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IJIR: Your Sexual Medicine Journal
https://doi.org/10.1038/s41443-020-0301-1
ARTICLE
Effects of ozone treatment on penile erection capacity and nitric
oxide synthase levels in diabetic rats
Aykut Colakerol1●Mustafa Zafer Temiz1●Hasan Huseyin Tavukcu2●Serdar Aykan3●Sule Ozsoy1●Ahmet Sahan4●
Engin Kandirali1●Atilla Semercioz1
Received: 11 February 2019 / Revised: 11 April 2020 / Accepted: 28 April 2020
© The Author(s), under exclusive licence to Springer Nature Limited 2020
Abstract
We aimed to determine the effects of ozone treatment on functional and biochemical changes in corpus cavernosum of
diabetic rats. A total of 18 rats were included in the study. The rats were divided into the three groups as control, diabetes
mellitus, and diabetes mellitus +ozone therapy groups. In the latter, ozone gas mixture was administered intraperitoneally
for 2 weeks after the induction of experimental diabetes model. Erectile response was evaluated by determining mean
intracavernosal pressure. Tissue neuronal, inducible and endothelial nitric oxide synthase levels were evaluated with
commercial ELISA kits. Immunohistochemical evaluation was also performed to determine the expression levels of nitric
oxide synthases semiquantatively. Mean intracavernosal pressure and intracavernosal pressure/systemic arterial blood
pressure ratio were significantly higher in the diabetes mellitus +ozone therapy group than those of diabetes mellitus group
(24.57 ± 6.36 mmHg vs. 5.98 ± 2.04 mmHg, p=0.005 and 0.81 ± 0.16 vs. 0.26 ± 0.11, p=0.0001, respectively). The level
of penile tissue endothelial nitric oxide synthase was significantly higher in diabetes mellitus +ozone therapy group
compared with others (19.28 ± 3.40 ng/mL vs. 13.47 ± 2.06 ng/mL and 13.28 ± 1.48 ng/mL, P=0.01). Endothelial nitric
oxide synthase expression increased significantly with ozone therapy. Our results suggest that ozone therapy may be
beneficial in reducing the negative effects of diabetes on erectile dysfunction as a result of enhanced enzymatic activity in
endothelial nitric oxide synthase levels.
Introduction
Diabetes-associated erectile dysfunction (ED) is an impor-
tant health problem worldwide. Vasculopathy, neuropathy,
visceral adiposity, insulin resistance, and hypogonadism are
considered as the main physiopathological events of ED
in diabetic males [1,2]. The most important part of the
vasculopathy is impaired endothelial function with the
inability of nitric oxide (NO) production [3]. NO is one of
the most important vasorelaxing messengers in erection
physiology, which provides vasodilation and vascular
homeostasis in penile tissue [4].
To date, two isoforms of NO directly playing role in
penile erection have been demonstrated as neuronal NO
(nNO) and endothelial NO (eNO) synthesized by mainly
neurons and endothelium, respectively. Another isoform,
inducible NO (iNO), synthesized by several tissues and
immune cells as immune response to different events such
as infection has also been reported [5,6].
Recently, ozone therapy has become a popular
treatment for the treatment of various acute and chronic
diseases. However, because of the controversial results
regarding the safety and efficacy of ozone therapy, it has not
been adopted in an everyday clinical practice yet. On the
basis of the mechanisms of action, ozone therapy induces a
biological response including the improvement of blood
circulation and oxygen delivery to a ischemic tissue as a
result of the an upregulation of cellular antioxidant enzymes
*Aykut Colakerol
aykutcolakerol@hotmail.com
1Department of Urology, University of Health Sciences Bagcilar
Training and Research Hospital, Istanbul, Turkey
2Department of Urology, University of Health Sciences Sultan
Abdulhamid Han Training and Research Hospital,
Istanbul, Turkey
3Department of Urology, University of Health Sciences Haydarpasa
Numune Training and Research Hospital, Istanbul, Turkey
4Department of Urology, University of Health Sciences Dr. Lutfi
Kirdar Kartal Training and Research Hospital, Istanbul, Turkey
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[7]. Several in vitro and in vivo studies, as well as some
clinical trials, have shown a positive effect in cardiovascular
disorders such as coronary artery disease, chronic heart
failure, myocardial infarction, and peripheral artery diseases
[8–10]. Moreover, increased red blood cell glycolysis rate
has been reported with ozone therapy in the literature [11].
