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reperfusion-induced injury in rat ovaries
European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 458–463
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Blockade of endothelin receptors with bosentan limits ischaemia/ reperfusion-induced injury in rat ovaries O. Sengul a,*, I. Ferah b, B. Polat b, Z. Halici c, Y. Bayir d, M. Yilmaz e, N. Kilic f, O.N. Keles g a
Etlik Zu¨beyde Hanım Women’s Health Education and Research Hospital, Etlik, Ankara, Turkey Department of Pharmacology, Ataturk University Faculty of Pharmacy, Erzurum, Turkey c Department of Pharmacology, Ataturk University Faculty of Medicine, Erzurum, Turkey d Department of Biochemistry, Ataturk University Faculty of Pharmacy, Erzurum, Turkey e Department of Obstetrics and Gynaecology, Ataturk University Faculty of Medicine, Erzurum, Turkey f Department of Obstetrics and Gynaecology, Kafkas University Faculty of Medicine, Kars, Turkey g Department of Histology and Embryology, Ataturk University Faculty of Medicine, Erzurum, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 December 2012 Received in revised form 10 April 2013 Accepted 28 June 2013
Objective: To investigate the role of endothelin receptors in ovarian ischaemia/reperfusion (I/R) injury in rats using the endothelin receptor antagonist bosentan. Study design: Group 1: sham operation; Group 2: sham operation and bosentan 60 mg/kg; Group 3: bilateral ovarian ischaemia; Group 4: 3-h period of ischaemia followed by 3 h of reperfusion; Groups 5 and 6: bosentan 30 and 60 mg/kg, respectively, with bilateral ovarian ischaemia applied 30 min later; the bilateral ovaries were removed after 3 h of ischaemia; Groups 7 and 8: 3 h of bilateral ovarian ischaemia was applied, with bosentan 30 and 60 mg/kg, respectively, administered 2.5 h after the induction of ischaemia; following the 3-h period of ischaemia, 3 h of reperfusion was applied, after which the ovaries were removed. Results: Ischaemia and I/R decreased superoxide dismutase (SOD) activity and the level of glutathione (GSH) in ovarian tissue, but increased the level of malondialdehyde (MDA) significantly compared with the sham operation group. Bosentan 30 and 60 mg/kg before ischaemia and I/R decreased the MDA level and increased SOD activity and the GSH level in the experimental groups. The serum levels of the inflammatory cytokines interleukin (IL)-1b, IL-6 and tumour necrosis factor-a were also measured in the I/R injury model in rat ovaries. The levels of these cytokines were significantly higher in the ischaemia and I/R groups compared with the sham operation and sham operation plus bosentan groups. The histopathological findings also demonstrated the protective role of bosentan against I/R-induced injury in rat ovaries. Conclusion: Administration of bosentan protects the ovaries against oxidative damage and I/R-induced injury. Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Bosentan Ischaemia/reperfusion Ovarian Oxidative stress Endothelin
1. Introduction Ovarian torsion is a condition in which an ovary twists or turns on its supporting ligament, causing occlusion of the ovarian vein or artery and resulting in obstruction of venous and lymphatic drainage. Many studies have been undertaken to investigate the use of medications that prevent ischaemia/reperfusion (I/R) injury in ovarian torsion [1–4].
* Corresponding author at: Etlik Zubeyde Hanım Women’s Health Education and Research Hospital, Etlik 06010, Ankara, Turkey. Tel.: +90 505 634 27 70. E-mail addresses: [email protected], [email protected] (O. Sengul).
Due to torsion of the adnexa, blood flow in the ovary decreases and eventually results in ischaemia [5,6]. Maintaining the circulation of the ovary after detorsion deteriorates the injury developed during reperfusion [7,8]. Restoration of blood supply to ischaemic tissues can cause additional damage due to the release of reactive oxygen species (ROS). Production of ROS is an important mechanism of injury in the I/R process which is characterized by oxidative stress. Moreover, ROS have been implicated in the pathogenesis of tissue injury during reperfusion [9]. It has been suggested that activation of neutrophils, accumulation of released cytokines and nitric oxide, platelet activation and apoptosis also play a paramount role in I/R injury [10]. The formation of cytokines and ROS is stimulated by endothelin (ET) [11]. ET-1, a potent vasoconstrictor peptide with 21 amino acids, is
0301-2115/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.06.040
O. Sengul et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 458–463
produced by a large variety of cells from a wide range of species including endothelial cells, leukocytes, macrophages and monocytes [12–14]. ET receptor type A (ETA) and type B (ETB), which have different molecular and pharmacological characteristics, are present in the ovaries [15–17]. In general, the two dissimilar ET receptors are expressed constitutively but are spatially confined to specific cell types in the ovary; a number of cells express both receptors, whereas other cells express one form or the other [18]. ET plays a role in the modulation of microcirculatory blood flow disturbances during ischaemia, and is one of the most important mediators in I/R [19–23]. Many studies have reported the release of ET during I/R injury [24,25], and there has been growing interest in the roles of ET receptor antagonists in I/R injury. ET antagonists have been reported to have beneficial effects in several I/R models including the heart, liver, lung, kidney and small bowel [26–28]. Bosentan is a potent, non-peptide, oral ETA and ETB antagonist, with higher affinity for the ETA subtype [29]. The ET antagonist bosentan has been reported to protect and improve microcirculatory blood flow in several splanchnic organs and in peripheral tissues [29]. The protective effect of bosentan in myocardial I/R injury [30], I/R-induced endothelial dysfunction [31] and I/R injury in rat skeletal muscle [32] has been observed previously in a number of experimental models. To date, however, no studies have shown a link between ET antagonists and ovarian I/R injury. The aim of this study was to evaluate the possible role of ET-1 in the pathogenesis of ovarian I/R injury. This study investigated whether a specific ET receptor antagonist, bosentan has a protective effect against ovarian I/R injury by evaluating alterations in the oxidant–antioxidant system, generation of cytokines and histopathological examinations. 