117 | Modulation of DNA methylation patterns and heat shock proteins following exercise and probiotic supplementation

A. Boatta^1|2, G. Messina¹, A. Pagliaro³, A. Alioto³, C. Rossi³, L. Paladino⁴, F. Rappa⁶, F. Naselli⁵, F. Cappello⁶, R. Barone⁶, P. Proia³ | ¹Department of Human Sciences and Promotion of Quality of Life, San Raffaele Roma Open University, Roma, Italy; ²Institute for Biomedical Research and Innovation, CNR, Palermo, Italy; ³Sport and Exercise Sciences Research Unit, Department of Psychology, Educational Sciences and Human Movement, University of Palermo, Palermo, Italy; ⁴Department of Theoretical and Applied Sciences DiSTA, eCampus University, Novedrate, Italy; ⁵Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy; ⁶Section of Human Anatomy, Department of Biomedicine, Neuroscience and Advanced Diagnostics BIND, University of Palermo, Palermo, Italy.

Physical activity is a stimulus that influences overall physiology and induces biochemical changes in various tissues, including modulation of genes expression. These effects can be mediated by epigenetic modifications, such as DNA methylation, which regulate chromatin structure and modulate its accessibility to transcription factors. Meanwhile, heat shock proteins (HSPs), including HSP60, HSP70 and HSP90, act as molecular chaperones that facilitate proper protein folding and protect cells from homeostatic imbalances. Physical exercise, as a physiological stressor, increases the expression of these HSPs in plasma, mononuclear cells and some tissues such as muscle, liver, heart and brain. Recent studies suggest that probiotic supplementation may influences these pathways further by modulating DNA methylation and the proteostatic response. However, experimental evidence in humans is still limited. This pilot study aimed to evaluate the influence of probiotic supplementation on DNA methylation profiles and the expression of HSP60, HSP70 and HSP90 in plasma exosomes of physically active individuals, while exploring the interaction between exercise and nutritional modulation. Participants followed a 12-week daily supplementation programme involving a probiotic containing Lactobacillus and Bifidobacterium strains. Blood samples were collected before and after the probiotic supplementation. DNA methylation was analysed using MESAP-PCR, while HSP levels in plasma exosomes were assessed using Western blot analysis. At the start of the study, the subjects showed a state of basal hypomethylation, which is consistent with adaptation to physical exercise. After 12 weeks of supplementation, however, a trend towards hypermethylation was observed, which suggests that probiotics may have a protective and anti-inflammatory effect. HSP analysis revealed different results. While HSP70 showed no significant changes, HSP60 and HSP90 revealed a downward trend, indicating a reduction in the activation of the cellular stress response. Overall, these data suggest that probiotic supplementation combined with exercise may contribute to greater epigenetic stability and more efficient functional and mitochondrial adaptation. These preliminary results highlight the complementary role of physical activity and probiotic supplementation in modulating epigenetic and proteostatic mechanisms to promote cellular plasticity and homeostasis. Further research involving larger cohorts and additional parameters of inflammation and performance is essential to confirm these effects and understand their physiological implications.