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reperfusion in rats
Acta Histochemica 116 (2014) 167–175
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The role of epidermal growth factor in prevention of oxidative injury and apoptosis induced by intestinal ischemia/reperfusion in rats Pelin Arda-Pirincci ∗ , Sehnaz Bolkent Department of Biology, Faculty of Science, Istanbul University, 34134 Vezneciler, Istanbul, Turkey
a r t i c l e
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Article history: Received 19 February 2013 Received in revised form 29 June 2013 Accepted 1 July 2013 Keywords: Epidermal growth factor Ischemia/reperfusion Intestinal injury Apoptosis Oxidative stress Antioxidant enzymes Rat
a b s t r a c t Intestinal ischemia/reperfusion is a major problem which may lead to multiorgan failure and death. The aim of the study was to evaluate the effects of epidermal growth factor (EGF) on apoptosis, cell proliferation, oxidative stress and the antioxidant system in intestinal injury induced by ischemia/reperfusion in rats and to determine if EGF can ameliorate these toxic effects. Intestinal ischemia/reperfusion injury was produced by causing complete occlusion of the superior mesenteric artery for 60 min followed by a 60-min reperfusion period. Animals received intraperitoneal injections of 150 g/kg human recombinant EGF 30 min prior to the mesenteric ischemia/reperfusion. Mesenteric ischemia/reperfusion caused degeneration of the intestinal mucosa, inhibition of cell proliferation, stimulation of apoptosis and oxidative stress in the small intestine of rats. In the ischemia/reperfusion group, lipid peroxidation was stimulated accompanied by increased intestinal catalase and glutathione peroxidase activities, however, glutathione levels and superoxide dismutase activities were markedly decreased. EGF treatment to rats with ischemia/reperfusion prevented the ischemia/reperfusion-induced oxidative injury by reducing apoptosis and lipid peroxidation, and by increasing antioxidant enzyme activities. These results demonstrate that EGF has beneficial antiapoptotic and antioxidant effects on intestinal injury induced by ischemia/reperfusion in rats. © 2013 Elsevier GmbH. All rights reserved.
Introduction Ischemia/reperfusion of the gut in humans is a serious condition occurring after trauma, burns, septic shock, and liver or small intestine transplantation. Ischemia/reperfusion-induced intestinal injury in experimental animals is a practical model commonly used in studies on pathogenesis of systemic inflammation, respiratory failure and multiple organ failure. Experimental intestinal ischemia/reperfusion is one of the important models used to investigate oxidative stress induced by reactive oxygen species especially during reperfusion in small intestine (Horton and Walker, 1993; Nilsson et al., 1994; Kacmaz et al., 1999). Intestinal ischemia/reperfusion damages the mucosa by impairing its barrier function, and by causing bacterial translocation (Biffl and Moore, 1996; Wu et al., 2004). Various mediators play a role in the pathogenesis of ischemia/reperfusion-induced intestinal injury including reactive oxygen/nitrogen species, proinflammatory cytokines, leukocyte adhesion and infiltration (Nilsson et al., 1994; Xia et al., 2002; El Assal and Besner, 2004). Recent evidence has indicated that apoptosis is increased significantly during
∗ Corresponding author. E-mail address: [email protected] (P. Arda-Pirincci). 0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.07.005
ischemia/reperfusion of the gut and may play a key role in the pathogenesis of ischemia/reperfusion-induced intestinal injury. The intestinal ischemia/reperfusion model has often been used to study apoptosis (Shah et al., 1997; Ikeda et al., 1998; Noda et al., 1998; Fujise et al., 2006). Although intestinal ischemia/reperfusion does not have a specific treatment, several antioxidants and monoclonal antibodies against adhesion molecules have shown to be protective against ischemia/reperfusion injury (Kacmaz et al., 1999; Kubes, 1999; Kazez et al., 2000). Epidermal growth factor (EGF) is a mitogenic peptide that is secreted into the lumen of the duodenum by Brunner’s glands. EGF is implicated in the regulation of a wide variety of physiological processes, including growth, cell proliferation, regeneration, differentiation, and wound repair. It is suggested that EGF, not only increases mucosal repair, but also behaves as a cytoprotective and trophic agent for gastrointestinal epithelium (Buret et al., 1998; Berlanga et al., 2002). Many in vivo or in vitro studies have demonstrated that members of the EGF family are among the important factors for healing after damage in diverse experimental models (Pillai et al., 1999; Michalsky et al., 2001; Xia et al., 2002; Jahovic et al., 2004; Martin et al., 2005; Clark et al., 2005; El-Assal et al., 2007, 2008). However, there are only a limited number of studies regarding the effects of EGF on apoptosis and on the antioxidant system in ischemia/reperfusion-induced intestinal injury. In this
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study, we aimed to investigate the role of EGF on apoptosis, cell proliferation, oxidative stress and antioxidant system in intestinal injury induced by ischemia/reperfusion in rats.
The sections obtained from different groups were examined under an Olympus CX 41 light microscope, and photomicrographs were recorded using an Olympus DP71 digital camera.
Materials and methods
TUNEL assay
Animals
In order to detect apoptotic cells, in situ labeling of the fragmented DNA generated by apoptosis associated endonucleases was performed with terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay in the paraffinized crosssections of jejunum. Tissue parts taken from the jejunum were fixed in 10% phosphate-buffered formalin. Paraffin sections at 5 m were dewaxed, rehydrated, and then treated with proteinase K (20 g/mL), and blocked for endogenous peroxidase activity with 3% hydrogen peroxide (H2 O2 ). The labeling procedure was performed with the commercially available ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA, USA; S7101) according to the manufacturer’s instructions. The apoptotic cells were made visible with 3,3’-diaminobenzidine (DAB) and the nuclei were counterstained with methyl green. Rat breast tissue taken 4 days after breast-feeding cessation was used as a positive control. For negative control, the TdT enzyme was replaced by phosphate-buffered saline. A dark brown nuclear staining was regarded as positive. Besides, the apoptotic bodies were also positively stained. TUNEL positive epithelial cells in villi and crypts were evaluated for each sample using an Olympus CX41 light microscope using a 40× objective. A minimum of 20 full crypts or 10 full-length villi were randomly selected for apoptotic index analysis in the sections of jejunum. The apoptotic index values were expressed as a percentage of apoptotic cells in the total cells counted in villi or crypts.
Two- to 2.5-month-old Sprague–Dawley male rats weighing 220–240 g (n = 39) obtained from DETAE (Experimental Medical Research Institute of Istanbul University) were used in the study. The experiments were reviewed and approved by the Animal Care and Use Committee of Istanbul University. The animals were fed with Purina Laboratory Rodent Diet 5001 and tap water ad libitium, but fasted overnight prior to the experiments. Induction of ischemia/reperfusion injury The rats were anesthetized with ketamin (100 mg/kg) (Ketalar, Pfizer, Istanbul, Turkey) and chlorpromazine (0.75 mg/kg) (Largactil, Eczacıbas¸ı, Istanbul) intraperitoneally. After induction of anesthesia, the abdomen was opened through a midline abdominal incision. Intestinal ischemia/reperfusion (I/R) injury was produced by causing complete occlusion of the superior mesenteric artery followed by a period of reperfusion. The superior mesenteric artery was clamped for 60 min. After 60 min of ischemia, the vascular clamp at the superior mesenteric artery was removed and three drops of 2% lidocaine (Aritmal, Osel, Istanbul, Turkey) were applied directly on the superior mesenteric artery to facilitate reperfusion. Blood circulation was restarted for a 60-min reperfusion period (Noda et al., 1998). At the end of the reperfusion period, the animals were killed by an overdose of anesthesia.
Active caspase-3 staining Experimental protocol Animals were selected randomly and divided into five groups. Group I (Sham): Sham-operated animals. These animals were subjected to abdominal incision and their organs exposed for 120 min, but without clamping of the mesenteric artery so as to distinguish the differences between the effects of intestinal I/R and the changes because of non-specific surgical stress (n = 7); Group II (Control): Control rats received i.p. injection of 10 mM acetic acid with 0.1% BSA (vehicle) (n = 7); Group III (EGF): Animals administered i.p. injection of 150 g/kg human recombinant EGF (Sigma-Aldrich, St. Louis, MO, USA; E 9644) (n = 7); Group IV (I/R): Rats received an i.p. injection of 10 mM acetic acid with 0.1% BSA (vehicle) 30 min prior to the intestinal I/R (n = 9); Group V (I/R + EGF): Animals injected intraperitoneally (i.p.) with 150 g/kg human recombinant EGF dissolved in 10 mM acetic acid solution with 0.1% BSA 30 min prior to the intestinal I/R (n = 9). The animals belonging to groups II, III, IV and V, to which EGF or vehicle were applied, were killed 2.5 h after the injections. The control rats in group I were killed 2 h after the sham operation in order to supply the same conditions as with group IV. At the end of the experiments, samples from the jejunum were taken from the animals for all examinations. Histological assessment of intestinal injury Samples from the jejunum were taken from the animals for histological examination. The tissues were fixed in Bouin’s solution for 24 h at room temperature and embedded in paraffin wax. Serial sections 5 m thick were cut and stained with Masson’s trichrome for general morphological evaluation and the detection of connective tissue. Periodic acid-Schiff (PAS) staining was performed to observe the changes in the goblet cells and brush border of villi.
