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reperfusion in rats
Life Sciences 86 (2010) 30–38
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Cellular and molecular mechanisms of 17β-estradiol postconditioning protection against gastric mucosal injury induced by ischemia/reperfusion in rats Dongshu Du a,b, Xiaobo Ma a,b, Jianfu Zhang a,b,⁎, Yongmei Zhang a,b, Xiaoyan Zhou a,b, Yu Li a,b a b
Department of Physiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China Department of Neurobiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China
a r t i c l e
i n f o
Article history: Received 17 June 2009 Accepted 30 October 2009 Keywords: 17β-estradiol Gastric ischemia/reperfusion Apoptosis Proliferation Bcl-2 Bax SOD activity MDA content Rat
a b s t r a c t Aims: To investigate the protective effects of 17β-estradiol postconditioning against ischemia/reperfusion (I–R)induced gastric mucosal injury in rats. Main methods: The animal model of gastric ischemia/reperfusion was established by clamping of the celiac artery for 30 min and reperfusion for 30 min, 1 h, 3 h, 6 h, 12 h or 24 h. 17β-estradiol at doses of 5, 50 or 100 μg/kg (rat) was administered via peripheral veins 2 min before reperfusion. In a subgroup of rats, the estrogen receptor antagonist fulvestrant (Ful, 2 mg/kg) was intravenously injected prior to 17β-estradiol administration. Histological and immunohistochemical methods were employed to assess the gastric mucosal injury index and gastric mucosal cell apoptosis and proliferation. The malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, xanthine oxidase (XOD) activity and hydroxyl free radical (–OH) inhibitory ability were determined by colorimetric assays. Subsequently, the expression of Bcl-2 and Bax in rat gastric mucosa was examined by western blotting. Key findings: 17β-estradiol dose-dependently inhibited gastric I–R (GI–R) injury, and 17β-estradiol (50 μg/kg) markedly attenuated GI–R injury 1 h after reperfusion. 17β-estradiol inhibited gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation in addition to increasing SOD activity and –OH inhibitory ability and decreasing the MDA content and XOD activity. The Bax protein level increased 1 h after GI–R and was markedly reduced by intravenous administration of 17β-estradiol. In contrast, the level of Bcl-2 protein decreased 1 h after GI–R and was restored to normal levels by intravenous administration of 17β-estradiol. These effects of 17β-estradiol were inhibited by pretreatment with fulvestrant. Significance: 17β-estradiol postconditioning should be investigated further as a possible strategy against gastric mucosal injury. © 2009 Elsevier Inc. All rights reserved.
Introduction Gastric ulceration is very prevalent in humans and is usually preceded by burns, sepsis, major surgery, ischemia, trauma and other heterogeneous forms of stress. In recent years, studies on gastric ischemia/reperfusion injury (GI–RI) have revealed that reactive oxygen species (ROS), endothelin, microvascular dysfunction, polymorphonuclear leukocyte infiltration, nitric oxide release, gastric acid secretion and decreased prostaglandin concentrations during reperfusion may play a role in the pathogenesis of gastric mucosal injury induced by I–R (Blebea et al. 1996; Kishimoto et al. 1997; Kitano et al. 1997; Brzozowski et al. 2000; Cabeza et al. 2001; Kim and Hwan 2001;
⁎ Corresponding author. Department of Physiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China. Tel.: +86 516 83262105; fax: +86 516 83262004. E-mail addresses: [email protected], [email protected] (J. Zhang). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.11.001
Tanaka et al. 2001). Mucosal integrity is maintained by the equilibrium between proliferation and apoptosis of the gastric mucosal cells. To better understand the pathogenesis of gastric lesions, it is of great importance to study the imbalance between proliferation and apoptosis. Many researchers have proposed approaches to the protection of the gastric mucosa against GI–RI, such as antacid drugs (Kitano et al. 1997; Cabeza et al. 2001), the endothelin-converting enzyme inhibitor phosphoramidon (Hassan et al. 1997), tetrahydrobiopterin (an anti-ROS drug) (Ishii et al. 2000), (+)-catechin, ghrelin (antioxidant drug) (Rao and Vijayakumar 2007; El Eter et al. 2007), prostaglandin (Brzozowski et al. 2005) and electrical stimulation of the paraventricular nucleus (Zhang et al. 2007). Estrogen is a steroid hormone whose actions involve genomic and non-genomic mechanisms (Acconcia et al. 2005). Previous research has shown that estrogen can induce endothelial progenitor cell proliferation (Zhao et al. 2008) and abrogate murine skeletal muscle cell apoptosis (Vasconsuelo et al. 2008). However, little is known about the systemic