2. Materials and methods 2.1. Animals Animals were housed in accordance with international guidelines, and the study was approved by and conducted in accordance with the Institutional Animal Care and Use Committee. In total, 64 adult female Wistar albino rats were used in this study. 2.2. Surgical technique The rats were randomized into eight groups and anaesthetized with intraperitonal thiopental sodium 25 mg/kg. The area of the abdomen to be operated was prepared and cleaned using betadine. A longitudinal incision (2.5 cm) was performed in the midline area of the lower abdomen. A small peritoneal incision was made, and the uterine horns and adnexa were located. In Group 1, a sham operation was performed. These rats only underwent laparotomy. In Group 2, rats were administered bosentan 60 mg/kg by oral gavage, and the bilateral ovaries were surgically removed 6 h later. In Group 3, bilateral ovarian ischaemia was created by applying vascular clips below the ovaries in the rats. Atraumatic vascular clamps were used in this study to produce ovarian ischaemia. The incision was closed with 4/0 nylon sutures, and the bilateral ovaries were surgically removed after 3 h. In Group 4, a 3-h period of ischaemia was followed by 3 h of reperfusion, following which the bilateral ovaries were removed. In Groups 5 and 6, rats were administered bosentan 30 and 60 mg/kg, respectively, by oral gavage before 30 min of ischaemia, and then bilateral ovarian ischaemia was created by applying vascular clips below the ovary in both groups. After a 3-h period of ischaemia, the bilateral ovaries were surgically removed. In Groups 7 and 8, a 3-h period of bilateral ovarian ischaemia was created by applying vascular clips below the ovary. Rats were administered bosentan 30 and 60 mg/kg, respectively, by oral gavage 2.5 h after the induction of ischaemia. At the end of 3-h
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period of ischaemia, the bilateral vascular clips were removed and 3 h of reperfusion was applied. The ovaries were subsequently removed for histological and biochemical examination. 2.3. Biochemical investigations 2.3.1. Cytokine serum measurements Sera from the eight groups were separated and stored at 80 8C until they were thawed for the assay. Interleukin (IL)-1b, IL-6 and tumour necrosis factor (TNF)-a from each sample were measured with highly sensitive enzyme-linked immunosorbent assay (ELISA) kits [BMS630, eBioscience (USA); KRC0061, Invitrogen (USA); and BMS622, eBioscience (USA); respectively]. Kits were specifically designed for rat cytokines, and all measurements were performed according to the manufacturers’ instructions. 2.3.2. Biochemical investigation of ovarian tissues After macroscopic analyses, rat tissues were kept at 80˚ C. Tissues weighing approximately 50 mg were homogenized with TissueLyser II (Qiagen) in 1 ml of an appropriate buffer. The tissues were then centrifuged at 4˚ C. Supernatants were used to determine the levels of glutathione (GSH) and malondialdehyde (MDA), and the enzyme activity of superoxide dismutase (SOD). All assays were carried out at room temperature. For the biochemical investigation, the activity of SOD and the levels of MDA and GSH from each supernatant were measured in duplicate using highly sensitive ELISA kits [Cayman-706002 (Ann Arbour, MI, USA), Cell Biolabs-STA-330 (San Diego, CA, USA) and Cell Biolabs-STA-312, respectively], which were specifically designed for rat tissue, according to the manufacturers’ instructions. 2.3.3. Histological examination Ovaries were rapidly fixed in 10% buffered formalin for 24–48 h for histological examination. After fixation, each ovarian tissue sample was routinely processed and embedded in paraffin. Fivemicrometre-thick sections were cut as paraffin-embedded tissue samples. After deparaffinization and rehydration, sections were stained with haematoxylin and eosin. All sections were studied and photographed using a light photomicroscope. For semi-thin sections, ovaries were fixed in buffered 3% glutaraldehyde in 0.1 M phosphate. Glutaraldehyde-fixed tissues were postfixed in 1% osmium tetroxide. Each ovarian tissue sample was dehydrated in a graded ethanol series and transferred to propylene oxide. After dehydration, each ovarian tissue sample was embedded in Araldite CY 212. Glutaraldehyde-fixed tissue was postfixed in 1% osmium tetroxide and embedded in thin viscosity resin (Spurr kit, Sigma Chemical Co., St. Louis, MO, USA). Semi-thin sections (1 mm) obtained with an ultramicrotome were stained with Toluidine blue. All sections were studied and photographed using a light photomicroscope. 2.3.4. Statistical analyses Data for the serum cytokine levels were measured by ELISA, and oxidant and antioxidant enzymes were subjected to one-way analysis of variance using Statistical Package for the Social Sciences Version 18.0 (SPSS Inc., Chicago, IL, USA). Differences between the groups were analyzed using Duncan’s multiple range test, and were considered significant at p < 0.05. All data were expressed as mean standard deviation (SD) in each group. 3. Results 3.1. Results of biochemical investigations SOD activity and the levels of MDA and GSH were studied in the I/R injury model in rat ovaries (Table 1). In rats exposed to
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Table 1 Effects of bosentan treatment on changes in activity of superoxide dismutase (SOD) and levels of malondialdehyde (MDA) and total glutathione (GSH) in rat ovaries. Group Sham operation Sham operation + bosentan 60 mg/kg Ischaemia Ischaemia/reperfusion Ischaemia + bosentan 30 mg/kg Ischaemia + bosentan 60 mg/kg I/R + bosentan 30 mg/kg I/R + bosentan 60 mg/kg
n
SOD activity (U/mg protein) ef
8 8 8 8 8 8 8 8
MDA (nmol/mg protein) a
32.56 5.71 34.19 5.88f 24.00 4.52bcd 16.52 2.65a 28.51 6.51de 28.07 2.97cde 22.59 4.20b 23.13 5.22bc
GSH (nmol/mg protein) 4.75 1.04e 4.27 0.58e 2.69 0.29bc 1.73 0.40a 3.46 0.77d 3.25 0.43cd 2.62 0.52bc 2.43 0.39b
2.07 0.53 1.93 0.21a 4.04 0.89c 4.99 1.55d 2.72 0.71ab 2.78 0.39ab 3.16 0.85b 3.61 0.78bc
n, number of rats; I/R, ischaemia/reperfusion. Means in the same column with the same letter are not significantly different using the Duncan test. (a = 0.05). Results are means standard deviation.
