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Effects of ozone therapy on haemostatic and oxidative stress index in coronary artery disease

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Coronary artery disease (CAD) is the most common cause of sudden death, and death of people over 20 years of age. Because ozone therapy can activate the antioxidant system and improve blood circulation and oxygen delivery to tissue, the aim of this study was to investigate the therapeutic efficacy of ozone in patients with CAD, treated with antithrombotic therapy, Aspirin and policosanol. A randomized controlled clinical trial was performed with 53 patients divided into two groups: one (n=27) treated with antithrombotic therapy and other (n=26) treated with antithrombotic therapy plus rectal insufflation of O(3). A parallel group (n=50) age and gender matched was used as reference for the experimental variables. The efficacy of the treatments was evaluated by comparing hemostatic indexes and biochemical markers of oxidative stress in both groups after 20 day of treatment. Ozone treatment significantly (P<0.001) improved prothrombin time when compared to the antithrombotic therapy only group, without modifying bleeding time. Combination antithrombotic therapy+O(3) improved the antioxidant status of patients reducing biomarkers of protein and lipid oxidation, enhancing total antioxidant status and modulating the level of superoxide dismutase and catalase with a 57% and 32% reduction in superoxide dismutase and catalase activities respectively, moving the redox environment to a status of low production of O(2)(•-) with an increase in H(2)O(2) detoxification. No side effects were observed. These results show that medical ozone treatment could be a complementary therapy in the treatment of CAD and its complications.
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Cardiovascular pharmacology
Effects of ozone therapy on haemostatic and oxidative stress index
in coronary artery disease
Gregorio Martı
´nez-Sa
´nchez
a,
n
, Livan Delgado-Roche
b
, Arquı
´mides
´az-Batista
c
,
Gema Pe
´rez-Davison
d
, Lamberto Re
a,d
a
Medinat srl Clinic, Via Fazioli 22, 60021 Camerano, Italy
b
Center of Studies for Research and Biological Evaluations, Pharmacy and Food Sciences College, University of Havana, Havana, Cuba
c
National Institute of Angiology and Vascular Surgery, Havana, Cuba
d
D.I.S.M.A.R., University of Ancona, Ancona, Italy
article info
Article history:
Received 20 February 2012
Received in revised form
25 June 2012
Accepted 2 July 2012
Available online 13 July 2012
Keywords:
Ozone
Coronary artery diseases
Oxidative stress
Aspirin
Policosanol
abstract
Coronary artery disease (CAD) is the most common cause of sudden death, and death of people over 20
years of age. Because ozone therapy can activate the antioxidant system and improve blood circulation
and oxygen delivery to tissue, the aim of this study was to investigate the therapeutic efficacy of ozone
in patients with CAD, treated with antithrombotic therapy, Aspirin
s
and policosanol. A randomized
controlled clinical trial was performed with 53 patients divided into two groups: one (n¼27) treated
with antithrombotic therapy and other (n¼26) treated with antithrombotic therapy plus rectal
insufflation of O
3
. A parallel group (n¼50) age and gender matched was used as reference for the
experimental variables. The efficacy of the treatments was evaluated by comparing hemostatic indexes
and biochemical markers of oxidative stress in both groups after 20 day of treatment. Ozone treatment
significantly (Po0.001) improved prothrombin time when compared to the antithrombotic therapy
only group, without modifying bleeding time. Combination antithrombotic therapy þO
3
improved the
antioxidant status of patients reducing biomarkers of protein and lipid oxidation, enhancing total
antioxidant status and modulating the level of superoxide dismutase and catalase with a 57% and 32%
reduction in superoxide dismutase and catalase activities respectively, moving the redox environment
to a status of low production of O
2
with an increase in H
2
O
2
detoxification. No side effects were
observed. These results show that medical ozone treatment could be a complementary therapy in the
treatment of CAD and its complications.
&2012 Elsevier B.V. All rights reserved.
1. Introduction
Cardiovascular diseases, comprising coronary artery (CAD) and
cerebro-vascular diseases, are currently the leading cause of
death globally, accounting for 21.9% of total deaths, and are
projected to increase to 26.3% by 2030 (WHO, 2008). The factors
that coalesce to increase the risk of developing atherosclerotic
CAD were demonstrated and have subsequently been shown to be
pervasive across ethnicities and regions all over the world (Yusuf
et al., 2004). These are not new risks, but the ubiquity of smoking,
dyslipidemia, obesity, diabetes, and hypertension has been gra-
dually escalating (Gupta et al., 2008), and is thought to be the
driving influence behind the epidemic of heart disease faced
today (Franco et al., 2011).
Ozone administered in an appropriate dose interval can
modulate many biochemical pathways with the activation of
second messengers (Bocci et al., 2011). Scientific evidence con-
nected the modulation of different biomarkers (e.g. antioxidant
enzymes, nitric oxide pathways, 2,3-diphosphoglycerate) as a
consequence of applying low ozone doses. Those facts support
some of the current clinical applications of ozone (Colombo et al.,
2000;Re et al., 2008;Steppan et al., 2010). In an experimental
model, rectal insufflation (a simple, easy and inexpensive method
of delivering ozone) showed a sustained improvement in ery-
throcytes deformability suggesting its systemic effects (Seda Artis
et al., 2010).
In this work, we considered that the treatment with ozone at
repeated low doses by rectal way plus regular anti-thrombotic
therapy in CAD patients, could improve blood homeostatic index,
reducing the oxidative stress and providing an antioxidant status,
which can compensate for the CAD-derived cardiovascular com-
plications. Our results confirmed this hypothesis and demon-
strated that ozone not only improved blood homeostatic indexes
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/ejphar
European Journal of Pharmacology
0014-2999/$ - see front matter &2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2012.07.010
n
Corresponding author. Tel.: þ39 71 731076; fax: þ39 71 731347.
E-mail address: gregorcuba@yahoo.it (G. Martı
´nez-Sa
´nchez).
European Journal of Pharmacology 691 (2012) 156–162
but also compensated for the antioxidant/pro-oxidant balance in
those patients.
2. Materials and methods
2.1. Study design
This randomized parallel controlled clinical trial was approved
by an institutional review board (Scientific and Ethics Committees
of the Institution) in accordance with the principle of the
Declaration of Helsinki (WMA, 2004). All patients gave their
informed consent to being enrolled after receiving adequate
information about the study (characteristics of the study, benefits
and possible side effects). Before enrolling, all participants
attended a training program to familiarize them with the study
objectives and treatment plans. The personnel involved empha-
sized that all participating physicians would treat each patient
according to the randomised scheme of treatment.
Adult patients of both gender and different ethnic origin with a
diagnosis of CAD who were hospitalized in the Institute of
Angiology and Vascular Surgery (La Habana, Cuba) were eligible
to participate in the study. Exclusion criteria were: severe septic
conditions, hypersensitivity to the medication to be used, hepatic
dysfunction, renal failure (serum creatinine level41.32
m
mol/l),
pregnancy, cancer or other serious disease, inability to cooperate
with the requirements of the study, recent history of alcohol or
drug abuse, current therapy with any immunosuppressive agent
or anticonvulsant, concurrent participation in another clinical
study, or current treatment with an investigational drug.
For the calculation of the size of the sample, the G
n
Power
3 system (version 3.1) (Faul et al., 2007) was used. The type
1 error was 5% and the type 2 error was 20%, with a minimal
difference between effect rates not higher than 25%. The target
level of enrollment was determined to be 23 patients per group.
Assuming that 10% of study patients would be lost to follow-up,
26 patients per group were studied. In the study were included 26
patients per treatment group and data from 50 gender- and age-
matched healthy subjects was used as reference to establish the
normal reference interval of the experimental variables (oxidative
stress bio-markers).