This leads to an increase in the amount of oxygen released
to the tissues by the stimulation of 2, 3-diphosphoglycerate.
As a result, the combination of increased antioxidant
enzymes and tissue oxygenation stimulated by ozone therapy
competes with the oxidative stress in the body [12]. In this
regard, diabetic complications attributed to the hyperglycemia
and oxidative stress in the body may be more suitable target
for ozone therapy. Some evidences have already supported
this opinion and preventive effects of ozone therapy with
enhanced antioxidant system affecting the level of glycemia
on several diabetic complications were reported in the litera-
ture [12,13].
Herein, we hypothesized that ozone therapy may have
some potential benefits on diabetic ED.
Therefore, we aimed to investigate the effects of ozone
therapy to the erectile capacity in streptozotocin (STZ)-
induced diabetic rats with the evaluation of intracavernosal
pressure (ICP) profile and tissue NO synthase (NOS) levels.
Materials and methods
A total of 18 healthy 5 weeks of age male Sprague-Dawley
rats weighing 250–330 g were included in the study. This
study was carried out in Istanbul Bağcılar Training and
Research Hospital Animal Experiments Ethics Committee
(Istanbul B.E.H. HADYEK) with the approval of the
project 2015–27 Istanbul B.E.H Experimental Research
and Skill Development Training Center (BADABEM).
A power analysis is used to determine the sample size in
this nonrandomized, nonblinded study. The rats had free
access to normal laboratory food and water ad libitum, and
were kept under controlled room temperature 22 °C and
12–12 h light–dark cycle. The rats were divided into
three groups (n=6 per group) as control, diabetes mellitus
(DM), and DM +ozone therapy groups. To provide
diabetic rat model, a single dose of intraperitoneal (IP)
60mg/kgSTZwasusedinDMandDM+ozone therapy
groups. The rats were accepted as diabetic when blood
glucose levels higher than 300 mg/dL after STZ adminis-
tration for 72 h, was administered for 2 weeks in DM +
ozone therapy group to investigate the effects of ozone
therapy on diabetes-associated ED [14]. Ozone was
obtained from medical grade oxygen and used immedi-
ately. Represents only 3% of the gas (O2 +O3) mixture
[15,16].Afteraperiodof8weekstoemergeanendo-
thelial damage in each diabetic rat, a 60 µl/ml (97% O2-3%
O3) gas mixture was administered intraperitoneally with
a 25 G injector everyday for 2 weeks (Hyper Medozon,
Herrmann Apparatebau GmbH, Baden, Germany). Tygon
polymer tubes and single-use silicon-treated poly-
propylene syringes (ozone resistant) were used throughout
the reaction to ensure containment of O3 and consistency
of concentrations [17].
After completion of the ozone therapy, at the 10th week of
the experimental procedure, body weight, mean arterial blood
pressure (MAP) (mmHg), and ICP (mmHg) of the each rat
were measured and noted. Left internal carotid artery of the
rats were dissected using cervical incision in supine position
and a PE50 tube was inserted into the internal carotid artery
to determine the MAP after providing anesthesia with IP
injections of 1 mg/kg dose of 10% ketamine (Ketaminhy-
drochlorid, Ketalar; Pfizer, Istanbul, Turkey) and 0.25 mg/kg
dose of 2% xylazine (Xylazinclorid, Rompun; Bayer Vital
GmbH, Leverkusen, Germany). Afterwards, penile tunica
albuginea was revealed after denuding the skin of penis and a
24 G needle which connected with a heparinized PE50 tube
was inserted into the right corpus cavernosum. A pressure
transducer and amplifier unit (COMBAT Pharmacology &
Physiology Instruments, Ankara, Turkey) were connected to
the PE50 tubes in the carotid artery and corpus cavernosum
to determine the MAP and ICP, respectively [18]. Finally,
cavernosal nerves were revealed with abdominal laparotomy
and were held just below the main ganglion with two parallel
bipolar stainless steel electrodes. The electrodes were
connected to the STPT02 stimulator (COMBAT Pharma-
cology & Physiology Instruments, Ankara, Turkey) and the
stimulation parameters were set to be; 1.5 milliamps, 20 Hz,
5 millisecond pulse interval, 35 millisecond delay, 7.5 volts
for 60 sec. Eventually, the measurements of MAP and ICP
were done during the penile erection with nerve stimulation
[18,19].