ischaemia and I/R, SOD activity and the GSH level were significantly lower compared with the sham operation and sham operation plus bosentan groups. Moreover, the MDA level was significantly higher in the ischaemia and I/R groups compared with the sham operation and sham operation plus bosentan groups. The MDA levels in the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups were significantly lower compared with the I/R group, and the difference between the two groups was not significant. In the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups, SOD activity and the level of GSH were significantly higher compared with the I/R group. Levels of the inflammatory cytokines IL-1b, IL-6 and TNF-a in the serum were also measured in the I/R injury model in rat ovaries (Table 2). In the ischaemia and I/R groups, the levels of these cytokines were significantly higher compared with the sham operation and sham operation plus bosentan groups. In the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups, IL-1b, IL-6 and TNF-a serum levels were significantly lower compared with the I/R group. 3.2. Results of histopathological investigations The ovaries from the sham operation and bosentan group showed no pathological changes (Fig. 1 A and B). Oocytes had normal morphology with intact zona pellucida. The morphology of the follicle cells surrounding oocytes, luteal cells of the ovine corpus luteum and connective tissue of tunica albuginea were normal (Fig. 1A and B). The oocytes from the ischaemia group showed brown pigmentation, and the thickness of the zona pellucida was significantly lower compared with the control group. In addition, hypertrophic changes of parenchymal and stromal cells were accompanied by leucocyte infiltration, haemorrhage and necrosis in the ischaemia group (Fig. 1C and D). The ovaries of the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups exhibited a thicker zona pellucida compared with the ischaemia group, and fewer cases of
haemorrhage and necrosis compared with the ischaemia group (Fig. 1E and F). Acute inflammatory processes such as neutrophil adhesion and migration, apoptotic and degenerative cells, stromal oedema and haemorrhage were severe in the I/R group (Fig. 2 A and B). Neutrophil migration through the endothelial cells was mild in the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups. The ovaries treated with bosentan showed mild oedema and mild vascular congestion of the ovaries without bleeding or leucocyte infiltration (Fig. 2C and D). In the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups, although minimal hypertrophic cell degeneration was observed in both follicular and stromal cells, most cellular structures did not show any pathological changes (Fig. 2C and D). 4. Comments To the authors’ knowledge, this is the first study to demonstrate the protective effect of bosentan on ovarian I/R injury. This study demonstrated that bosentan treatment attenuates I/R-induced lipid peroxidation and oxidative stress. This study is also the first to suggest the antioxidant properties of bosentan in rat ovary. It has been suggested that the ET system is involved in the pathophysiology of several ischaemia models in rats. I/R damage is very closely related to endothelial cell damage. In the pathophysiology of this damage, there is an imbalance of vasoconstrictor and vasodilator molecules, such as ET, ROS and nitric oxide [33]. This may lead to a no-reflow phenomenon with release of proinflammatory cytokines, neutrophils and oxidative stress [34]. It has been reported that ETA and ETA/B antagonists may reduce I/Rinduced damage in some tissues, such as myocardium, liver, kidney and skeletal muscle [32,35–37]. Loss of ET action may have a protective role in the I/R cascade by blocking the initiation of release of pro-inflammatory cytokines and ROS. During the detorsion process, reperfusion leads to formation of free oxygen radicals [9]. Production of ROS is an important mechanism causing injury in the I/R process, which is characterized
Table 2 Effects of bosentan treatment on changes in the serum levels of interleukin (IL)-1b, IL-6 and tumour necrosis factor (TNF)-a in sera of rats. Group Sham operation Sham operation + bosentan 60 mg/kg Ischaemia Ischaemia/reperfusion Ischaemia + bosentan 30 mg/kg Ischaemia + bosentan 60 mg/kg I/R + bosentan 30 mg/kg I/R + bosentan 60 mg/kg
n 8 8 8 8 8 8 8 8
IL-1b (pg/ml)
TNF-a (pg/ml)
IL-6 (pg/ml) a
49.86 12.85 52.63 11.19a 111.61 57.11bcd 205.59 41.61e 84.59 21.51abc 71.30 33.19ab 130.77 55.05d 116.27 49.31cd
a
142.62 17.55 144.71 58.31a 348.55 131.05b 820.31 128.58d 358.90 78.40b 225.85 37.65a 590.55 144.15c 512.90 165.46c
39.96 11.73a 42.03 24.62ab 120.79 45.16cd 277.73 43.03e 93.50 42.76bc 83.09 45.77abc 155.18 68.59d 155.98 77.64d
n, number of rats; I/R, ischaemia/reperfusion. Means in the same column with the same letter are not significantly different using the Duncan test, (a = 0.05). Results are means standard deviation.