Patients were randomized to two different groups (1) regular
anti-thrombotic therapy; 27 patients were treated with oral
anticoagulant (Aspirin
s
, acetyl salicylic acid 125 mg and Ater-
omixol
s
, policosanol 5 mg) once a day during 20 day, and (2)
ozone plus anti-thrombotic therapy; 26 patients were treated
daily with ozone and anti-thrombotic therapy as in group 1.
Patients of group 2 were treated with 200 ml of O
3
/O
2
containing
40
m
g/ml of ozone once a day during 20 day. Nelaton Robinson
catheter 40 cm was introduced 10/15 cm by rectal way to deliver
the gas and was place in site for 5 min. The patients were
encouraged to empty his or her bladder and bowels before the
procedure. The insufflation was done immediately after a colonic
enema in case of constipation.
Ozone was generated by OZOMED equipment (Center for
Ozone Research, Havana, Cuba), and was administered by rectal
insufflation. Ozone was obtained from medical grade oxygen, and
was used immediately upon generation and represented only
about 3% of the gas mixture (O
2
þO
3
). The ozone concentration
was measured by using a built-in UV spectrophotometer set at
254 nm (Delgado-Roche et al., 2011).
Blood samples for biochemical analysis were obtained after a
12 h overnight fast, at the beginning and 24 h after the last ozone
treatments. The samples were immediately centrifuged at 3000 g,
at 4 1C for 10 min. The serum was collected and aliquots were
stored at 70 1C until analysis (Delgado-Roche et al., 2011).
2.2. Biochemical determinations
All biochemical parameters were determined as previously
described (Delgado-Roche et al., 2011) by spectrophotometric
methods using an Ultrospect Plus Spectrophotometer from Phar-
macia LKB (Sweden). Catalase activity was measured by following
the decomposition of hydrogen peroxide at 240 nm at 10 s
intervals for 1 min (Haining and Legan, 1972). Superoxide dis-
mutase (SOD) activity was measured using kits supplied by
Randox Laboratories Ltd., Ireland (Cat. No. SD125 and No.
RS505). Concentrations of malondialdehyde (MDA) were analyzed
using the LPO-586 kit obtained from Calbiochem (La Jolla, CA,
USA). In the assay, the production of a stable chromophore, after
40 min of incubation at 45 1C, was measured at 586 nm. For
standards, freshly prepared solutions of malondialdehyde bis
[dimethyl acetal] (Sigma, St. Louis, MO, USA) were used and
assayed under identical conditions (Esterbauer and Cheeseman,
1990). Quantification of total hydroperoxides was measured by
Bioxytech H
2
O
2
-560 kit (Oxis International Inc., Portland, OR,
USA) using xylenol orange to form a stable colored complex,
which can be measured at 560 nm. The peroxidation potential
(PP) was measured by inducing lipid peroxidation by adding Cu
þ
(2 mM) to serum (incubated for 24 h at 37 1C), in order to know
the balance between prooxidant-antioxidant factors. The differ-
ence between MDA levels, measured at 0 and 24 h after induction,
for each sample, was calculated (Ozdemirler et al., 1995).
After precipitation of thiol proteins using trichloroacetic acid
10%, reduced glutathione was measured according to the method
of Sedlak and Lindsay (1968) with Ellman’s reagent [5
0
5 dithiobis
(2-nitrobenzoic acid) 10
2
M (Sigma St. Louis, MO, USA)]; absor-
bance was measured at 412 nm. The advanced oxidation protein
products (AOPP) were measured as the oxidation of iodide anion
to diatomic iodine by AOPP (Witko-Sarsat et al., 1998). Briefly, the
technique consists in treating 100
m
l of serum in PBS (1 ml) with
50
m
l of potassium iodide 1.16 M followed by 100
m
l of acetic acid.
The absorbance of the reaction mixture was immediately read at
340 nm. AOPP concentrations were expressed as
m
M of chlora-
mine-T (Sigma St Louis, MO, USA). Ferric Reducing Ability of
Plasma (FRAP) was assayed through the reduction of Fe
3þ
to Fe
2þ
by serum or reference (ascorbic acid). The Fe
2þ
–2,4,6-tripiridil-s-
triazine complex was detected at 593 nm (Benzie and Strain,
1996).
To assay prothrombin time, patient plasma was incubated
before addition of thromboplastin (Dade Thromboplastin C Plus;
Dade Behring, Marburg, Germany) in buffer and time in seconds
to clot formation was determined by using a Sigma Amelung KC4
coagulation instrument. The laboratory reference interval was
10.1–15.2 s (Schmaier, 2008).
Measurement of bleeding time was conducted during the
blood extraction procedures. Briefly, a blood pressure cuff was
inflated around the patient’s upper arm. Two small cuts on the
lower arm were done just deep enough to cause a tiny amount of
bleeding. The blood pressure cuff was immediately deflated.
Blotting paper was touched to the cuts every 20 s until the
bleeding stops. The bleeding time was recorded with a timer.
The laboratory reference interval was 1–3 min (Schmaier, 2008).
2.3. Statistical analysis
The OUTLIERS preliminary test for detection of error values
was initially applied. Afterward, data were analyzed by one-way
analysis of variance (ANOVA) followed by a homogeneity variance
test (Bartlett-Box). In addition, a multiple comparison test was
used (Duncan test). Results are presented as means7standard
deviation. The level of statistical significance used was Po0.05.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162 157
3. Results
3.1. General characteristics of the patients involved in the study
In relation to the baseline characteristics (Table 1), both
groups were similar at randomization (P40.05). Seventy-five
percent of patients in both groups were older than 60 years and
males were the majority. Their medical history was characterized
mainly by hypertension and ischemic cardiopathy.
Concomitant treatments were those used to control hyperten-
sion (captopril in 31%, nifedipine in 10% and nitropental in 2% of
patients respectively) with no significant difference in propor-
tions between groups. The prevalence of risk factors for cardio-
vascular diseases as hypercholesterolemia and obesity was
monitored in patients. No side effects were observed in patients
enrolled in the study.
3.2. Homeostatic indicators
At the beginning of the treatments prothrombin time and
bleeding time were in the normal reference interval values for all
the patients enrolled in the study, with no significant statistical
differences between the groups. At the end of the treatments
(20 day), prothrombin time was significantly (Po0.001) raised in
both group. The increment was significantly (Po0.001) higher in
the group treated with antithrombotic therapy þO
3
when com-
pared to the antithrombotic therapy group. However values of
bleeding time were non-modified at the end of the study in any
group (antithrombotic therapy or antithrombotic therapyþO
3
).
3.3. Biomarkers of antioxidant-prooxidant balance
The AOPP and MDA concentrations, the end products of
protein and lipid peroxidation respectively, were higher at the
beginning of the treatment in both groups than in the reference
normal subjects. At the end a significant decrease (Po0.05) was
achieved for both markers in the antithrombotic therapy þO
3
group compared to the group antithrombotic therapy. Even the
improvement of those biomarkers in antithrombotic therapy þO
3
group values remain significantly (Po0.05) higher than the
normal reference interval (Table 3). At the beginning of the study,
TH levels were high in both groups (antithrombotic therapy
142.977.7
m
M and antithrombotic therapyþO
3
155.478.6
m
M),
significantly higher (Po0.05) than the reference values
(103.7717.7
m
M). At the end of treatment values in both groups
Table 1
Baseline characteristics of CAD patients.