The major executioner caspase involved in apoptosis is caspase3, and activation of caspase-3 is one of the hallmarks of apoptosis. We examined apoptosis by using a polyclonal antibody to active caspase-3. Immunohistochemical active caspase-3 staining was performed using the streptavidin-biotin-peroxidase method. Jejunum tissues were fixed in 10% phosphate-buffered formalin. Following dewaxing and rehydration of paraffin-embedded sections, to facilitate antigen retrieval sections were permeabilized with 0.3% Triton X-100 for 10 min and then were heated in 10 mM citrate buffer (pH 6.0) for 15 min in a microwave oven at 700 W. Endogenous peroxidase activity was blocked by incubating with 3% H2 O2 for 10 min. The labeling procedure was performed with the Histostain Plus Broad Spectrum Kit (Invitrogen, Zymed Laboratories, San Francisco, CA, USA), followed by the application of polyclonal rabbit anti-active caspase-3 (1:50 in PBS, Millipore, Billerica, MA, USA; AB-3623) for 60 min at room temperature. Slides were then incubated with 3-amino-9-ethylcarbazole, and counterstained with Mayer’s hematoxylin. For negative control, phosphate-buffered saline was used instead of antibody. Cytoplasm with dark red staining was assessed as positive. Quantitative analysis of caspase-3 immunoreactive epithelial cells was made by using an Olympus CX41 light microscope with a 40× objective. For the labeling index, 20 full crypts or 10 full-length villi were randomly selected in the jejunum sections of each sample and caspase-3 positive cells were counted. The caspase-3 labeling index was calculated as a percentage of caspase-3 positive epithelial cell in the total cells counted in the crypts for each animal. Proliferating cell nuclear antigen assay The proliferating cells in intestine sections were detected by proliferating cell nuclear antigen (PCNA) immunohistochemistry.
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Fig. 1. Histology of jejunal mucosa in all groups. (A) Normal histological appearance of jejunal mucosa in a sham-operated control rat; (B) vehicle control rat; (C) rat given EGF; (D) rat subjected to ischemia/reperfusion injury indicates disruption in integrity of villi and villus loss ( ), infiltration of polymorphonuclear cells and lymphocytes ( ), disruption in integrity of crypts ( ), necrosis ( ) with severe mucosal damage. (E) EGF treated ischemia/reperfusion group demonstrate infiltration of polymorphonuclear cells and lymphocytes ( ) and hyperemia (h). Masson’s trichrome. Scale bar = 500 m.
Tissue samples from the jejunum were fixed in Bouin’s solution. After having completed the routine follow-up, the sections were boiled in 10 mM citrate buffer (pH 6.0) in microwave oven for 10 min at 700 W for antigen retrival. Then, the tissues were permeabilized with 0.3% Triton X-100 for 10 min. After blocking endogenous peroxidase activity with 3% H2 O2 , Ultra Vision Detection System (Lab Vision, Fremont, CA, USA) using a streptavidin-biotin-peroxidase technique for immunohistochemical analysis. In order to assess cell proliferation, the sections were incubated with primary antibody specific for PCNA (Clone PC10; NeoMarkers, Fremont, CA, USA; MS-106) at room temperature for 30 min. The antibody was used at a dilution of 1:50. Peroxidase activity of the tissue was demonstrated by 3-amino9-ethylcarbazole and then the sections were counterstained with Mayer’s hematoxylin. For negative control, the primary antibody was replaced by phosphate-buffered saline. Quantitative analysis of PCNA positive epithelial cells in crypts was made by using an Olympus CX41 light microscope using a 40× objective. In the jejunum sections of each sample, 20 full crypts were randomly selected and PCNA positive crypt cells were counted. The proliferation index was calculated as a percentage of PCNA positive epithelial cell in the total cells counted in the crypts for each section. Proliferation index =
PCNA-positive crypt cell count × 100 Total crypt cell count
Biochemical analysis Jejunum tissues were homogenized in cold 0.9% physiologic saline by means of a glass homogenizer (Yellow line OST basic
mixer, IKA, Staufen, Germany) to make up a 10% (w/v) homogenate. The homogenates were centrifuged at 20,000 × g for 15 min (+4 ◦ C) to obtain their supernatants. In the clear supernatants were determined activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced glutathione (GSH), malondialdehyde (MDA) and total protein by using a spectrophotometer (Shimadzu UV 1700, Kyoto, Japan). Catalase activity was estimated by the method described by Aebi (1974). Intestinal GPx activity was determined using the method of Paglia and Valentine (1967) modified by Wendel (1981). SOD activity in the intestinal homogenate was determined with the method of Sun et al. (1988). Reduced glutathione content was determined according to the method of Beutler et al. (1963). Intestinal oxidative stress was quantified in the homogenates by measuring malondialdehyde (MDA). MDA levels in the intestine were determined using a method described by Ledwozyw et al. (1986) with some modifications (Arda-Pirincci and Bolkent, 2011). Total protein content of the samples was established by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a standard. Statistical analyses Microscopic and biochemical data obtained were processed with SPSS 15 software (IBM, Chicago, IL, USA). Results of CAT, GPx and SOD activities were performed by using one-way ANOVA and unpaired Student’s t-test. Data of GSH and MDA levels, TUNEL assay, caspase-3 and PCNA immunohistochemistry were analyzed by non-parametric Kruskal–Wallis test and Mann–Whitney U-test. The results were given as mean ± standard error (SE). P < 0.05 was considered as statistically significant.
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Fig. 2. TUNEL-positive apoptotic cells in the jejunum of: (A) ( ) sham-operated control rat; (B) vehicle control group rat; (C) rat given EGF; (D) I/R group rat showing TUNEL-positive epithelial cells ( ); (E) rat of the I/R + EGF group showing apoptotic cell ( ) numbers significantly decreased when compared with the I/R group. Scale bar = 100 m.
Results Histology Light microscopic appearances of jejunum for all experimental groups are presented in Fig. 1. Histological examination of jejunal tissues from the sham-operated group, vehicle control group and EGF group revealed normal mucosal architecture. In the I/R group, disruption in brush border and integrity of villi, villi loss, widespread necrotic areas, infiltration of polymorphonuclear cells and lymphocytes, severe hemorrhage were commonly observed. In addition, we found increased number of the cells with pyknotic nuclei and densely eosinophilic cytoplasm in epithelium, mucosal edema, hemorrhage in the mucosa of in the same group rats. In the I/R group rats, PAS-positive reaction intensity in the goblet cells, in the brush border of villi was shown to decrease when compared with the control groups. On the other hand, treatment of EGF of the I/R group partially recovered intestinal mucosal architecture. However, in some jejunum sections of the I/R + EGF group
still showed disruption in the brush border and in integrity of villi, infiltration of lymphocytes and moderate hyperemia. PAS-positive reaction intensity was greater than in the I/R group. TUNEL immunostaining The photomicrographs and quantitative assessment of TUNELpositive epithelial cells in the jejunal mucosa are shown in Figs. 2 and 3. In this study, the apoptotic cells were observed in the crypts and villi of jejunum in all groups. In all control groups which Sham-operated, vehicle control and EGF-given was determined very few numbers of apoptotic cells in the jejunal mucosa by TUNEL assay. In the rats subjected to I/R injury, the number of TUNEL-positive apoptotic crypt and villus cells increased in a significant manner compared to sham and vehicle control groups (P < 0.05). The highest apoptotic index was detected in the villus compartment of the rats subjected to I/R. In this group, the apoptotic index was found as 1.65 ± 0.26% for the epithelium of villus. Apoptotic index decreased in a statistically meaningful way
Fig. 3. Apoptotic index (%) in epithelium of villus and crypt. The data are given as mean ± SE per group. The data for the epithelium of villus: Sham: 0.29 ± 0.06, Control: 0.19 ± 0.01, EGF: 0.14 ± 0.04, I/R: 1.65 ± 0.26, I/R + EGF: 0.65 ± 0.07. The data for the epithelium of crypt: Sham: 0.17 ± 0.01, Control: 0.14 ± 0.01, EGF: 0.13 ± 0.02, I/R: 0.62 ± 0.04, I/R + EGF: 0.26 ± 0.05. a P < 0.05 versus sham group, b P < 0.05 versus control group, c P < 0.05 versus EGF group, d P < 0.01 versus I/R group.
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Fig. 4. Active caspase-3 immunoreactive epithelial cells ( ) in the jejunum of: (A) sham-operated control rat; (B) vehicle control rat; (C) EGF-treated rat; (D) I/R group rat and (E) I/R + EGF group rat. Scale bar = 50 m.
in crypts and villi when EGF was applied to the I/R group (P < 0.01). However, in the EGF treated I/R group were observed a significantly increase in the number of TUNEL-positive apoptotic villus cells as compared to the sham group (P < 0.05) or control group (P < 0.05) or EGF group (P < 0.05).
EGF of the I/R group was significantly decreased the number of active caspase-3 positive crypt or villus cells according to the I/R group (P < 0.01).
PCNA immunohistochemistry Active caspase-3 immunohistochemistry The photomicrographs and quantitative assessment of immunolocalization of active caspase-3 in the jejunum are shown in Figs. 4 and 5. In the sham-operated group, vehicle control group and EGF-given group, caspase-3 was expressed only in a few crypt and villus epithelial cells. In this study, we showed an intense staining for caspase-3 in the cytoplasm of epithelial cells in the group subjected to I/R injury. Compatible with the TUNEL findings, in this group the numbers of active caspase-3 positive epithelial cells were markedly increased as compared to the sham group (P < 0.05) or control group (P < 0.05). However, treatment of
The photomicrographs and quantitative assessment of the proliferating epithelial cells in the jejunal crypts are shown in Figs. 6 and 7. The highest cell proliferation index in crypts of the jejunum was detected in the group administered EGF, whereas the lowest proliferation index was found in the I/R group. According to statistical results, the number of PCNA-positive crypt cells showed a significant decrease in the group subjected to I/R injury as compared with the sham-operated animals (P < 0.05). Preadministration of EGF to the I/R group increased the cell proliferation index, but this increase did not reach the ratio observed in the control groups.
Fig. 5. The caspase-3 labeling index (%) in epithelium of villus and crypt. The data were given as mean ± SE per group. The data for the epithelium of villus: Sham: 0.06 ± 0.03, Control: 0.05 ± 0.02, EGF: 0.05 ± 0.02, I/R: 0.77 ± 0.04, I/R + EGF: 0.12 ± 0.07. The data for the epithelium of crypt: Sham: 0.03 ± 0.02, Control: 0.03 ± 0.03, EGF: 0.03 ± 0.02, I/R: 0.33 ± 0.02, I/R + EGF: 0.05 ± 0.03. a P < 0.05 versus sham group, b P < 0.05 versus control group, c P < 0.01 versus I/R group.