After measurement of MAP and ICP, the rats were
sacrificed and the penile tissues were divided to proximal
and distal parts for the evaluation of tissue NOS levels using
ELISA and immunohistochemistry techniques, respectively.
Determination of NOS enzyme activity in the corpus
cavernosum were performed after the homogenizing the
tissue in 1 ml of 0.15 molar KCl buffer in pH 7.4. The
supernatant was obtained by centrifuging the homogenate at
+4 °C for 15 min at 14000 rpm. From the supernatants
obtained, nNOS, eNOS, and iNOS tissue levels were
determined with ELISA method using the Bio Tek
ELXx800 (BioTek®Instruments, IncUSA) device and YH-
Biosearch commercial kits (Shanghai YeHua Biological
Technology Co) (nNOS catalog number: YHB0774Ra,
eNOS catalog number: YHB0379Ra and iNOS catalog
number: YHB0574Ra). Distal parts of the resected rat penis
were sent to the pathological examination in a 10% buffered
formaldehyde solution. The entire tissues were implanted to
A. Colakerol et al.
paraffin blocks and 4 μm thick sections were provided.
nNOS, eNOS, and iNOS antibodies were applied to lysine
coated slides for immunohistochemical examination and
examinations were performed with light microscopy and
×10 objective (Olympus BX51, Olympus America, Inc.) by
the same pathologist who did not know that the specimens
were belonged to which group. The expression levels of
nNOS, eNOS, and iNOS were assessed by semiquantitative
method according to their prevalence in the tissue and
scored as grade 1, 2, or 3 (Table 1). Intensity of the
expressions were also scored (0–3) in terms of the staining
degree [20].
The statistical analyses were performed using SPSS
22 software (IBM Corp., Armonk, NY).
Test of normality was performed with the Shapiro–Wilk
method. The data were expressed as median and inter-
quartile range. The Kruskal–Wallis was used to test the
differences in weight and blood glucose levels before and
after the development of streptozotocin-induced diabetes.
The parameters MAP, ICP, ICP/MAP, ELISA, and immu-
nohistochemical results for NOS levels were compared
using the Kruskal–Wallis test in the groups.
Results
Body weights and blood glucose levels
The body weights of the rats in DM and DM +ozone
therapy groups were significantly decreased after the
development of experimental DM model, whereas those of
the control group significantly increased (Table 2). The
blood glucose levels were also significantly increased with
experimental DM model (Table 2).
MAP, ICP, and ICP/MAP levels
The mean MAP levels were comparable between the groups
(Fig. 1). However, the mean level of ICP was significantly
higher in the control group as expected. It was detected as
5.98 ± 2.04 and 24.57 ± 6.36 mmHg in the DM and DM +
ozone therapy groups, respectively (p=0.005). The char-
acteristics of ICP/MAP ratio were detected as 0.96 ± 0.19,
0.26 ± 0.11, and 0.81 ± 0.16 in the control, DM, and DM +
ozone therapy groups, respectively (p=0.0001) (Fig. 2).
Tissue levels of nNOS, eNOS, and iNOS
There was no statistically significant difference between the
groups in terms of penile tissue levels of nNOS and iNOS
(Fig. 3). However, the level of penile tissue eNOS was
significantly higher in the rats of DM +ozone therapy
group compared with others (p=0.01). Expression patterns
of nNOS, eNOS, and iNOS in penile tissue. No statistically
significant differences were observed in the prevalence and
intensity of immunohistochemical expression of iNOS in
penile tissue between the groups. While the intensity of
eNOS expression was similar in the groups, the prevalence
of eNOS expression significantly increased with ozone
therapy in the DM +ozone therapy group in comparison
with the DM group (Table 3) (Figs. 4–5).
Immunostaining with nNOS was not provided in the penile
tissues and statistical comparison could not been performed.
Discussion
For normal erection, endothelial cells, nerve structures,
and cavernosal smooth muscles should be under normal
Table 1 Semiquantitative assessement and scoring method of the expression levels of nNOS, eNOS, and iNOS in the tissue.
