Ozone: a natural bioactive molecule with antioxidant property as potential new strategy in aging and in neurodegenerative disorders

Abstract

Systems medicine is founded on a mechanism-based approach and identifies in this way specific therapeutic targets. This approach has been applied for the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Nrf2 plays a central role in different pathologies including neurodegenerative disorders (NDs), which are characterized by common pathogenetic features. We here present wide scientific background indicating how a natural bioactive molecule with antioxidant/anti-apoptotic and pro-autophagy properties such as the ozone (O₃) can represent a potential new strategy to delay neurodegeneration. Our hypothesis is based on different evidence demonstrating the interaction between O₃ and Nrf2 system. Through a meta-analytic approach, we found a significant modulation of O₃ on endogenous antioxidant-Nrf2 (p < 0.00001, Odd Ratio (OR) = 1.71 95%CI:1.17-2.25) and vitagene-Nrf2 systems (p < 0.00001, OR = 1.80 95%CI:1.05-2.55). O₃ activates also immune, anti-inflammatory signalling, proteasome, releases growth factors, improves blood circulation, and has antimicrobial activity, with potential effects on gut microbiota. Thus, we provide a consistent rationale to implement future clinical studies to apply the oxygen-ozone (O₂-O₃) therapy in an early phase of aging decline, when it is still possible to intervene before to potentially develop a more severe neurodegenerative pathology. We suggest that O₃ along with other antioxidants (polyphenols, mushrooms) implicated in the same Nrf2-mechanisms, can show neurogenic potential, providing evidence as new preventive strategies in aging and in NDs.

Keywords: Neurodegenerative Disorders; Oxygen-Ozone (O(2)-O(3)) therapy; Ozone (O(3)); antioxidant system; nuclear factor (erythroid-derived 2)–like 2 (Nrf2); stress oxidative biomarkers.

Fig. 1

Molecular mechanisms linked to antioxidant/pro-authophagy activities of ozone (O₃) via Nfr2 signalling. In the absence of stimuli, Nrf2 (nuclear factor erythroid 2–related factor 2) binds to its repressor Keap1 (kelch-like ECH-associated protein), an adapter between Nrf2 and Cullin 3 (Cul3) protein, which leads to ubiquitination followed by proteasome degradation. When O₃ is administrated, it dissolves immediately and it reacts with PUFA (Poly-Unsaturated Fatty Acids) leading to the formation of fundamental messengers such as hydrogen peroxide (H₂O₂), 4-hydroxynonenal (4HNE) and lipid oxidation products (LOPs). These messengers can influence the modifications of cysteine residues present in Keap1 (S-HNE or ―S―S) inhibiting ubiquitin conjugation to Nrf2 by the Keap1 complex and provoking the nuclear accumulation of Nrf2. Once in the nucleus, Nrf2 dimerizes and binds to cis-acting DNA AREs (Antioxidant Response Elements) in different genes: Heme Oxygenase 1 (HO-1), Superoxide dismutases (SOD), Glutathione peroxidase (GSH-Px), Glutathione-S-Transferase (GST), Catalase (CAT), GSH-reductase (GR), NADPH quinone oxidoreductase 1 (NQO1), Heat Shock Proteins (HSPs), Cytochrome P450 monooxygenase (CYP450), Thioredoxin reductase (TrxR), phase II enzymes (UDP-glucuronosyltransferases, UGTs; N-acetyltransferases, NATs, sulfotransferases, SULTs). A) O₃ involves casein kinase 2 (CK2), a regulator of the Nrf2 activity through its phosphorylation, and MAPK (mitogen-activated protein kinase) signalling pathway, that is inhibited with consequent inactivation of oxidative stress and apoptosis by O₃ administration. B) O₃ modulates the degradation protein systems (autophagy), via activation of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. C) O₃ downregulates inducible nitric oxide synthase (iNOS), which generates nitric oxide (NO) via NF-κB (Nuclear Factor Kappa B Subunit 1) pathway. (CO = carbon monoxide).

Fig. 2

Molecular mechanisms linked to anti-apoptotic property of ozone (O₃) via pro-apoptotic molecules inactivation. Various apoptotic stimuli, ischemia, Reactive Oxygen Species, ROS, ipoxia can activate directly p53 that in turn can play a role as transcription factor and activate the expression of pro-apoptotic genes. Among these, Bak (Bcl-2 homologous antagonist/killer) and Bax (Bcl-2-associated X protein) can stimulate in mitochondrial membrane the activation of Cytochrome C that in turn activates Apaf1 (Apoptotic protease activating factor-1) and caspase 9 to close the circle to stimulate the activity of caspase 3. Enzymes such as SOD (Superoxide dismutase), CAT (catalase), and GSH-Px (glutathione peroxidase), can regulate p53, Bax and Bcl-2. O₃ administration decreases the expression of caspases 1-3-9, Hypoxia-inducible factor (HIFα), Tumor Necrosis Factor-α (TNF-α), Bax and p53 genes. (BID (BH3-interacting domain death agonist), Nrf2 (Nuclear Factor Erytroid 2-related factor 2), CUL3 (Cullin 3), Keap 1 (Kelch-like ECH-associated Protein), H₂O₂ (Hydrogen Peroxide), 4HNE (4-hydroxynonenal), LOPs (Lipid Oxidation Products), Cyt C (Cytochrome C), PUFAs (Poly-Unsaturated Fatty Acid), AREs (Antioxidant Response Elements)).

Fig. 3

Forest plot for odds ratio from meta-analysis of the endogenous Nrf2- antioxidant pathway before and after ozone (O3) treatment. CI, confidence interval; Chi², χ² test of goodness of fit; Tau², estimate of the between-study variance in a random-effects meta-analysis. Superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GSH-Px), Glutathione (GSH), Glutathione S-transferase (GST), Glutathione reductase (GR). RI = rectal insufflations; MATH = major autohemotherapy

Fig. 4

Forest plot for odds ratio from meta-analysis of the endogenous Nrf2- vitagene pathway before and after ozone (O₃) treatment. CI, confidence interval; Chi², χ² test of goodness of fit; Tau², estimate of the between-study variance in a random-effects meta-analysis. Nuclear factor Nrf2, heme-oxigenase (HO-1), heat shock protein (HSP)