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Fig. 6. PCNA immunoreactivity ( ) in crypts of the jejunum in: (A) sham-operated control rat; (B) vehicle control group rat; (C) EGF-treated rat; (D) I/R group rats; (E) I/R + EGF group rat. The intensity and number of PCNA-positive crypt cells ( ) decreased in I/R group rats. In I/R + EGF group, the intensity and number of PCNA-positive crypt cells ( ) increased when compared with the I/R group. Scale bar = 200 m.
Biochemical results The activities of antioxidant enzymes, and levels of GSH and MDA in intestinal tissues are presented in Table 1. Intestinal activity of CAT insignificantly increased in the I/R group as compared with the control groups. However, CAT activity belonging to the I/R + EGF group showed a significant increase compared to the I/R group or the sham group (P < 0.01), or the vehicle control group (P < 0.001) or the EGF-treated animals (P < 0.05). Tissue GPx activities observed in the I/R + EGF group increased compared to the I/R group, but this increase was not significant. GPx activity in the EGF-administered rats with I/R was significantly higher than the sham and vehicle control group (P < 0.05), or the EGF control group (P < 0.01). Like the other antioxidant enzymes, the I/R + EGF group showed markedly higher tissue SOD activities than the I/R group (P < 0.001), and also intestinal SOD activities in the I/R + EGF group significantly
Fig. 7. Proliferation index was determined by using PCNA immunohistochemistry in the jejunum. The data were given as mean ± SE per group. Sham: 61.02 ± 6.13, Control: 59.37 ± 8.72, EGF: 67.55 ± 2.59, I/R: 42.84 ± 4.40, I/R + EGF: 48.83 ± 1.01. a P < 0.05 versus sham group.
increased when compared to the sham, vehicle control and EGF groups (P < 0.001). A significant decrease was noted in the intestinal GSH levels of both the I/R group (P < 0.05) and I/R + EGF group (P < 0.001) compared to the sham group. Also, EGF pretreatment of the I/R group markedly reduced the GSH levels when compared with the vehicle control group (P < 0.05). The intestinal MDA levels were highest in the I/R group, compared with the control groups (P < 0.05). MDA content significantly decreased by the administration of EGF as compared to the I/R group. Discussion The intestine is a fairly sensitive organ with regard to ischemia/reperfusion damage because of its high oxygen requirements. Intestinal I/R injury can cause structural disorders in the mucosa of the small intestine and functional losses, as well as multiorgan failure and death (El Assal and Besner, 2004). The response of the intestinal mucosa toward ischemia can be divided into two phases. In the hypoxic phase, lack of oxygen causes intestinal mucosal cell death and severe damage in the mucosa. In the second phase, reoxygenation occurring during reperfusion causes further damage to the surviving mucosal cells after hypoxia (Parks and Granger, 1986; El Assal and Besner, 2004). The first indicators of ischemic injury in the small intestinal mucosa are degeneration of the villus epithelium and desquamation of the epithelial lining. During reperfusion, due to the production of reactive oxygen species (ROS), villi and mucosa suffer more severe damage (Martin et al., 2005). In this study, rats undergoing 60-min superior mesenteric artery occlusion followed by 60-min reperfusion had degenerative histopathological changes, especially mucosal and villus damage, in their jejunal tissues. In our applied experimental model, we showed that apoptotic cell death was also markedly stimulated in the jejunal mucosa. In a study reported by Noda et al.
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Table 1 Intestinal activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), and levels of intestinal glutathione (GSH) and lipid peroxidation (MDA) for all groups. Group
n
CAT* (U/mg protein)
Sham Control EGF I/R I/R + EGF
7 7 7 9 9
5.36 4.94 6.64 7.20 12.24
a b c d e f g h i k *
± ± ± ± ±
1.04 0.83 1.21 0.75 1.53a , d , g , h
GPx* (U/g protein) 9.56 9.26 8.73 11.05 14.90
± ± ± ± ±
1.30 1.59 0.63 1.49 1.34c , f , i
SOD* (U/mg protein) 2.83 2.62 2.52 2.54 3.36
± ± ± ± ±
0.17 0.10 0.10 0.12 0.08b , d , g , k
GSH* (nmol/mg protein) 10.79 11.61 6.56 6.20 4.39
± ± ± ± ±
1.31 2.64 1.16 0.97c 0.40e , f
MDA* (nmol/mg protein) 0.40 0.38 0.40 0.87 0.39
± ± ± ± ±
0.05 0.03 0.09 0.11c , f 0.04a
P < 0.01 versus I/R group. P < 0.001 versus I/R group. P < 0.05 versus sham group. P < 0.01 versus sham group. P < 0.001 versus sham group. P < 0.05 versus control group. P < 0.001 versus control group. P < 0.05 versus EGF group. P < 0.01 versus EGF group. P < 0.001 versus EGF group. Mean ± SE, n = number of animals.
(1998), small intestines of rats undergoing 60-min ischemia were reperfused at different times and, they demonstrated that after 60 min of ischemia, apoptotic cell death was maximal. Our TUNEL findings in the I/R group parallel the apoptotic findings reported by Noda et al. (1998). The current study evaluated the role of exogeneous EGF on intestinal architecture and apoptosis in small intestinal injury produced by ischemia/reperfusion and, it also determined whether EGF has protective and antiapoptotic effects against intestinal ischemic injury. In our study, we observed that EGF applied to the animals at 150 g/kg dose 30 min before ischemia/reperfusion could partially prevent the severe damage occurring in the jejunal mucosa. Berlanga et al. (2002) reported that EGF administered at 2 mg/kg dose to rats reduced by 80% the microscopic damage in the small intestine induced by mesenteric ischemia/reperfusion. This dose of EGF is fairly high compared to the dose we used in our study. In the present study, however, the apoptotic index and labeling index of caspase-3, which is a key caspase involved in the apoptotic pathway, in the intestinal mucosa were markedly reduced 1 h after ischemia with an EGF dose of 150 g/kg. Our literature survey did not discover studies about the effects of EGF on apoptosis in intestinal injury produced by ischemia/reperfusion, yet in some experimental models such as small intestinal resection (Stern et al., 2000), necrotizing enterocolitis (Clark et al., 2005; Feng et al., 2006) and sepsis (Clark et al., 2008), EGF or heparin-binding EGF-like growth factor (HB-EGF) reduces intestinal apoptosis. Homeostasis of epithelial architecture in the small intestine is regulated by both cell proliferation and cell death including apoptosis (Potten and Booth, 1997). Since intestinal epithelial cells have short proliferation cycle and powerful growth ability, regeneration in the intestinal tissue is quite fast. Another factor which will can damage the integrity of the intestinal mucosal barrier is the inhibition of epithelial cell proliferation (Xu et al., 2005). It has been reported that intraluminal EGF administration to experimental animals has proliferative and trophic effects (Ulshen et al., 1986). In our study, in order to determine whether EGF is effective against cell proliferation in the intestinal ischemia/reperfusion injury model, PCNA staining was investigated immunohistochemically. In the jejunum crypts of the rats belonging to the I/R group, cell proliferation was inhibited when compared to the control groups, and it was seen that EGF administration prevented this inhibition to some extent, but it could not reach the proliferative index of the control groups. In a study performed by Xia et al. (2002), rats undergoing 90-min intestinal ischemia followed by 45-min reperfusion had a lower cell proliferation index in the intestinal mucosa, but the application of HB-EGF, a member of the EGF family, increased the cell proliferation. However, our literature survey did not discover
any studies on the effects of EGF on cell proliferation in intestinal injury induced by ischemia/reperfusion. It is known that the production of ROS and the other damaging free radical metabolites is increased during ischemia/reperfusion (Horton and Walker, 1993; Nilsson et al., 1994; Carden and Granger, 2000; Kazez et al., 2000). Uncontrolled production of these free radicals could lead to apoptosis and necrosis as well as lipid peroxidation, resulting in cellular damage. We measured the MDA levels, which are indicators of free radical-induced oxidative damage and lipid peroxidation. According to our MDA results, an increase was found in the tissue lipid peroxidation level in the I/R group, and it was concluded that oxidative damage was present in ischemic intestine. In a study performed by Berlanga et al.(2002) the MDA increase observed in ischemic intestinal tissue was reversed following administration of 2 mg/kg EGF to rats. In the present study, we observed a reduction in the intestinal MDA to the levels of control group members in the I/R + EGF group, with an intraperitoneal EGF dose of 150 g/kg. GSH is an important endogeneous antioxidant, participating in the detoxification of ROS and elimination of lipid peroxides (Aw, 2005). GSH has important functions in the protection of tissues against ROS-mediated injury. Previous studies have reported that intestinal ischemia/reperfusion injury causes changes in the GSH redox cycle and a decrease in GSH synthesis in the tissue (Harward et al., 1994; Sener et al., 2001; Sasaki and Joh, 2007; Kimura et al., 2009). In our study, in accordance with the other researchers’ findings, intestinal GSH levels in rats undergoing ischemia/reperfusion were observed to be markedly lower than those of the sham control group. Up to now, it was not known how EGF affects GSH levels after intestinal ischemia/reperfusion. In this study, 1 h following superior mesenteric artery occlusion, the decrease occurring in the GSH levels of the intestinal tissue could not be reversed by EGF application. While EGF application before ischemia to the rats prevents LPO production in the intestine, the fact that GSH levels are lower than those in the control group members caused us to consider that oxidative stress could be removed by the other factors of antioxidative defense mechanism. For this purpose, the role of EGF on antioxidant enzymes in ischemia/reperfusion-induced intestinal injury was also investigated in this study. It is known that antioxidant enzymes such as CAT, GPx and SOD play important roles in the prevention or reduction of free radical damage. Oxidative stress is suggested to possibly increase or suppress, in a timely manner, the gene expression of antioxidant enzymes (Kacmaz et al., 1999). In our study, CAT and GPx activities after 1-h-ischemia/1-h-reperfusion in the small intestine responded to oxidative stress by showing a slight increase, as compared to the sham group. In intestinal ischemia/reperfusion