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Fig. 1. (A and B) Tunica albuginea, corpus luteum, primary oocyte, zona pellucida (white arrow) and granulosa cells (black arrow) of ovaries with normal structures from control group. (C and D) Histopathological observations of ovaries from ischaemia group. Dense and prevalent haemorrhage in corpus luteum (Cl) and stroma (S) in the ovarian tissue. Ovarian tissue also showed hypertrophy in granulosa cells (black arrows), pigmentation in oocyte cytoplasm, and dense loss in zona pellucida (white arrow). (E and F) Histopathological observations of ovaries from ischaemia plus bosentan 60 mg/kg group. Minimal haemorrhage in corpus luteum (Cl) and stroma (S) in the ovarian tissue. Ovarian tissue also showed minimal loss in zona pellucida (white arrow), granulosa cells (black arrow) and tunica albugenia (Ta) cells with normal appearance. Scale bars: 60 mm.
by oxidative stress [9]. One of the most important processes in oxidative-stress-induced cellular damage in I/R injury is lipid peroxidation [38]. MDA is an end product in the lipid peroxidation chain reaction, and an important indicator of the degree of lipid peroxidation [39,40]. It has been reported previously that I/R injury enhances lipid peroxidation and MDA [41]. In the present study, the MDA level was markedly higher in the I/R group compared with the control group. Increased MDA levels in the ovary suggests increased lipid peroxidation, leading to tissue damage and the failure of antioxidant defence mechanisms to prevent formation of excessive free radicals. These results showed that the MDA level was significantly decreased by bosentan pretreatment. Therefore, this study suggests that bosentan has a beneficial effect on lipid peroxidation and oxidative stress. To protect tissues against free radical damage, the body has a defence system involving numerous enzymatic and non-enzymatic antioxidants. The antioxidant defence systems are extensive and consist of multiple layers. Although there are mechanisms for scavenging ROS to prevent oxidative stress, this enzymatic antioxidant defence system appears to be vulnerable to free radical damage. When the balance between oxidants and antioxidants shifts in favour of the former, the antioxidant defence system weakens, leading to lipid peroxidation, and protein and DNA damage [42].
One of the most important antioxidant enzymes that scavenge ROS is SOD. Decreased serum activity of SOD in ovarian tissue damaged by I/R has been demonstrated in previous studies [3,41,43]. In the present study, a period of ischaemia followed by reperfusion led to a significant decrease in SOD activity. The reduction in SOD activity during I/R was probably due to depletion of this antioxidant enzyme by the high amount of ROS with the resumption of blood flow during reperfusion. SOD activity in the I/R group was found to be reduced compared with the other groups in this study. Groups treated with bosentan showed improved SOD activity. This indicates that bosentan can reduce ROS that might attenuate oxidative damage to the tissues and improve the activities of the antioxidant enzymes. Oxidative stress can be regulated by not only enzymatic defence systems but also by non-enzymatic antioxidant defence systems [44]. GSH is an endogenous non-enzymatic antioxidant that plays an important role in the non-enzymatic antioxidant defence and cell signalling systems [45]. The GSH level has been reported to decrease after I/R injury in several previous studies [41,46,47]. In the current study, the GSH level was lower in the I/R group compared with the control group, and a significant increase in GSH level was observed in the livers of animals treated with bosentan compared with the I/R group. Administration of bosentan significantly increased the GSH level. As such, it appears that
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Fig. 2. (A and B) Histopathological observations of ovaries from ischaemia/reperfusion group. Apoptotic deaths (black arrows) in granulosa cells and neutrophil migration (black arrows) through the endothelium of blood vessel (Bv) in the ovaries. (C and D) Histopathological observations of ovaries from ischaemia/reperfusion plus bosentan 60 mg/kg group. There was no migration of neutrophils to endothelium in ovaries from ischaemia/reperfusion plus bosentan 60 mg/kg group. Ovarian tissue also showed hypertrophy in granulosa cells (black arrows), but no apoptotic deaths. Scale bars: 60 mm.
