Accepted Manuscript

The effect of ozone and naringin on intestinal ischemia/reperfusion injury in anexperimental model

Arda Isik, Kemal Peker, Cebrail Gursul, Ilyas Sayar, Deniz Firat, Ismayil Yilmaz,Ismail Demiryilmaz

PII: S1743-9191(15)00378-7

DOI: 10.1016/j.ijsu.2015.07.012

Reference: IJSU 2023

To appear in: International Journal of Surgery

Received Date: 12 June 2015

Accepted Date: 14 July 2015

Please cite this article as: Isik A, Peker K, Gursul C, Sayar I, Firat D, Yilmaz I, Demiryilmaz I, The effectof ozone and naringin on intestinal ischemia/reperfusion injury in an experimental model, InternationalJournal of Surgery (2015), doi: 10.1016/j.ijsu.2015.07.012.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTTITLE PAGE

The effect of ozone and naringin on intestinal ischemia/reperfusion injury in an experimental model.

ARDA ISIK1*, KEMAL PEKER1 , CEBRAIL GURSUL2 , ILYAS SAYAR3 , DENIZ FIRAT1 , ISMAYIL YILMAZ1 , ISMAIL DEMIRYILMAZ1

1: General Surgery Department, Erzincan University, Erzincan-Turkey 2: Physiology Department, Erzincan University, Erzincan-Turkey 3: Pathology Department, Erzincan University, Erzincan-Turkey

*: Corresponding author: General Surgery Department, Erzincan University, Erzincan-Turkey +90 533 0580707, kararda@yahoo.com

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTThe effect of ozone and naringin on intestinal ischemia/reperfusion injury in an experimental model.

Background: The aim of the study was to evaulate the effect of ozone and naringin on the

intestine after intestinal ischemia-reperfusion(II/R) injury.

Methods: Thirty five rats divided into 5 groups of 7 animals: control, II/R, ozone, naringin

and naringin+ozone. Only laparotomy and exploration of the superior mesenteric

artery(SMA) were done in control group. In the experimental groups, SAM was occluded for

1 h and reperfused for 1 h. 15 min after ischemia, ozone(25 µg/ml, 0.5 mg/kg), naringin(80

mg/kg) and naringin+ozone(80 mg/kg+25 µg/ml, 0.5 mg/kg) were infused intraperitoneally to

each groups. Ileum tissues were harvested to determine intestinal mucosal injury and

oxidative stress markers. For SMA occlusion, different than literature, silk suture binding was

used.

Results: Oxidative stress markers were significantly low in experimental groups compared

with II/R group(p < 0.05). Histopathologically, the injury score was significantly low at

experimental groups compared with II/R group(p < 0.05). The lowest injury score was

encountered at naringine+ozone group.

Conclusions: Ozone alone or combined with naringin has a protective effect for mesenteric

ischemia. Instead of using instruments such as clamps in the II/R rat model, silk binding may

be used safely.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTThe effect of ozone and naringin on intestinal ischemia/reperfusion injury in an experimental model.

Introduction

Intestinal ischemia is a fatal clinical condition and intestinal ischemia-reperfusion (II/R)

causes a decrease in vital nutrients, especially O2. As a result, vascular and inflammatory

mediators increase, which causes adhesion, migration and activation of leukocytes. During

reperfusion of the superior mesenteric artery (SMA), reactive oxygen species (ROS) and

reactive nitrogen species (RNS) increase, causing biomolecules such as membrane lipids,

nucleic acids, enzymes and receptors in the tissues to be damaged. This phenomenon is

known as reperfusion injury phenomenon. ROS adhere to membrane-associated

polyunsaturated fatty acids after this peroxidation starts. Increased vascular leakages (protein

and liquid leakages) may cause multi-organ dysfunction syndrome due to the increased

response to the local and systemic response1,2,3. During resuscitation, oxygenated blood,

which goes to ischemic tisssues, may paradoxially increase the degree of damage due to the

increase in free oxygen radicals4.

During II/R treatment, pharmacologic agents such as drugs resisting ischemic injury, drugs

inhibiting the formation of free oxygen radicals and drugs that cause rehabilitation of the

intestine were used. Ozone treatment consisting of an ozone and oxygen mixture can be used

safely and economically especially for chronic ischemic diseases, peritonitis, infected

wounds, chronic skin ulcers, gangrenes and burns. The exact positive effect mechanism of

ozone has not yet been established but is probably due to the increased circulation of blood

that delivers increased O2 to damaged tissues. This process increases general metabolism,

boosts cellulary antioxidant enzyme capacity, activates the immune system and promotes the

release of growth factors from platelets5,6,7.