Characteristics Group antithrombotic therapy
(n¼27)
Group antithrombotic therapyþO
3
(n¼26)
n%n%
Age (years) 50–60 5 18.51 8 30.76
61–70 10 37.03 13 50.00
71–80 8 29.62 4 15.38
480 4 14.81 1 3.84
Gender Female 8 29.62 9 34.61
Male 19 70.37 17 65.38
Previous history Hypertension
a
21 77.77 20 76.92
Myocardial stroke 3 11.11 2 7.69
Ischemic cardiopathy 25 92.59 24 92.30
Risk factors Hypertension 19 70.37 20 76.92
Hypercholesterolemia
b
8 29.62 9 34.61
Obesity
c
10 37.03 13 50.00
Smoking 15 55.55 13 50.00
Complementary diagnosis criteria TC (mM) 7.0570.90 6.8571.33
BMI (kg/m
2
) 32.1577.31 34.21 79.63
No significant statistical differences between groups (P40.05) for these variables were achieved. CAD, coronary artery disease. TC: total
cholesterol. Anti-thrombotic therapy (acetyl salicylic acid 125 mg and policosanol 5 mg). Antithrombotic therapy þO
3
, anti-thrombotic
therapy plus ozone therapy (200 ml of O
3
/O
2
40
m
g/ml) BMI, body mass index: weight(kg)/height(m
2
).
a
Hypertension was defined as elevation of systolic ( 4140 mmHg) and/or diastolic ( 490 mmHg) blood pressure.
b
Hypercholesterolemia: increase in total cholesterol 46.7 mM.
c
BMI430.
Table 2
Hemostatic indexes, during the course of the study.
Parameter Start (X7S.D.) End (X7S.D.)
Prothrombin time (s) Antithrombotic therapy, n¼27 11.070.4 18.474.6
a
Antithrombotic therapyþO
3
,n¼26 11.270.6 24.6 76.6
a,b
Bleeding time (min) Antithrombotic therapy, n¼27 1.470.1 1.6 70.2
Antithrombotic therapyþO
3
,n¼26 1.570.1 1.7 70.2
Start and end, beginning and end of treatment (after 20 day) with antithrombotic therapy (acetyl salicylic acid 125 mg and policosanol
5 mg) or antithrombotic therapy þO
3
: antithrombotic therapy plus ozone therapy (200 ml of O
3
/O
2
40
m
g/ml). Data are means7S.D.
For prothrombin time the laboratory reference interval was 10.1–15.2 s and for bleeding time 1–3 min.
a
Po0.001 vs. compared to start point between the same group.
b
Po0.001 vs. prothrombin time in group antithrombotic therapy at the end of the treatment.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162158
(antithrombotic therapy and antithrombotic therapyþO
3
) were
not significantly different from reference values.
Variables of total antioxidant activities in lipid (PP) or hydro-
soluble fractions (FRAP) at the beginning of the study in both groups
were significantly different from reference values. However, at the
end of the study, a significant (Po0.05) decrease in PP was achieved
in antithrombotic therapyþO
3
, which tend to reach normal values.
FRAP was improved in both treatment groups but final values did
not reach the reference normal values.
After or before treatment, the level of reduced glutathione was
lower in both treatment groups when compared to the reference
group. However, after the treatment the concentration of GSH
was higher in the antithrombotic therapy þO
3
group (with sig-
nificant (Po0.05) differences from the initial levels) than in
antithrombotic therapy patients.
Enzymatic activities of catalase and SOD were significantly
(Po0.05) increased in both groups before treatment when
compared to the reference control group, but maintained the
same ratio (catalase/SOD) compared to the reference. After treat-
ment, a reduction in 67.2% of catalase activities, without mod-
ification of SOD activities, was observed in antithrombotic
therapy group with a consequent significant (Po0.05) reduction
in catalase/SOD ratio compared to the reference group. In con-
trast, in antithrombotic therapyþO
3
group a reduction of 57.1% in
SOD and 32.2% in catalase activities was noted, corresponding
with a significant (Po0.05) increment of the catalase/SOD ratio
compared to the reference group.
4. Discussion
Cardiovascular diseases is a major factor in mortality rates
around the world and contributes to more than one-third of
deaths in the USA (Roger et al., 2011). It has been predicted that
atherosclerosis will be the primary cause of death in the world
by 2020 (Scott, 2002). Consequently, developing a treatment
regimen that can slow or even reverse the atherosclerotic process
is imperative. In this context ozone therapy, simultaneously
applied with regular drugs, may represent a promising approach
for correcting oxidative stress and improving the uncertain
prognosis of many patients (Bocci et al., 2009;Mason, 2011).
Abnormal platelet aggregation has direct consequences in CAD
that can be avoided by antithrombotic agent. As shown in Table 2
a combination of Aspirin
s
and policosanol significant increase the
prothrombin time without modification of the bleeding time, in
line with previous results (Arruzazabala et al., 1997). Low dose
regimens (50–325 mg) of the antiplatelet agent Aspirin
s
(acet-
ylsalicylic acid) are a standard treatment for the secondary
prevention of cardiovascular outcomes (Campbell et al., 2007).
Meta-analysis of randomized controlled trials has shown that low
doses of Aspirin
s
is protective in most types of patients that are
at increased risk of occlusive vascular events (Collaboration,
2002). Guidelines recommend long term use of low dose aspirin
(75–150 mg/day) as an effective antiplatelet regimen for patients
with cardiovascular diseases, unless contraindicated (Graham
et al., 2007).
A randomized, double-blind, placebo-controlled study com-
pared the effects of policosanol, Aspirin
s
and combination
therapy on platelet aggregation. Meanwhile, Aspirin
s
signifi-
cantly reduced platelet aggregation induced by collagen (61.4%)
and epinephrine (21.9%) but not ADP-induced aggregation. Com-
bined therapy significantly inhibited aggregation induced by all
the agonists reaching the highest reductions of platelet aggrega-
tion induced by collagen (71.3%) and epinephrine (57.5%). It was
demonstrated that policosanol was as effective as Aspirin
s
.
Moreover, combination therapies have shown some advantages
compared to the respective monotherapies (Arruzazabala et al.,
1997). The rationale for using two or more types of antiplatelet
agents is that platelets can be activated by various pathways.
Aspirin
s
targets cyclooxygenase-related activation pathway,
while policosanol acts by other mechanisms (Noa et al., 2007).
According to the present results, combined therapy (Aspirin
s
plus
Table 3
Biomarkers of oxidative damage and antioxidant-pro/oxidant balance.
Groups Reference Antithrombotic therapy Antithrombotic therapyþO
3
Biomarkers (n¼50) (n¼27) (n¼26)
AOPP (
m
M of chloramine) Time 0 9.19 70.64
a
23.1275.31
b
23.9070.84
b
20 day 24.2577.18
b
18.2673.61
c
MDA (
m
M) Time 0 3.8070.07
a
13.7173.01
b
12.0470.44
b
20 day 10.8272.52
b
8.3873.63
c
TH (
m
M) Time 0 103.78717.71
a
142.9677.71
b
155.4378.61
b
20 day 98.08712.29
a
104.65713.27
a
PP (
m
M) Time 0 9.1770.82
a
20.4178.16
b
16.2573.62
b
20 day 19.0077.03
b
6.1672.51
a
FRAP (
m
M) Time 0 1017.17206.0
a
146.2762.3
b
191.30757.0
b
20 day 386.07104. 3
c
471.97115.8
d
GSH (mM) Time 0 2.56 70.31
a
1.5070.23
b
1.1870.23
b
20 day 1.0870.45
b
1.5570.63
c
SOD (U/ml) Time 0 11.3571.97
a
40.48710.12
b
37.7577.13
b
20 day 35.27714.21
b
16.2778.39
c
CAT (U/l) Time 0 231.80711.33
a
880.47250.1
b
839.57165.3
b
20 day 288.0799.6
a
543.47172.3
c
CAT/SOD Time 0 0.020470.0057
a
0.022070.0306
b
0.0213 70.0152
b
20 day 0.0081 70.0070
c
0.033370.0205
d
Antithrombotic therapy (acetyl salicylic acid 125 mg and policosanol 5 mg); antithrombotic therapy þO
3
, antithrombotic therapy plus ozone therapy (200 ml of O
3
/O
2
40
m
g/ml); AOPP, advanced oxidation protein products; CAT, catalase; GSH, reduced glutathione; FRAP, ferric reducing ability of plasma; MDA, malondialdehyde; PP,
peroxidation potential; SOD, superoxide dismutase; TH, total hydroperoxides. Data are means 7S.D. Means having different superscript letters indicate significant
difference (Po0.05) comparing reference, initial and final time of each groups values between the same set.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162 159
policosanol) can induce an antithrombotic response, however it only
partially modifies some markers of the oxidative stress system
(normalized catalase and TH and improved FRAP) (Table 3).