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cellular and molecular mechanisms of 17β-estradiol postconditioning against ischemia/reperfusion (I/R)-induced gastric mucosal injury in rats. In this study, to investigate the cellular and molecular mechanisms of 17β-estradiol in protecting against GI–R injury, the characteristics of gastric mucosal cellular apoptosis and proliferation induced by GI–R were observed. To further examine whether the mitochondrial anti-apoptotic pathway is involved in the effects of 17β-estradiol postconditioning against GI–R injury, variations of Bcl-2 and Bax protein levels were detected by western blotting. Progress in this field may shed new light on the pathology of GI–R injury at both the cellular and molecular levels. Materials and methods Reagents The reagents and detection kits are described as follows: 17βestradiol (ABCR Co., Germany); fulvestrant (Sigma Co., USA); ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., Temecula, CA, USA); mouse anti-proliferative nucleolus monoclonal antibody (proliferative nuclear antigen, PCNA); PowerVisionTM two-step immunohistochemistry detection kit and 3,3′-diaminobenzidine (DAB) (Maixin-Bio Co., Fuzhou, China); substrate solution (Zhongshan Golden Bridge Biotech Co., Beijing, China); mouse anti-Bcl-2, mouse anti-Bax and mouse anti-β-actin antibodies (Zhongshan Golden Bridge Biotech Co., Beijing, China); malondialdehyde (MDA), superoxide dismutase (SOD), xanthine oxidase (XOD) and Hydroxyl Free Radical Detection Kit (Nanjing Jiancheng Bioengineering Institute, China). Animals Adult male Sprague–Dawley (SD) rats (Xuzhou Medical College Experimental Animal Center, Xuzhou, China) weighing 200 ± 15 g were used in all studies (usage certificate NO.: SYXK (su) 20020038).
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Experimental protocol The animals were randomized into five groups (n = 6): sham (with the same surgical procedures except clamping the celiac artery); GI–R (reperfusion for 1 h after 30 min of ischemia); 17β-E2 (17β-estradiol + GI–R); vehicle (DMSO + GI–R); and 17β-E2 + Ful (17β-estradiol + GI–R + fulvestrant). Each group was housed in wire mesh cages at room temperature and in a 12/12 hours day/ night cycle. The project protocol was approved by Xuzhou Medical College Animal Care and Use Committee. Prior to the experiment, all rats were fasted for 24 h and allowed access to tap water ad libitum. The animals were anesthetized under sodium pentobarbital (40 mg/kg, i.p.). Their abdomens were incised along the midline, and the celiac artery and its adjacent tissues were carefully isolated. The animals were included into different protocols as illustrated in Fig. 1. The celiac artery was clamped with a small non-traumatic vascular clamp for 30 min to induce gastric ischemia and then released for 30 min, 1 h, 3 h, 6 h, 12 h, or 24 h to allow reperfusion. Following reperfusion, the rats were sacrificed and the stomachs were immediately removed. 17β-estradiol (dissolved in DMSO) was intravenously injected in doses of 5 μg/kg, 50 μg/kg and 100 μg/kg 2 min before reperfusion in the postconditioning group. Fulvestrant (2 mg/kg, estrogen receptor antagonist) (Tissier et al. 2007) was intravenously injected 5 min before injection of 17β-estradiol. In the 17β-estradiol preconditioning group, 17β-estradiol was intravenously injected at a dose of 50 μg/kg before ischemia. Measurement of gastric mucosal injury index According to Zhang et al. (2007), the murine stomach was incised along the greater curvature and flushed with ice-cold PBS (0.1 mol/l). The index is based on a cumulative-length scale on which an individual lesion limited to the mucosal epithelium (including pinpoint erosions, ulcers, and hemorrhagic spots) is scored according to its length as follows: 1, ≤1 mm; 2, N1 mm and ≤2 mm; 3, N2 mm and ≤3 mm. For lesions N1 mm in width, the lesion score was
Fig. 1. Experimental protocol. CAO, celiac artery occlusion; CAR, celiac artery reperfusion; vehicle and genistein were administrated 2 min prior to reperfusion; fulvestrant was administered before injection of 17β-estradiol (17β-E2).