bosentan prevented the depletion of antioxidant enzymes, including GSH, in the present study. Aside from ROS, I/R involves the release of cytokines [10]. Leucocyte activation, expression of adhesion molecules and cytokines have been found to play an important role in a number of experimental I/R models [48–50]. In addition, TNF-a, a proinflammatory cytokine, has been implicated in the pathophysiology of I/R injury [51]. Some studies have demonstrated that TNF and other cytokines promote ET gene expression in a variety of cell types [52,53]. On the other hand, ET stimulation can increase the production of proinflammatory mediators such as TNF, IL-1, IL-6 and IL-8 [53]. Therefore, there may be a positive feedback loop between TNF-a and ET. This study found that TNF-a, IL-1 and IL-6 levels were significantly up-regulated after ovarian torsion, which was in agreement with previous studies [54]. In addition, bosentan treatment decreased serum TNF-a, IL-1 and IL-6 levels compared with the I/R groups. The reduction in cytokine levels during I/R was probably due to blockage of the ET receptors by bosentan. These results indicate that bosentan may reduce the production of TNF-a and other cytokines by blocking ET receptors. The effects of bosentan on rat ovaries have also been evaluated histopathologically. Bosentan treatment decreased the I/R-induced histological damage in the ovaries. In the present study, bosentan
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Blockade of endothelin receptors with bosentan limits ischaemia/ reperfusion-induced injury in rat ovaries O. Sengul a,*, I. Ferah b, B. Polat b, Z. Halici c, Y. Bayir d, M. Yilmaz e, N. Kilic f, O.N. Keles g a
Etlik Zu¨beyde Hanım Women’s Health Education and Research Hospital, Etlik, Ankara, Turkey Department of Pharmacology, Ataturk University Faculty of Pharmacy, Erzurum, Turkey c Department of Pharmacology, Ataturk University Faculty of Medicine, Erzurum, Turkey d Department of Biochemistry, Ataturk University Faculty of Pharmacy, Erzurum, Turkey e Department of Obstetrics and Gynaecology, Ataturk University Faculty of Medicine, Erzurum, Turkey f Department of Obstetrics and Gynaecology, Kafkas University Faculty of Medicine, Kars, Turkey g Department of Histology and Embryology, Ataturk University Faculty of Medicine, Erzurum, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 December 2012 Received in revised form 10 April 2013 Accepted 28 June 2013
Objective: To investigate the role of endothelin receptors in ovarian ischaemia/reperfusion (I/R) injury in rats using the endothelin receptor antagonist bosentan. Study design: Group 1: sham operation; Group 2: sham operation and bosentan 60 mg/kg; Group 3: bilateral ovarian ischaemia; Group 4: 3-h period of ischaemia followed by 3 h of reperfusion; Groups 5 and 6: bosentan 30 and 60 mg/kg, respectively, with bilateral ovarian ischaemia applied 30 min later; the bilateral ovaries were removed after 3 h of ischaemia; Groups 7 and 8: 3 h of bilateral ovarian ischaemia was applied, with bosentan 30 and 60 mg/kg, respectively, administered 2.5 h after the induction of ischaemia; following the 3-h period of ischaemia, 3 h of reperfusion was applied, after which the ovaries were removed. Results: Ischaemia and I/R decreased superoxide dismutase (SOD) activity and the level of glutathione (GSH) in ovarian tissue, but increased the level of malondialdehyde (MDA) significantly compared with the sham operation group. Bosentan 30 and 60 mg/kg before ischaemia and I/R decreased the MDA level and increased SOD activity and the GSH level in the experimental groups. The serum levels of the inflammatory cytokines interleukin (IL)-1b, IL-6 and tumour necrosis factor-a were also measured in the I/R injury model in rat ovaries. The levels of these cytokines were significantly higher in the ischaemia and I/R groups compared with the sham operation and sham operation plus bosentan groups. The histopathological findings also demonstrated the protective role of bosentan against I/R-induced injury in rat ovaries. Conclusion: Administration of bosentan protects the ovaries against oxidative damage and I/R-induced injury. Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Bosentan Ischaemia/reperfusion Ovarian Oxidative stress Endothelin
1. Introduction Ovarian torsion is a condition in which an ovary twists or turns on its supporting ligament, causing occlusion of the ovarian vein or artery and resulting in obstruction of venous and lymphatic drainage. Many studies have been undertaken to investigate the use of medications that prevent ischaemia/reperfusion (I/R) injury in ovarian torsion [1–4].
* Corresponding author at: Etlik Zubeyde Hanım Women’s Health Education and Research Hospital, Etlik 06010, Ankara, Turkey. Tel.: +90 505 634 27 70. E-mail addresses: [email protected], [email protected] (O. Sengul).
Due to torsion of the adnexa, blood flow in the ovary decreases and eventually results in ischaemia [5,6]. Maintaining the circulation of the ovary after detorsion deteriorates the injury developed during reperfusion [7,8]. Restoration of blood supply to ischaemic tissues can cause additional damage due to the release of reactive oxygen species (ROS). Production of ROS is an important mechanism of injury in the I/R process which is characterized by oxidative stress. Moreover, ROS have been implicated in the pathogenesis of tissue injury during reperfusion [9]. It has been suggested that activation of neutrophils, accumulation of released cytokines and nitric oxide, platelet activation and apoptosis also play a paramount role in I/R injury [10]. The formation of cytokines and ROS is stimulated by endothelin (ET) [11]. ET-1, a potent vasoconstrictor peptide with 21 amino acids, is
0301-2115/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.06.040
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produced by a large variety of cells from a wide range of species including endothelial cells, leukocytes, macrophages and monocytes [12–14]. ET receptor type A (ETA) and type B (ETB), which have different molecular and pharmacological characteristics, are present in the ovaries [15–17]. In general, the two dissimilar ET receptors are expressed constitutively but are spatially confined to specific cell types in the ovary; a number of cells express both receptors, whereas other cells express one form or the other [18]. ET plays a role in the modulation of microcirculatory blood flow disturbances during ischaemia, and is one of the most important mediators in I/R [19–23]. Many studies have reported the release of ET during I/R injury [24,25], and there has been growing interest in the roles of ET receptor antagonists in I/R injury. ET antagonists have been reported to have beneficial effects in several I/R models including the heart, liver, lung, kidney and small bowel [26–28]. Bosentan is a potent, non-peptide, oral ETA and ETB antagonist, with higher affinity for the ETA subtype [29]. The ET antagonist bosentan has been reported to protect and improve microcirculatory blood flow in several splanchnic organs and in peripheral tissues [29]. The protective effect of bosentan in myocardial I/R injury [30], I/R-induced endothelial dysfunction [31] and I/R injury in rat skeletal muscle [32] has been observed previously in a number of experimental models. To date, however, no studies have shown a link between ET antagonists and ovarian I/R injury. The aim of this study was to evaluate the possible role of ET-1 in the pathogenesis of ovarian I/R injury. This study investigated whether a specific ET receptor antagonist, bosentan has a protective effect against ovarian I/R injury by evaluating alterations in the oxidant–antioxidant system, generation of cytokines and histopathological examinations. 