The significant (Po0.001) increment in prothrombin time
observed in antithrombotic therapyþO
3
compared to antithrom-
botic therapy (Table 2), could be as consequence of the up-
regulation of adenosine A
2
receptors, induced by conditions such
as oxidative stress (Johnston-Cox and Ravid, 2011). It was
demonstrated that ozone oxidative preconditioning can activate
adenosine A
1
receptors (Leon Fernandez et al., 2008). If the same
mechanisms happen also for adenosine A
2
receptor in platelet the
increment of adenosine signaling mechanisms can act stimulating
platelets adenylate cyclase and increasing platelet cyclic-3
0
,5
0
-
adenosine monophosphate levels. Throughout this mechanism,
platelet aggregation is inhibited in response to various stimuli
such as platelet activating factors, collagen, and adenosine
diphosphate.
Clinical practice has consolidated the use of ozone therapy in
the treatment of peripheral occlusive arterial disease (POAD). It
has been demonstrated that ozonized autohemotransfusion may
be useful to improve both the poor rheological properties of the
blood and the oxygen delivery to tissues in patients suffering
from POAD (Giunta et al., 2001;Turczynski et al., 1991;Verrazzo
et al., 1995). Even if the mechanism of action is not fully under-
stood so far, it seems evident that ozone does not affect the blood
coagulation parameters (Biedunkiewicz et al., 2006). However, it
is now proven that low doses of ozone can modulate the
antioxidant system and improve the redox status (Bocci et al.,
2011). As shown in Table 2, ozone potentiates the anticoagulant
effect of Aspirin
s
plus policosanol, without modification of the
bleeding time. In addition, the combination antithrombotic
therapyþO
3
improves the antioxidant status of the patients
(Table 3).
Increasing evidence has highlighted the roles of oxidative stress
and inflammation in the promotion of atherosclerotic cardiovascular
diseases (Rao and Kiran, 2011). Recent pathological studies have
elucidated specific mediators that appear to link these pathways to
the progression and rupture of the atherosclerotic plaque in the
artery wall (Uno and Nicholls, 2010). Although the symptoms and
signs of CAD are noted in the advanced state of disease, most
individuals with CAD show no evidence of disease for decades. Over
time, vulnerable plaque rupture could activate blood clotting and
consequently an arterial stenosis, which in turn could promote heart
attack, stroke, POAD and major debilitating events (Rao and Kiran,
2011). In addition to the influence on blood coagulation parameters,
modulation of oxidative stress by ozone can be preventive for a
cardiovascular event.
AOPP levels were down-regulated in antithrombotic therapyþO
3
compared to the antithrombotic therapy group. AOPP is a novel
marker of oxidative stress correlating tightly with the degree of
monocyte activation, dityrosine, advanced glycation end products,
neopterin and inflammatory cytokines (i.e.TNF-
a
and its soluble
receptor) levels (Witko-Sarsat et al., 1999). The chlorinated oxidants
are closely related with the mechanism of generation of AOPP.
Moreover, AOPP-induced reactive oxygen species (ROS) production
in endothelial cells is partially mediated by NADPH oxidase activa-
tion. AOPP appeared to act as true inflammatory mediators since
they are able to trigger the oxidative burst and the synthesis of
cytokines (Martinez-Sanchez et al., 2005b). The effect of ozone
regulating AOPP is an indirect index connecting with the control
of the inflammatory process.
Improvement of MDA and PP in the antithrombotic therapyþO
3
group is indicative of the paradoxical antioxidant effect of ozone
therapy, and it is of paramount importance due to the connection of
lipid oxidation with CAD (Rao and Kiran, 2011). Improvement of TH
in antithrombotic therapy and antithrombotic therapyþO
3
groups
after treatment should be connected with the antioxidant effect of
acetyl salicylic acid (Baltazar et al., 2011) or policosanol (Castano
et al., 2002). However, improvement in GSH was only reached in
antithrombotic therapyþO
3
group. The FRAP, indicator of total
antioxidant capacity (Huang et al., 2005), was increased only in
the antithrombotic therapy þO
3
group. FRAP parameter is indicative
of the reducing ability of the sample involving the complex
integration of all compounds with antioxidant activity (Benzie and
Strain, 1996), its rise could be due to the increment of GSH and other
hydro soluble antioxidants. The preconditioning actions of ozone
(Chen et al., 2008;Rodriguez et al., 2009;Stadlbauer et al., 2008), as
well as the improvement in the antioxidant defense systems and the
reduction in ROS, favors an appropriate redox balance, suggesting
that CAD patients could be better protected from an eventual
cardiovascular accident.
It is important to know that the biological effect of rectal
insufflation of O
3
has been demonstrated extensively both experi-
mentally (Gonzalez et al., 2004;Guanche et al., 2010;Seda Artis
et al., 2010) and clinicaly (Calunga et al., 2011;Hernandez Rosales
et al., 2005;Martinez-Sanchez et al., 2005a;Romero Valdes et al.,
1993;Zaky et al., 2011a,2011b). Furthermore, preclinical studies
demonstrated its low toxicity (Diaz-Llera et al., 2009;Guanche
et al., 2010). For this reason, the application of O
3
by rectal way
has been now extended to treat many diseases. Because of the
oxidative preconditioning effect of ozone therapy, a cycle of 20
treatments will be enough to sustain the effect for approximately
3 months, depending on the oxidative stress status of the patients
(Schwartz et al., 2011).
Etiologic inference about the role of oxidative stress on
cardiovascular diseases are complicated by the complexity of
the relation between oxidative stress and antioxidant enzyme
activity (Flores-Mateo et al., 2009). SOD is a scavenger of O
2
,
transforming it in H
2
O
2
. But if H
2
O
2
is a ROS, then SOD activities
cannot be interpreted as a single antioxidant, but as a part of the
antioxidant system. Thus a better understanding of the role of
SOD and catalase could be easily evaluated from the analysis of
the ratio catalase/SOD (Martinez-Sanchez et al., 2005a). According
to the present results (Table 2), CAD patients without treatment
continue to maintain catalase/SOD homeostasis, but experienced
an increment of 3.42 or 3.64 fold in SOD and catalase activities
respectively. After treatment two different scenarios appeared. In
the antithrombotic therapy group, levels of SOD remain high with
normalization of catalase activities compared to non CAD sub-
jects. This scenario implied high level of O
2
production with
normal level of H
2
O
2
detoxification capacity. This fact may be due
to the lowering effect of acetyl salicylic acid in catalase levels
(Kowalczyk et al., 2006). In contrast, in antithrombotic
therapyþO
3
a reduction of 57% and 32% of SOD and catalase
activities respectively moves the system to a status of low
production of O
2
with an increment in H
2
O
2
detoxification.
Those findings suggest a better status for antithrombotic
therapyþO
3
patients. It is well known that in atherosclerotic
plaques, O
2
seems to play an important role in the local
inflammatory response, in migration and proliferation of vascular
smooth muscle and in endothelial dysfunction. Several of these
effects might be mediated by an increased oxidative stress,
reducing the bioavailability of NO and promoting an uncoupled
of nitric oxide sintase (NOS) activity (Miller et al., 2008). In
addition, determining the role of H
2
O
2
in the pathophysiology of
cardiovascular diseases has proven to be difficult. One reason is
that altering the expression of enzymes related to the catabolism
of H
2
O
2
is likely to alter the expression of other genes (e.g.,
through epidermal growth factor receptor transactivation). Over-
expression of CuZnSOD paradoxically accelerates the develop-
ment of atherosclerosis in apolipoprotein E
/
mice and can be
‘‘rescued’’ by overexpression of catalase (Yang et al., 2004). Taking
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162160
these elements into consideration suggests that H
2
O
2
might play
an important role in the development of atherosclerosis. The
modulation of SOD and catalase enzyme by ozone was noted in
other clinical trials (Martinez-Sanchez et al., 2005a) and probably
involves gene expression modulation (Cardile et al., 1995).