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injury, the effects of EGF on antioxidant enzyme system are not known fully. In our study, EGF injection to rats 30 min prior to ischemia/reperfusion application caused increases in CAT and GPx activities, both in control animals and I/R group members. In some studies, there is strong evidence concerning xanthine oxidase, which is considered to be the real source of oxidants which occur during ischemia/reperfusion in the gastrointestinal tract. It is reported that, during reperfusion of ischemic intestine, xanthine oxidase, which reaches high concentrations in the tissue, causes oxidative damage in the tissue by increasing especially superoxide radical production (Parks et al., 1982; McCord, 1985; Granger et al., 1986; Sasaki and Joh, 2007). Being a highly specific scavenger of superoxide, the potential of SOD to protect against gastrointestinal injury induced by ischemia/reperfusion is an important indicator that this antioxidant enzyme plays a major role in the first defence against oxidative stress which occurs during ischemia/reperfusion (Parks et al., 1982; Parks and Granger, 1986; Matés et al., 1999). In this study, the decrease in the intestinal SOD activity of rats belonging to the I/R group also verifies the literature data. We believe that the increase of intestinal SOD activity (even more than those of the control group) after administration of EGF to rats undergoing ischemia/reperfusion, is indicative of EGF trying to neutralize the superoxide radicals which emerge during ischemia/reperfusion injury, and also that EGF demonstrates an antioxidant effect in this injury model. As a result of this study, it appears that the EGF can play important roles in protection against oxidative damage induced by ischemia/reperfusion in the small intestine by decreasing apoptotic cell death, by preventing lipid peroxidation, by increasing antioxidant enzyme activities. Acknowledgments This study was funded by the Scientific Research Project Coordination Unit of Istanbul University (Grant numbers: 152/20082003 and UDP-1159). References Aebi H. Bergmeyer HU, editor. Catalase: Methods of enzymatic analysis. NewYork: Academic Press; 1974. p. 673–83. Arda-Pirincci P, Bolkent S. The role of glucagon-like peptide-2 on apoptosis, cell proliferation, and oxidant-antioxidant system at a mouse model of intestinal injury induced by tumor necrosis factor-alpha/actinomycin D. Mol Cell Biochem 2011;350:13–27. Aw TY. Intestinal glutathione: determinant of mucosal peroxide transport, metabolism, and oxidative susceptibility. Toxicol Appl Pharmacol 2005;204:320–8. Berlanga J, Prats P, Remirez P, Gonzalez R, Lopez-Saura P, Aguiar J, et al. Prophylactic use of epidermal growth factor reduces ischemia/reperfusion intestinal damage. Am J Pathol 2002;161:373–9. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882–8. Biffl WL, Moore EE. Splanchnic ischemia/reperfusion and multiple organ failure. Br J Anaesth 1996;77:59–70. Buret A, Olson ME, Gall DG, Hardin JA. Effects of orally administered epidermal growth factor on enteropathogenic Escherichia coli infection in rabbits. Infect Immun 1998;66:4917–23. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 2000;190:255–66. Clark JA, Clark AT, Hotchkiss RS, Buchman TG, Coopersmith CM. Epidermal growth factor treatment decreases mortality and is associated with improved gut integrity in sepsis. Shock 2008;30:36–42.
Clark JA, Lane RH, Maclennan NK, Holubec H, Dvorakova K, Halpern MD, et al. Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2005;288:G755–62. El Assal ON, Besner GE. Heparin-binding epidermal growth factorlike growth factor and intestinal ischemia-reperfusion injury. Semin Pediatr Surg 2004;13:2–10. El-Assal ON, Paddock H, Marquez A, Besner GE. Heparin-binding epidermal growth factor-like growth factor gene disruption is associated with delayed intestinal restitution, impaired angiogenesis, and poor survival after intestinal ischemia in mice. J Pediatr Surg 2008;43:1182–90. El-Assal ON, Radulescu A, Besner GE. Heparin-binding EGF-like growth factor preserves mesenteric microcirculatory blood flow and protects against intestinal injury in rats subjected to hemorrhagic shock and resuscitation. Surgery 2007;142: 234–42. Feng J, El-Assal ON, Besner GE. Heparin-binding epidermal growth factor-like growth factor reduces intestinal apoptosis in neonatal rats with necrotizing enterocolitis. J Pediatr Surg 2006;41:742–7. Fujise T, Iwakiri R, Wu B, Amemori S, Kakimoto T, Yokoyama F, et al. Apoptotic pathway in the rat small intestinal mucosa is different between fasting and ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol 2006;291:G110–6. Granger DN, McCord JM, Parks DA, Hollwarth ME. Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine. Gastroenterology 1986;90:80–4. Harward TR, Coe D, Souba WW, Klingman N, Seeger JM. Glutamine preserves gut glutathione levels during intestinal ischemia/reperfusion. J Surg Res 1994;56:351–5. Horton JW, Walker PB. Oxygen radicals, lipid peroxidation, and permeability changes after intestinal ischemia and reperfusion. J Appl Physiol 1993;4:1515–20. Ikeda H, Suzuki Y, Suzuki M, Koike M, Tamura J, Tong J, et al. Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium. Gut 1998;42:530–7. Jahovic N, Güzel E, Arbak S, Ye˘gen BC. The healing-promoting effect of saliva on skin burn is mediated by epidermal growth factor (EGF): role of the neutrophils. Burns 2004;30:531–8. Kacmaz M, Ozturk HS, Karaayvaz M, Guven C, Durak I. Enzymatic antioxidant defence mechanism in rat intestinal tissue is changed after ischemia-reperfusion. Effects of an allopurinol plus antioxidant combination. Can J Surg 1999;42:427–31. Kazez A, Demirba˘g M, Ustünda˘g B, Ozercan IH, Sa˘glam M. The role of melatonin in prevention of intestinal ischemia-reperfusion injury in rats. J Pediatr Surg 2000;35:1444–8. Kimura Y, Pierro A, Eaton S. Glutathione synthesis in intestinal ischaemia-reperfusion injury: effects of moderate hypothermia. J Pediatr Surg 2009;44:353–7. Kubes P. The role of adhesion molecules and nitric oxide in intestinal and hepatic ischaemia/reperfusion. Hepatogastroenterology 1999;46:1458–63. Ledwozyw A, Michalak J, Stepien A, Kadziolka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986;155:275–83. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. Martin AE, Luquette MH, Besner GE. Timing, route, and dose of administration of heparin-binding epidermal growth factor-like growth factor in protection against intestinal ischemiareperfusion injury. J Pediatr Surg 2005;40:1741–7.
P. Arda-Pirincci, S. Bolkent / Acta Histochemica 116 (2014) 167–175
˜ de Castro I. Antioxidant enzymes Matés JM, Pérez-Gómez C, Núnez and human diseases. Clin Biochem 1999;32:595–603. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159–63. Michalsky MP, Kuhn A, Mehta V, Besner GE. Heparin-binding EGFlike growth factor decreases apoptosis in intestinal epithelial cells in vitro. J Pediatr Surg 2001;36:1130–5. Nilsson UA, Schoenberg MH, Aneman A, Poch B, Magadum S, Beger HG, et al. Free radicals and pathogenesis during ischemia and reperfusion of the cat small intestine. Gastroenterology 1994;106:629–36. Noda T, Iwakiri R, Fujimoto K, Matsuo S, Aw TY. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 1998;274:G270–6. Paglia DE, Valentine WN. Studies on the quantitative and qalitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69. Parks DA, Bulkley GB, Granger DN, Hamilton SR, McCord JM. Ischemic injury in the cat small intestine: role of superoxide radicals. Gastroenterology 1982;82:9–15. Parks DA, Granger DN. Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 1986;250:G749–53. Pillai SB, Hinman CE, Luquette MH, Nowicki PT, Besner GE. Heparin-binding epidermal growth factor-like growth factor protects rat intestine from ischemia/reperfusion injury. J Surg Res 1999;87:225–31. Potten CS, Booth C. The role of radiation-induced and spontaneous apoptosis in the homeostasis of the gastrointestinal epithelium: a brief review. Comp Biochem Physiol B Biochem Mol Biol 1997;118:73–8. Sasaki M, Joh T. Oxidative stress and ischemia-reperfusion injury in gastrointestinal tract and antioxidant, protective agents. J Clin Biochem Nutr 2007;40:1–12.
175
Sener G, Akgün U, Satiro˘glu H, Topalo˘glu U, Keyer-Uysal M. The effect of pentoxifylline on intestinal ischemia/reperfusion injury. Fundam Clin Pharmacol 2001;15:19–22. Shah K, Shuray S, Green C. Apoptosis after intestinal ischemiareperfusion injury: a morphologic study. Transplantation 1997;64:1393–7. Stern LE, Erwin CR, O’Brien DP, Huang F, Warner BW. Epidermal growth factor is critical for intestinal adaptation following small bowel resection. Microsc Res Tech 2000;51: 138–48. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500. Ulshen MH, Lyn-Cook LE, Raasch RH. Effects of intraluminal epidermal growth factor on mucosal proliferation in the small intestine of adult rats. Gastroenterology 1986;91:1134–40. Wendel A. Glutathione peroxidase. Methods Enzymol 1981;77:325–33. Wu GH, Wang H, Zhang YW, Wu ZH, Wu ZG. Glutamine supplemented parenteral nutrition prevents intestinal ischemia/reperfusion injury in rats. World J Gastroenterol 2004;10:2592–4. Xia G, Martin AE, Michalsky MP, Besner GE. Heparin-binding EGF-like growth factor preserves crypt cell proliferation and decreases bacterial translocation after intestinal ischemia/reperfusion injury. J Pediatr Surg 2002;37: 1081–7. Xu LF, Li J, Sun M, Sun HW. Expression of intestinal trefoil factor, proliferating cell nuclear antigen and histological changes in intestine of rats after intrauterine asphyxia. World J Gastroenterol 2005;11:2291–5.