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doubled. The sum total of the scores of all lesions represents the gastric mucosal injury index. To avoid bias, the index was determined by a researcher who was blind to the treatments. Observation of gastric mucosal pathological changes by light microscopy Following gastric injury index measurements, two pieces of tissue from the gastric mucosa were immersed in 4% formaldehyde solution at 4 °C for paraffin tissue slicing. The resulting slices (5 μm) were inconsecutively pasted to polylysine-treated slides, which were then stained with hematoxylin–eosin. Gastric mucosal injury and repair in different groups were observed and photographed under a light microscope. Immunohistochemical assay of gastric mucosal apoptosis and proliferation following GI–R For immunohistochemistry, gastric mucosal cell apoptosis was visualized with ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., Temecula, CA, USA). PCNA was used as a marker for gastric mucosal cell proliferation, and the immunohistochemical staining was performed with PowerVisionTM two-step immunohistochemistry detection kit (Zhongshan Golden Bridge Biotech Co., Beijing, China). The sections were stained with 3,3′-diaminobenzidine (DAB), counterstained using hematoxylin and then analyzed under an inverted research microscope (Olympus, Model IX71, Japan). Cells with brown granules visible in the cytoplasm or nucleus were considered positive. The number of positive cells per section was counted in ten random high-power (× 40) or lower-power (× 10) fields, and the percentage of positive cells (positive cells/total cells×100%) was calculated. Three inconsecutive sections were selected from each specimen and those indexes were averaged. Measurement of malondialdehyde content and antioxidant enzyme activity As an index of lipid peroxidation, MDA was determined by the thiobarbituric acid reaction method. Briefly, the gastric mucosa was excised and subsequently homogenized in saline at 4 °C. The homogenate was centrifuged at 3000 g for 10 min, and the supernatant was retained. The protein concentrations were determined by Coomassie brilliant blue protein assay. The level of lipid peroxidation was indicated by thiobarbituric acid-reactive substances and was determined spectrophotometrically at 532 nm.
Fig. 2. Dose-dependent effects of 17β-E2 injection (i.v.) on gastric ischemia/reperfusion (GI–R) injury in rats. Before GI–R, vehicle or 17β-E2 (5 μg/kg, 50 μg/kg, 100 μg/kg) was intravenously injected. GI/R injury was induced by 30 min ischemia and 1 h reperfusion. Mean ± SD; n = 6; ⁎P b 0.05 compared with the GI/R group.
Fig. 3. Effects of 17β-E2 on GI/R injury at different time points of reperfusion in rats. Vehicle or 17β-E2 (50 µg/kg rat) was intravenously injected 2 min before reperfusion. GI/R injury was induced by 30 min ischemia and reperfusion for 30 min, 1 h, 3 h, 6 h, 12 h, 24 h. Mean ± SD; n = 6; ⁎ P b 0.05 compared with the Vehicle group at corresponding time points.
SOD activity and –OH inhibitory ability were determined spectrophotometrically at 550 nm by the xanthine/xanthine oxidase and the fenton reaction method, respectively. SOD activity and –OH inhibitory ability are expressed in Units/mg of protein. XOD was determined spectrophotometrically at 530 nm using a commercial XOD kit, and XOD activity is expressed in Units/g of protein. Western blotting Tissue samples were homogenized in ice-cold buffer A (mmol/l) [3(N-morpholino) propanesulfonic acid (MOPS) 50; MgCl2 0.5; KCl 10; sucrose 320; dithiothreitol (DTT) 1; Na3VO4 1; EDTA 1; EGTA 1; benzamidine 1; phenylmethylsulfonyl fluoride (PMSF) 1; aprotinin 10; leupeptin 10; and pepstain A 10 mg/l at pH 7.4]. The homogenate was centrifuged at 8000 g for 15 min at 4 °C, and the supernatant containing the cytoplasmic components was retained. Samples that had been stored at −80 °C were thawed. Protein concentrations were determined by Coomassie brilliant blue protein assay. Samples were mixed with loading buffer and boiled for 5 min. The proteins were isolated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The blots were incubated with 4% bovine serum albumin in TBST (10 mmol/l Tris, pH 7.5; 150 mmol/l NaCl, 0.05% Tween-20) at room temperature for 3 h and probed with primary antibodies at 4 °C overnight. Membranes were rinsed and incubated with secondary antibody for 2 h and were detected with an NBT/BCIP assay kit. At the end of immunoblotting, bands were scanned and analyzed by Image J software.