In summary, combined treatment using Aspirin
s
, policosanol
and ozone in patients with CAD improved homeostatic and
antioxidant indexes. Integrative therapy with ozone could be a
future opportunity for correcting the chronic oxidative stress
caused by antithrombotic therapy and improving the uncertain
prognosis of CAD patients. In addition, this pilot study, establish
the foundation for a longer time study in order to determine:
(1) clinical implications of antiagregants and ozonetherapy com-
bination on CAD, (2) clinical implications of antiagregant and
ozonetherapy combination on risk factors and (3) possible low-
ering dose of antiagregant.
Acknowledgments
We gratefully acknowledge the support of the staff from the
Laboratory of Biochemistry of the Center of Studies for Research
and Biological Evaluations, Pharmacy and Food Sciences College,
University of Havana, Havana, Cuba. We are grateful and thank
Mr. Lou Dietrich for revising the English manuscript.
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... Recent evidence should support the hypothesis that ozone is able to improve cardiovascular disorders. A recent study by Martínez-Sánchez et al. 63 reported the effective use of ozone in 53 patients with CAD previously treated with an antithrombotic therapy protocol. In this randomized controlled trial, 27 patients were treated with only antithrombotic therapy (Aspirin®, acetyl-salicylic acid (125 mg) and Ateromixol®, policosanol (5 mg), once a day for 20 days), and 26 patients were treated with the same antithrombotic protocol plus 40 μg/mL via rectal insufflation of 3% ozone in 200 mL of an oxygen-ozone calibrated mixture. ...
... In this randomized controlled trial, 27 patients were treated with only antithrombotic therapy (Aspirin®, acetyl-salicylic acid (125 mg) and Ateromixol®, policosanol (5 mg), once a day for 20 days), and 26 patients were treated with the same antithrombotic protocol plus 40 μg/mL via rectal insufflation of 3% ozone in 200 mL of an oxygen-ozone calibrated mixture. 63 Patients undergoing the ozone protocol showed that ozone enhanced the antithrombotic effect of acetyl salicylate with policosanol, improving the antioxidant status of patients and ameliorating their clinical status, although they reported mild platelet activation, which was attributed to the ozone effect on platelet adenosine receptors. 63,64 Bocci et al. 65 reported in past reports that the use of heparin, instead of calcium chelators such as sodium citrate, prior to oxygenozone autohemotherapy may cause platelet aggregation rather than inhibiting platelet activation, whereas oxygenozone autohemotherapy with 50 μg/mL O 3 in the presence of sodium citrate as an anticoagulant did not induce spontaneous or adenosine-induced platelet aggregation. ...
... 63 Patients undergoing the ozone protocol showed that ozone enhanced the antithrombotic effect of acetyl salicylate with policosanol, improving the antioxidant status of patients and ameliorating their clinical status, although they reported mild platelet activation, which was attributed to the ozone effect on platelet adenosine receptors. 63,64 Bocci et al. 65 reported in past reports that the use of heparin, instead of calcium chelators such as sodium citrate, prior to oxygenozone autohemotherapy may cause platelet aggregation rather than inhibiting platelet activation, whereas oxygenozone autohemotherapy with 50 μg/mL O 3 in the presence of sodium citrate as an anticoagulant did not induce spontaneous or adenosine-induced platelet aggregation. 66 This raises the fundamental concern that protocols in ozone therapy must be particularly tailored and standardized to achieve the best outcome and prevent any adverse effects. ...
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Objetivo: Analisar a influência da ozonioterapia no desfecho sintomático de pacientes arterioscleróticos e o impacto na qualidade de vida dos pacientes. Método: Revisão integrativa, realizada nos bancos de dados U.S. National Institutes of Health's National Library of Medicine (NIH/NLM – PUBMED), Biblioteca Virtual em Saúde e Cochrane Library. Após avaliação criteriosa foram selecionados 13 artigos para a realização do estudo. Resultados: Constatou-se que a maioria dos estudos atestaram a eficácia (92%; n=12) da ozonioterapia no desfecho e qualidade de vida de pacientes com doenças arterioscleróticas. Mesmo com os achados expressivos, incluindo redução da taxa absoluta de amputações em 20%, o número de estudos relacionados ao objeto de estudo é limitado, inversamente proporcional à gravidade das doenças ateroscleróticas e aos resultados significativamente positivos obtidos pela quase totalidade dos estudos selecionados. Conclusão: Conforme os dados apresentados nos ensaios clínicos, notou-se que a ozonioterapia apresentou resultados positivos e significativos em pacientes com diversos níveis de aterosclerose; não foi demonstrado um método de excelência para aplicação de ozônio nos pacientes.
... One way to expand the list of procedures is with ozone therapy. Ozone therapy is a bio-oxidative therapy that, when dosed correctly, promotes blood circulation, tissue healing and stimulates the immune system [9][10][11] . It is considered a very efficient collagen biostimulator that improves skin texture through hydration and tissue stretching 12 . ...
... The authors believe that blood circulation, tissue healing and stimulation of the immune system are the keys to this clinical success. Some authors hypothesize the importance of ozone therapy on oxidative stress 10,16 . Stimulating the immune system must be very useful in treating dark circles due to its association with chronic allergy. ...
Article
Objectives: Dark circles under the eyes are a common condition worldwide with multiple treatment options. The aim of this paper is to report a case of dark circles treated with ozone therapy which showed a very reasonable clinical improvement. Case report: A case of dark circles under the eyes treatment with ozone therapy concentrations and doses was reported. We searched MEDLINE treatment options for dark circles. Literature on this subject is scarce. CARE guidelines have been adopted for this case report RESULTS: Ozone therapy is a really effective biostimulator, that reduces spots and wrinkles. In this case, the patient and healthcare professionals were satisfied with just two ozone therapy sessions. This is an interesting alternative treatment for this common condition. Conclusions: Ozone therapy is a safe, inexpensive, and quick treatment for dark circles under the eyes. It can be used alone or in combination with other methods.
... However, evidence suggests that these alternative modalities are all effective in treating CVD and HFrEF, hence, our use of these modalities. [7][8][9][10][11][12][13][14][17][18][19] Nonetheless, nutrition is the foundation of our integrative heart failure therapy, and therefore, it is the common denominator of treatments. These clinical observations raise important questions about the potential benefits of these integrative therapies in the HFrEF population. ...
Article
Heart failure with reduced ejection fraction (HFrEF) is a major contributor of premature cardiovascular-related deaths. Patients are typically on numerous medications to manage this condition; however, patients continue to experience poor quality of life. Alternative therapeutic approaches are needed to treat HFrEF. The clinical course of seven patients with Stage C and D HFrEF who failed guideline-directed medical therapy were retrospectively analyzed based on medical chart data. All patients consumed a defined, plant-based diet as part of their clinical treatment, and a subset also underwent alternative treatment modalities: External Counterpulsation therapy, BEMER therapy, infrared sauna therapy, ozone therapy, or PlaqueX® therapy. Chart review of these patients indicated improvement in left ventricular ejection fraction (LVEF) and right ventricular systolic pressure (RVSP). All patients also had a significant reduction in medication needs and body weight. Further, all patients reported significant improvements in their quality of life. These data suggest that a defined, plant-based diet combined with other alternative modalities may be efficacious in reducing HFrEF medications and treating Stage C or D HFrEF patients who failed guideline-direct medical therapies. Observations from this case series indicate a need for rigorous prospective studies to confirm these effects.