Naringin (4',5,7-Trihydroxyflavanone-7-rhamnoglucoside) is an bioflavonoid and

polyphenolic compound that is found in grapefruit and is related to citrus herbs species, the

roots of cudrania cochinchinensis and poncirus fruits. It has a biologically and

pharmacologically wide spectrum. It possesses antioxidant, antiulcer, anti-inflammatory,

antiapoptotic, antidiabetic and hepatoprotective effects. It furthermore has effects against the

proliferation of breast cancers. Naringin changes to an absorbable form called naringenin

(4',5,7-Trihydroxyflavanone) with the help of intestinal microflora8,9.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTHere, we evaulate the effect of ozone and naringin on the intestine after II/R injury. After

PubMed and Google Scholar searches we found that there have been no other studies focused

on searching for both of these variables. This study is also the first investigation of SMA

binding via silk sutures instead of clamping.

Materials and Methods

Animals

This study was conducted with the approval of the Ataturk University Animal

Experimentation Ethical Committee (protocol number: 06/06/2014-98) and was performed in

accordance with the guidelines of the National Animal Experiments Ethical Committee. The

rats used in the experiments were obtained from the Ataturk University Medical Practice and

Research Center and were fed on a 12 h day/night cycle in aerated plastic breeding cages at a

room temperature of 22°C. The rats were fed using a standard ad libitum pellet chow and tap

water. The study was conducted with 35 adult male (Sprague Dawley) rats weighing 250 ± 50

g.

Experimental Design

We made an abdominal midline incision in all groups under aseptic conditions and revealed

the SMA (Figure 1a) by opening the abdomen. We divided the rats into 5 groups of 7 animals

as follows:

1. Control Group: Animals were anesthetized with 50 mg/kg ketamine (Ketalar, Eczacıbaşı,

Lüleburgaz, Turkey) and 10 mg/kg xylazine (Rompun, Bayer, Istanbul, Turkey)

intraperitoneally (IP) and placed under the heat lamps for the same duration of ischemia

without any intervention except for laparotomy and exploration of the SMA.

2. II/R Group: Animals were subjected to 1 h of mesenteric (SMA occlusion) ischemia and

sacrificed 1 h after reperfusion. The protocol was similar to that of Boybeyi et al. (2014)4.

Mesenteric ischemia was conducted by binding the SMA with 3-0 silk sutures with the help of

10 Fr- 1 cm plastic catheter (Figure 1b). When there was no pulsation at the SMA and the

color of the ileum changed, we accepted that the SMA was occluded.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT3. II/R+Ozone Group: Animals were subjected to 1 h of ischemia and sacrificed 1 h after

reperfusion but treated with ozone, administered IP one time 45 minutes before reperfusion.

4. II/R+Naringin Group: Animals were subjected to 1 h of ischemia and sacrificed 1 h after

reperfusion but treated with naringin, administered IP one time 45 minutes before reperfusion.

5. II/R+Ozone+Naringin Group: Animals were subjected to 1 h of ischemia and sacrificed

1 h after reperfusion but treated with ozone and naringin, administered IP one time 45

minutes before reperfusion.

The rats were placed on a homeothermic table in order to maintain their core body

temparetures at 37°C. At the time of sacrification via exsanguination under anesthesia, 1 cm

ileum tissue samples from 1 cm proximal to the ileoceacal valve (Figure 2) were obtained and

frozen at −80°C to determine catalase (CAT), superoxide dismutase (SOD), glutathione

reductase (GR) activities and malondialdehyde (MDA) levels. Ileum biopsies were also fixed

in buffered formalin for histological examinations.

Biochemical Procedure

Preparation of tissue

On the experiment day, the ileum tissues were homogenized (IKA Ultra-Turrax T25 basic

homogenizer, Germany) with 0.2 mM pH 7.4 Tris-HCl buffer. We measured the tissue MDA

level in these examples. The homogenate was centrifuged at 4000 rpm for 55 min. Next, the

clean portion of the upper remnant of the supernatant was separated and stored as aliquots to

measure CAT, SOD and GR activities. For the measurement of SOD levels, the supernatant

was extracted with an equal volume of ethanol-chloroform (5/3, v/v) mixture. A UV-

Shimadzu 1600 (Shimadzu, Kyoto, Japan) was used for the spectrophotometric

measurements.

