Gabriele Tabaracci

In the last decades, ozone (O₃)-based medical treatments have become a widely applied complementary therapy for several pathological conditions. O₃ is administered at low dosages since the induction of a mild oxidative stress does not cause damage but stimulates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2). Mitochondria are sensitive to even mild oxidative stress, thus being a responsive target for O₃. This study aimed to evaluate the mitochondrial response to low O₃ doses used for medical treatments. As the skeletal muscle represents a primary target in local O₃ treatments, a murine non-tumoral muscle cell line was selected as an appropriate in vitro model. Transmission electron microscopy, biochemistry, and flow cytometry provided original information on the O₃ dose-dependent modifications of mitochondrial structural and molecular features. Low O₃ doses promoted an increase in mitochondrial area and in cristae extension, as well as an enhancement of the electron transport chain complexes and of antioxidant catalase and manganese-dependent superoxide dismutase. Nrf2 maintained its association with the outer mitochondrial membrane, thus exerting its protective role. All mitochondrial modifications were observed 24 h after treatment and disappeared after 48 h, demonstrating that cells promptly respond to the O₃-driven oxidative stress, effectively restoring homeostasis.

Ozone (O₃) is an unstable, highly oxidative gas that rapidly decomposes into oxygen. The therapeutic use of O₃ dates back to the beginning of 20th century and is currently based on the application of low doses, inducing moderate oxidative stress that stimulates the antioxidant cellular defences without causing cell damage. In recent decades, experimental investigations allowed the establishment of some basic mechanisms accounting for the therapeutic effects of eustress-inducing low-dose O₃. In this review, special attention was given to the impact of O₃ administration on the cell oxidant–antioxidant status, O₃ anti-inflammatory and analgesic properties, efficacy in improving tissue regeneration, and potential anticancer action. Low O₃ concentrations proved to drive the cell antioxidant response mainly by activating nuclear factor erythroid 2-related factor 2. The anti-inflammatory effect relies on the downregulation of pro-inflammatory factors and the modulation of cytokine secretion. The painkilling action is related to anti-inflammatory processes, inhibition of apoptosis and autophagy, and modulation of pain receptors. The regenerative potential depends on antioxidant, anti-inflammatory, anti-apoptotic, and pro-proliferative capabilities, as well as fibroblast activation. Finally, the anticancer potential is based on oxidant and anti-inflammatory properties, as well as the inhibition of cell proliferation, invasion, and migration and the induction of apoptosis.

Medical treatment with low ozone concentrations proved to exert therapeutic effects in various diseases by inducing a cytoprotective antioxidant response through the nuclear factor erythroid derived-like 2 (Nrf2) transcription factor pathway. Low ozone doses are increasingly administered to oncological patients as a complementary treatment to mitigate some adverse side-effects of antitumor treatments. However, a widespread concern exists about the possibility that the cytoprotective effect of Nrf2 activation may confer drug resistance to cancer cells or at least reduce the efficacy of antitumor agents. In this study, the effect of low ozone concentrations on tamoxifen-treated MCF7 human breast cancer cells has been investigated in vitro by histochemical and molecular techniques. Results demonstrated that cell viability, proliferation and migration were generally similar in tamoxifen-treated cells as in cells concomitantly treated with tamoxifen and ozone. Notably, low ozone concentrations were unable to overstimulate the antioxidant response through the Nfr2 pathway, thus excluding a possible ozone-driven cytoprotective effect that would lead to increased tumor cell survival during the antineoplastic treatment. These findings, though obtained in an in vitro model, support the hypothesis that low ozone concentrations do not interfere with the tamoxifen-induced effects on breast cancer cells.

Platelet-rich plasma (PRP) is gaining more and more attention in regenerative medicine as an innovative and efficient therapeutic approach. The regenerative properties of PRP rely on the numerous bioactive molecules released by the platelets: growth factors are involved in proliferation and differentiation of endothelial cells and fibroblasts, angiogenesis and extracellular matrix formation, while cytokines are mainly involved in immune cell recruitment and inflammation modulation. Attempts are ongoing to improve the therapeutic potential of PRP by combining it with agents able to promote regenerative processes. Two interesting candidates are ozone, administered at low doses as gaseous oxygen-ozone mixtures, and procaine. In the present study, we investigated the effects induced on platelets by the in vitro treatment of PRP with ozone or procaine, or both. We combined transmission electron microscopy to obtain information on platelet modifications and bioanalytical assays to quantify the secreted factors. The results demonstrate that, although platelets were already activated by the procedure to prepare PRP, both ozone and procaine induced differential morpho-functional modifications in platelets resulting in an increased release of factors. In detail, ozone induced an increase in surface protrusions and open canalicular system dilation suggestive of a marked α-granule release, while procaine caused a decrease in surface protrusions and open canalicular system dilation but a remarkable increase in microvesicle release suggestive of high secretory activity. Consistently, nine of the thirteen platelet-derived factors analysed in the PRP serum significantly increased after treatment with ozone and/or procaine. Therefore, ozone and procaine proved to have a remarkable stimulating potential without causing any damage to platelets, probably because they act through physiological, although different, secretory pathways.

