S13-6
On the Action Mechanism of Ozone Therapy
オゾン療法の作用メカニズムについて
Toshiaki Miura
三浦敏明
Hokkaido University
Nishi-8, West-5, Kita-ku, Sapporo 060-0808, Japan
北海道大学	  〒060-0808	  札幌市北区北 8 条西 5 丁目
The low-dose ozone acts as mild oxidative stress and induces both inflammatory and anti-inflammatory
responses which are mainly regulated by two transcriptional factors, NFκB and Nrf2. NF-κB is involved inthe expressions of cyclooxygenase-2 and various inflammatory cytokines whereas Nrf2 is involved in theup-regulations of numerous antioxidant enzymes and cytoprotective proteins. In major autohemotherapy,
the most used application form in ozone therapy, ozone in contact with blood instantaneously reacts withunsaturated fatty acids generating mainly H2O2 and 4-hydroxynonenal which are responsible for activationsof NFκB and Nrf2, respectively.

1. Ozone Therapy
Owing to its potential oxidant properties, ozone
exerts damaging effects on the respiratory tract [1].
However, the low-dose ozone has been used fortherapy of various diseases in European countries
during the last six decades. In ozone therapy, theozone-oxygen mixture is administered by various
routes: topical application on skin and mucosa,
parenteal injections. Of these various routes, majorautohemotherapy (MAH) is the most used routefor the administration of ozone and it involves thewithdrawal of blood (100-200 mL) from subjects
and then reinfusion of the blood back into a vein ofsubjects after mixing with the same volume ofozone -oxygen gas (10-80 µgO3/ mL). Now MAH
is a common therapy in Europe, South Americaand Cuba and is performed to treat inflammation,
circulatory disturbances, infections and pain.
Clinical studies have demonstrated that MAH
induces the following biological effects [2]:(1) Improvement in blood circulation and oxygen
delivery to ischemic tissues.
(2) Enhancement in general metabolism throughimproved oxygen delivery.
(3) Upregulation of cellular antioxidant enzymes
and cytoprotective proteins.
(4) Induction of mild activation of the immune
system and enhanced release of growth factors.
Action mechanism of ozone responsible forsome of these biological effects can be explained
form the recent studies [3, 4].
2. Action of Ozone on Blood
Because human blood plasma contains various
antioxidants such as ascorbic acid, tocopherols,

uric acid and thiol (SH) containing substances(albumin, glutathione, cysteine, etc.) with highreactivity to ozone, most of ozone is consumed
by these antioxidants upon contact with blood.
However, a small portion of ozone escaped fromplasma antioxidant defenses can react with
unsaturated fatty acids to give reactive oxygen
species and lipid oxidation products [5]. Bocci,
et al. revealed the formation of hydrogen
peroxide (H2O2) and 4-hydroxy-2 nonenal(4-HNE) in the reactions of the low-dose ofozone with human blood and serum [3, 4]. Since
H2O2 and 4-HNE are unionized and their lifespans are longer than ozone, they can freely
diffuse into cells and trigger several biochemical
reactions. Therefore, H2O2 and 4-HNE act as
short-lived and long-lived second messengers,respectively, in MAH.

Fig.1. Structure of 4-hydroxy-2-nonenal

3. Roles of Second Messengers in MAH
It is now well-documented that both H2O2 and4-HNE are “double edged” molecules. They canhave either protective (adaptive response) or
noxious effect on the cells and their effects
depend on the dosage [6]. In MAH, the exposureof human blood to the low-dose ozone induces
adaptive and beneficial responses through theformation of small amounts of H2O2 and 4-HNE.
Recent studies demonstrated that two nuclear
transcriptional factors, nuclear transcriptional

factor κB (NF-κB) and nuclear factor erythroid
2-related factor 2 (Nrf2) are involved in such
adaptive responses induced by H2O2 and 4-HNE.
NF-κB is involved in inflammatory or immune
responses by regulating expression of inflammatory
mediators such as cyclooxygenase-2 (COX-2) andvarious inflammatory cytokines. Whereas Nrf2 isinvolved in anti-inflammatory responses by
up-regulations of numerous antioxidant enzymes
and cytoprotective proteins.
In fact, ozonation of human blood produces
various cytokines such as α-, β- and γ-interferons,
tumor necrosis factor-α, interleukin-2,-4, -6-, -8,-10 and nitric oxide (NO). Blood also producessome growth factors (GM-CSF, PDGF, TGF-β1) byozonation (10-80 µgO3/ mL). Up-regulations ofthese cytokines can be explained by activation ofNFκB of leucocytes and platelets by H2O2. H2O2
also accelerates the glycolysis in erythrocyes byenhancing the recycle of the reduced glutathione,
which increases ATP and 2,3-diphosphoglycerate(2,3-DPG). Mild induction of immune responses
may be usful in immune-depressed patients after
chemotherapy, or in chronic infectious diseases
and the increase of growth factors in circulation
may be beneficial for enhancing the wound healing.
In addition, NO and 2,3-DPG are effective for theimprovement in blood circulation and oxygen
delivery to ischemic tissues.
On the other hand, 4-HNE induces theup-regulation of antioxidant enzymes throughactivation of Nrf2. The mechanism of activationof Nrf2 is well known. Briefly, Nrf2 is present incytoplasm as the complex with its inhibitory
protein, Keap1, in the absence of a specificstimulus. Many sources of stimulus such asoxidants and electrophiles activate Nrf2 byreacting with cysteine residues of Keap1 andrelease Nrf2 from the Complex. Once released,Nrf2 migrates into nucleus and bonds to the DNA
at the location of the ARE which is the master
regulator of the entire antioxidant system locatedin all human cells [7]. Since 4-HNE is powful
electrophile, it activates Nrf2 by alkylating thiolgroups of cysteine residue in Keap1 andconsequently induces antioxidant enzymes andcytoprotective proteins.
Recently, Pecorelli, et al. have revealed thatozonated human serum containing 4-HNE activatesNrf2 of human endothelial cells in a dose dependent
manner with the subsequent induction of heme
oxygenase-1 (HO-1) and NADPH quinine oxidoreductase (NQO-1) [4]. The results suggest that
4-HNE can distribute in all body tissues andactivate Nrf2 after refusion of ozonated blood inMAH. More recently, Re et al. demonstrated thatlevel of Nrf2 in peripheral blood mononuclear cells
increased immediately after MAH. Thus, MAH is
beneficial for the treatment of the diseases thatinvolved a chronic disruption of redox status(oxidative stress) by up-regulation of antioxidant
enzymes.
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