Fig. 4. Reversal of the protective effect of 17β-E2 by the estrogen receptor antagonist fulvestrant. Before to intravenous injection of vehicle or 17β-E2 (50 µg/kg rat), fulvestrant (2 mg/kg rat) was first injected. Summarized data are reported as mean ± SD; n = 6; # P b 0.05 compared with the Vehicle group; ⁎P b 0.05 compared with the 17β-E2 group.
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gastric mucosa, whereas in the sham group no lesions were observed. Following I–R, the gastric mucosal injury index was 126.4 ± 18.15. When 17β-estradiol at three doses (5, 50, or 100 μg/kg) was administered subsequent to ischemia, the gastric mucosal injury index decreased to 112.4 ± 25.95, 82.0 ± 27.97 (P b 0.05 versus I–R group) and 53.0 ± 26.83 (P b 0.01 versus I–R group). The results indicate that 50 μg/kg of 17β-estradiol was the optimal protective dose, which was applied to the following experiments. 17β-estradiol significantly improved gastric mucosal integrity against GI–R. Effects of 17β-estradiol on GI–R injury in rats at different time points The effects of 17β-estradiol on GI–R injury were determined at various time points after reperfusion. As Fig. 3 shows, gastric mucosal lesions were evident at 30 min, peaked at 1 h, and thereafter lessened at 3 h, 6 h and 12 h and were restored to normal at 24 h. 17β-estradiol significantly attenuated the injury at 1 h and 3 h (*P b 0.05) with a greater attenuation at 1 h. Thus, GI–R injury 1 h after reperfusion was observed. Fig. 5. Protective effects of 17β-E2 postconditioning on GI/R injury compared with 17β-E2 preconditioning. 17β-E2 (50 µg/kg rat) was intravenously injected 2 min before reperfusion in the postconditioning group while 17β-E2 (50 µg/kg rat) was intravenously injected before ischemia. ⁎P b 0.05 compared with the Vehicle group. #P N 0.05 compared with the 17β-E2 preconditioning group.
Statistical analysis Data are presented as mean ± SD (n = 6). Statistical analyses were performed with SPSS13.0 software and multiple-group analyses were conducted by the one-way analysis of variance (ANOVA). In all cases, P b 0.05 was considered statistically significant. Results
Effect of the estrogen receptor antagonist fulvestrant on the protective effect of 17β-estradiol To ascertain whether the estrogen receptor antagonist fulvestrant could reverse the protective effect of 17β-estradiol against GI–R injury, fulvestrant was injected via the femoral vein prior to 17β-estradiol administration (i.v.). As shown in Fig. 4, fulvestrant completely blocked the protective effect of 17β-estradiol against GI–R injury (*P b 0.05). These results indicate that the protective effect of 17βestradiol might be mediated by the estrogen receptor. Effects of 17β-estradiol postconditioning compared with 17β-estradiol preconditioning
Effects of different doses of 17β-estradiol on GI–R injury in rats Fig. 2 shows the total area of the erosions expressed as a morphological index of gastric injury. GI–R caused erosions in the
In order to further ascertain the protective effects of 17β-estradiol postconditioning against GI–R injury, a 17β-estradiol preconditioning group was added as a comparison. As Fig. 5 shows, 17β-estradiol
Fig. 6. Photomicrographs showing the gastric mucosa after GI/R and 17β-E2 treatment. Sham: normal gastric mucosa; GI/R: GI/R alone; Vehicle: vehicle injection + GI–R; 17β-E2: 17β-estradiol (50 μg/kg rat) injection + GI–R; 17β-E2 + Ful: fulvestrant (2 mg/kg) injection prior to 17β-E2 + GI/R. Scale bar, 100 μm.
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postconditioning prevented GI–R injury, and this effect of 17β-estradiol postconditioning was similar to 17β-estradiol preconditioning (*P N 0.05).
against GI–R, but this effect was blocked by estrogen receptor antagonist fulvestrant (Fig. 6 17β-E2 + Ful).