Measurement of Catalase Enzyme Activity

We assessed catalase (CAT, EC 1.11.1.6) enzyme activity using a method described by Aebi

(1974)10 in which the consumption of hydrogen peroxide(H2O2) by the catalase in the medium

was measured spectrophotometrically at 240 nm. The buffer was adjusted to an optical

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTdensity(OD) of 0.500 by adding H2O2 to a 50 mM phosphate buffer. The decrease in the

absorbance with the addition of the sample were recorded every 15 sec. The rate of consumed

H2O2 in 1 min was expressed as k/g protein (k = (2.3 × log (OD1/OD2)) / 30 sec).

Measurement of Superoxide Dismutase Enzyme Activity

We measured superoxide dismutase (SOD, EC 1.15.1.1) enzyme activity

spectrophotometrically at 560 nm based on the principle of reduction of O2!- with nitroblue

tetrazolium (NBT). Enzyme activity was assessed based on the activity of the enzyme

inhibiting 50.0% NBT reduction and was expressed as U/mg of protein (% enzyme inhibition

= (Abscontrol – Abssample) / Abscontrol × 100)11.

Measurement of Glutatyon Reductase Enzyme Activity

Glutathione reductase (EC 1.8.1.7) catalyzes the reduction of glutathione disulfide to the

sulfhydryl form glutathione. We measured the enzyme activity of glutathione reductase using

reduction of NADPH at 340 nm. One enzyme unit is defined as the oxidation of 1 mmol

NADPH per min under assay conditions (25°C, pH 8.0)12. GR were expressed as U/mg

protein.

Measurement of the Amounts of Thiobarbituric Acid-Reactive Substances We measured the amount of thiobarbituric acid-reactive substances (TBARS) in the sample

spectrophotometrically at 532 nm by assessing the reactivity of thiobarbituric acid in the

acidic medium at 90–95°C based on the method described by Esterbauer and Cheeseman

(1990)13. The results were calculated according to the standard and expressed as nmol/g wet

tissue.

Determination of protein

We analyzed the protein for supernatant and extracted samples using the method of Lowry et

al(1951)14.

Histopathological Preparation

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTThe specimens for histopathological examination were collected immediately after

euthanasia. The samples tissues were fixed in 10.0% neutral buffered formalin solution for 2

days. The tissues were washed in running water and were dehydrated with ethanol. After

dehydration, the specimens were placed into xylene to obtain transparency and then were

embedded in paraffin. We cut the embedded tissues into 5 µm-thick sections and stained them

with hematoxylin/eosin. Histopathological examinations of the rat tissue damage were

conducted by blinded pathologist using the methodology of Chiu et al. (1970)15, as shown in

Table 1. Histopathological evaluations were done using a light microscope (Olympus BX53,

Tokyo, Japan).

Ozone preparation

Ozone was prepared by Medozon Compact-YX980D(Herrmann -Germany). Ozone was

prepared from medical-grade oxygen by means of a silent electric discharge representing

about 3% of the ozone/oxygen gas mixture. We measured the ozone concentration using an

ultraviolet spectrophotometer at 254 nm. The ozone solution dissolved in serum physiologic

(0.9% NaCl) was applied by IP as 25 µg/ml, 0.5 mg/kg16.

Naringin preparation

We dissolved the naringin in serum physiologic. A dosage of 80 mg/kg IP was administered

to each rat17.

Statistical Analysis

We present our results as means ± standard deviation (SD). We express the histopathological

results as median (min-max). First, we applied the non-parametric Kruskal-Wallis analysis of

variance test to evaluate the data. Then, we performed pairwise comparisons with the Mann-

Whitney U test. A P value of P<0.05 was considered to be statistically significant. We

performed the calculations using the SPSS 15.0 package program (SPSS Inc, Chicago, IL)

compatible with Windows.

Results

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTThe results of the oxidative stress markers are listed in Table 2. The significance levels

according to each parameter are shown in Figure 3. The significance levels among control and

II/R, II/R and treatment groups, and finally among the treatment groups are listed in Table 3.

The histological examinations of the tissues of the control group rats were normal and were

grade 0 according to Chiu et al.'s scoring system. The macroscopic view of material from the

experimental groups were fragile, edematous and color distorted. The Chiu et al. scores were

consistent with the macroscopic evaulation of the specimens. The intestinal microscopic

views of each groups are shown in Figure 4. The median Chiu et al. scores of each group are

listed in Table 2. There were significant differences among groups according to pathological

evaulation (P=.000). As shown microscopically and classified according to the Chiu et al.

scores, most of the injuries were encountered in the II/R group.

