Accepted Manuscript Effects of Ukrain in Rats with Intestinal Ischemia and Reperfusion Raziye Akcılar, Aydın Akcılar, Bircan Savran, Ceylan Ayada, Cengiz Koçak, F. Emel Koçak, Osman Genç PII:

S0022-4804(14)01158-5

DOI:

10.1016/j.jss.2014.12.040

Reference:

YJSRE 13073

To appear in:

Journal of Surgical Research

Received Date: 7 August 2014 Revised Date:

23 October 2014

Accepted Date: 18 December 2014

Please cite this article as: Akcılar R, Akcılar A, Savran B, Ayada C, Koçak C, Koçak FE, Genç O, Effects of Ukrain in Rats with Intestinal Ischemia and Reperfusion, Journal of Surgical Research (2015), doi: 10.1016/j.jss.2014.12.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Effects of Ukrain in Rats with Intestinal Ischemia and Reperfusion

Raziye Akcılar1, Aydın Akcılar2, Bircan Savran3, Ceylan Ayada1, Cengiz Koçak4, F. Emel

University of Dumlupınar, Faculty of Medicine, Department of Physiology, Kütahya, Turkey 2 3

University of Dumlupınar, Faculty of Medicine, Kütahya, Turkey

University of Dumlupınar, Faculty of Medicine, Department of Pediatric Surgery, Kütahya, Turkey

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Koçak5, Osman Genç1

University of Dumlupınar, Faculty of Medicine, Department of Pathology, Kütahya, Turkey 5

University of Dumlupınar, Faculty of Medicine, Department of Biochemistry, Kütahya,

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Turkey

Running title: Does ukrain alter oxidant status in rats with intestinal ischemia and

Correspondence to:

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Raziye AKCILAR

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reperfusion?

Dumlupınar University, Medical Faculty, Department of Physiology, Kütahya, Turkey

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E-mail: [email protected] Telephone: 0507 953 94 74 Fax: +90-274-265 22 85

Authors' contributions:

R.A. conceived and designed the article, analyzed the data and wrote the article. R.A., A.A., B.S., C.A., F.E.K., C.K. contributed reagents/materials/analysis tools. R.A. and A.A. performed the experiments. R.A., A.A., B.S., C.A., F.E.K., O.G. approved the final version of the manuscript.

ACCEPTED MANUSCRIPT ABSTRACT Background: The purpose of this study is to investigate the potential protective effect of the ukrain on ischemia-reperfusion (IR) injury in rat intestine, which has not previously been studied. Methods: Thirty-one male Sprague-Dawley rats were randomly assigned to four groups, each

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consisting of 8 rats: (1) a sham group (S) (laparotomy, but no IR injury); (2) Ukrain group (U) (no IR, and Ukrain was administered intraperitoneally one hour before laparotomy); (3) intestinal ischemia-reperfusion (II/R) group (30 min occlusion of the superior mesenteric artery (SMA) then two hour reperfusion); (4) Ukrain + II/R group (U + II/R) (30 min

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occlusion of the SMA then two hour reperfusion; Ukrain was administered intraperitoneally one hour before ischemia-reperfusion).

Results: Serum total oxidant status (TOS) and total antioxidant status (TAS) were measured

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using Erel's method. Oxidative stress index (OSI) was calculated using the TOS/TAS ratio. TAS levels increased and TOS serum levels were also significantly decreased in the Ukrain + IR group compared with the IR group (respectively p = 0.000 and p= 0.015). Conclusions: In this study, we demonstrated for the first time in the literature that ukrain helps to prevent intestinal tissue breakdown against intestinal ischemia-reperfusion injury and

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that this effect can be achieved by antioxidant activities.