Effects of 17β-estradiol postconditioning on pathological changes of the gastric mucosa
Immunohistochemical assay of quantitative variations of apoptotic cells in gastric mucosa
Fig. 6 shows light microscopic photographs of the gastric mucosa. The control sample (sham-operated) obtained prior to the ischemic period showed a normal appearance (Fig. 6, Sham). Lesions, widespread hemorrhage with a loss of surface epithelium and infiltration of leukocytes in the deeper mucosal strata of the stomach were observed microscopically in the GI–R group. As shown in Fig. 6, 17β-estradiol lessened these lesions and protected gastric mucosa
As shown in Fig. 7A, apoptotic cells (with brown staining in nuclei) were predominantly distributed in the strata of the gastric glands and the epithelium. In the Sham group, apoptotic cells were scarce. As shown in Fig. 7B, in the GI–R group the percentage of apoptotic cells significantly increased (P b 0.01). There was no significant difference between the gastric I/R group and the vehicle group (P N 0.05). Intravenous administration of estradiol markedly
Fig. 7. Effect of 17β-E2 on GI/R-induced apoptosis of gastric mucosal cells in rats. A: The apoptotic-like cells were probed with anti-M30 Cyto DEATH antibody and counterstained with hematoxylin in the rat gastric mucosa. Sham: normal gastric mucosa; GI/R: GI/R alone; Vehicle: vehicle injection + GI/R; 17β-E2: 17β-E2 (50 μg/kg rat) injection + GI–R; 17β-E2 + Ful: fulvestrant (2 mg/kg rat) injection prior to 17β-E2 + GI–R. Scale bar, 25 μm. B: The percentage of apoptotic cells was calculated by counting the cells in 10 microscopic fields (×400). Sham: normal gastric mucosa; GI/R: reperfusion for 60 min following 30 min ischemia; Vehicle: vehicle injection + GI/R; 17β-E2: 17β-E2 injection + GI/R; 17β-E2 + Ful: fulvestrant injection before 17β-E2 + GI/R. ⁎P b 0.01 compared with the Vehicle group; #P b 0.01 compared with the 17β-E2 group.
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decreased the percentage of apoptotic gastric mucosal cells (Fig. 7B, 17β-E2) while intravenous administration of the estradiol receptor antagonist fulvestrant increased the apoptotic percentage as compared to the 17β-E2 group (P b 0.01) (Fig. 7B). Immunohistochemical assay of quantitative variations of the proliferative cells in the gastric mucosa As shown in Fig. 8A, the PCNA-positive cells (i.e., proliferative cells) were predominantly distributed in the neck region of the gastric glands. In the Sham group, the PCNA-positive cells were abundant. As shown in Fig. 8B, in the GI–R groups where animals were subjected to 30 min ischemia prior to the 1 h reperfusion, the percentage of PCNApositive cells decreased rapidly. The percentage of PCNA-positive cells was significantly higher in the 17β-E2 group than in the I–R group
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(P b 0.01), but the proliferation percentage was markedly decreased in the 17β-E2 + Ful group compared with the 17β-E2 group (P b 0.01). Effects of 17β-estradiol on SOD, XOD activity, –OH inhibitory ability and MDA content in gastric mucosa with I/R-induced injury MDA and XOD are regarded as indexes to mucosal injuries from ROS. Scarcity of MDA and low activity of XOD were detected in normal mucosa. One hour after GI–R, the MDA content and the XOD activity significantly increased (P b 0.05) but were attenuated by 17β-estradiol administration via the peripheral vein (P b 0.05). Conversely, fulvestrant (2 mg/kg) pretreatment increased the MDA content and XOD ability in the gastric mucosa of the 17β-E2 group. The enzymatic activity of SOD is an index to the antioxidative properties of cells. As Table 1 shows, the SOD activity of gastric
Fig. 8. Effect of 17β-E2 on I/R-induced proliferation of gastric mucosal cells in rats. A: Proliferative-positive cells (brown) were detected with mouse anti-PCNA antibody followed by hematoxylin staining in the gastric mucosa (×400). Sham: normal gastric mucosa; GI/R: GI/R alone; Vehicle: vehicle injection + GI/R; 17β-E2: 17β-E2 (50 μg/kg rat) injection + GI–R; 17β-E2 + Ful: fulvestrant (2 mg/kg rat) injection prior to 17β-E2 + GI/R. Scale bar, 25 μm. B: The percentage of proliferative cells was calculated by the cell counts in 10 microscopic fields (× 400). ⁎P b 0.01 compared with the Vehicle group; #P b 0.01 compared with the 17β-E2 group.
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Table 1 Effects of 17β-E2 on SOD, XOD activity, –OH inhibitory ability and MDA content in gastric mucosa with GI/R-induced injuries.
