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Potential Cytoprotective Activity of Ozone Therapy in SARS-CoV-2/COVID-19

(1) Background: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) in China at the end of 2019 has caused a large global outbreak. Systemic ozone therapy (OT) could be potentially useful in the clinical management of several complications secondary to SARS-CoV-2. The rationale and mechanism of action has already been proven clinically in other viral infections and has been shown in research studies to be highly effective at decreasing organ damage mediated by inflammation and oxidative stress. This review summarizes the OT studies that illustrate the possible cytoprotective mechanism of action of ozone and its physiological by-products in target organs affected by SARS-CoV-2. (2) Methods: This review encompasses a total of 74 peer-reviewed original articles. It is mainly focused on ozone as a modulator of the NF-κB/Nrf2 pathways and IL-6/IL-1β expression. (3) Results: In experimental models and the few existent clinical studies, homeostasis of the free radical and antioxidant balance by OT was associated with a modulation of NF-κB/Nrf2 balance and IL-6 and IL-1β expression. These molecular mechanisms support the cytoprotective effects of OT against tissue damage present in many inflammatory diseases, including viral infections. (4) Conclusions: The potential cytoprotective role of OT in the management of organ damage induced by COVID-19 merits further research. Controlled clinical trials are needed.


Introduction
Coronaviruses are important human and animal pathogens. At the end of 2019, a novel coronavirus was identified as the cause of a cluster of pneumonia cases in Wuhan (Hubei Province of China) and caused a large global outbreak representing a major public health issue [1]. It rapidly spread, resulting in an epidemic throughout China, with increasing cases reported globally. In February 2020, the World Health Organization designated the disease COVID-19, which stands for coronavirus disease 2019 [2]. The virus that causes COVID-19 is designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); previously, it was referred to as 2019-nCoV. SARS-CoV-2 is closely related to two bat-derived severe acute respiratory syndrome-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, in particular BetaCoV/bat/Yunnan/RaTG13/2013 are similar to the human SARS-CoV-2 [3]. It is shown to have large genetic diversity and rapid evolution [4].
SARS-CoV-2 is spread by human-to-human transmission via respiratory droplets or direct contact, and infection has been estimated to have a mean incubation period of 6.4 days and a basic reproduction number of (2.24-3.58) days [1]. Among patients with pneumonia caused by SARS-CoV-2, fever was the most common symptom, followed by cough, malaise and dry cough at the prodromal phase [5]. Bilateral lung involvement with ground-glass opacity was the most common finding from computed clinical evidence to support the potential role of OT in the prevention and management of cytotoxicity induced by different drugs and diseases including viral diseases [21][22][23][24]. The main mechanism is related to the modulation of the oxidative stress and pro-inflammatory cytokines. The Evidence Acquisition Terms included in the information search were: COVID-19, SARS-CoV-2, SARS, ozone, OT, viral pneumonia. Bibliographic databases consulted: MEDLINE/PubMed, SciELO, LILACS, PAHO, EMBASE, ZOTERO ISCO3, WHO International Clinical Trials Registry Platform and NIH. U.S. National Library of Medicine. The type of documents reviewed were published between 1980 to 2020, in Russian or English and included: Original articles, published thesis, clinical reports, ongoing clinical trials and bibliographic reviews. The exclusion criteria were the lack of free access to complete text due to financial constraints and/or, studies presenting inadequate scientific evidence.

Potential Therapeutic Actions of Ozone in Viral Diseases
Ozone can inactivate viruses via direct oxidation of its components [25]. However, the viricidal activity in vivo becomes uncertain when viruses are in biological fluids or, even worse, when they are intracellular (pneumocytes, hepatocytes, epithelia, CD4 + lymphocytes, monocytes, glial and neuronal cells) because the cell's potent antioxidant system protects viral integrity [15]. That is why it is irrational to use direct I.V. injection of gas or other non-recommended methods of application of ozone [16,26]. OT represents a useful adjunctive and complementary therapy but neither ozone, nor H 2 O 2 (one of the main O 3 mediators) can reach sufficient concentrations in tissues because the plasma antioxidant capacity protects free pathogens and intracellular viruses are inaccessible [27]. In order to explore the efficacy of OT in viral diseases, Bocci and Paulesu [28] explained the possibility of how ozone may act in vivo. The following mechanisms may have some relevance:

1.
A prolonged ozone therapeutic treatment appears able to induce an adaptation to oxidative stress, hence a re-equilibration of the cellular redox state, which is a fundamental process for inhibiting viral replication. The paradoxical mechanism by which ozone (a potent oxidant) can induce an antioxidant response, is currently demonstrated not only at a proteomic level, but also at a genomic one. Ozone applied at a therapeutic dose modulates the nuclear factor Nrf2 and NF-κB and induces the homeostasis of the antioxidant environment [18,[29][30][31][32]. Oxidative stress and innate immunity have a key role in lung injury pathways that control the severity of acute lung cytotoxicity during viral infections like SARS [33].

2.
The induction of antiviral cytokines such as IFN and the modulation of pro-inflammatory cytokines as IL-6, have been demonstrated by ozonating blood such as major autohemotherapy (MAH). Although ozone is a weak inducer, reinfused lymphocytes and monocytes during mayor autohemotherapy (MAH), can migrate through the lymphoid system, and activate other cells that, in time, will lead to the stimulation of the immune system [32,34]. This may represent an important process because it is known that an acute viral disease becomes more severe because the virus is particularly virulent, the heterogeneous viral population evolves rapidly and escapes immune control, or because the immune system becomes tolerant to viral antigens and becomes unable to counteract the infection. Moreover, besides the induction of HO-1 [18], a protective enzyme, there is also the release of some heat shock proteins (HSP) such as HSP60, HSP70 and HSP90 that also have an influence on viricidal activity. These proteins are potent activators of the innate immune system, and are able to induce the monocyte-macrophage system and the activation of antigen-presenting cells [15,35]. The evidence shows that, during the COVID-19 epidemic, the severe deterioration of some patients has been closely related to a dysregulated inflammatory process referred to as "the cytokine storm" [36,37].

3.
Oxygen-ozone therapy improves oxygenation [38,39], especially in poorly oxygenated tissues [40]. Patients with SARS are prone to have mild non-specific hepatitis [41], lung fibrosis [42] and renal failure [43]. OT stabilizes hepatic metabolism and tend to normalize fibrinogen and prothrombin plasma levels in infected patients, suggesting an improvement of the hepatic protein synthesis [15]. There is extensive research demonstrating the cytoprotective effect of ozone to prevent oxidative Antioxidants 2020, 9, 389 4 of 12 damage to the heart [44,45], liver [46,47], lungs [48] and renal tissues [49]. The authors have described in a recent review, the cytoprotective effect of ozone to prevent and even to treat chemotherapy-induced damage in heart, liver, renal and lung tissue [22].

4.
During blood ozonation ex vivo for the minor autohemotherapy, using ozone concentrations near 90 µg/mL per mL of blood, it may be feasible to induce the oxidation of free viral components, which could theoretically represent an inactivated and immunogenic vaccine [15,50,51].

5.
Ozonized Saline Solution (O 3 SS). This method was formalized by the Ministry of Health of the Russian Federation in the early 1980s and has been officially implemented in public health hospitals, specifically for the specialties of orthopedics, dermatology, gynecology and obstetrics [16,52]. In 2004, it was also officially recognized in Ukraine. The beneficial effects of this therapy are supported by a large amount of scientific papers and strong clinical experience. [53]. The method consists of bubbling and saturating a physiological solution (0.9%) with ozone-oxygen mixture at concentrations that are calculated depending on the patient's weight (ranging 1-5 µg/kg b.w.).
Its administration takes about 20-30 min. Unlike MAH, the O 3 SS has been used as complementary therapy in viral diseases such as Epstein Barr, Cytomegalovirus, Papillomavirus, HIV, Herpes zoster, Herpes simplex, etc. Since the saline solution is a plasma expander, O 3 SS represents a greater amount of blood being treated than MAH and therefore, theoretically, the number of sessions could be reduced.
Korolev, B.A., Boyarinov, G.A. and Sokolov, V.V. [54,55] showed that when an O 3 SS was used during cardiopulmonary bypass, the cells of the patient's organs use more glucose compared to basal levels. Therefore, it is concluded that the therapeutic effects of ozonated physiological solutions, is determined by the dissolved O 2 /O 3 mixture, free radicals, hydrogen peroxide and hexagonal aqueous structures formed during the bubbling of aqueous NaCl solutions with a mixture of O 2 /O 3 gas.