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Keyword: Ukrain, intestine ischemia- reperfusion, TAS, TOS, OSI

ACCEPTED MANUSCRIPT 1. INTRODUCTION Mesenteric ischemia is a severe disease of the gastrointestinal tract that can result in varying degrees of tissue damage, which results in intestinal necrosis. Ischemia-reperfusion injury is often observed in abdominal organ transplantations, arterial and venous occlusive diseases of the digestive tract, and circulatory shock (1). When therapeutic measures are

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performed to restore blood flow, the paradoxical worsening of ischemic damage by the action of cytokines, interleukins, and especially of reactive oxygen species (ROS) can ocur (2, 3). Toxic oxygen metabolites have emerged as a major common pathway of tissue injury in a wide variety of diseases including ischemia-reperfusion (I/R) injury, because many

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constituents of the cell are potentially subjected to a free radical attack (4).

Ukrain (NSC 631570) contains alkaloids of greater celandine (Chelidonium majus, a

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member of the Papaveraceae family) (5), made water soluble by derivatization with thiotepa, displaying a putative proapoptotic effect. This drug has previously been reported to be an effective anti-cancer agent with minimal side-effects, because of its selective toxicity towards malignant cells as demonstrated in vitro. (6). The molecular mechanisms of Ukrain-induced antineoplastic effects, however, are not yet completely understood, although an immunostimulating and immunomodulating (7), antiangiogenic on human endothelial cells in

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vitro, and antiapoptotic actions (8, 9) of Ukrain have been reported. Due of the antioxidative properties of ukrain, it seems possible that using ukrain before ischemia-reperfusion may protect the intestinal tissue against oxidative intestinal I/R injury.

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To the best of our knowledge, there has not been such a report in the literature. The mechanism of action of Ukrain is as yet unknown. The purpose of this study was to determine

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the mechanism of action of Ukrain and whether ukrain could prevent intestinal tissue injury induced by I/R by measuring oxidant parameters, such as total oxidative status (TOS), oxidative stress index (OSI), and antioxidative parameters such as total antioxidant status (TAS) in the serum samples of rats.

ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS 2.1. Animals Sprague-Dawley 10-12-wk-old male rats (250–300 g) were obtained from the Experimental Research Unit of the University of Dumlupınar (Kütahya, Turkey). They were

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maintained on a 12/12 h light–dark cycle under controlled temperature and humidity. The animals were fed standard rat chow and given water ad libitum. All protocols used in this study were approved by the Dumlupınar University Ethics Committee on animal research.

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2.2. Experimental Protocol and Model of Intestinal I/R

A rat's small-intestinal I/R model was created, as described previously, by some investigators (10). All rats were fasted for 18 h before the experiments, but were allowed free

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access to water until 20–30 min before starting the experiment. All experiments were started between 10 and 11 a.m. The rats were anesthetized with an intraperitoneal injection of ketamin/xylazine HCl (75 mg/kg/10 mg/kg). The rats were placed on a heating mat, to keep the body temperature at 36 ± 1 °C. They were placed in the supine position and secured in the dissection tray. The abdominal region was shaved, cleaned with povidone–iodine 10%

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antiseptic solution and 2–3 cm abdominal mid-line incision was applied. Laparotomy was performed. This procedure was sufficient to keep the animals under anaesthesia until the end of the experiment. In the intestinal ischemia-reperfusion injury model, the superior mesenteric artery was exposed with care and occluded with a nontraumatic microvascular clamp, for 30

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min. At the end of this period, the clamp was removed, mesenteric artery pulsation was observed, and the intestine reperfused for 120 min (10). During I/R period the area of

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operation was covered with a warm moist dressing to prevent hypothermia. Rats were allocated to 4 experimental groups: (1) a sham group (S; n=7) that underwent sham surgery with isolation of the superior mesenteric artery without occlusion were treated in an identical fashion with 0.5 ml sterile, pyrogen-free saline; (2) Ukrain group (U, n=8) (no IR, and Ukrain (7 mg/kg in a volume of 0.5 ml/100g) was administered intraperitoneally one hour before laparotomy); (3) an intestinal ischemia-reperfusion group (II/R; n=8) that was subjected to intestinal ischemia and reperfusion after the superior mesenteric artery had been isolated and occluded; (4) ukrain treatment plus ischemia-reperfusion group (U+II/R, n=8) subjected to intraperitoneally injection of ukrain (7 mg/kg in a volume of 0.5 ml/100g) one hour before intestinal ischemia-reperfusion.