Grading of the prevalence
of NOS expression
nNOS eNOS iNOS
Score 1 0–1 positive cell at ×10 magnification 0–25% positive cells at ×10 magnification 0–1 positive cell at ×10 magnification
Score 2 2–3 positive cells at ×10 magnification 25–75% positive at ×10 magnification 2–3 positive cells at ×10 magnification
Score 3 ≥4 positive cells at ×10 magnification 75–100% positive at ×10 magnification ≥4 positive cells at ×10 magnification
Table 2 Body weights and blood glucose levels of the rats before and after the diabetes model.
Control group DM group DM +ozone therapy group
Body weight (gr) Before 303.63 ± 6.33 After 344.58 ± 10.23
p=0.0001aBefore 313.37 ± 14.01 After 259.59 ± 10.02
p=0.0001aBefore 312.01 ± 15.58 After 253.21 ± 10.68
p=0.0001a
Blood glucose (mg/dl) Before 90.67 ± 4.18 After 90.67 ± 4.27
p=0.9aBefore 90.33 ± 2.16 After 409.17 ± 15.54
p=0.0001aBefore 89 ± 2.19 After 413 ± 24.31
p=0.0001a
DM diabetes mellitus.
aKruskal–Wallis test.
Effects of ozone treatment on penile erection capacity and nitric oxide synthase levels in diabetic rats
conditions. Complex interactions between these structures
are required to provide and maintain a successful penile
erection. Actually, the key element responsible for pro-
voking the erection is NO. It leads to penile tumescence
with the relaxation of cavernosal smooth muscle by
increasing intrecellular cyclic guanosine monophosphate
and decreasing the cytosolic calcium concentrations
[21,22]. The enzyme NO synthase (NOS), which produce
NO from the conversion of L‐arginine to L‐citrulline, has
been localized to neuronal tissue, endothelium, and epi-
thelium within several tissues and immune cells [23,24]. In
the literature, neuronal NO (nNO) has been assumed as the
main NO type playing a key role in the penile erection by
inducing increased cavernosal relaxation and blood flow.
While nNO is considered to be an important enzyme during
erection, endothelial NO (eNO) is also necessary for the
achievement and maintenance of erection [23,25]. Role of
the third NO isoform, the inducible NO (iNO), does not
Fig. 1 MAP and ICP
parameters in the groups.
aMAP, ICP max levels in the
groups. bICP/MAP ratios of
groups.
A. Colakerol et al.
appear directly in physiological penile erection but plays
role during some pathological processes such as the aging
or injury-related fibrosis [26].
Impaired NO metabolism is an important part of the
diabetes-associated ED. It is commonly associated with
neuropathy and vasculopathy with reduced production of
NO by neurons and endothelium and clearly caused by
hyperglycemia [27,28]. Hyperglycemia directly activates
oxidative stress and proinflammatory cytokine activity, as
well [29]. As a result, persisting oxidative stress will cause
impaired enzymatic activity in eNOS in diabetic males and
contribute to reduced production of NO [30,31].
The ozone is a gas molecule consist of three oxygen
atoms, which can oxidize organic compounds has also
therapeutic effects when administered in therapeutic doses
with appropriate administration methods [32,33]. In 2014,
Saleh S et al. [34] investigated the effects of adjunctive
ozone therapy to insulin on diabetes outcomes in a rat
model and reported the additive multiprotective antioxidant
effect of ozone in the treatment of diabetes.
In addition, ozone stimulates the transmembrane flow of
oxygen in the cells and increased cellular oxygen levels
with ozone therapy can cause increase in NOS. Several
previous studies have been reported that ozone has ability to
activate and upregulate the genes associated with NOS, and
increases NO levels in several tissues including liver, heart,
muscular tissue, kidney, and testis [35–37]. In the present
experimental study, we aimed to induce enzymatic activity
of the eNOS and other isoforms with antioxidative ozone
therapy in a rat model of streptozotocin-induced diabetic
ED. We found that mean ICP and ICP/MAP ratio
were significantly increased with ozone therapy in diabetic
rats compared with therapy naive diabetic rats (24.57 ±
6.36 mmHg vs. 5.98 ± 2.04 mmHg, p=0.005 and 0.81 ±
0.16 vs. 0.26 ± 0.11, p=0.0001, respectively). The level
of penile tissue eNOS was significantly higher in the
rats treated with ozone compared with others (19.28 ±
3.40 ng/mL vs. 13.47 ± 2.06 ng/mL and 13.28 ± 1.48 ng/mL,
p=0.01).