Ozone Therapy as Redox Modulator
During a systemic application of O 2 /O 3 (mainly MAH, O 3 SS, vaginal and rectal insufflation), part of the O 3 is removed by the antioxidants of the medium. Further reaction of O 3 with biomolecules generates aldehyde (e.g., 4-hydroxynonenal (4-HNE)) and peroxide (H 2 O 2 and organic peroxides). These byproducts of the reaction act as secondary messengers and induce a further adaptive hormetic responses [56][57][58]. Ozone at a therapeutic dose "only acts" as a modulator or pro-drug and, by inducing secondary messengers, will enhance subsequent adaptive responses [21].
Mediators such as 4-HNE and H 2 O 2 are among the most relevant secondary messengers producing beneficial effects during medical applications, they induce a gradual oxidative stimulus, that produces the synthesis of endogenous antioxidants such as SOD, CAT and GPx [18,59]. This fact implies that O 2 /O 3 is a paradoxical pro-oxidant therapy that invokes an endogenous antioxidant response. Moreover, low quantities of H 2 O 2 formed as consequence of O 2 /O 3 have a key role in the molecular mechanism. H 2 O 2 is crucial and a common activator of the modulation of NF-κB and Nrf2 pathways [60][61][62]. In addition, 4-HNE also sends a signal of transient oxidative stress and its effects depend on concentration as well as cell/tissue origin. This pathway can activate the synthesis of several substances such as: γ-glutamyl transferase, γ-glutamyl transpeptidase, HSP-70, HO-1, and antioxidant enzymes such as SOD, GPx, CAT and glucose-6-phosphate dehydrogenase [21]. In addition, these pluripotent effects of 4-HNE can be explained by its concentration-dependent interactions with the cytokine networks and complex cellular antioxidant systems also showing cell and tissue specificities [59,63].
Experimental results demonstrated that ozone at therapeutic dosages ex vivo or in vivo can activate Nrf2 [20,32] that involve an indirect modulation (inhibition) of the NF-κB pathway. In addition, Nrf2 suppresses NF-κB activity by eliminating ROS, which may cause NF-κB activation via antioxidative protein induction, such as HO-1 and NQO1. Moreover, Nrf2 suppresses NF-κB activity through some protein-protein interactions, and also suppresses an inflammatory cytokine gene expression through binding to their gene promoter directly [64]. NF-κB pathway activates the release of pro-inflammatory cytokines like: TNFα, IFNγ, IL1β, IL6, IL8, as well as pro-inflammatory genes likecyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [65]. It is well known that the regulation of both pathways, NF-κB and Nrf2, involved a crosstalk to bring a coordinated inflammatory response [66,67]. The modulation of the inflammatory response by ozone was evident in a clinical trial l on patients with multiple sclerosis (ME) treated with O 2 /O 3 by rectal insufflation for 30 days [19]. Taking the original data of this trail and recalculating the values in terms of the ratio Nrf2 phosphorylation (as expression of activation of Nrf2 pathway) and IL-1β (as marked of the NF-κB pathway) this modulation became evident (Figure 1). In patients with ME without treatment, the balance Nrf2/NF-κB favor the inflammatory process, and O 2 /O 3 restores the balance of those pathways. the regulation of both pathways, NF-κB and Nrf2, involved a crosstalk to bring a coordinated inflammatory response [66,67]. The modulation of the inflammatory response by ozone was evident in a clinical trial l on patients with multiple sclerosis (ME) treated with O2/O3 by rectal insufflation for 30 days [19]. Taking the original data of this trail and recalculating the values in terms of the ratio Nrf2 phosphorylation (as expression of activation of Nrf2 pathway) and IL-1β (as marked of the NF-κB pathway) this modulation became evident (Figure 1). In patients with ME without treatment, the balance Nrf2/NF-κB favor the inflammatory process, and O2/O3 restores the balance of those pathways.
An individual analysis of the variable assayed in this study shown a significant (p < 0.05) increase in Nrf2 values in ozone treated patients compared to control group (0.93 vs. 0.75 densitometry unit, respectively), that restore the significant downregulated values of Nrf2 at basal level in ME patients (0.56 densitometric unit). The increment in Nrf2 was in line with the significant reduction in 61 % of the level of the pro inflammatory cytokine IL-1β in ME-treated patients compared with basal levels, even IL-1β levels in ME remain 94 % higher than values in normal subjects. An imbalance between Nrf2/NF-κB has been proposed in other diseases like diabetic neuropathy [68] in which ozone experimentally demonstrated its efficacy equilibrating this disruption [62]. Similar trends were found in viral diseases, NF-κB pathways can support influenza A virus infection and promote pneumonia. Through the Activation of the Nrf2 signaling some drug as emodin can increased the survival rate, reduce lung edema, pulmonary viral titer and inflammatory cytokines, and improve lung histopathological changes [69]. In addition, it has been shown that the rabbit hemorrhagic disease virus (that causes lethal fulminant hepatitis in rabbits) has a pathological mechanism that involves is the repression of Nrf2 pathway [70].