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Acta Histochemica journal homepage: www.elsevier.de/acthis
The role of epidermal growth factor in prevention of oxidative injury and apoptosis induced by intestinal ischemia/reperfusion in rats Pelin Arda-Pirincci ∗ , Sehnaz Bolkent Department of Biology, Faculty of Science, Istanbul University, 34134 Vezneciler, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 19 February 2013 Received in revised form 29 June 2013 Accepted 1 July 2013 Keywords: Epidermal growth factor Ischemia/reperfusion Intestinal injury Apoptosis Oxidative stress Antioxidant enzymes Rat
a b s t r a c t Intestinal ischemia/reperfusion is a major problem which may lead to multiorgan failure and death. The aim of the study was to evaluate the effects of epidermal growth factor (EGF) on apoptosis, cell proliferation, oxidative stress and the antioxidant system in intestinal injury induced by ischemia/reperfusion in rats and to determine if EGF can ameliorate these toxic effects. Intestinal ischemia/reperfusion injury was produced by causing complete occlusion of the superior mesenteric artery for 60 min followed by a 60-min reperfusion period. Animals received intraperitoneal injections of 150 g/kg human recombinant EGF 30 min prior to the mesenteric ischemia/reperfusion. Mesenteric ischemia/reperfusion caused degeneration of the intestinal mucosa, inhibition of cell proliferation, stimulation of apoptosis and oxidative stress in the small intestine of rats. In the ischemia/reperfusion group, lipid peroxidation was stimulated accompanied by increased intestinal catalase and glutathione peroxidase activities, however, glutathione levels and superoxide dismutase activities were markedly decreased. EGF treatment to rats with ischemia/reperfusion prevented the ischemia/reperfusion-induced oxidative injury by reducing apoptosis and lipid peroxidation, and by increasing antioxidant enzyme activities. These results demonstrate that EGF has beneficial antiapoptotic and antioxidant effects on intestinal injury induced by ischemia/reperfusion in rats. © 2013 Elsevier GmbH. All rights reserved.
Introduction Ischemia/reperfusion of the gut in humans is a serious condition occurring after trauma, burns, septic shock, and liver or small intestine transplantation. Ischemia/reperfusion-induced intestinal injury in experimental animals is a practical model commonly used in studies on pathogenesis of systemic inflammation, respiratory failure and multiple organ failure. Experimental intestinal ischemia/reperfusion is one of the important models used to investigate oxidative stress induced by reactive oxygen species especially during reperfusion in small intestine (Horton and Walker, 1993; Nilsson et al., 1994; Kacmaz et al., 1999). Intestinal ischemia/reperfusion damages the mucosa by impairing its barrier function, and by causing bacterial translocation (Biffl and Moore, 1996; Wu et al., 2004). Various mediators play a role in the pathogenesis of ischemia/reperfusion-induced intestinal injury including reactive oxygen/nitrogen species, proinflammatory cytokines, leukocyte adhesion and infiltration (Nilsson et al., 1994; Xia et al., 2002; El Assal and Besner, 2004). Recent evidence has indicated that apoptosis is increased significantly during
∗ Corresponding author. E-mail address: [email protected] (P. Arda-Pirincci). 0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.07.005
ischemia/reperfusion of the gut and may play a key role in the pathogenesis of ischemia/reperfusion-induced intestinal injury. The intestinal ischemia/reperfusion model has often been used to study apoptosis (Shah et al., 1997; Ikeda et al., 1998; Noda et al., 1998; Fujise et al., 2006). Although intestinal ischemia/reperfusion does not have a specific treatment, several antioxidants and monoclonal antibodies against adhesion molecules have shown to be protective against ischemia/reperfusion injury (Kacmaz et al., 1999; Kubes, 1999; Kazez et al., 2000). Epidermal growth factor (EGF) is a mitogenic peptide that is secreted into the lumen of the duodenum by Brunner’s glands. EGF is implicated in the regulation of a wide variety of physiological processes, including growth, cell proliferation, regeneration, differentiation, and wound repair. It is suggested that EGF, not only increases mucosal repair, but also behaves as a cytoprotective and trophic agent for gastrointestinal epithelium (Buret et al., 1998; Berlanga et al., 2002). Many in vivo or in vitro studies have demonstrated that members of the EGF family are among the important factors for healing after damage in diverse experimental models (Pillai et al., 1999; Michalsky et al., 2001; Xia et al., 2002; Jahovic et al., 2004; Martin et al., 2005; Clark et al., 2005; El-Assal et al., 2007, 2008). However, there are only a limited number of studies regarding the effects of EGF on apoptosis and on the antioxidant system in ischemia/reperfusion-induced intestinal injury. In this
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study, we aimed to investigate the role of EGF on apoptosis, cell proliferation, oxidative stress and antioxidant system in intestinal injury induced by ischemia/reperfusion in rats.
The sections obtained from different groups were examined under an Olympus CX 41 light microscope, and photomicrographs were recorded using an Olympus DP71 digital camera.
Materials and methods
TUNEL assay
Animals
In order to detect apoptotic cells, in situ labeling of the fragmented DNA generated by apoptosis associated endonucleases was performed with terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay in the paraffinized crosssections of jejunum. Tissue parts taken from the jejunum were fixed in 10% phosphate-buffered formalin. Paraffin sections at 5 m were dewaxed, rehydrated, and then treated with proteinase K (20 g/mL), and blocked for endogenous peroxidase activity with 3% hydrogen peroxide (H2 O2 ). The labeling procedure was performed with the commercially available ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA, USA; S7101) according to the manufacturer’s instructions. The apoptotic cells were made visible with 3,3’-diaminobenzidine (DAB) and the nuclei were counterstained with methyl green. Rat breast tissue taken 4 days after breast-feeding cessation was used as a positive control. For negative control, the TdT enzyme was replaced by phosphate-buffered saline. A dark brown nuclear staining was regarded as positive. Besides, the apoptotic bodies were also positively stained. TUNEL positive epithelial cells in villi and crypts were evaluated for each sample using an Olympus CX41 light microscope using a 40× objective. A minimum of 20 full crypts or 10 full-length villi were randomly selected for apoptotic index analysis in the sections of jejunum. The apoptotic index values were expressed as a percentage of apoptotic cells in the total cells counted in villi or crypts.
Two- to 2.5-month-old Sprague–Dawley male rats weighing 220–240 g (n = 39) obtained from DETAE (Experimental Medical Research Institute of Istanbul University) were used in the study. The experiments were reviewed and approved by the Animal Care and Use Committee of Istanbul University. The animals were fed with Purina Laboratory Rodent Diet 5001 and tap water ad libitium, but fasted overnight prior to the experiments. Induction of ischemia/reperfusion injury The rats were anesthetized with ketamin (100 mg/kg) (Ketalar, Pfizer, Istanbul, Turkey) and chlorpromazine (0.75 mg/kg) (Largactil, Eczacıbas¸ı, Istanbul) intraperitoneally. After induction of anesthesia, the abdomen was opened through a midline abdominal incision. Intestinal ischemia/reperfusion (I/R) injury was produced by causing complete occlusion of the superior mesenteric artery followed by a period of reperfusion. The superior mesenteric artery was clamped for 60 min. After 60 min of ischemia, the vascular clamp at the superior mesenteric artery was removed and three drops of 2% lidocaine (Aritmal, Osel, Istanbul, Turkey) were applied directly on the superior mesenteric artery to facilitate reperfusion. Blood circulation was restarted for a 60-min reperfusion period (Noda et al., 1998). At the end of the reperfusion period, the animals were killed by an overdose of anesthesia.
Active caspase-3 staining Experimental protocol Animals were selected randomly and divided into five groups. Group I (Sham): Sham-operated animals. These animals were subjected to abdominal incision and their organs exposed for 120 min, but without clamping of the mesenteric artery so as to distinguish the differences between the effects of intestinal I/R and the changes because of non-specific surgical stress (n = 7); Group II (Control): Control rats received i.p. injection of 10 mM acetic acid with 0.1% BSA (vehicle) (n = 7); Group III (EGF): Animals administered i.p. injection of 150 g/kg human recombinant EGF (Sigma-Aldrich, St. Louis, MO, USA; E 9644) (n = 7); Group IV (I/R): Rats received an i.p. injection of 10 mM acetic acid with 0.1% BSA (vehicle) 30 min prior to the intestinal I/R (n = 9); Group V (I/R + EGF): Animals injected intraperitoneally (i.p.) with 150 g/kg human recombinant EGF dissolved in 10 mM acetic acid solution with 0.1% BSA 30 min prior to the intestinal I/R (n = 9). The animals belonging to groups II, III, IV and V, to which EGF or vehicle were applied, were killed 2.5 h after the injections. The control rats in group I were killed 2 h after the sham operation in order to supply the same conditions as with group IV. At the end of the experiments, samples from the jejunum were taken from the animals for all examinations. Histological assessment of intestinal injury Samples from the jejunum were taken from the animals for histological examination. The tissues were fixed in Bouin’s solution for 24 h at room temperature and embedded in paraffin wax. Serial sections 5 m thick were cut and stained with Masson’s trichrome for general morphological evaluation and the detection of connective tissue. Periodic acid-Schiff (PAS) staining was performed to observe the changes in the goblet cells and brush border of villi.