... Quanto aos efeitos terapêuticos do ozônio medicinal, Zhang et al. (2014) do artigo 02, recrutaram dois grupos totalizando em 50 pacientes que estavam fazendo tratamento de oxigênio-ozônio e percebeu-se da melhora do controle glicêmico do pé diabético, redução da lesão, inibição do estresse oxidativo e o mais importante, menos amputações em pacientes que realizaram tratamento com ozônio por 20 dias via inflamação retal. Conforme Martínez-Sánchez et al. (2012) os efeitos antioxidantes do ozônio contribuem no melhoramento das alterações bioquímicas em relação à idade. A ozonoterapia por insuflação em pacientes com doença arterial coronariana (DAC), melhora significativamente o tempo de protrombina, reduzindo os biomarcadores de oxidação de proteínas e lipídios, e aumento do antioxidante total. ...
Book
O presente livro aborda sobre a importância da ozonioterapia na saúde social como tratamento alternativo de feridas. Que teve como objetivo geral demonstrar a eficácia do ozônio medicinal no tratamento de feridas. E teve como objetivos específicos, analisar os efeitos terapêuticos do O³ na regeneração tecidual, abordar sobre a melhora da qualidade de vida após a ozonioterapia, a diminuição de internações e a redução de intervenções cirúrgicas. Os resultados encontrados foram baseados em 20 artigos científicos que mostraram a eficácia do ozônio medicinal, que promoveu a regeneração tecidual e diminuiu a proliferação de microrganismo.
... Dentro de las múltiples complicaciones de la DM-Tipo 2 anteriormente mencionadas, la nefropatía diabética, la hiperlipidemia, la hepatopatía, el estrés oxidativo y la polineuropatía se hacen de difícil manejo terapéutico y en un gran porciento los pacientes no responden de forma satisfactoria a la terapia convencional, este hecho refleja la necesidad de buscar terapias complementarias con el objetivo de mejorar la calidad de vida de estos pacientes portadores de DM-Tipo 2 (Paez et al, 2016). Atendiendo a este planteamiento, la Ozonoterapia la cual ha mostrado efectos biológicos beneficiosos como: el incremento del metabolismo del oxígeno, la protección endotelial, su efecto antinflamatorio, la estimulación del sistema antioxidante endógeno, su efecto germicida de amplio espectro, su acción como modulador del sistema inmune y la actividad neuroprotectora de la ozonoterapia reportado a nivel clínico como preclínico (Martínez et al, 2012;Bocci et al, 2009;Martínez et al, 2005;Menéndez et al, 2008;García et al, 2005), hacen de este proceder terapéutico un potencial complemento de otras terapias convencionales. Diversos estudios, reportados en la literatura, evidencian los efectos beneficiosos de la ozonoterapia en la diabetes mellitus tanto a nivel preclínico como en ensayos clínicos, algunos de estos son: mejor control de la glicemia, disminución del estrés oxidativo, disminución de los productos avanzados de la glicosilación de proteínas e incremento de los niveles de óxido nítrico (Martínez et al, 2005;Menéndez et al, 2008;García et al, 2005). ...
Article
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Diabetes Mellitus Type 2 presents with major serious complications such as chronic kidney failure, liver disease, diabetic neuropathy, cerebrovascular disease created by metabolic dysfunction that leads to a state of chronic inflammation in these patients. With the objective of evaluating the influence of rectal ozone therapy as complementary therapy in patients with Type 2 diabetes Mellitus. 38 patients suffering Diabetes Mellitus Type 2 were selected and randomly divided into two groups. The first group (Ozone) received conventional treatment plus rectal ozone therapy and the second (Conventional) only received conventional treatment. Blood glucose, glycosylated hemoglobin, LDL-cholesterol, VLDL-cholesterol and triglycerides were measured in blood, markers of liver and kidney damage and the DNA-4 scale of neuropathic pain were measured at the beginning and at end of treatments with rectal ozone therapy. The group of patients who received rectal ozone therapy plus conventional treatment showed a significant decrease in glycemic, glycated hemoglobin, VLDL-cholesterol, triglyceride, LDL-cholesterol values compared to the group with conventional treatment. It reflected significant improvement in the liver and kidney damage markers as well as the levels of the DNA.-4 pain scale, compared to the group that only received conventional treatment. The complementary application of rectal ozone therapy showed a protective effect on metabolic, liver and kidney function in patients with Type 2 Diabetes Mellitus, and also showed a certain analgesic character by reducing pain in said disease.
... [14][15][16][17][18][19]33 Moreover, taking into account that in patients with venous leg ulcers the cause of pain is related mainly to hypoxemia and venous hypertension the other mechanism of analgesic effect of ozone therapy could be increasing the oxygen contents in the tissues surrounding the ulcer due to the activation of the Krebs cycle in erythrocytes and the intensification of cytochrome C oxidation, as it was described above, as well as the upregulation of vasoactive substances release (increased production of nitric oxide, adenosine and prostaglandins, resulting in stimulation of vasodilatation process), enhanced erythrocyte flexibility and improved blood rheology related to lowering blood viscosity. [33][34][35] On the other hand it was confirmed in experimental and clinical studies that ozone can prevent or improve the peripheral neuropathy resulting in reduction among others the intensity of neuropathic pain due to adaptive antioxidant and anti-inflammatory response. 33,36,37 Based on the results of the presented study and literature data, it should be assumed that the use of modern physical methods in comprehensive treatment, including local ozone therapy may in many cases be of significant importance in the treatment of difficult to heal ulcers, including venous ulcers. ...
Article
Full-text available
A significant health problem in many countries of the world is the occurrence of hard to heal leg ulcers. In recent years modern methods of physical medicine in comprehensive treatment have been used often including ozone therapy. The study included 54 patients, 25 male and 29 female in age between 39 and 87 years (mean age: 66.7 ± 11.9 years) with venous leg ulcers who underwent a cycle of local ozone therapy. The progress in wound healing was evaluated by computerized planimetry and pain intensity was assessed with use a visual analog scale (VAS) . As a result of the applied local ozone therapy a statistically significant reduction of the ulcer area was achieved from median 7.1 (5.6-9.4) cm ² to 4.4 (3-7) cm ² ( P = .000001), which was on median 38.74 (27.27-51.42)% compared to the baseline values before the start of the therapy. In 2 patients (3.7%) the ulcers were completely healed. 18 patients (33.3%) achieved a reduction in ulcer area of more than 50% of the baseline value and the remaining 34 patients (63%) also achieved a reduction in ulcer area. A statistically significant in the percentage of surface area was observed in the group of 19 patients suffering from > 5 years of age compared to the group of 35 patients suffering from ≤5 years (median 50 (32.03-67.16)% versus 33.96 (23.71-45); P = .033178), while percentage changes in ulcer surface area did not differ significantly between all other subgroups of patients. There was also a statistically significant reduction in the intensity of pain in VAS scale in all patients, median 6 (5-7) points before treatment versus 4.4 (3-7) points after treatment, P = .000001). Local ozone therapy of venous leg ulcers accelerate the healing process of ulcers in objective planimetric assessment and reduce the intensity of pain ailments.
... Therefore, the decrease in D-dimer levels due to the administration of medicinal ozone is a fundamental aspect in the therapy against COVID-19, since thromboembolic events have been manifested in these patients [26,27]. Likewise, and in support of vascular function, it has been reported that the ozone therapy favors the regeneration of the microcirculation and the gaseous exchanges (through the increase of the blood flow, a decrease of the blood viscosity and the platelet aggregation) and, at the same time, to reduce thrombotic and fibrotic processes [28]. ...