2. Materials and methods 2.1. Animals Animals were housed in accordance with international guidelines, and the study was approved by and conducted in accordance with the Institutional Animal Care and Use Committee. In total, 64 adult female Wistar albino rats were used in this study. 2.2. Surgical technique The rats were randomized into eight groups and anaesthetized with intraperitonal thiopental sodium 25 mg/kg. The area of the abdomen to be operated was prepared and cleaned using betadine. A longitudinal incision (2.5 cm) was performed in the midline area of the lower abdomen. A small peritoneal incision was made, and the uterine horns and adnexa were located. In Group 1, a sham operation was performed. These rats only underwent laparotomy. In Group 2, rats were administered bosentan 60 mg/kg by oral gavage, and the bilateral ovaries were surgically removed 6 h later. In Group 3, bilateral ovarian ischaemia was created by applying vascular clips below the ovaries in the rats. Atraumatic vascular clamps were used in this study to produce ovarian ischaemia. The incision was closed with 4/0 nylon sutures, and the bilateral ovaries were surgically removed after 3 h. In Group 4, a 3-h period of ischaemia was followed by 3 h of reperfusion, following which the bilateral ovaries were removed. In Groups 5 and 6, rats were administered bosentan 30 and 60 mg/kg, respectively, by oral gavage before 30 min of ischaemia, and then bilateral ovarian ischaemia was created by applying vascular clips below the ovary in both groups. After a 3-h period of ischaemia, the bilateral ovaries were surgically removed. In Groups 7 and 8, a 3-h period of bilateral ovarian ischaemia was created by applying vascular clips below the ovary. Rats were administered bosentan 30 and 60 mg/kg, respectively, by oral gavage 2.5 h after the induction of ischaemia. At the end of 3-h
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period of ischaemia, the bilateral vascular clips were removed and 3 h of reperfusion was applied. The ovaries were subsequently removed for histological and biochemical examination. 2.3. Biochemical investigations 2.3.1. Cytokine serum measurements Sera from the eight groups were separated and stored at 80 8C until they were thawed for the assay. Interleukin (IL)-1b, IL-6 and tumour necrosis factor (TNF)-a from each sample were measured with highly sensitive enzyme-linked immunosorbent assay (ELISA) kits [BMS630, eBioscience (USA); KRC0061, Invitrogen (USA); and BMS622, eBioscience (USA); respectively]. Kits were specifically designed for rat cytokines, and all measurements were performed according to the manufacturers’ instructions. 2.3.2. Biochemical investigation of ovarian tissues After macroscopic analyses, rat tissues were kept at 80˚ C. Tissues weighing approximately 50 mg were homogenized with TissueLyser II (Qiagen) in 1 ml of an appropriate buffer. The tissues were then centrifuged at 4˚ C. Supernatants were used to determine the levels of glutathione (GSH) and malondialdehyde (MDA), and the enzyme activity of superoxide dismutase (SOD). All assays were carried out at room temperature. For the biochemical investigation, the activity of SOD and the levels of MDA and GSH from each supernatant were measured in duplicate using highly sensitive ELISA kits [Cayman-706002 (Ann Arbour, MI, USA), Cell Biolabs-STA-330 (San Diego, CA, USA) and Cell Biolabs-STA-312, respectively], which were specifically designed for rat tissue, according to the manufacturers’ instructions. 2.3.3. Histological examination Ovaries were rapidly fixed in 10% buffered formalin for 24–48 h for histological examination. After fixation, each ovarian tissue sample was routinely processed and embedded in paraffin. Fivemicrometre-thick sections were cut as paraffin-embedded tissue samples. After deparaffinization and rehydration, sections were stained with haematoxylin and eosin. All sections were studied and photographed using a light photomicroscope. For semi-thin sections, ovaries were fixed in buffered 3% glutaraldehyde in 0.1 M phosphate. Glutaraldehyde-fixed tissues were postfixed in 1% osmium tetroxide. Each ovarian tissue sample was dehydrated in a graded ethanol series and transferred to propylene oxide. After dehydration, each ovarian tissue sample was embedded in Araldite CY 212. Glutaraldehyde-fixed tissue was postfixed in 1% osmium tetroxide and embedded in thin viscosity resin (Spurr kit, Sigma Chemical Co., St. Louis, MO, USA). Semi-thin sections (1 mm) obtained with an ultramicrotome were stained with Toluidine blue. All sections were studied and photographed using a light photomicroscope. 2.3.4. Statistical analyses Data for the serum cytokine levels were measured by ELISA, and oxidant and antioxidant enzymes were subjected to one-way analysis of variance using Statistical Package for the Social Sciences Version 18.0 (SPSS Inc., Chicago, IL, USA). Differences between the groups were analyzed using Duncan’s multiple range test, and were considered significant at p < 0.05. All data were expressed as mean standard deviation (SD) in each group. 3. Results 3.1. Results of biochemical investigations SOD activity and the levels of MDA and GSH were studied in the I/R injury model in rat ovaries (Table 1). In rats exposed to
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Table 1 Effects of bosentan treatment on changes in activity of superoxide dismutase (SOD) and levels of malondialdehyde (MDA) and total glutathione (GSH) in rat ovaries. Group Sham operation Sham operation + bosentan 60 mg/kg Ischaemia Ischaemia/reperfusion Ischaemia + bosentan 30 mg/kg Ischaemia + bosentan 60 mg/kg I/R + bosentan 30 mg/kg I/R + bosentan 60 mg/kg
n
SOD activity (U/mg protein) ef
8 8 8 8 8 8 8 8
MDA (nmol/mg protein) a
32.56 5.71 34.19 5.88f 24.00 4.52bcd 16.52 2.65a 28.51 6.51de 28.07 2.97cde 22.59 4.20b 23.13 5.22bc
GSH (nmol/mg protein) 4.75 1.04e 4.27 0.58e 2.69 0.29bc 1.73 0.40a 3.46 0.77d 3.25 0.43cd 2.62 0.52bc 2.43 0.39b
2.07 0.53 1.93 0.21a 4.04 0.89c 4.99 1.55d 2.72 0.71ab 2.78 0.39ab 3.16 0.85b 3.61 0.78bc
n, number of rats; I/R, ischaemia/reperfusion. Means in the same column with the same letter are not significantly different using the Duncan test. (a = 0.05). Results are means standard deviation.