Ozone Therapy and Cytoprotection
Antioxidants are important for the maintenance of cellular integrity and cytoptotection. Modulating the balance Nrf2/NF-κB, O2/O3 not only increases the endogenous antioxidant system but also modulates the expression of pro-inflammatory cytokines and has an impact in cytoprotection. COVID-19 infects the upper and lower respiratory tracts and causes mild to highly acute respiratory syndrome with consequent over-expression of pro-inflammatory cytokines, including interleukin IL-1β and IL-6. Activation of toll-like receptors by SARS Cov-2 RNA lead to the release of pro-IL-1β which is cleaved by caspase-1, followed by inflammatory activation and production of active mature IL-1β which is a mediator of lung inflammation, fever and fibrosis [71]. However, suppression but An individual analysis of the variable assayed in this study shown a significant (p < 0.05) increase in Nrf2 values in ozone treated patients compared to control group (0.93 vs. 0.75 densitometry unit, respectively), that restore the significant downregulated values of Nrf2 at basal level in ME patients (0.56 densitometric unit). The increment in Nrf2 was in line with the significant reduction in 61% of the level of the pro inflammatory cytokine IL-1β in ME-treated patients compared with basal levels, even IL-1β levels in ME remain 94% higher than values in normal subjects.
An imbalance between Nrf2/NF-κB has been proposed in other diseases like diabetic neuropathy [68] in which ozone experimentally demonstrated its efficacy equilibrating this disruption [62]. Similar trends were found in viral diseases, NF-κB pathways can support influenza A virus infection and promote pneumonia. Through the Activation of the Nrf2 signaling some drug as emodin can increased the survival rate, reduce lung edema, pulmonary viral titer and inflammatory cytokines, and improve lung histopathological changes [69]. In addition, it has been shown that the rabbit hemorrhagic disease virus (that causes lethal fulminant hepatitis in rabbits) has a pathological mechanism that involves is the repression of Nrf2 pathway [70].