ACCEPTED MANUSCRIPT Ukrain ampoules were donated by Nowicky Pharmaceuticals (Margaretenstrasse 7, 1040 Vienna, Austria). The ampoules were consistently protected from light and stored at 7°C until use. 2.3. Biochemical Analyses

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2.3.1. Blood homogenization At the end of the experiment, blood samples were collected in polypropylene tubes from the abdominal artery of rats. The blood samples were centrifuged at 5000 rpm for 5 min for analyses of TAS, TOS levels and serum samples were then separated and stored at -80°C until

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examination. Serum TAS and TOS levels were measured using a Rel Assay commercial kit (Mega Tip, Gaziantep, Turkey) in Beckman Coulter AU680 analyzer (Beckman Coulter,

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Miami, FL, USA)

2.3.2. Measurement of Total Antioxidant Status (TAS)

The novel automated TAS method is based on antioxidants in the sample reducing dark blue-green colored 2,20-Azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical to colorless reduced ABTS form. The change of absorbance at 660 nm is related to total

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antioxidant level of the sample. The assay is calibrated with a stable antioxidant standard solution which is called as Trolox Equivalent that is a vitamin E analog. The results were reported as mmol Trolox equivalents/L (11).

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2.3.3. Measurement of Total Oxidant Status (TOS) The TOS assay is based on oxidants that are present in the sample and oxidize the ferrous

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ion chelator complex to ferric ion. The oxidation reaction was prolonged by glycerol molecules abundantly present in the reaction medium. The ferric ion produces a colored complex with chromogen in an acidic medium and the color intensity, which was measured spectrophotometrically, is related to the total amount of oxidant molecules present in the samples (12). The assay was calibrated with hydrogen peroxide and the results were expressed in terms of micromolar hydrogen peroxide equivalent per liter (µmol H2O2 equiv./L) for serum.

ACCEPTED MANUSCRIPT 2.3.4. Calculation of Oxidative Stress Index (OSI) The ratio of TOS to TAS was accepted as the OSI. To perform the calculation, the unit of TAS, mmol Trolox equivalent/L, was converted to µmol Trolox equivalent/L, and OSI was calculated as follows: OSI = [(TOS, µmol H2O2 equivalent/L)/(TAS, µmol Trolox equivalent/L) × 100] (13). We used OSI as another indicator of oxidative stress. OSI has been

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suggested that it may reflect the state of oxidative status more accurately than TOS (14). 2.4. Histopathological Analysis

All animals were sacrificed in appointed interval. Small intestine samples 1–2 cm long were

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taken 10 cm from the Trietz ligament. Biopsies were washed with cold saline and fixed in 10% neutral formaldehyde for 24 hours. The tissues were then embedded in paraffin, cut into

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4-5 µm thick sections, and mounted. After deparaffinization, the tissue sections were stained with hematoxylin & eosin (H&E) and were examined by the pathologist. All sections were studied using optic microscopy (Olympus BX–51, Tokyo, Japan). Histopathological changes were evaluated using Chiu’s score, which is utilized to describe and quantify the degree of mucosal injury associated with IR injury of the intestine (15). Criteria of intestinal injury were

2.5. Statistical Analysis

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shown in Table 1.

Statistical analysis was done with SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) 16.0 pocket program. All results were given as means ± standard error (SE).

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Comparisons among multiple groups were done with Kruskal-Wallis test, and between two groups were with Mann-Whitney U test. Values smaller than p≤0.05 were accepted as

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statistically significant.

