raw data20220706

Biochemical index assay Blood lactic acid (Bla) was measured in whole blood with an EFK semi-automatic lactic acid analyzer (EFK, German). The total number of red blood cells (RBC), hematocrit (HCT%) and the total number of white blood cells (WBC) in immune cells were measured in 2 mL blood samples, using the automatic blood cell analyzer（mindray, BC-5130, China). Creatine kinase (CK) and blood urea (BUN) in serum were detected using a fully automatic biochemical analyzer (Chemray-420, Rayto, China). ELISA Serum samples were taken out from the -80 °C freezer, thawn on ice and centrifuged at 4℃ with 2,000 g for 5 min. The level of 5-HT in serum was assayed by ELISA (ABN-KA1894 Serotonin ELISA Kit) using a Multiskan Spectrum (Thermo Scientific, USA). Samples were analysed in duplicates. Physiological index assay RR-intervals (or Normal to Normal intervals-NN), standard deviation of RR intervals (SDNN) Heart rate (HR) at rest and after exercise, heart rate variability (HRV), were measured with ECG collector (EL-191 Good friend holter, China). Measurements of HRV were: standard deviation of RR intervals (SDNN), root mean square of successive differences (RMSSD), low-frequency power (LF) , high-frequency power (HF) and LF/HF. SDNN reflects the total change of heart rate variability, and RMSSD reflects the activity of parasympathetic nerves. LF reflects sympathetic nerve activity, and HF reflects Parasympathetic nerve activity, LF/HF reflects the balance of sympathetic nerves and parasympathetic nerves. For 100-meter section results, a stopwatch was used to record the time of each 100-meter segment during the 1500-m freestyle swimming session. A wingate anaerobic power test was completed before the 1500-m freestyle exercise and after the 5-min HRV was detected, and the POLAR RC3 (POLAR, Finland) heart rate monitor was used to monitor the heart rate in real-time. The 30s fastest pedaling power test was conducted on a MONARK-894E power bicycle (Sweden), at the power load the test athlete’s weight (kg)´0.075. The maximum power (PP) and the relative maximum power(PP/KG) were recorded. power decline (PD), relative power decline (PD/KG), power decline rate (FI), and fatigue percentage (PD%) are used to evaluate the anaerobic capacity of subjects. Response Time (RT) and Accuracy (ACC) during Stroop Task According to the experimental purpose, the color matching word Stroop task was designed by the E-prime software, and the marking signal data formed by stimulating the brain was recorded. For this trial, the color word ‘red’, ‘green’, or ‘blue’ were printed in the congruent color that is the correct response (e.g., red was printed in red). Otherwise, the color word was printed in an incongruent color that is an incorrect response (e.g., red was printed in blue). In the NIRsport portable near-infrared spectrum test image (fNIRs), the Stroop task was performed before exercise and 15 min after exercise to obtain the response time and accuracy data. EVs isolation The Umibio Extracellular vesicles extraction kits (Umibio, China) were employed to isolate the EVs from plasma. Briefly, 3 ml of pre-chilled PBS and 1 ml of Blood Pure Exo Solution was added to the stored supernatant. The mixture was vortexed for 1 min, incubated at 4 °C for 2 h and centrifuged at 4 °C, 10,000 x g for 60 min. The supernatant was discarded, while the pellet rich in EVs particles was resuspended with 0.5 ml of PBS. After it is dissolved, the resuspension was transferred the resuspension to a new centrifuge tube. Identification of EVs with microscope A transmission electron microscope (TEM) was used to directly observe the characteristics and morphology of the EVs for identification. After resuspending the extracted EVs with 50-100 ul of 2% paraformaldehyde, 50 ul of the EVs suspension was placed on the copper mesh and allowed to stand still at room temperature for 20 min. 1% glutaraldehyde was fixed for 5 min, 4% uranyl acetate was used to negatively stain for 5 min, then the EVs pictures were photographed. The EVs were tracked using Nanosight platform nanoparticle tracking analysis technology (NTA), and distinguished from other particles, and finally the concentration and particle size distribution of EVs were detected. Western blot The isolated EVs were lysed with RIPA lysis buffer (Umibio, China), and the protein concentration was determined by the BCA method. SDS-PAGE was performed on a 10% polyacrylamide gel and transferred to a PVDF membrane. Sealed with 5% skimmed milk powder at room temperature for 1 hour, incubated overnight at 4 °C with primary antibody (CD63, ALIX) solution, followed by secondary antibody to block for 1 hour at room temperature, and then the protein bands were visualized using enhanced chemi-luminescence (ECL) reagent. Total RNA extraction and concentration assay In brief, 200 ul of EVs sample was taken into RNase-Free centrifuge tube, mixed with an equal volume of 2 x Denaturing Solution and placed on ice for 5 min. 200 ul of phenol: chloroform solution was added into the tube, and then vortexed for 30 seconds, centrifuged at 10,000 x g for 5 min at 4°C. The supernatant was transfered to a new centrifuge tube, mixed with 1.25-fold volume of absolute ethanol, then 700 ul of the solution was transfered to the spin column, centrifuged at 10,000 x g for 30 seconds at 4°C until the solution passed through the spin column. 700 ul of miRNA washing solution 1 was add, and centrifuged at 10,000 x g for 30 seconds at 4°C until the solution passed through the spin column. 500 ul of washing solution 2 was added, and centrifuged at 10,000 x g for 30 s at 4 °C until the solution passed through the spin column. The supernatant was discarded and the spin column was put back into the collection tube. Centrifuged at 10,000 x g for 1 min at 4 °C until all the solution passed through the spin column. The adsorption column was put into a new collection tube and 50 ul of preheated washing solution was added at 95 °C. The precipitate was collected by centrifugation for 30s, which is total RNA, and the concentration of total RNA extracted from EVs was detected by an Agilent 2100 Bioanalyzer System. miRNA Quality Detection and Library Construction The 3' and 5'adapters was connected, 2 ul of QIAseq miRNA NGS RT Initiator was added for RNA reverse transcription, mixed with QIAseq Beads and QIAseq miRNA NGS Bead Binding Buffer thoroughly for magnetic beads (QMN Beads) preparation. cDNA synthesis and purification was performed on ice, and then library amplification. PCR product fragments were screened, and the concentration of the library was detected using Qubit dsDNA HS Assay. The Agilent 2100 Bioanalyzer High Sensitivity DNA Assay was used to detect the fragment distribution range of the library. The main peak of the library was ~170-180 bp. Finally, the high-throughput Illumina 2x150 bp platform was used to detect the total miRNA extracted from EV. Differential expressions of miRNAs Using the Expdiff method, the known miRNAs in EVs was counted to determine whether there were significant differences in the expression levels between EVs, and the expression levels of miRNAs co-expressed between samples were compared using Log2(fold-change) and Scatter plots.