Study of the Modulation of Soccer Players’ Salivary Extracellular Vesicles in Response to a Training Session: an Innovative Source of Exercise Biomarkers?

Purpose: Physical exercise represents a highly complex metabolic stimulus which leads to systemic adaptations that improve cardiorespiratory fitness and consequently the capacity for oxygen uptake by working muscles. For this reason, investigating these mechanisms on a molecular level would be of particular importance for the development of personalized exercise protocols to increase sport performance. In this context, extracellular vesicles (EVs) released following an exercise bout and isolated from accessible biological fluids (such as saliva) could represent a non-invasive, complex and dynamic diagnostic tool. Therefore, the present project aims to develop an effective EV isolation protocol from saliva samples, to characterize and utilize these vesicles as a novel source of exercise biomarkers. Methods: Saliva samples were collected from 18 professional soccer players (Ternana Football Club – BKT Italian League) pre- and post-training (2h, 15h and 24h recovery) and then processed by serial ultracentrifugation. In the preseason period, the training session consisted in a strength workout (30 min.) and small side games (36 min.). In order to characterize salivary EVs, protein content (BCA), particle counting (NTA) and vesicles marker detection (Dot Blot) were performed. Results: The obtained NTA results showed typical EV size concentration and distribution. Interestingly, Dot Blot highlighted higher CD63 and HSP60 positivity levels in EXOs compared to MVs, and assessed a tendency of the highest CD63 and HSP60 positivity at 15 and 24h hours post-exercise, suggesting that there is an increase in EVs in response to physical exercise, which could reveal metabolic changes and muscle adaptation to exercise. Conclusions: The ultracentrifugation protocol applied allowed us to isolate salivary EVs and to further reveal an increase in EVs following the training session. Hence, Salivary EVs could represent an innovative source of exercise biomarkers. For this purpose, further studies would be needed to confirm these preliminary data.