ACCEPTED MANUSCRIPT 3. RESULTS Serum TOS, TAS and OSI Serum TOS levels levels differed a significantly among the groups of S, U, II/R, and U + II/R (p=0.002). Also serum TOS levels in the II/R group were significantly higher than in the

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S, U and U+II/R groups (respectively p=0.004, p=0.001 and 0.015). The high levels of serum TOS were found in U + II/R group compared with U group, p=0.05 (Table 2).

There were statistically significant differences among the groups of S, U, II/R, and U + II/R regarding the serum TAS levels (p=0.000). The levels of serum TAS in II/R group

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compared with the U and U+II/R groups were found to be significantly lower, p=0.001. In addition, The TAS levels were significantly higher in the Ukrain application groups than in

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the sham group (respectively p= 0.004 and p= 0.000). Also, the levels of serum TAS in the U + II/R group were significantly higher than U and II/R groups, respectively p=0.001, and p=0.000 (Table 2).

OSI levels differed a significantly among the groups of S, U, II/R, and U + II/R (p=0.001). Serum OSI levels were significantly higher in the II/R group than in the S, U and

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U+II/R groups, respectively p= 0.001, p= 0.000 and p=0.007 (Table 2). Histopathological Analysis

There were no pathological changes in the intestinal tissue of the sham and ukrain groups

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upon histopathological evaluation. In these groups, all the animals did not have injuries (level 0) (Figure 1). The intestinal damage score was significantly higher in II/R group than in the S, U, U+II/R groups (Table 3, 4). Intestinal specimens from rats after II/R exhibited necrotic

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material, presence of dilated capillaries and inflamed cells. There was severe mucosal damage consistent with ischemic injury, ranging from loss of villi to mucosal infarction in animals that received II/R. In this group, three rats had level 2 injuries (Figure 3), four had level 3 injuries (Figure 4) and one had level 4 injury (Figure 5). In the U+II/R group, the mean value of the tissue injury level was 1.5. Five rats had level 1 injuries (Figure 2), two had level 2 injuries an one had level 3 injury.

ACCEPTED MANUSCRIPT 4. DISCUSSION Ischemia-reperfusion (IR) injury is a major problem associated with high morbidity and mortality following trauma and hemorrhagic shock (16). Injury of the small intestine result from inadequate blood supply (17, 18). Causes of the reduced blood flow can include changes in the systemic circulation or local factors such as constriction of blood vessels or a blood

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clot. It is more common in the elderly (19, 20). Well-established outcomes of IR injury include acute lung injury, adult respiratory distress syndrome, depressed cardiac function, altered kidney function, and absorptive capacity of the intestine as well as intestinal barrier derangement leading to increased cytokine production and eventually multiorgan failure

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Among the internal organs, the intestine is probably the most sensitive to IR injury (21). The mechanisms of ischemia-reperfusion injury are complex and several treatment modalities

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have been used to limit intestine reperfusion injury in animals (16). The pathogenesis and prevention of intestinal ischemia-reperfusion injury have been studied (22). Oxidative stress induced by free radicals may be the primary cause of tissue injury and may occur in inflammation (23). Oxidative stress and depletion of antioxidants may play a key role in the pathogenesis of intestinal damage. Oxidative stress associated with lipid peroxidation is one of the causes of intestinal I/R, which causes changes in injury to the small

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intestines (16). Therefore, oxidative stress may contribute to the inflammatory response after intestinal ischemia-reperfusion injury (24).