ischaemia and I/R, SOD activity and the GSH level were significantly lower compared with the sham operation and sham operation plus bosentan groups. Moreover, the MDA level was significantly higher in the ischaemia and I/R groups compared with the sham operation and sham operation plus bosentan groups. The MDA levels in the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups were significantly lower compared with the I/R group, and the difference between the two groups was not significant. In the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups, SOD activity and the level of GSH were significantly higher compared with the I/R group. Levels of the inflammatory cytokines IL-1b, IL-6 and TNF-a in the serum were also measured in the I/R injury model in rat ovaries (Table 2). In the ischaemia and I/R groups, the levels of these cytokines were significantly higher compared with the sham operation and sham operation plus bosentan groups. In the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups, IL-1b, IL-6 and TNF-a serum levels were significantly lower compared with the I/R group. 3.2. Results of histopathological investigations The ovaries from the sham operation and bosentan group showed no pathological changes (Fig. 1 A and B). Oocytes had normal morphology with intact zona pellucida. The morphology of the follicle cells surrounding oocytes, luteal cells of the ovine corpus luteum and connective tissue of tunica albuginea were normal (Fig. 1A and B). The oocytes from the ischaemia group showed brown pigmentation, and the thickness of the zona pellucida was significantly lower compared with the control group. In addition, hypertrophic changes of parenchymal and stromal cells were accompanied by leucocyte infiltration, haemorrhage and necrosis in the ischaemia group (Fig. 1C and D). The ovaries of the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups exhibited a thicker zona pellucida compared with the ischaemia group, and fewer cases of
haemorrhage and necrosis compared with the ischaemia group (Fig. 1E and F). Acute inflammatory processes such as neutrophil adhesion and migration, apoptotic and degenerative cells, stromal oedema and haemorrhage were severe in the I/R group (Fig. 2 A and B). Neutrophil migration through the endothelial cells was mild in the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups. The ovaries treated with bosentan showed mild oedema and mild vascular congestion of the ovaries without bleeding or leucocyte infiltration (Fig. 2C and D). In the I/R plus bosentan 30 mg/kg and I/R plus bosentan 60 mg/kg groups, although minimal hypertrophic cell degeneration was observed in both follicular and stromal cells, most cellular structures did not show any pathological changes (Fig. 2C and D). 4. Comments To the authors’ knowledge, this is the first study to demonstrate the protective effect of bosentan on ovarian I/R injury. This study demonstrated that bosentan treatment attenuates I/R-induced lipid peroxidation and oxidative stress. This study is also the first to suggest the antioxidant properties of bosentan in rat ovary. It has been suggested that the ET system is involved in the pathophysiology of several ischaemia models in rats. I/R damage is very closely related to endothelial cell damage. In the pathophysiology of this damage, there is an imbalance of vasoconstrictor and vasodilator molecules, such as ET, ROS and nitric oxide [33]. This may lead to a no-reflow phenomenon with release of proinflammatory cytokines, neutrophils and oxidative stress [34]. It has been reported that ETA and ETA/B antagonists may reduce I/Rinduced damage in some tissues, such as myocardium, liver, kidney and skeletal muscle [32,35–37]. Loss of ET action may have a protective role in the I/R cascade by blocking the initiation of release of pro-inflammatory cytokines and ROS. During the detorsion process, reperfusion leads to formation of free oxygen radicals [9]. Production of ROS is an important mechanism causing injury in the I/R process, which is characterized
Table 2 Effects of bosentan treatment on changes in the serum levels of interleukin (IL)-1b, IL-6 and tumour necrosis factor (TNF)-a in sera of rats. Group Sham operation Sham operation + bosentan 60 mg/kg Ischaemia Ischaemia/reperfusion Ischaemia + bosentan 30 mg/kg Ischaemia + bosentan 60 mg/kg I/R + bosentan 30 mg/kg I/R + bosentan 60 mg/kg
n 8 8 8 8 8 8 8 8
IL-1b (pg/ml)
TNF-a (pg/ml)
IL-6 (pg/ml) a
49.86 12.85 52.63 11.19a 111.61 57.11bcd 205.59 41.61e 84.59 21.51abc 71.30 33.19ab 130.77 55.05d 116.27 49.31cd
a
142.62 17.55 144.71 58.31a 348.55 131.05b 820.31 128.58d 358.90 78.40b 225.85 37.65a 590.55 144.15c 512.90 165.46c
39.96 11.73a 42.03 24.62ab 120.79 45.16cd 277.73 43.03e 93.50 42.76bc 83.09 45.77abc 155.18 68.59d 155.98 77.64d
n, number of rats; I/R, ischaemia/reperfusion. Means in the same column with the same letter are not significantly different using the Duncan test, (a = 0.05). Results are means standard deviation.