Sham GI–R 17β-E2 17β-E2 + Ful Vehicle
SOD (U/mg prot)
–OH (U/mg prot)
XOD (U/g prot)
MDA (nmol/mg prot)
176.35 ± 6.71 120.03 ± 4.12 161.87 ± 5.89⁎⁎ 131.01 ± 4.92# 122.83 ± 7.98
267.06 ± 20.32 93.89 ± 12.01 189.34 ± 10.78⁎⁎ 123.81 ± 13.36## 104.98 ± 9.43
22.39 ± 3.05 31.67 ± 2.12 25.31 ± 1.27⁎ 29.31 ± 2.32# 30.82 ± 4.36
3.54 ± 0.83 6.09 ± 1.08 4.54 ± 0.53⁎ 5.79 ± 1.11# 5.98 ± 0.92
⁎P b 0.05, ⁎⁎P b 0.01, the 17β-E2 group compared with the GI/R group; #P b 0.05, ##P b 0.01, the 17β-E2 group compared with the 17β-E2 + Ful group.
mucosa in the Sham group remained at a high level while it was markedly decreased in the GI–R group. In contrast to the GI–R group, 17β-estradiol administration via the peripheral vein significantly enhanced SOD activity (P b 0.05). Fulvestrant pretreatment attenuated SOD activity as compared to the 17β-E2 group. The pattern of the variations of –OH inhibitory ability in gastric mucosa was similar to that of the SOD activity in each group. Effects of 17β-estradiol on the expression of Bcl-2 and Bax in gastric mucosa with I/R-induced injuries As shown in Fig. 9, Bcl-2 and Bax were expressed in normal gastric mucosa in rats. Compared with the Vehicle group, the expression of Bcl-2 increased in the 17β-estradiol group (P b 0.01) whereas the expression of Bax decreased (P b 0.05). In addition, in the 17β-E2 + Ful group the expression of Bcl-2 decreased (P b 0.01), while the expression of Bax increased (P b 0.05) in comparison to the 17β-E2 group. Discussion The present study demonstrates that administration of pharmacological doses of estradiol via the peripheral vein attenuates GI–R injury in a dose and time-dependent manner. This effect was supported by the evidence that the estrogen receptor antagonist fulvestrant reversed the effect of estradiol. Fulvestrant, a new type of estrogen receptor antagonist with no agonist effects, blocks and causes degradation of the estrogen receptor, culminating in complete abrogation of estrogen-sensitive gene transcription (Dowsett et al. 2005). In addition, it can competitively bind to the estrogen receptor with a much greater affinity than tamoxifen (McKeage et al. 2004). It is well known that gastric mucosal cells have a strong capacity to self-restore. Our previous study (Qiao et al. 2006) demonstrated that gastric mucosal injury occurs primarily in the early stages of reperfusion, and repair begins quickly during reperfusion. Ischemia for 30 min alone caused only slight gastric injury; reperfusion led to increased injury that peaked after 1 h and then gradually declined to the basal level after 72 h of reperfusion. In addition, the effect of 17β-estradiol was gradually attenuated during reperfusion. Therefore, 17β-estradiol only plays a role in protecting gastric injury at the 1 h and 3 h time points. All of these results support the notion that 17β-estradiol can protect gastric mucosal integrity from GI–R, which has been implicated in regulating the genesis of gastric mucosal injury in gastric response to noxious visceral stimuli (Filaretova et al. 1998) and in stressinduced gastric ulceration (Zhang and Zheng 1997). Our present study demonstrates that the protective effect of estradiol on GI–R injury might be mediated by the estrogen receptor. Estrogen receptors, which are expressed in the gastric epithelium (Campbell-Thompson et al. 2001) in normal rats and can be regulated by estradiol (Murphy et al. 2009), mediate estrogen protection against gastric acid-induced duodenal injury (Smith et al. 2008). Therefore,
Fig. 9. Effects of 17β-E2 on the expression of Bcl-2 and Bax in rat gastric mucosa. The western immunoblot from cellular protein extracts was probed with antibodies to Bcl-2 and Bax. Quantitative assessment, values are means ± SD of optical density of Bcl-2 and Bax bands from western immunoblots (n = 6). A: Bcl-2 expression; B: Bax expression. ⁎P b 0.05 compared with the Vehicle group; #P b 0.05 compared with the 17β-E2 group.