Pranata [38] published a preliminary results about the
effects of ozone therapy on patients with type 2 diabetes-
associated ED in 2010. The study investigated 14 men with
diabetes-associated ED who received nutrition therapy with
adjunctive ozone therapy. At the end of the study protocol,
decreased blood glucose and glycosylated hemoglobin
levels and decreased oxidative stress were reported with
adjunctive ozone therapy. The author reported a sig-
nificantly increased International Index of Erectile Function
(IIEF-5) score from 12.55 ± 2.51 to 17.57 ± 2.04 with ozone
therapy (p< 0.001), as well. The study concluded that the
beneficial effects of ozone therapy may be with the
improvement of tissue oxygenations, improved endothelial
function, and NO production. However, these parameters
did not determined by Pranata [38] and the author high-
lighted that it is necessary to perform further study to
Fig. 2 Representative ICP recordings of each experimental study group. The red line in the time axis shows 60 sec stimulation of cavernosal
nerves. aICP curve for control group. bICP curve for DM group. cICP curve for DM +Ozone group. (color figure online).
Fig. 3 Tissue levels of the parameters by ELISA. Tissue levels of
nNOS, eNOS and iNOS by ELISA analysis in the groups.
Effects of ozone treatment on penile erection capacity and nitric oxide synthase levels in diabetic rats
investigate the improvement of endothelial function after
ozone therapy as cause for the restoration. Unfortunately,
neither Pranata nor other authors reported further experi-
mental results [38]. Similarly, to our knowledge, there is no
any clinical study investigating the effects of ozone therapy
on endothelial function in patients with ED. In this respect,
the present study have an importance because of its pre-
cursor nature. We determined a significant increase in tissue
expression levels of the eNOS and in tissue nNOS levels
with ozone therapy in the corpus cavernosum of the diabetic
rats. Moreover, our ICP measurements proved that these
favorable effects of ozone therapy on eNOS levels enhanced
the erection capacity of the rats with higher pressure in
penile corpus cavernosum. Our results were consistent with
those of Pranata’s[38]findings. In our opinion, present
study can be accepted as a confirmative experimental
investigation of the claim made by Pranata as IIEF-5 score
increases with ozone therapy in diabetic population.
The present study had some limitations. We provided
semiquantative determination of protein expression levels in
penile tissue. However, advanced quantative protein ana-
lysis with western blot or RT-PCR methods might have
enhance our results and the lack of the advanced protein
analysis methods is the major limitation of the current
study. The lack of the wash out period which provides
adequate time interval between ozone therapy and func-
tional evaluation is the second major limitation, as well. The
other one may be accepted as the lack of the comparison of
blood reactive oxygen specimens and antioxidant enzymes
with blood glucose panel between the groups. In conclu-
sion, our results suggest that ozone therapy may be bene-
ficial in reducing the negative effects of diabetes on ED as a
result of enhanced enzymatic activity in eNOS. Future
researches will have to point out the benefits clearly if the
use of ozone therapy will be initiated in humans in the near
future.
Fig. 4 Tissue eNOS
expressions. Cavernosal tissue
expression levels of eNOS in
terms of intencity (aScore 1,
bScore 3) at ×400 magnificaiton
and prevalence (cScore 1,
dScore 3) at 200 magnification.
Red arrows indicate
eNOS staining areas.
(color figure online).
Table 3 Tissue expression levels
of iNOS and eNOS by
immunohistochemical analysis
in the groups.
Control group DM group DM +ozone therapy group Pa
iNOS Prevalance 1.5 ± 0.55 2 ± 0.63 2.17 ± 0.98 0.302
Intencity 1.33 ± 0.52 1.5 ± 0.55 1.5 ± 0.55 0.827
eNOS Prevalance 2.33 ± 0.52 1.17 ± 0.41 1.67 ± 0.82 0.015
Intencity 1.83 ± 0.75 1.33 ± 0.52 2.17 ± 0.75 0.139
DM diabetes mellitus.
aKruskal–Wallis test.
Statistically significant p< 0.05 values are in bold.
A. Colakerol et al.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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