The major executioner caspase involved in apoptosis is caspase3, and activation of caspase-3 is one of the hallmarks of apoptosis. We examined apoptosis by using a polyclonal antibody to active caspase-3. Immunohistochemical active caspase-3 staining was performed using the streptavidin-biotin-peroxidase method. Jejunum tissues were fixed in 10% phosphate-buffered formalin. Following dewaxing and rehydration of paraffin-embedded sections, to facilitate antigen retrieval sections were permeabilized with 0.3% Triton X-100 for 10 min and then were heated in 10 mM citrate buffer (pH 6.0) for 15 min in a microwave oven at 700 W. Endogenous peroxidase activity was blocked by incubating with 3% H2 O2 for 10 min. The labeling procedure was performed with the Histostain Plus Broad Spectrum Kit (Invitrogen, Zymed Laboratories, San Francisco, CA, USA), followed by the application of polyclonal rabbit anti-active caspase-3 (1:50 in PBS, Millipore, Billerica, MA, USA; AB-3623) for 60 min at room temperature. Slides were then incubated with 3-amino-9-ethylcarbazole, and counterstained with Mayer’s hematoxylin. For negative control, phosphate-buffered saline was used instead of antibody. Cytoplasm with dark red staining was assessed as positive. Quantitative analysis of caspase-3 immunoreactive epithelial cells was made by using an Olympus CX41 light microscope with a 40× objective. For the labeling index, 20 full crypts or 10 full-length villi were randomly selected in the jejunum sections of each sample and caspase-3 positive cells were counted. The caspase-3 labeling index was calculated as a percentage of caspase-3 positive epithelial cell in the total cells counted in the crypts for each animal. Proliferating cell nuclear antigen assay The proliferating cells in intestine sections were detected by proliferating cell nuclear antigen (PCNA) immunohistochemistry.
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Fig. 1. Histology of jejunal mucosa in all groups. (A) Normal histological appearance of jejunal mucosa in a sham-operated control rat; (B) vehicle control rat; (C) rat given EGF; (D) rat subjected to ischemia/reperfusion injury indicates disruption in integrity of villi and villus loss ( ), infiltration of polymorphonuclear cells and lymphocytes ( ), disruption in integrity of crypts ( ), necrosis ( ) with severe mucosal damage. (E) EGF treated ischemia/reperfusion group demonstrate infiltration of polymorphonuclear cells and lymphocytes ( ) and hyperemia (h). Masson’s trichrome. Scale bar = 500 m.
Tissue samples from the jejunum were fixed in Bouin’s solution. After having completed the routine follow-up, the sections were boiled in 10 mM citrate buffer (pH 6.0) in microwave oven for 10 min at 700 W for antigen retrival. Then, the tissues were permeabilized with 0.3% Triton X-100 for 10 min. After blocking endogenous peroxidase activity with 3% H2 O2 , Ultra Vision Detection System (Lab Vision, Fremont, CA, USA) using a streptavidin-biotin-peroxidase technique for immunohistochemical analysis. In order to assess cell proliferation, the sections were incubated with primary antibody specific for PCNA (Clone PC10; NeoMarkers, Fremont, CA, USA; MS-106) at room temperature for 30 min. The antibody was used at a dilution of 1:50. Peroxidase activity of the tissue was demonstrated by 3-amino9-ethylcarbazole and then the sections were counterstained with Mayer’s hematoxylin. For negative control, the primary antibody was replaced by phosphate-buffered saline. Quantitative analysis of PCNA positive epithelial cells in crypts was made by using an Olympus CX41 light microscope using a 40× objective. In the jejunum sections of each sample, 20 full crypts were randomly selected and PCNA positive crypt cells were counted. The proliferation index was calculated as a percentage of PCNA positive epithelial cell in the total cells counted in the crypts for each section. Proliferation index =
PCNA-positive crypt cell count × 100 Total crypt cell count
Biochemical analysis Jejunum tissues were homogenized in cold 0.9% physiologic saline by means of a glass homogenizer (Yellow line OST basic
mixer, IKA, Staufen, Germany) to make up a 10% (w/v) homogenate. The homogenates were centrifuged at 20,000 × g for 15 min (+4 ◦ C) to obtain their supernatants. In the clear supernatants were determined activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced glutathione (GSH), malondialdehyde (MDA) and total protein by using a spectrophotometer (Shimadzu UV 1700, Kyoto, Japan). Catalase activity was estimated by the method described by Aebi (1974). Intestinal GPx activity was determined using the method of Paglia and Valentine (1967) modified by Wendel (1981). SOD activity in the intestinal homogenate was determined with the method of Sun et al. (1988). Reduced glutathione content was determined according to the method of Beutler et al. (1963). Intestinal oxidative stress was quantified in the homogenates by measuring malondialdehyde (MDA). MDA levels in the intestine were determined using a method described by Ledwozyw et al. (1986) with some modifications (Arda-Pirincci and Bolkent, 2011). Total protein content of the samples was established by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a standard. Statistical analyses Microscopic and biochemical data obtained were processed with SPSS 15 software (IBM, Chicago, IL, USA). Results of CAT, GPx and SOD activities were performed by using one-way ANOVA and unpaired Student’s t-test. Data of GSH and MDA levels, TUNEL assay, caspase-3 and PCNA immunohistochemistry were analyzed by non-parametric Kruskal–Wallis test and Mann–Whitney U-test. The results were given as mean ± standard error (SE). P < 0.05 was considered as statistically significant.
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Fig. 2. TUNEL-positive apoptotic cells in the jejunum of: (A) ( ) sham-operated control rat; (B) vehicle control group rat; (C) rat given EGF; (D) I/R group rat showing TUNEL-positive epithelial cells ( ); (E) rat of the I/R + EGF group showing apoptotic cell ( ) numbers significantly decreased when compared with the I/R group. Scale bar = 100 m.
Results Histology Light microscopic appearances of jejunum for all experimental groups are presented in Fig. 1. Histological examination of jejunal tissues from the sham-operated group, vehicle control group and EGF group revealed normal mucosal architecture. In the I/R group, disruption in brush border and integrity of villi, villi loss, widespread necrotic areas, infiltration of polymorphonuclear cells and lymphocytes, severe hemorrhage were commonly observed. In addition, we found increased number of the cells with pyknotic nuclei and densely eosinophilic cytoplasm in epithelium, mucosal edema, hemorrhage in the mucosa of in the same group rats. In the I/R group rats, PAS-positive reaction intensity in the goblet cells, in the brush border of villi was shown to decrease when compared with the control groups. On the other hand, treatment of EGF of the I/R group partially recovered intestinal mucosal architecture. However, in some jejunum sections of the I/R + EGF group
still showed disruption in the brush border and in integrity of villi, infiltration of lymphocytes and moderate hyperemia. PAS-positive reaction intensity was greater than in the I/R group. TUNEL immunostaining The photomicrographs and quantitative assessment of TUNELpositive epithelial cells in the jejunal mucosa are shown in Figs. 2 and 3. In this study, the apoptotic cells were observed in the crypts and villi of jejunum in all groups. In all control groups which Sham-operated, vehicle control and EGF-given was determined very few numbers of apoptotic cells in the jejunal mucosa by TUNEL assay. In the rats subjected to I/R injury, the number of TUNEL-positive apoptotic crypt and villus cells increased in a significant manner compared to sham and vehicle control groups (P < 0.05). The highest apoptotic index was detected in the villus compartment of the rats subjected to I/R. In this group, the apoptotic index was found as 1.65 ± 0.26% for the epithelium of villus. Apoptotic index decreased in a statistically meaningful way
Fig. 3. Apoptotic index (%) in epithelium of villus and crypt. The data are given as mean ± SE per group. The data for the epithelium of villus: Sham: 0.29 ± 0.06, Control: 0.19 ± 0.01, EGF: 0.14 ± 0.04, I/R: 1.65 ± 0.26, I/R + EGF: 0.65 ± 0.07. The data for the epithelium of crypt: Sham: 0.17 ± 0.01, Control: 0.14 ± 0.01, EGF: 0.13 ± 0.02, I/R: 0.62 ± 0.04, I/R + EGF: 0.26 ± 0.05. a P < 0.05 versus sham group, b P < 0.05 versus control group, c P < 0.05 versus EGF group, d P < 0.01 versus I/R group.
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Fig. 4. Active caspase-3 immunoreactive epithelial cells ( ) in the jejunum of: (A) sham-operated control rat; (B) vehicle control rat; (C) EGF-treated rat; (D) I/R group rat and (E) I/R + EGF group rat. Scale bar = 50 m.
in crypts and villi when EGF was applied to the I/R group (P < 0.01). However, in the EGF treated I/R group were observed a significantly increase in the number of TUNEL-positive apoptotic villus cells as compared to the sham group (P < 0.05) or control group (P < 0.05) or EGF group (P < 0.05).
EGF of the I/R group was significantly decreased the number of active caspase-3 positive crypt or villus cells according to the I/R group (P < 0.01).
PCNA immunohistochemistry Active caspase-3 immunohistochemistry The photomicrographs and quantitative assessment of immunolocalization of active caspase-3 in the jejunum are shown in Figs. 4 and 5. In the sham-operated group, vehicle control group and EGF-given group, caspase-3 was expressed only in a few crypt and villus epithelial cells. In this study, we showed an intense staining for caspase-3 in the cytoplasm of epithelial cells in the group subjected to I/R injury. Compatible with the TUNEL findings, in this group the numbers of active caspase-3 positive epithelial cells were markedly increased as compared to the sham group (P < 0.05) or control group (P < 0.05). However, treatment of
The photomicrographs and quantitative assessment of the proliferating epithelial cells in the jejunal crypts are shown in Figs. 6 and 7. The highest cell proliferation index in crypts of the jejunum was detected in the group administered EGF, whereas the lowest proliferation index was found in the I/R group. According to statistical results, the number of PCNA-positive crypt cells showed a significant decrease in the group subjected to I/R injury as compared with the sham-operated animals (P < 0.05). Preadministration of EGF to the I/R group increased the cell proliferation index, but this increase did not reach the ratio observed in the control groups.
Fig. 5. The caspase-3 labeling index (%) in epithelium of villus and crypt. The data were given as mean ± SE per group. The data for the epithelium of villus: Sham: 0.06 ± 0.03, Control: 0.05 ± 0.02, EGF: 0.05 ± 0.02, I/R: 0.77 ± 0.04, I/R + EGF: 0.12 ± 0.07. The data for the epithelium of crypt: Sham: 0.03 ± 0.02, Control: 0.03 ± 0.03, EGF: 0.03 ± 0.02, I/R: 0.33 ± 0.02, I/R + EGF: 0.05 ± 0.03. a P < 0.05 versus sham group, b P < 0.05 versus control group, c P < 0.01 versus I/R group.