Discussion

Intestinal ischemia can have several origins such as mesenteric embolism, volvulus,

invagination and small intestine transplantation4. Intestinal ischemia is clinically complex to

diagnose and treat and an early diagnosis is necessary to avoid mortality. At the same time,

there is no treatment algorithm. Because of these reasons many researchers were intended to

evaulate the injury and treatment options. The main goal of treatment is to stop the ischemia

and inflammatory process at an early time and therefore decrease the degree of injury. The

degree of intestinal injury depends on the II/R duration. II/R injuries have two components:

ischemia and reperfusion. Although reperfusion is needed to counteract the injury of

ischemia, reperfusion itself causes more cellular injury. Reperfusion injury occurs in two

steps. The first one is reversible and occurs early after reperfusion and lasts for a few hours.

This reperfusion injury causes more damage than the original ischemic injury18. The second

phase lasts for days and characterized by tissue injury19,20,21.

After the administration of ozone it dissolves in the biological fluids (plasma, lymph and

urine) and reacts quickly with polyunsaturated fatty acids and antioxidants, thereby resulting

in glutathione and albumine decreases. These compounds act as electron donors and oxidizers

to form H2O2 and lipid oxidation products. H2O2, as a basic ROS molecule, acts as an ozone

messenger to cause biological and therapeutic effects. Although H2O2 generally thought to be

detrimental it exerts a regulator effect on signal transduction and plays an important role in

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTboth patient defense and the immune response. Since H2O2 acts early and disappears in an

amount of short time (early and transient messenger), lipid oxidation products are late to

arrive and are long-acting messengers spreading along the body via circulation. This process

strongly warns the immune system and keeps the cells in a damaged state22. Ozonated

erythrocytes increase ATP and 2,3 DPG levels by increasing glycolysis and shifting the HbO2

dissociation curve to the right. This process increases the O2 delivering capacity to tissues.

Ozone generally changes vasoconstrictor effects as vasodilatation and therefore cause

repression of no-reflow phenomenon19. Ozone affects hypoxic tissues by decreasing

neutrophil adherence, erythrocyte aggregation and blood viscosity and increasing

microcirculation23. Ozone improves erytrocyte rheology, increases both flexibility and

electric charge of erythrocytes and accordingly causes a decrease in blood viscosity and

platelet aggregation. As a result, blood flow increases to the ischemic tissue24. In a necrotizing

enterocolitis model, ozone improves the healing phase by decreasing TNFα levels and by anti-

inflammatory effects22. Additionally, at repeated low doses (treatment dosage), cellular

durability improves by inducing antiapoptotic pathways and increasing cellular plasticity.

In the previous studies of the I/R model, the levels of antioxidants enzymes (CAT, SOD and

GR) either decreased or increased25,26,27,28. In our study, we found a significant increase in

enzyme activity in the II/R groups. A significant decrease in enzyme activity was also

observed in the ozone, naringin and ozone+naringin groups in ileum tissue. In our study, the

antioxidant enzyme levels were high in the II/R group compared with those of the control

group due to the shortness of the I/R duration. When the duration of ischemia and reperfusion

increases, the degradation of antioxidant enzymes will increase and the levels of antioxidant

enzymes in the I/R group will diminish below the enzyme level of the control groups25. In

another study, after 1 h of ischemia and 2 h of reperfusion of skeletal muscles, the nitric oxide

(NO), MDA and SOD levels were increased compared with those of the control group26.

Recently, another study obtained a similar result to our own. In this study, the control group’s

SOD and MDA levels were lower than those of the testicular ischemia reperfusion group.

After applying a punica granatum extract to the testicular ischemia reperfusion group, the

authors showed that the antioxidant enzyme levels decreased, consistent with our results27.

Another similar result was shown by Bosco and et al. (2007)28. These authors showed that

MDA, SOD and CAT levels in I/R skeletal muscles increased and that these levels decreased

after 1 h of hyperbaric oxygen treatment. Also the increase in enzyme levels in our study can

be considered to be a reflex response of the antioxidant enzyme levels to oxidative stress29.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTBorrego et al. (2004)30 showed with cisplatin injured renal tissue of rats that when the dosage

of ozone increases the SOD and similar antioxidant enzyme levels will decrease due to

increased levels of H2O2. In vivo investigations of high dosages of ozone usage result in

decreased antioxidant enzyme levels via a similar mechanism31,32,33. In our study of ozone

therapy, the antioxidant enzyme levels decreased just like in the studies described above.

Ozone as a scavenger directly sweeps up free radicals and thereby decreases the need for

antioxidative enzymes. On the other hand, ozone may increase antioxidant enzyme levels and

help to diminish free radicals via increased antioxidant enzymes. Another mechanism by

which enzyme levels can be elevated levels is the remaining ozone (after sweeping up the free

radicals) that persists after high dosages. The remaining ozone may have an antioxidant

enzyme-increasing effect34. In this case, the hormesis phenomenon of ozone comes to mind19.

