Low-dose ozone administration in vitro: analysing the cellular and molecular mechanisms of its therapeutic effect

Ozone (O₃), a potent oxidizing molecule, has gained increasing attention in medicine for its therapeutic applications in various fields, including dermatology, internal medicine, orthopaedics, and immunology. The effectiveness of O₃ therapy is attributed to its ability to induce a controlled oxidative stress. By generating reactive oxygen species and lipid oxidation products, O₃ stimulates adaptive responses that enhance the body's endogenous antioxidant defence and modulate inflammation. O₃ exerts its therapeutic effects through hormesis, where low-level oxidative stress elicits beneficial cellular adaptations without inducing damage via Nrf2 activation, which leads to the transcription of genes involved in antioxidant and anti-inflammatory responses. Despite the wide use of O₃ as adjuvant/complementary therapy in many diseases, the cellular and molecular mechanisms accounting for its beneficial effects remain largely unclear. The aim of my PhD work was to contribute to the knowledge of these mechanisms by investigating the effects of low-dose O3 treatment in different in vitro models. We conducted a metabolomic analysis combined with using haemogasometry, light microscopy, and bioanalytical assays on blood treated in vitro with O3 to profile molecular changes. This integrated approach offered novel insights into metabolic adaptations involving amino acids, carbohydrates, lipids, and nucleotides. These alterations support the maintenance of redox homeostasis and energy production, underscoring the broad biochemical impact of low-dose O₃ treatment on blood. To understand the beneficial effects of O3 observed in neurodegenerative diseases, we employed a multimodal microscopy approach and molecular assays to investigate structural and functional modification of activated microglial HMC3 cells following O3 treatment. We found that O₃ reduces microglial cell motility and inflammatory cytokine secretion via Nrf2 activation. To enhance the regenerative and anti-inflammatory potential of platelet-rich plasma (PRP), we treated it with O₃ or procaine, and investigated the effects on platelets with transmission electron microscopy and bioanalytical assays. Both treatments enhanced the release of key platelet-derived factors without damaging platelets, suggesting that O3 and procaine may act synergistically through complementary secretory pathways, increasing the PRP's therapeutic potential. We examined the impact of low-dose O₃ on tamoxifen-treated MCF7 breast cancer cells, utilizing histochemical and molecular methods. In fact, concern exists regarding potential interference of O3 therapy, used in cancer patients to alleviate side effects of treatments, with anticancer drugs. Results showed that O₃ does not enhance tumour cell viability, proliferation, or migration, nor does it increase antioxidant responses to levels that might confer cytoprotective benefits, thus supporting O₃ administration to cancer patients without promoting drug resistance. Finally, given the susceptibility of mitochondria to oxidative stress, we examined the mitochondrial response in C2C12 muscle cells exposed to low-dose O₃ using fluorescence and electron microscopy alongside biochemical assays. Findings demonstrated that low-dose O₃ modulates mitochondrial structure and function in a dose dependent manner, increasing their size, cristae extension and respiratory chain enzymes. Moreover, O3 was found to favour the association of Nrf2 with the outer membrane of mitochondria, probably protecting these organelles against oxidative stress. Collectively, our studies emphasize the central role of Nrf2 in cellular response to O3 treatment. In fact, Nrf2 activates antioxidant defences, modulates inflammation and supports mitochondrial function, maintaining cell homeostasis under stress conditions. This is consistent with the therapeutic efficacy of O3 in treating diseases characterised by oxidative stress and inflammation.