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Fig. 6. PCNA immunoreactivity ( ) in crypts of the jejunum in: (A) sham-operated control rat; (B) vehicle control group rat; (C) EGF-treated rat; (D) I/R group rats; (E) I/R + EGF group rat. The intensity and number of PCNA-positive crypt cells ( ) decreased in I/R group rats. In I/R + EGF group, the intensity and number of PCNA-positive crypt cells ( ) increased when compared with the I/R group. Scale bar = 200 m.
Biochemical results The activities of antioxidant enzymes, and levels of GSH and MDA in intestinal tissues are presented in Table 1. Intestinal activity of CAT insignificantly increased in the I/R group as compared with the control groups. However, CAT activity belonging to the I/R + EGF group showed a significant increase compared to the I/R group or the sham group (P < 0.01), or the vehicle control group (P < 0.001) or the EGF-treated animals (P < 0.05). Tissue GPx activities observed in the I/R + EGF group increased compared to the I/R group, but this increase was not significant. GPx activity in the EGF-administered rats with I/R was significantly higher than the sham and vehicle control group (P < 0.05), or the EGF control group (P < 0.01). Like the other antioxidant enzymes, the I/R + EGF group showed markedly higher tissue SOD activities than the I/R group (P < 0.001), and also intestinal SOD activities in the I/R + EGF group significantly
Fig. 7. Proliferation index was determined by using PCNA immunohistochemistry in the jejunum. The data were given as mean ± SE per group. Sham: 61.02 ± 6.13, Control: 59.37 ± 8.72, EGF: 67.55 ± 2.59, I/R: 42.84 ± 4.40, I/R + EGF: 48.83 ± 1.01. a P < 0.05 versus sham group.
increased when compared to the sham, vehicle control and EGF groups (P < 0.001). A significant decrease was noted in the intestinal GSH levels of both the I/R group (P < 0.05) and I/R + EGF group (P < 0.001) compared to the sham group. Also, EGF pretreatment of the I/R group markedly reduced the GSH levels when compared with the vehicle control group (P < 0.05). The intestinal MDA levels were highest in the I/R group, compared with the control groups (P < 0.05). MDA content significantly decreased by the administration of EGF as compared to the I/R group. Discussion The intestine is a fairly sensitive organ with regard to ischemia/reperfusion damage because of its high oxygen requirements. Intestinal I/R injury can cause structural disorders in the mucosa of the small intestine and functional losses, as well as multiorgan failure and death (El Assal and Besner, 2004). The response of the intestinal mucosa toward ischemia can be divided into two phases. In the hypoxic phase, lack of oxygen causes intestinal mucosal cell death and severe damage in the mucosa. In the second phase, reoxygenation occurring during reperfusion causes further damage to the surviving mucosal cells after hypoxia (Parks and Granger, 1986; El Assal and Besner, 2004). The first indicators of ischemic injury in the small intestinal mucosa are degeneration of the villus epithelium and desquamation of the epithelial lining. During reperfusion, due to the production of reactive oxygen species (ROS), villi and mucosa suffer more severe damage (Martin et al., 2005). In this study, rats undergoing 60-min superior mesenteric artery occlusion followed by 60-min reperfusion had degenerative histopathological changes, especially mucosal and villus damage, in their jejunal tissues. In our applied experimental model, we showed that apoptotic cell death was also markedly stimulated in the jejunal mucosa. In a study reported by Noda et al.
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Table 1 Intestinal activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), and levels of intestinal glutathione (GSH) and lipid peroxidation (MDA) for all groups. Group
n
CAT* (U/mg protein)
Sham Control EGF I/R I/R + EGF
7 7 7 9 9
5.36 4.94 6.64 7.20 12.24
a b c d e f g h i k *
± ± ± ± ±
1.04 0.83 1.21 0.75 1.53a , d , g , h
GPx* (U/g protein) 9.56 9.26 8.73 11.05 14.90
± ± ± ± ±
1.30 1.59 0.63 1.49 1.34c , f , i
SOD* (U/mg protein) 2.83 2.62 2.52 2.54 3.36
± ± ± ± ±
0.17 0.10 0.10 0.12 0.08b , d , g , k
GSH* (nmol/mg protein) 10.79 11.61 6.56 6.20 4.39
± ± ± ± ±
1.31 2.64 1.16 0.97c 0.40e , f
MDA* (nmol/mg protein) 0.40 0.38 0.40 0.87 0.39
± ± ± ± ±
0.05 0.03 0.09 0.11c , f 0.04a
P < 0.01 versus I/R group. P < 0.001 versus I/R group. P < 0.05 versus sham group. P < 0.01 versus sham group. P < 0.001 versus sham group. P < 0.05 versus control group. P < 0.001 versus control group. P < 0.05 versus EGF group. P < 0.01 versus EGF group. P < 0.001 versus EGF group. Mean ± SE, n = number of animals.
(1998), small intestines of rats undergoing 60-min ischemia were reperfused at different times and, they demonstrated that after 60 min of ischemia, apoptotic cell death was maximal. Our TUNEL findings in the I/R group parallel the apoptotic findings reported by Noda et al. (1998). The current study evaluated the role of exogeneous EGF on intestinal architecture and apoptosis in small intestinal injury produced by ischemia/reperfusion and, it also determined whether EGF has protective and antiapoptotic effects against intestinal ischemic injury. In our study, we observed that EGF applied to the animals at 150 g/kg dose 30 min before ischemia/reperfusion could partially prevent the severe damage occurring in the jejunal mucosa. Berlanga et al. (2002) reported that EGF administered at 2 mg/kg dose to rats reduced by 80% the microscopic damage in the small intestine induced by mesenteric ischemia/reperfusion. This dose of EGF is fairly high compared to the dose we used in our study. In the present study, however, the apoptotic index and labeling index of caspase-3, which is a key caspase involved in the apoptotic pathway, in the intestinal mucosa were markedly reduced 1 h after ischemia with an EGF dose of 150 g/kg. Our literature survey did not discover studies about the effects of EGF on apoptosis in intestinal injury produced by ischemia/reperfusion, yet in some experimental models such as small intestinal resection (Stern et al., 2000), necrotizing enterocolitis (Clark et al., 2005; Feng et al., 2006) and sepsis (Clark et al., 2008), EGF or heparin-binding EGF-like growth factor (HB-EGF) reduces intestinal apoptosis. Homeostasis of epithelial architecture in the small intestine is regulated by both cell proliferation and cell death including apoptosis (Potten and Booth, 1997). Since intestinal epithelial cells have short proliferation cycle and powerful growth ability, regeneration in the intestinal tissue is quite fast. Another factor which will can damage the integrity of the intestinal mucosal barrier is the inhibition of epithelial cell proliferation (Xu et al., 2005). It has been reported that intraluminal EGF administration to experimental animals has proliferative and trophic effects (Ulshen et al., 1986). In our study, in order to determine whether EGF is effective against cell proliferation in the intestinal ischemia/reperfusion injury model, PCNA staining was investigated immunohistochemically. In the jejunum crypts of the rats belonging to the I/R group, cell proliferation was inhibited when compared to the control groups, and it was seen that EGF administration prevented this inhibition to some extent, but it could not reach the proliferative index of the control groups. In a study performed by Xia et al. (2002), rats undergoing 90-min intestinal ischemia followed by 45-min reperfusion had a lower cell proliferation index in the intestinal mucosa, but the application of HB-EGF, a member of the EGF family, increased the cell proliferation. However, our literature survey did not discover
any studies on the effects of EGF on cell proliferation in intestinal injury induced by ischemia/reperfusion. It is known that the production of ROS and the other damaging free radical metabolites is increased during ischemia/reperfusion (Horton and Walker, 1993; Nilsson et al., 1994; Carden and Granger, 2000; Kazez et al., 2000). Uncontrolled production of these free radicals could lead to apoptosis and necrosis as well as lipid peroxidation, resulting in cellular damage. We measured the MDA levels, which are indicators of free radical-induced oxidative damage and lipid peroxidation. According to our MDA results, an increase was found in the tissue lipid peroxidation level in the I/R group, and it was concluded that oxidative damage was present in ischemic intestine. In a study performed by Berlanga et al.(2002) the MDA increase observed in ischemic intestinal tissue was reversed following administration of 2 mg/kg EGF to rats. In the present study, we observed a reduction in the intestinal MDA to the levels of control group members in the I/R + EGF group, with an intraperitoneal EGF dose of 150 g/kg. GSH is an important endogeneous antioxidant, participating in the detoxification of ROS and elimination of lipid peroxides (Aw, 2005). GSH has important functions in the protection of tissues against ROS-mediated injury. Previous studies have reported that intestinal ischemia/reperfusion injury causes changes in the GSH redox cycle and a decrease in GSH synthesis in the tissue (Harward et al., 1994; Sener et al., 2001; Sasaki and Joh, 2007; Kimura et al., 2009). In our study, in accordance with the other researchers’ findings, intestinal GSH levels in rats undergoing ischemia/reperfusion were observed to be markedly lower than those of the sham control group. Up to now, it was not known how EGF affects GSH levels after intestinal ischemia/reperfusion. In this study, 1 h following superior mesenteric artery occlusion, the decrease occurring in the GSH levels of the intestinal tissue could not be reversed by EGF application. While EGF application before ischemia to the rats prevents LPO production in the intestine, the fact that GSH levels are lower than those in the control group members caused us to consider that oxidative stress could be removed by the other factors of antioxidative defense mechanism. For this purpose, the role of EGF on antioxidant enzymes in ischemia/reperfusion-induced intestinal injury was also investigated in this study. It is known that antioxidant enzymes such as CAT, GPx and SOD play important roles in the prevention or reduction of free radical damage. Oxidative stress is suggested to possibly increase or suppress, in a timely manner, the gene expression of antioxidant enzymes (Kacmaz et al., 1999). In our study, CAT and GPx activities after 1-h-ischemia/1-h-reperfusion in the small intestine responded to oxidative stress by showing a slight increase, as compared to the sham group. In intestinal ischemia/reperfusion