The ozone-decreased histopathological injury that we observed is similar to that of other

studies18.

In a study by Gao et al. (2005)23, after mesenteric I/R on rabbits, the rabbits were fed by

hyperoxygenated solution. Structural and functional improvements were seen in the

enterocyte mitochondria. Similarly, after mesenteric ischemic operations, patients may be fed

via oral ozone solution and their recovery may be accelarated. Even so, ozone administration

via IP is often more effective than oral administration. Because ozone is given at ischemic

conditions, the high O2 gradient will cause the ozone to transfuse to the bowel wall via passive

diffusion. As a result, the effect of the ozone will be visible in the entire ischemic bowel23. In

intrarectal ozone treatment in a hepatic I/R model, the ozone provides protection. This

protection depends on adenosine accumulation and blockage of the xanthine/xanthine oxidase

pathway and decreased ROS generation after reperfusion35. In atherosclerotic patients who

have low mesenteric flows, ozone administration via oral, intravenous, IP or rectal routes can

improve the the vitality of the intestinal mucosa36.

Naringin works by inhibiting lipid peroxidation and arachinodoic acid metabolism. This

process results in anti-inflammatory and antithrombotic effects. Akondi et al. (2011)37 showed

the protective effect of naringin on testicular torsion, similar to our study. In the study of Rani

et al. (2013)38, the dose-dependent effectiveness of naringin on myocard was shown. When

the dosage was increased up to 80 mg/kg/day per oral administration, the infarct size at the

myocard infarctus group decreased to the level of the sham group. In our study, the

effectiveness of naringin was as high as in other studies but not as high as that of ozone. The

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTeffect of naringin may be increased by increasing the dosage. Thus, it may be used for treating

patients who have mesenteric ischemia due to microembolism and as a prophylaxis for

patients with a high risk of ischemia. At the same time, naringin increases gene expression of

SOD, catalase and glutathione peroxidase9. Gaur et al. (2009)39 studied naringin in 50–100

mg/kg doses in a cerebral I/R model and demonstrated the protective effects of naringin on

ischemic brain injuries, particularly at increased dosages. Similarly, a study performed by

Aggarwal et al. (2010)40 with 50–100 mg/kg IP naringin dosages was able to prevent post-

stroke depression via NO mechanism. The histopathological effectiveness of naringin in our

study was similar to that of other studies37. In our study, ozone and naringin sometimes had

additive effects and sometimes had synergistic effects but there was no antagonistic effects.

Thus, ozone and naringin may be combined for the treatment of mesenteric ischemia.

In our study, SMA occlusion for the II/R model was different from that of previous studies.

Silk sutures with the help of 10 Fr- 1 cm plastic catheter were used and cut for reperfusion. By

this method, the treatment regimes via IP did not leake from the abdomen during the ischemia

period. This II/R model is important because it is described here for the first time. In addition

to silk there are several other suturing methods such as a pledgeted suture which is one that is

supported by a pledget, essentially a small flat non-absorbent pad normally composed of

Polytetrafluoroethylene, used as buttresses under sutures when there is a possibility of sutures

tearing through tissue. Topical cyanoacrylate adhesives a.k.a. medicinal grade super glue,

have also been used in combination with, or as an alternative to, sutures in wound closure.

For validation of the animal experiments, real-time patients may be used for various

gastrointestinal surgeries; however, this will cause ethical problems. After preclinical trials on

animals, subtherapeutic doses may be used on patients, as phase 0-1 clinical trials, to evaluate

the agent or drug. In a prospective clinical trial organized at our clinic, we will use ozone and

naringin simultaneously during an operation. Meanwhile, the therapeutic actions of ozone and

naringin will be evaluated for confounding diseases, such as diabetes mellitus type II, sepsis,

and autoimmune disorders (such as lupus erythematosus, rheumatoid arthritis, etc.). In the

upcoming study, we will evaluate the effects of ozone and naringin on real-time patients who

have confounding diseases, such as diabetes mellitus, sepsis, or others. In the prospective

study, we will consider evaluating the levels of a panel of inflammatory markers, such as

interleukins, which would have generated mechanistic insights into the disease mechanisms.

There may be side effects of high doses of ozone and naringin on intestinal

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTischemia/reperfusion injury. These agents may reverse antioxidant effects into oxidative

effects. At therapeutic dosages, however, these kinds of side effects could be neglected.

As a result, ozone alone or combined with naringin has a protective effect for mesenteric

ischemia patients. Instead of using instruments such as clamps in the II/R rat model, silk

binding may be used safely.