Article
Full-text available
Background: SARS-CoV-2-induced inflammation in COVID-19 is mediated by cytotoxic and pro-oxidant effects that potentiate alveolar, endothelial and immune tissue damage. Objective: We investigated the effect of medicinal ozone administration on the oxidative stress markers; in addition to D-dimer, lactic acid and interleukin-6 as markers of endothelial injury and inflammation process. Methodology: Medicinal ozone with oligo metals was administered in vivo (major autohemotherapy) and in vitro (peripheral blood), to subsequently determine the levels of: H2O2, NO, GPx, CAT, TAP, TBARs, D-dimer, lactic acid and interleukin-6. Results: Medicinal ozone administration with oligo metals induced changes in oxidative stress markers both in vitro and in vivo. The H2O2 and TBARs levels decreased, in turn, NO levels increased (cardiovascular function marker). On the other hand, the levels of the antioxidant enzymes (GPx and CAT) show slightly increase, which indicates an antioxidant enzyme system regulation that counteracts the pro-oxidative effect of the infection. Furthermore, interleukin-6 levels decreased indicating the regulation of the systemic inflammatory process. Finally, lactic acid and D-dimer levels were decreased, establishing an improvement of energy metabolism and endothelial function respectively. Conclusion: The medicinal ozone administration induce decrease in the markers levels of oxidative stress, inflammation and cellular damage, improving the enzymatic antioxidant capacity and cellular metabolism with decrease plaque aggregation that contribute to reducing the risk of vascular endothelial damage. These benefits could be feasible to integrate in the treatment of endothelial injury in COVID-19 patients.
Article
Background Chemotherapy-induced peripheral neuropathy (CIPN) is a severe adverse reaction to chemotherapeutics, which seriously affects the outcome of chemotherapy and patients’ quality of life. Although it is commonly seen, it lacks effective treatment. Our previous study found that ozone could alleviate neuropathic pain. Damage-associated molecular patterns (DAMPs) or Toll-like receptor 4 (TLR4) or tissue factor (TF)-mediated neuroinflammation and microcirculation disturbance is the main reason for CIPN. Suppressors of cytokine signaling (SOCS) 3 is an endogenous negative feedback regulator of inflammation via TLR4 inhibition. Materials and Methods Oxaliplatin (L-OHP) was used to establish mice’s CIPN model. Nociceptive responses were assessed by observing the ICR mice’s incidence of foot regression in mechanical indentation response experiments. Cell signaling assays were performed by Western blotting and immunohistochemistry. The mouse leukemia cells of monocyte–macrophage line RAW 264.7 were cultured to investigate the effects of ozone administration on macrophage. Results Ozone decreased the expression of TF in the blood and sciatic nerve. It upregulated the adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK)-SOCS3 axis to relieve CIPN and inhibit TF expression in vivo. SOCS3 expression was induced by ozone to inhibit the p38/TF signaling in RAW 246.7 cells. Ozone also prevented L-OHP-induced sciatic nerve demyelination. Microglia activation was inhibited, and c-Fos and calcitonin gene-related peptide (CGRP) expression was decreased in the spinal dorsal horn via ozone. Conclusions In this study, we demonstrated that ozone could alleviate CIPN by upregulating the AMPK-SOCS3 axis to inhibit TF expression, which is a potential treatment for CIPN.
Article
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Cardiovascular (CV) disease is a major factor in mortality rates around the world and contributes to more than one-third of deaths in the US. The underlying cause of CV disease is atherosclerosis, a chronic inflammatory process that is clinically manifested as coronary artery disease, carotid artery disease, or peripheral artery disease. It has been predicted that atherosclerosis will be the primary cause of death in the world by 2020. Consequently, developing a treatment regimen that can slow or even reverse the atherosclerotic process is imperative. Atherogenesis is initiated by endothelial injury due to oxidative stress associated with CV risk factors including diabetes mellitus, hypertension, cigarette smoking, dyslipidemia, obesity, and metabolic syndrome. Since the renin– angiotensin–aldosterone system (RAAS) plays a key role in vascular inflammatory responses, hypertension treatment with RAAS-blocking agents (angiotensin-converting enzyme inhibitors [ACEIs] and angiotensin II receptor blockers [ARBs]) may slow inflammatory processes and disease progression. Reduced nitric oxide (NO) bioavailability has an important role in the process of endothelial dysfunction and hypertension. Therefore, agents that increase NO and decrease oxidative stress, such as ARBs and ACEIs, may interfere with atherosclerosis. Studies show that angiotensin II type 1 receptor antagonism with an ARB improves endothelial function and reduces atherogenesis. In patients with hypertension, the ARB olmesartan medoxomil provides effective blood pressure lowering, with inflammatory marker studies demonstrating significant RAAS suppression. Several prospective, randomized studies show vascular benefits with olmesartan medoxomil: reduced progression of coronary atherosclerosis in patients with stable angina pectoris (OLIVUS); decreased vascular inflammatory markers in patients with hypertension and micro- (pre-clinical) inflammation (EUTOPIA); improved common carotid intima-media thickness and plaque volume in patients with diagnosed atherosclerosis (MORE); and resistance vessel remodeling in patients with stage 1 hypertension (VIOS). Although CV outcomes were not assessed in these studies, the observed benefits in surrogate endpoints of disease suggest that RAAS suppression with olmesartan medoxomil may potentially have beneficial effects on CV outcomes in these patient populations.
Article
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Coronary artery disease is considered a major cause of death in the western world and its primary pathological manifestation is myocardial damage due to ischemia-reperfusion phenomena. Ozone has been used as a therapeutic agent and beneficial effects against the damage induced by renal or hepatic ischemia-reperfusion have been observed in vivo. The present study evaluated the behaviour of oxidative stress biomarkers in coronary artery disease patients after 20 sessions of ozone (50 μg/mL ; 200 mL) by rectal insufflation. Blood samples of 40 patients and 50 healthy subjects were tested by spectrophotometric techniques. Indicators of biomolecular damage, enzymatic antioxidant activity and total antioxidant status were determined. We demonstrated that ozone therapy reduced the oxidative stress index as reflected by the increase in superoxide dismutase and catalase activities, the reduction of malondialdehyde, total hydroperoxides and advanced oxidation protein products concentration. Furthermore, an increase in glutathione levels was noted. The results of the present study show that repeated administrations of ozone in non-toxic doses play a positive role in the control of antioxidant/pro-oxidant balance in coronary artery disease patients.
Article
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The in vivo genotoxic effect of ozone/oxygen mix (O2-O 3) was studied in leukocytes and exfoliated colorectal cells of SD rats using the Comet assay (single cell gel electrophoresis assay, SCGE). O 2-O3 (O3 final dose 42 g/kg b.w.) was applied for four days by colorectal insufflation simulating human ozone therapy. Bleomycin (15 mg/kg b.w., i.p.) was used as a positive control. The genotoxic effect of ozone was measured in exfoliated colorectal cells at 24, 48 and 72 h and in leukocytes at 0, 2, 6, 24, 48 and 72 h after the last exposure to O 2-O3. As a result, a significant (p<0.025) increase in the primary DNA damage was observed in exfoliated colorectal cells and in the peripheral blood leukocytes. The highest values of DNA damage were observed at 48 h and 24 h after the last exposure to O2-O3 in exfoliated colorectal cells and in leukocytes respectively. However, 72 h after the last exposure a significant (p<0.05) decrease of DNA damage was observed in both cell types, indicating an evident recovery of the DNA primary damage induced by the treatment.