In the study performed by Torun et. Al. (25) the TAS, TOS and OSI measurement do not only

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show the oxidative/anti-oxidative condition during the diagnosis, but they also play a role in the monitoring of the treatment (25). The TOS/TAS ratio, in other words the OSI, is an

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indicator of the oxidative stress degree and shows the anti-oxidation and oxidation redox balance (25). The measurement of TOS provides a sensitive index of lipid peroxidation and oxidative stress (26). We measured total oxidant and antioxidative capacites at the same time to more accurately assess oxidative stress. In this study, an increase was observed in serum TOS and OSI levels in the intestinal I/R group compared with the other groups. Recent studies have shown that intestinal I/R augments oxidative stress by either modulating the production of free radicals, reactive oxygen species, and toxic cytokines leading to inflammation and leukocyte infiltration, or via direct tissue damage (27). In the study performed by Sasaki et al. (28) found that the intestine may be the greatest source of oxidants compared with other organs (28). The generation of reactive oxygen species and reactive

ACCEPTED MANUSCRIPT oxygen species-mediated damage may be important in intestinal ischemia-reperfusion injury. Many oxygen-free radicals are generated during ischemia and reperfusion, causing excessive superoxide dismutase consumption (29). Zhang et al. (30) have demonstrated that oxidative stress occurs in intestinal ischemia-reperfusion injury in mice, manifested by a significant increase in serum malondialdehyde and decrease in serum superoxide dismutase (30).

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For many decades researchers have studied many markers of oxidative stress-associated tissue damage and antioxidant defense, including measurement of antioxidant enzymes– superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), ceruloplasmin, and proteins such as metalothionins (31). TAS, as an

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antioxidant, can protect tissue from the oxidative damage induced by I/R through scavenging reactive oxygen species. In our study, TAS levels decreased in the II/R group, and we believe

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that this response was due to oxidative damage caused by the production of free radicals and reactive oxygen species to II/R. In a study performed on experimental intestinal ischemiareperfusion injury, down-regulation of CAT levels were reported in intestinal I/R (32). In this study, we investigated the effects of ukrain on the parameters demonstrating oxidative and antioxidative status. In our study, we observed significantly increased TAS

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levels and decreased TOS and OSI levels with the administration of ukrain. There has not been any study in the literature between oxidative-antioxidative factors and ukrain that can be compared with the results of this study. In a study by Panzer et al. (33), it was observed that Ukrain is a chemotherapeutic drug which does not seem to cause serious side-effects (33). In

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another study, it was shown that a relatively higher affinity for Ukrain was observed in tumor tissue and liver, while affinity was lowest in the brain and muscles. The presence of tumors

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decreased Ukrain concentrations in plasma and normal tissues in comparison with those in control animals (34).

In the study, our data confirm that intestinal ischemia-reperfusion increase TOS and OSI levels, an effect that not only produces direct tissue damage, but also provides production of toxic cytokines leading to necrosis and inflammation. Histologically, treatment with Ukrain reduced the degree of intestinal tissue injury compared with II/R rats. These results further support the hypothesis that Ukrain can be protective against intestinal injury induced by intestinal I/R and that Ukrain could be a novel endogenous antioxidative factor.

ACCEPTED MANUSCRIPT Acknowledgments: Authors' contributions: R.A. conceived and designed the article, analyzed the data and wrote the article. R.A., A.A., B.S., C.A., F.E.K. and C.K. contributed reagents/materials/analysis tools. R.A. and A.A. performed the experiments. Disclosure: The authors reported no proprietary or commercial interest in any product

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mentioned or concept discussed in this article. The authors declared no conflict of interest. References

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31. Ferrari CKB. Oxidative stress pathophysiology: Searching for an effective antioxidant

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34. Doroshenko YM, Karavay AV, Hodysh YY, Uglyanitsa KN, Nowicky WM, Nefyodov

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LI. The dynamics of concentration of the main fluorescent component of Ukrain in the tissues and blood plasma of rats with W-256 tumor after a single intravenous injection.

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Drugs Exp Clin Res 2000;26(5–6):171–7.