Conflicts of Interest – None

Funding – None

Ethical Approval – Ataturk University, 2014

Registration / Trial Registry Number – Not applicable

Author Contribution - AI, KP, CG, İS, DF, İY, İD: study design, data collections, data

analysis, writing

Guarantors - AI, KP, CG, İS, DF, İY, İD

References:

1- Lin ZL, Yu WK, Tan SJ, Duan KP, Dong Y, Bai XW, Xu L, Li N. Protective effects of

terminal ileostomy against bacterial translocation in a rat model

of intestinalischemia/reperfusion injury.World J Gastroenterol. 2014;20:17905-13.

2- Granger DN, Höllwarth ME, Parks DA. Ischemia-reperfusion injury: role of oxygen-

derived free radicals. Acta Physiol Scand Suppl. 1986;548:47-63

3- Ocak T, Duran A, Özyalvaçli G, Ocak Z, Terzi EH, Tosuns M, Erdem K. Protective effects

of montelukast and Hypericum perforatum against intestinal ischemia-reperfusion injury in

hamsters. Turk J Med Sci. 2014;44:381-6

4- Boybeyi Ö, Gunal YD, Atasoy P, Kısa U, Aslan MK. The effect of colchicine and low-

dose methotrexate on intestinal ischemia/reperfusion injury in an experimental model.

J Pediatr Surg. 2014;49:1471-4

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

5- Al-Saadi H, Potapova I, Rochford ET, Moriarty TF, Messmer P. Ozonated saline shows

activity against planktonic and biofilm growing Staphylococcus aureus in vitro: a potential

irrigant for infected wounds. Int Wound J. 2015 Jan 14.

6- Bocci V, Zanardi I, Huijberts MS, Travagli V. It is time to integrate

conventional therapy by ozone therapy in type-2 diabetes patients. Ann Transl Med.

2014;2:117.

7- Re L, Mawsouf MN, Menéndez S, León OS, Sánchez GM, Hernández F. Ozone therapy:

clinical and basic evidence of its therapeutic potential. Arch Med Res. 2008;39:17-26.

8- Sharma AK, Bharti S, Ojha S, Bhatia J, Kumar N, Ray R, Kumari S, Arya DS. Up-

regulation of PPARγ, heat shock protein-27 and -72 by naringin attenuates insulin resistance,

β-cell dysfunction, hepatic steatosis and kidney damage in a rat model of type 2 diabetes. Br J

Nutr. 2011;106:1713-23.

9- Singh D, Chopra K. The effect of naringin, a bioflavonoid on ischemia-reperfusion induced

renal injury in rats. Pharmacol Res.;50:187-93

10- Aebi M. Catalase. In: Methods of enzymatic analysis. Bergmeyer HU (ed.). New York:

Verlag Chemie–Academic Press, 1974; 2: 673–84.

11. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase.

Clinical chemistry.1988;34:497-500.

12- Beutler, E., 1971. Red Cell Metabolism. A Manual of Biochemical Methods (1st) Grune

Stratton. New York, USA.

13. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products:

malonaldehyde and 4-hydroxynonenal. Methods in enzymology. 1990;186:407-21.

14.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin

phenol reagent. The Journal of biological chemistry. 1951;193:265-75.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT15- Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-

flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg.

1970;101:478-83.

16- Aslan MK, Boybeyi Ö, Şenyücel MF, Ayva Ş, Kısa Ü, Aksoy N, Soyer T, Cesur

Ö, Çakmak M.

Protective effect of intraperitoneal ozone application in experimental ovarian ischemia/reperfu

sion injury J Pediatr Surg. 2012;47:1730-4

. 17- Ali MM, El Kader MA The influence of naringin on

the oxidative state of rats with streptozotocin-induced acute hyperglycaemia. Z Naturforsch

C. 2004;59:726-33.

18- Gao C, Chai W, Xu L, Zhang G, Zhang H, Han L, Sun X Protective effects of hyperoxygenated solution preconditioning on intestinal ischemia-

reperfusion injury in rabbits. J Surg Res. 2006;135:268-74.

19- Ekici S, Doğan Ekici AI, Öztürk G, Benli Aksungar F, Sinanoğlu O, Turan G, Lüleci N.

Comparison of melatonin and ozone in the prevention of reperfusion injury following

unilateral testicular torsion in rats. Urology. 2012;804:899-906

20- Xing B, Chen H, Wang L, Weng X, Chen Z, Li X. Ozone oxidative preconditioning

protects the rat kidney from reperfusion injury via modulation of the TLR4-NF-κB pathway.