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injury, the effects of EGF on antioxidant enzyme system are not known fully. In our study, EGF injection to rats 30 min prior to ischemia/reperfusion application caused increases in CAT and GPx activities, both in control animals and I/R group members. In some studies, there is strong evidence concerning xanthine oxidase, which is considered to be the real source of oxidants which occur during ischemia/reperfusion in the gastrointestinal tract. It is reported that, during reperfusion of ischemic intestine, xanthine oxidase, which reaches high concentrations in the tissue, causes oxidative damage in the tissue by increasing especially superoxide radical production (Parks et al., 1982; McCord, 1985; Granger et al., 1986; Sasaki and Joh, 2007). Being a highly specific scavenger of superoxide, the potential of SOD to protect against gastrointestinal injury induced by ischemia/reperfusion is an important indicator that this antioxidant enzyme plays a major role in the first defence against oxidative stress which occurs during ischemia/reperfusion (Parks et al., 1982; Parks and Granger, 1986; Matés et al., 1999). In this study, the decrease in the intestinal SOD activity of rats belonging to the I/R group also verifies the literature data. We believe that the increase of intestinal SOD activity (even more than those of the control group) after administration of EGF to rats undergoing ischemia/reperfusion, is indicative of EGF trying to neutralize the superoxide radicals which emerge during ischemia/reperfusion injury, and also that EGF demonstrates an antioxidant effect in this injury model. As a result of this study, it appears that the EGF can play important roles in protection against oxidative damage induced by ischemia/reperfusion in the small intestine by decreasing apoptotic cell death, by preventing lipid peroxidation, by increasing antioxidant enzyme activities. Acknowledgments This study was funded by the Scientific Research Project Coordination Unit of Istanbul University (Grant numbers: 152/20082003 and UDP-1159). References Aebi H. Bergmeyer HU, editor. Catalase: Methods of enzymatic analysis. NewYork: Academic Press; 1974. p. 673–83. Arda-Pirincci P, Bolkent S. The role of glucagon-like peptide-2 on apoptosis, cell proliferation, and oxidant-antioxidant system at a mouse model of intestinal injury induced by tumor necrosis factor-alpha/actinomycin D. Mol Cell Biochem 2011;350:13–27. Aw TY. Intestinal glutathione: determinant of mucosal peroxide transport, metabolism, and oxidative susceptibility. Toxicol Appl Pharmacol 2005;204:320–8. Berlanga J, Prats P, Remirez P, Gonzalez R, Lopez-Saura P, Aguiar J, et al. Prophylactic use of epidermal growth factor reduces ischemia/reperfusion intestinal damage. Am J Pathol 2002;161:373–9. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882–8. Biffl WL, Moore EE. Splanchnic ischemia/reperfusion and multiple organ failure. Br J Anaesth 1996;77:59–70. Buret A, Olson ME, Gall DG, Hardin JA. Effects of orally administered epidermal growth factor on enteropathogenic Escherichia coli infection in rabbits. Infect Immun 1998;66:4917–23. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 2000;190:255–66. Clark JA, Clark AT, Hotchkiss RS, Buchman TG, Coopersmith CM. Epidermal growth factor treatment decreases mortality and is associated with improved gut integrity in sepsis. Shock 2008;30:36–42.
Clark JA, Lane RH, Maclennan NK, Holubec H, Dvorakova K, Halpern MD, et al. Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2005;288:G755–62. El Assal ON, Besner GE. Heparin-binding epidermal growth factorlike growth factor and intestinal ischemia-reperfusion injury. Semin Pediatr Surg 2004;13:2–10. El-Assal ON, Paddock H, Marquez A, Besner GE. Heparin-binding epidermal growth factor-like growth factor gene disruption is associated with delayed intestinal restitution, impaired angiogenesis, and poor survival after intestinal ischemia in mice. J Pediatr Surg 2008;43:1182–90. El-Assal ON, Radulescu A, Besner GE. Heparin-binding EGF-like growth factor preserves mesenteric microcirculatory blood flow and protects against intestinal injury in rats subjected to hemorrhagic shock and resuscitation. Surgery 2007;142: 234–42. Feng J, El-Assal ON, Besner GE. Heparin-binding epidermal growth factor-like growth factor reduces intestinal apoptosis in neonatal rats with necrotizing enterocolitis. J Pediatr Surg 2006;41:742–7. Fujise T, Iwakiri R, Wu B, Amemori S, Kakimoto T, Yokoyama F, et al. Apoptotic pathway in the rat small intestinal mucosa is different between fasting and ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol 2006;291:G110–6. Granger DN, McCord JM, Parks DA, Hollwarth ME. Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine. Gastroenterology 1986;90:80–4. Harward TR, Coe D, Souba WW, Klingman N, Seeger JM. Glutamine preserves gut glutathione levels during intestinal ischemia/reperfusion. J Surg Res 1994;56:351–5. Horton JW, Walker PB. Oxygen radicals, lipid peroxidation, and permeability changes after intestinal ischemia and reperfusion. J Appl Physiol 1993;4:1515–20. Ikeda H, Suzuki Y, Suzuki M, Koike M, Tamura J, Tong J, et al. Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium. Gut 1998;42:530–7. Jahovic N, Güzel E, Arbak S, Ye˘gen BC. The healing-promoting effect of saliva on skin burn is mediated by epidermal growth factor (EGF): role of the neutrophils. Burns 2004;30:531–8. Kacmaz M, Ozturk HS, Karaayvaz M, Guven C, Durak I. Enzymatic antioxidant defence mechanism in rat intestinal tissue is changed after ischemia-reperfusion. Effects of an allopurinol plus antioxidant combination. Can J Surg 1999;42:427–31. Kazez A, Demirba˘g M, Ustünda˘g B, Ozercan IH, Sa˘glam M. The role of melatonin in prevention of intestinal ischemia-reperfusion injury in rats. J Pediatr Surg 2000;35:1444–8. Kimura Y, Pierro A, Eaton S. Glutathione synthesis in intestinal ischaemia-reperfusion injury: effects of moderate hypothermia. J Pediatr Surg 2009;44:353–7. Kubes P. The role of adhesion molecules and nitric oxide in intestinal and hepatic ischaemia/reperfusion. Hepatogastroenterology 1999;46:1458–63. Ledwozyw A, Michalak J, Stepien A, Kadziolka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986;155:275–83. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. Martin AE, Luquette MH, Besner GE. Timing, route, and dose of administration of heparin-binding epidermal growth factor-like growth factor in protection against intestinal ischemiareperfusion injury. J Pediatr Surg 2005;40:1741–7.
P. Arda-Pirincci, S. Bolkent / Acta Histochemica 116 (2014) 167–175
˜ de Castro I. Antioxidant enzymes Matés JM, Pérez-Gómez C, Núnez and human diseases. Clin Biochem 1999;32:595–603. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159–63. Michalsky MP, Kuhn A, Mehta V, Besner GE. Heparin-binding EGFlike growth factor decreases apoptosis in intestinal epithelial cells in vitro. J Pediatr Surg 2001;36:1130–5. Nilsson UA, Schoenberg MH, Aneman A, Poch B, Magadum S, Beger HG, et al. Free radicals and pathogenesis during ischemia and reperfusion of the cat small intestine. Gastroenterology 1994;106:629–36. Noda T, Iwakiri R, Fujimoto K, Matsuo S, Aw TY. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 1998;274:G270–6. Paglia DE, Valentine WN. Studies on the quantitative and qalitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69. Parks DA, Bulkley GB, Granger DN, Hamilton SR, McCord JM. Ischemic injury in the cat small intestine: role of superoxide radicals. Gastroenterology 1982;82:9–15. Parks DA, Granger DN. Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 1986;250:G749–53. Pillai SB, Hinman CE, Luquette MH, Nowicki PT, Besner GE. Heparin-binding epidermal growth factor-like growth factor protects rat intestine from ischemia/reperfusion injury. J Surg Res 1999;87:225–31. Potten CS, Booth C. The role of radiation-induced and spontaneous apoptosis in the homeostasis of the gastrointestinal epithelium: a brief review. Comp Biochem Physiol B Biochem Mol Biol 1997;118:73–8. Sasaki M, Joh T. Oxidative stress and ischemia-reperfusion injury in gastrointestinal tract and antioxidant, protective agents. J Clin Biochem Nutr 2007;40:1–12.
175
Sener G, Akgün U, Satiro˘glu H, Topalo˘glu U, Keyer-Uysal M. The effect of pentoxifylline on intestinal ischemia/reperfusion injury. Fundam Clin Pharmacol 2001;15:19–22. Shah K, Shuray S, Green C. Apoptosis after intestinal ischemiareperfusion injury: a morphologic study. Transplantation 1997;64:1393–7. Stern LE, Erwin CR, O’Brien DP, Huang F, Warner BW. Epidermal growth factor is critical for intestinal adaptation following small bowel resection. Microsc Res Tech 2000;51: 138–48. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500. Ulshen MH, Lyn-Cook LE, Raasch RH. Effects of intraluminal epidermal growth factor on mucosal proliferation in the small intestine of adult rats. Gastroenterology 1986;91:1134–40. Wendel A. Glutathione peroxidase. Methods Enzymol 1981;77:325–33. Wu GH, Wang H, Zhang YW, Wu ZH, Wu ZG. Glutamine supplemented parenteral nutrition prevents intestinal ischemia/reperfusion injury in rats. World J Gastroenterol 2004;10:2592–4. Xia G, Martin AE, Michalsky MP, Besner GE. Heparin-binding EGF-like growth factor preserves crypt cell proliferation and decreases bacterial translocation after intestinal ischemia/reperfusion injury. J Pediatr Surg 2002;37: 1081–7. Xu LF, Li J, Sun M, Sun HW. Expression of intestinal trefoil factor, proliferating cell nuclear antigen and histological changes in intestine of rats after intrauterine asphyxia. World J Gastroenterol 2005;11:2291–5.