ACCEPTED MANUSCRIPT Table 1. Criteria of intestinal tissue injury evaluation according to Chiu et al (15). Level

1

2

3

Mucosa without changes Well-constituted villosities, no cellular lysis or inflammatory process, although there is formation of Grunhagen’s sub-epithelial space Presence of cellular lysis, formation of Grunhagen’s sub-epithelial space and increased spacing among the villosities

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0

Microscopic Findings

Destruction of the free villosities section, presence of dilated capillaries and inflamed cells

4

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Structural destruction of the villosities, only traces of some villosities,

formed by inflamed cells and necrotic material, with hemorrhage and basal

Destruction of all the mucosa, no glandular structure can be seen, only the amorphous material laying on the sub-mucosa tissue

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5

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glandular ulceration

ACCEPTED MANUSCRIPT Table 2. The levels of Total Oxidant Status, Total Antioxidant Status, Oxidative Stress Index in Sham (S), Ukrain (U), Intestinal Ischemia Reperfusion (II/R), Ukrain + Intestinal Ischemia Reperfusion (U + II/R) groups (mean ± SE)

S (n=7)

U (n=8)

II/R (n=8)

U + II/R (n=8)

p

TOS (µmol/L)

12.2 ± 2.40a

10.0 ± 1.15b

51.1 ± 12.8ab

22.4 ± 8.36b

0.002

TAS (mmol/L)

1.61 ± 0.03a

1.79 ± 0.04ab

1.60 ± 0.02bc

OSI (TOS/TAS)

7.57 ± 1.41a

5.63 ± 0.67b

31.5 ± 7.79abc

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Gruplar

2.49 ± 0.18ac

0.000

10.3 ± 3.30c

0.001

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p: Shows the differences between all groups (Kruskal Wallis test). a,b,c ; In each line, the difference between the means with same letters are significant, p≤0.05 (Mann-Whitney U test). TOS: Total Oxidant Status, TAS: Total Antioxidant Status, OSI: Oxidative Stress Index

ACCEPTED MANUSCRIPT Table 3. Intestinal tissue injury scores of groups according to CHIU and col.

LEVELS OF MUCOSAL INJURY Rats U

II/R

U+II/R

1

0

0

3

1

2

0

0

2

1

3

0

0

3

4

0

0

3

5

0

0

2

6

0

0

7

0

0

8

0

Mean Value

0

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S

1

1

2

3

1

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2

0

4

3

0

2.75

1.5

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2

ACCEPTED MANUSCRIPT Table 4. Histopathologic evaluation of intestinal tissue for each group, that is, in Sham (S), Ukrain (U), intestinal ischemiareperfusion (II/R) and Ukrain+ intestinal ischemia-reperfusion (U + II/R) groups (mean ± SE)

Groups

S (n=8)

U (n=8)

II/R (n=8)

U+II/R (n=8)

p

Intestinal Tissue Injury score

0.00±0.00a,b

0.00±0.00c,d

2.75±0.25a,c,e

1.50±0.26b,d,e

0.000

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p: shows the differences between all groups (Kruskal Wallis test). a,b,c,d,e ; In each line, the difference between the means with same letters are significant, p≤0.05 (Mann-Whitney U test).

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Figure 1. Microscopic appearance of level 0 of Chiu’s score (H&Ex100, H&Ex200)

Figure 1. Microscopic appearance of level 0 of Chiu’s score (H&Ex100, H&Ex200)

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Figure 2. Microscopic appearance of level 1 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

Figure 2. Microscopic appearance of level 1 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

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Figure 3. Microscopic appearance of level 2 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

Figure 3. Microscopic appearance of level 2 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

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Figure 4. Microscopic appearance of level 3 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

Figure 4. Microscopic appearance of level 3 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

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Figure 5. Microscopic appearance of level 4 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

Figure 5. Microscopic appearance of level 4 of Chiu’s Score of intestinal mucosa (H&Ex100, H&Ex200)

Effects of ukrain in rats with intestinal ischemia and reperfusion.

The purpose of this study is to investigate the potential protective effect of the ukrain on ischemia-reperfusion (IR) injury in rat intestine, which ...
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