Treatment of case

  Oxygen-ozone immunoceutical therapy in COVID-19 outbreak: facts and figures




  Giovanni Ricevuti,1 Marianno Franzini,2 Luigi Valdenassi3

  1Department of Drug Science, University of Pavia, Italy, High School in Oxygen Ozone Therapy, University of Pavia;

  2SIOOT International, Comunian Clinic, Gorle (BG), High School in Oxygen Ozone Therapy, University of Pavia;

  3SIOOT, Italian Society of Oxygen Ozone Therapy, High School in Oxygen Ozone Therapy, University of Pavia, Italy

  The problems related to the approach to the outbreak of COVID-19 in the world require that all possible effective treatment options be explored. The clinical criterion of the researcher is not to refuse a priori, but to verify and evaluate the proposals that are made. Italian Society of Oxygen Ozone Therapy (SIOOT) proposed to the Italian ISS (Italian Institute of Health) to use oxygen ozone therapy (O2O3) in patients with COVID-19. The ISS has said on 24 March 2020 that it is possible to use it in the light of scientific considerations:





  Although the proposal is supported by a certain rational basis, the data actually available in relation to the proposed indication consist essentially in demonstrating the effectiveness of ozone in killing SARS virus in monkey cells, and clinical experience of the benefit in patients with bronchopulmonia. However, as the proposal appears to be shared and supported by clinical centers experienced in the treatment of viral pneumonia, treatment could, if deemed appropriate, be carried out under the responsibility of the physician, after obtaining the informed consent of the patient. Considering the “experimental” character of the use this indication, which also requires specific medical experience and the availability of appropriate instruments, it is considered appropriate to acquire also the opinion of the Ethics Committee.




  Some hospitals in Lombardy, the region most affected by COVID-19 in Italy, have already started its use with initial positive results.

  As little is known about O2O3 in the world, although there is a lot of scientific evidence published in the recent years on the anti-inflammatory, immunomodulatory and organ-protective validity of O2O3 therapy, we consider useful to follow the suggestions of the article Activating Immunity to Fight a Foe – A New path, by Richard S. Hotchkiss and Steven M. Opal, and proposing the O2O3 therapy as immunoceutical therapy according to the indications of the article. The O2O3 therapy has many biological effects, but the most relevant is its ability to modulate the activation of Nrf2 (an important nuclear message transductor), the downregulation of NFkB, and it also modulates the NLRP3 inflammasome.

  意大利受COVID-19影响最大的伦巴第地区的一些医院已经开始使用COVID-19,并取得了初步的积极成果。世界上对三氧知之甚少,虽然近年来发表了大量关于三氧治疗的抗炎、免疫调节和器官保护有效性的科学证据,但我们认为遵循Richard S.Hotchkiss和并根据本文的适应症提出了三氧作为免疫药物治疗。三氧治疗有许多生物学效应,但最相关的是它能调节Nrf2(一种重要的核信息传导器)的激活,NFkB的下调,它还调节NLRP3炎症。

  In this letter, I wish to present this therapeutic opportunity, outline the important activities of the Oxygen Ozone Therapy (O2O3) and explain the rationale for this treatment in COVID-19 patients.

  Spike protein and Angiotensin-Converting Enzyme 2 (ACE2) cell receptors have been identified as putative receptors for SARS-CoV-2; they could promote the proliferation of COVID-19 (Figure 1). It is known that these receptors can be blocked with some specific monoclonals but also through the control of Nrf2 that regulates and blocks the activity of Spike and ACE2. Because O2O3 acts directly on Nrf2, stimulating them, it seems very likely that this is the most important physiological mechanism to block endogenous COVID-19 reduplication by preventing contact with putative receptors of SARS-CoV-19.




  Oxygen-ozone has a high solubility in plasma and induces formation of two second messengers, H2O2 and ozonoids and alchenals (Figure 2). These are the ones who are mainly competent, interacts with the membrain proteins and receptors of the cells, especially the immunocompetent ones, and enter the cells and interact with signal transduction proteins on the nucleus and mitochondria level. The key action mechanism of O2O3 therapy is its action on proteasome and inflammation cascade, to control inflammatory process, by stimulating the nuclear factor Nrf2 and by inhibiting nuclear factor NFkB (Figure 3).






  The O2O3 therapy can restore the right immune response by stimulating signal transduction molecules via Nrf2 and thus stimulating the nuclear transduction via specific microRNAs restoring the normal antioxidant and immunostimulating reaction. The action mechanism of O2O3 therapy has very well summarized by Noel L. Smith et al. in 2017 By reacting with Polyunsaturated Fatty Acids (PUFA) and water, O3 creates hydrogen peroxide (H2O2), a Reactive Oxygen Species (ROS). Simultaneously, O3 forms a mixture of Lipid Ozonation Products (LOP).