Acta Cir Bras. 2015 ;30:60-6

21- Aydos TR, Başar MM, Kul O, Atmaca HT, Uzunalıoğlu T, Kisa Ü, Efe OE. Effects of

ozone therapy and taurine on ischemia/reperfusion-induced testicular injury in a rat testicular

torsion model.. Turk J Med Sci. 2014;44:749-55.

22- Guven A, Gundogdu G, Vurucu S, Uysal B, Oztas E, Ozturk H, Korkmaz A Medical

ozone therapy reduces oxidative stress and intestinal damage in an experimental model of

necrotizing enterocolitis in neonatal rats. J Pediatr Surg. 2009;44:1730-5.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT23- Gao C, Xu L, Chai W, Sun X, Zhang H, Zhang G. Amelioration of intestinal ischemia-

reperfusion injury with intraluminal hyperoxygenated solution: studies on structural and

functional changes of enterocyte mitochondria. J Surg Res. 2005 ;129:298-305.

24- Fernández Iglesias A, González Núñez L, Calunga Fernández JL, Rodríguez Salgueiro S,

Santos Febles E Ozone postconditioning in renal ischaemia-reperfusion model. Functional

and morphological evidences. Nefrologia. 2011;31:464-70.

25- Aslan MK, Boybeyi Ö, Şenyücel MF, Ayva Ş, Kısa Ü, Aksoy N, Soyer T, Cesur Ö,

Çakmak M. Protective effect of intraperitoneal ozone application in experimental ovarian

ischemia/reperfusion injury. J Pediatr Surg. 2012 ;47:1730-4.

26- Ozyurt H, Ozyurt B, Koca K, Ozgocmen S Caffeic acid phenethyl ester (CAPE) protects rat skeletal muscle against ischemia-

reperfusion-induced oxidative stress. Vascul Pharmacol. 2007 ;47:108-12

27-Atilgan D, Parlaktas B, Uluocak N, Gencten Y, Erdemir F, Ozyurt H, Erkorkmaz U, Aslan

H. Pomegranate (Punica granatum) juice reduces oxidative injury and improves sperm

concentration in a rat model of testicular torsion-detorsion. Exp Ther Med. 2014;8:478-482.

28- Bosco G, Yang ZJ, Nandi J, Wang J, Chen C, Camporesi EM. Effects of hyperbaric

oxygen on glucose, lactate, glycerol and anti-oxidant enzymes in the skeletal muscle of rats

during ischaemia and reperfusion. Clin Exp Pharmacol Physiol. 2007;34:70-6

29- Kondo H, Miura M, Itokawa Y. Antioxidant enzyme systems in skeletal muscle atrophied

by immobilization. Pflugers Arch. 1993;422:404-6.

30- Borrego A, Zamora ZB, Gonzalez R, Romay C, Menéndez S, Hernández F, Montero T,

Rojas E. Protection by ozone preconditioning is mediated by the antioxidant system in

cisplatin-induced nephrotoxicity in rats. Mediat Inflamm 2004; 13:13.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT31- Bhor VM, Raghuram N, Sivakami S. Oxidative damage and altered antioxidant enzyme

activities in the small intestine of streptozotocin-induced diabetic rats. Int J Biochem Cell B

2004;36:89.

32- Whiteside C, Hassan HM. Induction and inactivation of catalase and superoxide

dismutase of Escherichia coli by ozone. Arch Biochem Biophys 1987;257:464.

33- Zamora ZB, Borrego A, Lopez OY, Delgado R, González R, Menéndez S, Hernández F,

Schulz S. Effects of ozone oxidative preconditioning on TNF-_ release and antioxidant-

prooxidant intracellular balance in mice during endotoxic shock. Mediat Inflamm 2005;14:16.

34-Haj B, Sukhotnik I, Shaoul R, Pollak Y, Coran AG, Bitterman A, Matter I Effect of ozone

on intestinal recovery following intestinal ischemia-reperfusion injury in a rat. Pediatr Surg

Int. 2014;30:181-8.

35- Peralta C, Xaus C, Bartrons R Leon OS, Gelpi E, Roselló-Catafau J. Effect of ozone

treatment on reactive oxygen species and adenosine production during hepatic ischemia-

reperfusion. Free Radic Res 2000;33:595.

36- Foglieni C, Fulgenzi A, Belloni D, Sciorati C, Ferrero E, Ferrero ME. Ozonated

autohemotherapy: protection of kidneys from ischemia in rats subjected to unilateral

nephrectomy. BMC Nephrol. 2011;12:61.