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Fig. 1. (A and B) Tunica albuginea, corpus luteum, primary oocyte, zona pellucida (white arrow) and granulosa cells (black arrow) of ovaries with normal structures from control group. (C and D) Histopathological observations of ovaries from ischaemia group. Dense and prevalent haemorrhage in corpus luteum (Cl) and stroma (S) in the ovarian tissue. Ovarian tissue also showed hypertrophy in granulosa cells (black arrows), pigmentation in oocyte cytoplasm, and dense loss in zona pellucida (white arrow). (E and F) Histopathological observations of ovaries from ischaemia plus bosentan 60 mg/kg group. Minimal haemorrhage in corpus luteum (Cl) and stroma (S) in the ovarian tissue. Ovarian tissue also showed minimal loss in zona pellucida (white arrow), granulosa cells (black arrow) and tunica albugenia (Ta) cells with normal appearance. Scale bars: 60 mm.
by oxidative stress [9]. One of the most important processes in oxidative-stress-induced cellular damage in I/R injury is lipid peroxidation [38]. MDA is an end product in the lipid peroxidation chain reaction, and an important indicator of the degree of lipid peroxidation [39,40]. It has been reported previously that I/R injury enhances lipid peroxidation and MDA [41]. In the present study, the MDA level was markedly higher in the I/R group compared with the control group. Increased MDA levels in the ovary suggests increased lipid peroxidation, leading to tissue damage and the failure of antioxidant defence mechanisms to prevent formation of excessive free radicals. These results showed that the MDA level was significantly decreased by bosentan pretreatment. Therefore, this study suggests that bosentan has a beneficial effect on lipid peroxidation and oxidative stress. To protect tissues against free radical damage, the body has a defence system involving numerous enzymatic and non-enzymatic antioxidants. The antioxidant defence systems are extensive and consist of multiple layers. Although there are mechanisms for scavenging ROS to prevent oxidative stress, this enzymatic antioxidant defence system appears to be vulnerable to free radical damage. When the balance between oxidants and antioxidants shifts in favour of the former, the antioxidant defence system weakens, leading to lipid peroxidation, and protein and DNA damage [42].
One of the most important antioxidant enzymes that scavenge ROS is SOD. Decreased serum activity of SOD in ovarian tissue damaged by I/R has been demonstrated in previous studies [3,41,43]. In the present study, a period of ischaemia followed by reperfusion led to a significant decrease in SOD activity. The reduction in SOD activity during I/R was probably due to depletion of this antioxidant enzyme by the high amount of ROS with the resumption of blood flow during reperfusion. SOD activity in the I/R group was found to be reduced compared with the other groups in this study. Groups treated with bosentan showed improved SOD activity. This indicates that bosentan can reduce ROS that might attenuate oxidative damage to the tissues and improve the activities of the antioxidant enzymes. Oxidative stress can be regulated by not only enzymatic defence systems but also by non-enzymatic antioxidant defence systems [44]. GSH is an endogenous non-enzymatic antioxidant that plays an important role in the non-enzymatic antioxidant defence and cell signalling systems [45]. The GSH level has been reported to decrease after I/R injury in several previous studies [41,46,47]. In the current study, the GSH level was lower in the I/R group compared with the control group, and a significant increase in GSH level was observed in the livers of animals treated with bosentan compared with the I/R group. Administration of bosentan significantly increased the GSH level. As such, it appears that
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Fig. 2. (A and B) Histopathological observations of ovaries from ischaemia/reperfusion group. Apoptotic deaths (black arrows) in granulosa cells and neutrophil migration (black arrows) through the endothelium of blood vessel (Bv) in the ovaries. (C and D) Histopathological observations of ovaries from ischaemia/reperfusion plus bosentan 60 mg/kg group. There was no migration of neutrophils to endothelium in ovaries from ischaemia/reperfusion plus bosentan 60 mg/kg group. Ovarian tissue also showed hypertrophy in granulosa cells (black arrows), but no apoptotic deaths. Scale bars: 60 mm.
bosentan prevented the depletion of antioxidant enzymes, including GSH, in the present study. Aside from ROS, I/R involves the release of cytokines [10]. Leucocyte activation, expression of adhesion molecules and cytokines have been found to play an important role in a number of experimental I/R models [48–50]. In addition, TNF-a, a proinflammatory cytokine, has been implicated in the pathophysiology of I/R injury [51]. Some studies have demonstrated that TNF and other cytokines promote ET gene expression in a variety of cell types [52,53]. On the other hand, ET stimulation can increase the production of proinflammatory mediators such as TNF, IL-1, IL-6 and IL-8 [53]. Therefore, there may be a positive feedback loop between TNF-a and ET. This study found that TNF-a, IL-1 and IL-6 levels were significantly up-regulated after ovarian torsion, which was in agreement with previous studies [54]. In addition, bosentan treatment decreased serum TNF-a, IL-1 and IL-6 levels compared with the I/R groups. The reduction in cytokine levels during I/R was probably due to blockage of the ET receptors by bosentan. These results indicate that bosentan may reduce the production of TNF-a and other cytokines by blocking ET receptors. The effects of bosentan on rat ovaries have also been evaluated histopathologically. Bosentan treatment decreased the I/R-induced histological damage in the ovaries. In the present study, bosentan
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