Ozone Therapy and Cytoprotection
Antioxidants are important for the maintenance of cellular integrity and cytoptotection. Modulating the balance Nrf2/NF-κB, O 2 /O 3 not only increases the endogenous antioxidant system but also modulates the expression of pro-inflammatory cytokines and has an impact in cytoprotection. COVID-19 infects the upper and lower respiratory tracts and causes mild to highly acute respiratory syndrome with consequent over-expression of pro-inflammatory cytokines, including interleukin IL-1β and IL-6. Activation of toll-like receptors by SARS Cov-2 RNA lead to the release of pro-IL-1β which is cleaved by caspase-1, followed by inflammatory activation and production of active mature IL-1β which is a mediator of lung inflammation, fever and fibrosis [71]. However, suppression but not depletion of the pro-inflammatory IL-1 family and IL-6 have been shown to have a therapeutic effect in many inflammatory diseases, including viral infections for instance, Mice lacking IL-1 signaling expression, elevated viral replication of coronavirus [72]. In addition, IL-6-deficient mice infected with influenza virus exhibited a higher lethality, more body weight loss and had higher fibroblast accumulation and lower extracellular matrix (ECM) turnover in the lungs than their wild-type counterparts [73]. The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection. The cytokines secreted, following inflammasome activation, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. Therefore, not suppressive but a modulator of cytokines may impact efficiently in vital cytotoxicity. A representative data about the downregulated of cytokines IL-1β, IL-6, IL-8 and TNF-α are shown in Table 1. Table 1. Effect of ozone therapy as modulator of pro-inflammatory cytokines.

Type of Study Model/Target Organ Respond in Term of Cytokines Main Result/Reference
In vivo (rats) Sepsis/Spleen and liver IL-1β↓/TNF-α↓ Increased survival rate in 33% [74].
The expression of cytokine responses to a previous signal is closely connected with the action of nuclear factors. An in vitro experiment, conducted in cardiomyocytes and skin fibroblasts, analysed the role of ozone at the level of Nrf2 and NF-κB induced by doxorubicin [44]. The authors analysed the individual role of different doses of ozone in this model. A re-analysis of this data, calculating the rate NF-κB/Nrf2, showed the evident downregulation of the effect of ozone but not suppression ( Figure 2). The same trend was observed also in skin fibroblasts cultures treated with doxorubicin and O 2 /O 3 . An analysis of the individual values showed that the restoration of the equilibrium NF-κB/Nrf2 was reached essentially by the preservation of the level of Nrf2 in ozone-treated cells (0.8 vs. 0.9 fold of chance, in control group, respectively), compared with the depleted values of Nrf2 observed in doxorubicin-treated cells (0.5, fold of chance, with respect to control cell culture). This maintenance of Nrf2 levels avoids the 100% increment in NF-κB that takes place during the doxorubicin treatment. The experiment showed that the intervention with ozone, preserve Nrf2 essential facts to avoid and up-regulate NF-κB.
The hormetic response, oxidative preconditioning or the adaptation to the chronic oxidative stress, during OT, has been now demonstrated experimentally [78]. The concept of doses is very remarkable in OT, the administration route and clinical protocols are also important. At higher doses the described effect of ozone could drastically change. High doses of ozone induce the gene transcription of the pro-inflammatory cytokine, its receptor, and inflammatory proteins. At the same time, they invoke a negative regulation of type 1 Interferon and the response to viral infections pathways [79].
The recommended systemic administration routes are: O 3 SS, MAH and Extracorporeal Blood Oxygenation-Ozonation (EBOO). Clinical protocols should comply with the standard doses and procedures defined in the Madrid Declaration of OT [16]. At least three clinical trials using major autohemotherapy are in progress in China and more clinical trials are needed to confirm the efficacy of OT as complementary therapy in the treatment of COVID-19 diseases. It is a complementary therapy because, while the infected patient is treated with allopathic medicine, at the same time the patient will also receive the complementary proposed treatment. It should be noted that, even if ozone has no effect on the virus infection, the demonstrated modulation of oxidative stress and inflammatory cytokines by ozone therapy could offer a relevant and beneficial clinical effect. Additionally, a small impact in the requirements of inpatients days, especially on intensive care units, could lead to a high benefit in the current critical situation that many countries are suffering.

Conclusions
Systemic OT can be potentially useful in SARS-CoV-2. The rationale and mechanism of action have already been proven clinically with other viral infections and have been shown to be highly effective in research studies. The mechanisms of action involved are the modulation of the NF-κB/Nrf2 pathway and IL-6/IL-1β expression. The modulation of these pathways by OT have an impact in the cytoptotection and blockage of viral replication. Future clinical trials are needed to prove the complementary use of OT in COVID-19.