37- Akondi BR, Challa SR, Akula A. Protective effects of rutin and naringin in testicular

ischemia-reperfusion induced oxidative stress in rats. J Reprod Infertil. 2011 ;12:209-14

38- Rani N, Bharti S, Manchanda M, Nag TC, Ray R, Chauhan SS, Kumari S, Arya DS

Regulation of heat shock proteins 27 and 70, p-Akt/p-eNOS and MAPKs by Naringin

Dampens myocardial injury and dysfunction in vivo after ischemia/reperfusion. PLoS One.

2013;8:e82577.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT39- Gaur V, Aggarwal A, Kumar A. Protective effect of naringin against ischemic reperfusion

cerebral injury: possible neurobehavioral, biochemical and cellular alterations in rat brain. Eur

J Pharmacol. 2009;616:147-54

40- Aggarwal A, Gaur V, Kumar A. Nitric oxide mechanism in the protective effect of

naringin against post-stroke depression (PSD) in mice. Life Sci. 2010;86:928-35.

Figure 1 a) SMA in control group b) 10 Fr- 1 cm plastic catheter used for occlusion of SMA in II/R and treatment groups

Figure 2

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

(a)

(b)

(c)

(d)

Figure 3. (a) CAT activity, (b) SOD activity, (c) GR activity, (d) MDA levels. ( *: p <0.05 vs.

C, # : p <0.05 vs. I / R)

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

Figure 4 a)Grade 0 injury- control group. Normal mucosa. (HE; x200) b)Grade 1- treatment group. Development of a supepithelial space, usually at the tip of the villus(black arrow). Mild congestion. (HE; x200) c) Grade 2 injury- treatment group. Extension of the subepithelial space with moderate lifting of the epithelial layer(red arrow). Congestion and edema at stroma. (HE; x200) d)Grade 3 injury- II/R group. Massive epithelial lifting down the sides of villi(black arrow). Erosion and denudation at epithelium (HE; x200) e)Grade 4 injury- II/R group. Denuded villi with lamina propria(red arrow), dilated capillaries exposed(green arrow), increased cellularity of the lamina propria(white arrow). Erosion at surface epithelium, congestion, hemorrhage and edema at stroma. (HE; x200)

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTTable 1 Histopathologic grades of intestinal tissue at I/R model(Chiu scoring system)

Grade Findings

0 Normal mucosa

1 Development of a supepithelial space, usually at the tip of the villus,

with capillary congestion

2 Extension of the subepithelial space with moderate lifting of the

epithelial layer

3 Massive epithelial lifting down the sides of villi

4 Denuded villi with lamina propria, dilated capillaries exposed, increased

cellularity of the lamina propria

5 Digestion and disintegration of the lamina propria, hemorrhage and

ulceration

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTTable 2 Results of oxidative stress markers and Chiu scores

C I/R Ozone Naringin Ozone+Naringin

CAT(mean)(K/g) 0,079±0,022 0,218±0,029 0,137±0,024 0,198±0,026 0,092±0,028

SOD(mean)(U/mg) 0,142±0,022 0,263±0,034 0,160±0,031 0,167±0,049 0,133±0,025

GR(mean)(U/mg) 0,055±0,006 0,100±0,007 0,078±0,010 0,071±0,020 0,053±0,011

MDA(mean)(nmol/g) 9,977±1,054 21,663±2,904 11,722±1,177 9,288±1,645 11,340±2,357

Chiu scores (median) 0(0-0) 3(3-4) 1(1-2) 1(1-2) 1(1-1)

C: Control, I/R: ischemia-reperfusion, CAT: Catalase, SOD: Superoxide Dismutase, GR: Glutatyon Reductase, MDA: Malondialdehyde

Table 3-p values among groups Groups p(CAT) p(SOD) p(GR) p(MDA) p(Chiu scores ) C- I/R .000 .000 .000 .000 .001 I/R - Ozone .000 .000 .001 .000 .001 I/R - Naringin .967 .001 .001 .000 .001 I/R - Naringin/Ozone .000 .000 .000 .000 .001

Ozone - Naringin .432 .741 .302 .003 .710 Ozone - Naringin/Ozone .000 .097 .001 .684 .383

Naringin – Naringin/Ozone .232 .098 .045 .024 .209

C: Control, I/R: ischemia-reperfusion, CAT: Catalase, SOD: Superoxide Dismutase, GR: Glutatyon Reductase, MDA: Malondialdehyde

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTHere we want to evaulate the effect of ozone and naringin on the intestine after intestinal ischemia-reperfusion injury. Also for superior mesenteric artery occlusion, different than literature, silk suture binding was used in our study. Since the management of mesenteric ischemia requires a multidisciplinary approach, we believe our manuscript will draw attention of abdominal surgeons, gastroenterologists, nutritionists and pharmacologists.