The mild oxidative stress induced by low doses of gaseous ozone (O₃) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O₃ target. In this in vitro study, we investigated the mitochondrial response to low O₃ doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O₃ doses. The O₃ concentration of 10 μg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 μg O₃-treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O₃ concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function.

Oxygen–ozone (O₂–O₃) therapy is increasingly applied as a complementary/adjuvant treatment for several diseases; however, the biological mechanisms accounting for the efficacy of low O₃ concentrations need further investigations to understand the possibly multiple effects on the different cell types. In this work, we focused our attention on fibroblasts as ubiquitous connective cells playing roles in the body architecture, in the homeostasis of tissue-resident cells, and in many physiological and pathological processes. Using an established human fibroblast cell line as an in vitro model, we adopted a multimodal approach to explore a panel of cell structural and functional features, combining light and electron microscopy, Western blot analysis, real-time quantitative polymerase chain reaction, and multiplex assays for cytokines. The administration of O₂–O₃ gas mixtures induced multiple effects on fibroblasts, depending on their activation state: in non-activated fibroblasts, O₃ stimulated proliferation, formation of cell surface protrusions, antioxidant response, and IL-6 and TGF-β1 secretion, while in LPS-activated fibroblasts, O₃ stimulated only antioxidant response and cytokines secretion. Therefore, the low O₃ concentrations used in this study induced activation-like responses in non-activated fibroblasts, whereas in already activated fibroblasts, the cell protective capability was potentiated.

Autohemotherapy is the most used method to administer O₂-O₃ systemically. It consists in exposing a limited amount of blood to a gaseous O₂-O₃ and reinfusing it, thus activating a cascade of biochemical pathways involving plasma and blood cells that gives rise to antioxidant and anti-inflammatory responses. The therapeutic effects strictly depend on the O₃ dose; it is therefore necessary to understand the relationship between the O₃ concentration and the effects on blood cells involved in antioxidant and immune response. Here we performed a basic study on the effects of the low O₃ concentrations used for autohemotherapy on the structural and functional features of the human T-lymphocyte-derived Jurkat cells. Ultrastructural, biomolecular, and bioanalytic techniques were used. Our findings showed that 10, 20, and 30 µg O₃ concentrations were able to trigger Nrf2-induced antioxidant response and increase IL-2 secretion. However, viability and proliferation tests as well as ultrastructural observations revealed stress signs after treatment with 20 and 30 µg O₃, thus designating 10 µg O₃ as the optimal concentration in combining cell safety and efficient antioxidant and immune response in our in vitro system. These data offer novel evidence of the fine regulatory role played by the oxidative stress level in the hormetic response of T lymphocytes to O₂-O₃ administration.

In clinical practice, administration of low ozone (O₃) dosages is a complementary therapy for many diseases, due to the capability of O₃ to elicit an antioxidant response through the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-dependent pathway. Nrf2 is also involved in the adipogenic differentiation of mesenchymal stem cells, and low O₃ concentrations have been shown to stimulate lipid accumulation in human adipose-derived adult stem cells in vitro. Thus, O₃ treatment is a promising procedure to improve the survival of explanted adipose tissue, whose reabsorption after fat grafting is a major problem in regenerative medicine. In this context, we carried out a pilot study to explore the potential of mild O₃ treatment in preserving explanted murine adipose tissue in vitro. Scanning and transmission electron microscopy, Western blot, real-time polymerase chain reaction and nuclear magnetic resonance spectroscopy were used. Exposure to low O₃ concentrations down in the degradation of the explanted adipose tissue and induced a concomitant increase in the protein abundance of Nrf2 and in the expression of its target gene Hmox1. These findings provide a promising background for further studies aimed at the clinical application of O₃ as an adjuvant treatment to improve fat engraftment.

Ozone (O₃) is a natural, highly unstable atmospheric gas that rapidly decomposes to oxygen. Although not being a radical molecule, O₃ is a very strong oxidant and therefore it is potentially toxic for living organisms. However, scientific evidence proved that the effects of O₃ exposure are dose-dependent: high dosages stimulate severe oxidative stress resulting in inflammatory response and tissue injury, whereas low O₃ concentrations induce a moderate oxidative eustress activating antioxidant pathways. These properties make O₃ a powerful medical tool, which can be used as either a disinfectant or an adjuvant agent in the therapy of numerous diseases. In this paper, the cellular mechanisms involved in the antioxidant response to O₃ exposure will be reviewed with special reference to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its role in the efficacy of ozone therapy.