Article
OBJECTIVE: To determine the effects of antiplatelet therapy among patients at high risk of occlusive vascular events. DESIGN: Collaborative meta-analyses (systematic overviews). INCLUSION CRITERIA: Randomised trials of an antiplatelet regimen versus control or of one antiplatelet regimen versus another in high risk patients (with acute or previous vascular disease or some other predisposing condition) from which results were available before September 1997. Trials had to use a method of randomisation that precluded prior knowledge of the next treatment to be allocated and comparisons had to be unconfounded-that is, have study groups that differed only in terms of antiplatelet regimen. STUDIES REVIEWED: 287 studies involving 135 000 patients in comparisons of antiplatelet therapy versus control and 77 000 in comparisons of different antiplatelet regimens. MAIN OUTCOME MEASURE: "Serious vascular event": non-fatal myocardial infarction, non-fatal stroke, or vascular death. RESULTS: Overall, among these high risk patients, allocation to antiplatelet therapy reduced the combined outcome of any serious vascular event by about one quarter; non-fatal myocardial infarction was reduced by one third, non-fatal stroke by one quarter, and vascular mortality by one sixth (with no apparent adverse effect on other deaths). Absolute reductions in the risk of having a serious vascular event were 36 (SE 5) per 1000 treated for two years among patients with previous myocardial infarction; 38 (5) per 1000 patients treated for one month among patients with acute myocardial infarction; 36 (6) per 1000 treated for two years among those with previous stroke or transient ischaemic attack; 9 (3) per 1000 treated for three weeks among those with acute stroke; and 22 (3) per 1000 treated for two years among other high risk patients (with separately significant results for those with stable angina (P=0.0005), peripheral arterial disease (P=0.004), and atrial fibrillation (P=0.01)). In each of these high risk categories, the absolute benefits substantially outweighed the absolute risks of major extracranial bleeding. Aspirin was the most widely studied antiplatelet drug, with doses of 75-150 mg daily at least as effective as higher daily doses. The effects of doses lower than 75 mg daily were less certain. Clopidogrel reduced serious vascular events by 10% (4%) compared with aspirin, which was similar to the 12% (7%) reduction observed with its analogue ticlopidine. Addition of dipyridamole to aspirin produced no significant further reduction in vascular events compared with aspirin alone. Among patients at high risk of immediate coronary occlusion, short term addition of an intravenous glycoprotein IIb/IIIa antagonist to aspirin prevented a further 20 (4) vascular events per 1000 (P<0.0001) but caused 23 major (but rarely fatal) extracranial bleeds per 1000. CONCLUSIONS: Aspirin (or another oral antiplatelet drug) is protective in most types of patient at increased risk of occlusive vascular events, including those with an acute myocardial infarction or ischaemic stroke, unstable or stable angina, previous myocardial infarction, stroke or cerebral ischaemia, peripheral arterial disease, or atrial fibrillation. Low dose aspirin (75-150 mg daily) is an effective antiplatelet regimen for long term use, but in acute settings an initial loading dose of at least 150 mg aspirin may be required. Adding a second antiplatelet drug to aspirin may produce additional benefits in some clinical circumstances, but more research into this strategy is needed.
Article
Arterial occlusive disease: pharmacological therapy and ozone therapy. Background. The therapeutic effects of intravenous ozone in the treatment of arterial occlusive disease have been known for some time. Various authors have shown how this treatment improves the transport and release of oxygen to tissues. Methods. Among the main mechanisms underlying this result, the most important is increased 2,3 diphosphoglycerate (2,3 DPG). This enzyme results in a diminished affinity of the hemoglobin for oxygen, thereby enabling the latter's increased release to the tissues. Results. We compared the results in a sample of patients receiving conventional pharmacological therapy and a group of patients receiving ozone therapy according to our usual protocols. All the patients were treated using a major own-blood transfusion. The dose used for every transfusion was 5000 ng ozone added to 200 cc uncoagulated blood with citrate. Applications were continued twice weekly for a total of 5 sessions. A total of 50 pharmacologically-treated patients were included at Fontaine's second stage. Another group of 50 patients commenced ozone treatment at the same time. Patients receiving pharmacological treatment presented improved symptoms in 50% of cases. Those who were treated with ozone treatment showed improved symptoms in 86% of cases. Changes in 2,3 DPG evaluated before and after treatment were correlated with the improved symptoms achieved. In general low indices of 2,3 DPG corresponded to enhanced levels after treatment and improved symptoms. However, significant improvements were noted even in the event of high enzyme levels before treatment. Conclusions. The predictive criterion for cases with low indices of 2,3 DPG at the start of treatment is indicative and not absolute. Ozone activity induces improvements that exceed all expectations when the patients to be treated present a metabolic reserve requiring stimulation and strengthening.
Article
Published research in English-language journals are increasingly required to carry a statement that the study has been approved and monitored by an Institutional Review Board in conformance with 45 CFR 46 standards if the study was conducted in the United States. Alternative language attesting conformity with the Helsinki Declaration is often included when the research was conducted in Europe or elsewhere. The Helsinki Declaration was created by the World Medical Association in 1964 (ten years before the Belmont Report) and has been amended several times. The Helsinki Declaration differs from its American version in several respects, the most significant of which is that it was developed by and for physicians. The term "patient" appears in many places where we would expect to see "subject." It is stated in several places that physicians must either conduct or have supervisory control of the research. The dual role of the physician-researcher is acknowledged, but it is made clear that the role of healer takes precedence over that of scientist. In the United States, the federal government developed and enforces regulations on researcher; in the rest of the world, the profession, or a significant part of it, took the initiative in defining and promoting good research practice, and governments in many countries have worked to harmonize their standards along these lines. The Helsinki Declaration is based less on key philosophical principles and more on prescriptive statements. Although there is significant overlap between the Belmont and the Helsinki guidelines, the latter extends much further into research design and publication. Elements in a research protocol, use of placebos, and obligation to enroll trials in public registries (to ensure that negative findings are not buried), and requirements to share findings with the research and professional communities are included in the Helsinki Declaration. As a practical matter, these are often part of the work of American IRBs, but not always as a formal requirement. Reflecting the socialist nature of many European counties, there is a requirement that provision be made for patients to be made whole regardless of the outcomes of the trial or if they happened to have been randomized to a control group that did not enjoy the benefits of a successful experimental intervention.
Chapter
This chapter discusses the methods used for the qualitative and quantitative determination of aldehydes in biological systems. It focuses on 4-hydroxynonenal (HNE) and malondialdehyde (MDA). 4-Hydroxynonenal is produced as a major product of the peroxidative decomposition of polyunsaturated fatty acids (PUFA) and possesses cytotoxic, hepatotoxic, mutagenic, and genoroxic properties. Increased levels of HNE are found in plasma and various organs under conditions of oxidative stress. In addition to HNE, lipid peroxidation generates many other aldehydes that may also be of toxicological significance. Malondialdehyde is in many instances the most abundant individual aldehyde resulting from lipid peroxidation, and its determination by thiobarbituric acid (TBA) is one of the most common assays in lipid peroxidation studies. In vitro MDA can alter proteins, DNA, RNA, and many other biomolecules. Recently, it has been demonstrated with monoclonal antibodies that malonaldehyde-altered protein occurs in atheroma of hyperlipidemic rabbits.
Article
Guidelines and Expert Consensus Documents summarize and evaluate all currently available evidence on a particular issue with the aim to assist physicians in selecting the best management strategies for a typical patient, suffering from a given condition, taking into account the impact on outcome, as well as the risk–benefit ratio of particular diagnostic or therapeutic means. Guidelines are not substitutes for textbooks. The legal implications of medical guidelines have been discussed previously. A great number of Guidelines and Expert Consensus Documents have been issued in recent years by the European Society of Cardiology (ESC) as well as by other societies and organizations. Because of the impact on clinical practice, quality criteria for development of guidelines have been established in order to make all decisions transparent to the user. The recommendations for formulating and issuing ESC Guidelines and Expert Consensus Documents can be found on the ESC web site (http://www.escardio.org/knowledge/guidelines/rules). In brief, experts in the field are selected and undertake a comprehensive review of the published evidence for management and/or prevention of a given condition. A critical evaluation of diagnostic and therapeutic procedures is performed, including assessment of the risk–benefit ratio. Estimates of expected health outcomes for larger societies are included, where data exist. The level of evidence and the strength of recommendation of particular treatment options are weighed and graded according to predefined scales, as outlined in the tables below. The experts of the writing panels have provided disclosure statements of all relationships they may have which might be perceived as real or potential sources of conflicts of interest. These disclosure forms are kept on file at the European Heart House, headquarters of the ESC. Any changes in conflict of interest that arise during the writing period must be notified to the ESC. The Task Force report was entirely …