous blood samples. Blood samples were kept at room temperature for 20 minutes, then centrifuged at 2000 g for 15 minutes and their serums were separated. Serum samples were stored at -82°C until required for further analysis. CK, AST, and ALT activities, TC, HDL-C and LDL-C were assessed using standard enzymatic-colorimetric methods with an autoanalyzer (Roche Cobas 8000, Switzerland). Serum MMP9 and TIMP1 levels were determined using enzyme-linked immunosorbent assay (ELISA) by measuring at 450 nm on a microplate reader (Dialab ELx800, Austria). Commercial kits (Human ELISA Kit Elabscience, USA) were used for these procedures. The MMP9/TIMP1 ratio was calculated using the molecular weight of MMP9 (92kDa) and TIMP1 (28kDa).
MMP9 -1562 C/T and TIMP1 372 T/C polymorphisms were determined from DNA samples isolated from peripheral blood leukocytes using standard techniques according to the manufacturer’s (QIAGEN®-Hilden, Germany) instructions. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) was used for the determination of genotypes. The primers used were 5’-GCCTGGCACATAGTAGGCCC-3’ (forward) and 5’-CTTCCTAGCCAGCCGGCATC-3’ (reverse) for the MMP9 gene; 5’-GCACATCACTACCTGCAGTC-3’ (forward) and 5’-GAAACAAGCCCACGATTTAG-3’ (reverse) for the TIMP1 gene. PCR components used per sample were as follows: 11.5 µL of DdH2O, 2.5 µL of 10X PCR buffer, 1 µL of dNTP mix, 2 µL of MgCl2, 1.25 µL of forward primer, 1.25 µL of reverse primer, 0.5 µL of Taq polymerase, 5 µL of DNA, 25 µL in total. The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, primer annealing for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. The primer annealing temperature was set to be 61°C for the MMP gene and 54.5°C for the TIMP1 gene. Following amplification, the PCR products (MMP9’s 436 bp, TIMP1’s 175 bp) were digested with the restriction endonuclease (MMP9- SphI, TIMP1- BssSI). Fragments were separated through electrophoresis in 6% agarose gel at 100V for 120 min and visualized using ultraviolet transillumination after ethidium bromide staining. A single band at 436 bp represented C homozygous, two bands at 194–242 represented T homozygous, and three bands at 294-242-436 bp represented heterozygous for MMP gene. For the TIMP1 gene, a single band at 175 bp represented T hemizygous, and two bands at 20–155 bp represented C hemizygous. Additionally, 10% of the samples were sequenced using a next-generation system (Nextseq) to confirm the genotyping results.
Statistical analyses were performed using SPSS (IBM SPSS Statistics for Windows, version 24.0. Armonk, NY). Results are presented mean ± standard deviation except in the tables, and p < 0.05 was considered for significant statistical difference. According to the results of the normality test (Shapiro-Wilk test), comparisons of physical and physiologic characteristics and comparisons of the measurements of the groups before acute exercise were performed using the Mann-Whitney U test. Comparisons of the measurements before and after the acute exercise in the same group were performed using the Wilcoxon test. Genotype and allele frequencies were calculated using the Hardy-Weinberg equation. Comparisons of genotype frequencies of the sedentary and athletic groups were performed using the Pearson Chi-square test. Comparison of the pre- and post-exercise measurements of the sedentary and athletic groups according to MMP9 -1562 C/T and TIMP1 372 T/C polymorphism genotypes was performed using three-way repeated-measures analysis of variance (ANOVA) [exercise (pre/post Yo-Yo IR1) x group (sedentary/athlete) x polymorphism genotype/allele group].