  三氧治疗可通过Nrf2刺激信号转导分子,从而通过特定的microRNAs刺激核转导,恢复正常的抗氧化和免疫刺激反应,从而恢复正常的免疫应答。Noel L.Smith等人对三氧治疗的作用机制进行了很好的总结。2017年通过与多不饱和脂肪酸(PUFA)和水反应,三氧生成过氧化氢(H2O2),一种活性氧物种(ROS)。同时,O3形成脂质臭氧氧化产物(LOP)的混合物。

  The LOPs created after O3 exposure include lipoperoxyl radicals, hydroperoxides, malonyldialdeyde, isoprostanes, the ozonide and alkenals, and 4-Hydroxynonenal (4-HNE). Moderate oxidative stress caused by O3 increases activation of the transcriptional factor mediating nuclear factor-erythroid 2-related factor 2 (Nrf2). Nrf2’s domain is responsible for activat ing the transcription of Antioxidant Response Elements (ARE). Upon induction of ARE transcription, an assortment of antioxidant enzymes gains increased concentration levels in response to the transient oxidative stress of O3. The antioxidants created include, but are not limited to, Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx), Glutathione S-Transferase (GST), Catalase (CAT), Heme Oxygenase-1 (HO-1), NADPHquinone-Oxidoreductase (NQO-1), Heat Shock Proteins (HSP), and phase II enzymes of drug metabolism. Many of these enzymes act as free radical scavengers clinically relevant to a wide variety of diseases.




  Masaru Sagai et al. in 2011 described the biological responses induced via the activation of Nrf2/ARE with mild oxidative stress (O2O3 therapy) that can be summarized in:

  i) Increasing the levels of direct antioxidants, such as GSH, CO, and bilirubin;

  ii) Stimulating GSH regeneration via glutathione and thioredoxin reductase;

  iii) Increasing the levels of enzymes that detoxify oxidants and electrophils (i.e. catalase, SOD, GPx, GSTr, NADPHQuinone Oxidoreductase (NQO1), HO-1, HSP70, etc);

  iv) Increasing the levels of phase II enzymes;

  v) Inhibiting cytokine-mediated inflammation via the induction of leukotriene B4 reductase;

  vi) Reducing iron overload, and subsequent oxidative stress induced via elevated ferritin;

  vii) Recognizing, repairing, and removing damaged proteins;

  viii) Protection from apoptosis induced via oxidative stress;

  ix) Increasing DNA repair activity.

  Masaru Sagai等人,描述了通过激活Nrf2/ARE和轻度氧化应激(O2O3疗法)诱导的生物反应,可概括为:

  1) 提高直接抗氧化剂的水平,如GSH、CO和胆红素;




  5) 通过诱导白三烯B4还原酶抑制细胞因子介导的炎症反应;





  In addition, Jacqueline Diaz-Luis et al. in 2015demonstrated that ozone was able to modulate the phagocytic cells in peripheral blood and the mechanisms on how messengers can activate immunological response leading to the therapeutic biological effects. Furthermore, it was demonstrated that there is a range of ozone concentrations where we can obtain the highest positive results, while lower doses are ineffective and higher doses can produce lower effects. Accordingly, ozone, in a dose-dependent behavior, may stimulate the phagocytic function of the peripheral blood cells.

  此外,Jacqueline Diaz Luis等人。2015年,证明臭氧能够调节外周血中的吞噬细胞,以及信使如何激活免疫反应从而产生治疗性生物效应的机制。此外,研究还表明,在臭氧浓度范围内,我们可以获得最高的阳性结果,而低剂量是无效的,高剂量可以产生较低的效果。因此,臭氧在剂量依赖性行为中,可能刺激外周血细胞的吞噬功能。

  Another important effect of O2O3 therapy than can explain its effects in improving the therapeutic approach to COVID-19 infected patients is its important action on NLRP3 inflammasome that is recognized to play a crucial part in the initiation and continuance of inflammation in various diseases. Gang Yu et al. in 2016 demonstrated that the protective effect of ozone therapy was achieved by its anti-inflammatory property through the modulation of the NLRP3 inflammasome. Ozone-oxygen mixture at low concentration could effectively improve organ ischemia-reperfusion that is what happens in the lungs of patients affected by COVID-19 infection.



  Ischemia-Reperfusion Injury (IRI) is a major cause of lung dysfunction during many pathological diseases. Zhiwen Wang et al. in 2018 demonstrated that ozone oxidative treatment protects the lung from IRI by attenuating nucleotide-binding oligomerization domainlike receptor containing pyrin domain 3 (NLRP3)-mediated inflammation, enhancing the antioxidant activity of Nrf2 and inhibiting apoptosis.


  In conclusion, as systemic oxygen therapy has all these positive effects: control of inflammation, stimulation of immunity, antivirus ability, protection from ischemia-reperfusion damage, action on proteasome and inflammation.7,9 Oxygen-ozone therapy can be said to be a new method of immunoceutical therapy and therefore its use in combination with other treatments in COVID-19 positive patients may be justified, helpful and synergic.

  Further studies and tests are needed, but we hope to soon have confirmation that O2O3 therapy is synergistic and effective in controlling COVID-19-infection.





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