estradiol might regulate GI–R injury mediated by the estrogen receptor. However, estrogen inhibition of ischemia-induced apoptosis may be mediated by the Fas pathway (Jia et al. 2009). In addition, estrogen also limits ischemic cell death by modulating caspase-12mediated apoptotic pathways following middle cerebral artery occlusion (Crosby et al. 2007). Another finding of the present study is that estradiol significantly inhibited I/R-induced apoptosis of gastric mucosal cells and promoted gastric mucosal cell proliferation, which is consistent with previous reports (Drago et al. 1999; Gunal et al. 2003) that estrogen can ameliorate acetic acid-induced colon injuries. However, some reports (Luo et al. 2005) have indicated that 17β-estradiol exacerbates stomach injuries induced by cysteamine administration. Previous relevant studies (Kwiecien et al. 2002; Brzozowski et al. 2003; Tarnawski 2005; Li et al. 2007; Bao et al. 2008) have demonstrated
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that SOD, MDA, ROS and XOD are crucial in the protection against organ injuries, which was in accord with one of our above findings that estradiol had an antioxidative effect against GI–R injury. In order to ascertain the molecular mechanism of estradiol attenuating GI–R injury via the inhibition of apoptosis, the protein expression of Bcl-2 and Bax was determined. Our study indicates that the expression of Bcl-2 protein decreased 1 h after GI–R and was restored to normal levels by 17β-estradiol administration; in contrast, the level of Bax protein increased 1 h after GI–R and was markedly reduced by 17β-estradiol administration. Therefore, the protective effect of estradiol might be mediated by the upregulation of Bcl-2 and the downregulation of Bax. Another important innovation of the present study is the novel research method. Pharmacological postconditioning was employed in gastric ischemic–reperfusion injury, which avoided not only ischemic preconditioning, but also ischemic postconditioning, that might cause mechanical injury to the organism. The research methods of previous relevant studies (Dembiński et al. 2003; Narin et al. 2006; Bulbul et al. 2008; Liu et al. 2009) have focused on ischemic preconditioning, ischemic postconditioning and pharmacological preconditioning. A recent study (Wang et al. 2008) demonstrated that stem cells can inhibit cellular necrosis and apoptosis induced by reperfusion of ischemic kidneys in rats. However, in this report the stem cells were difficult to obtain. In summary, the present project demonstrates that administration of 17β-estradiol, which helped to restore and maintain the structural integrity of I–R gastric mucosa, attenuated GI–R injury. This protective effect might be mediated by the inhibition of gastric mucosal cell apoptosis and the promotion of gastric mucosal cell proliferation. Not only does our study provide insights into the mechanism of gastric mucosal tissue repair; it also provides information that could potentially guide development of a new therapeutic strategy. Conclusion 17β-estradiol exhibited a gastroprotective effect against ischemia/ reperfusion-induced injury, and these protective effects of 17β-estradiol are mediated by the estrogen receptor. Acknowledgements This project was supported by grants from the National Natural Science Foundation of China (No. 30570671) and the Foundation of Education Ministry of Jiangsu Province (No. 02KJD310008). The authors are grateful for this financial support. References Acconcia F, Totta P, Ogawa S, Cardillo I, Inoue S, Leone S, Trentalance A, Muramatsu M, Marino M. Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling. Journal of Cellular Physiology 203 (1), 193–201, 2005. Bao L, Yao XS, Tsi D, Yau CC, Chia CS, Nagai H, Kurihara H. Protective effects of bilberry (Vaccinium myrtillus L.) extract on KBrO3-induced kidney damage in mice. Journal of Agricultural and Food Chemistry 56 (2), 420–425, 2008. Blebea J, Bacik B, Strothman G, Myatt L. Decreased nitric oxide production following extremity ischemia and reperfusion. American Journal of Surgery 172 (2), 158–161, 1996. Brzozowski T, Konturek PC, Konturek SJ, Drozdowicz D, Kwiecien S, Pajdo R, Bielanski W, Hahn EG. Role of gastric acid secretion in progression of acute gastric erosions induced by ischemia/reperfusion into gastric ulcers. European Journal of Pharmacology 398 (1), 147–158, 2000. Brzozowski T, Konturek PC, Konturek SJ, Kwiecien S, Sliwowski Z, Pajdo R, Duda A, Ptak A, Hahn EG. Implications of reactive oxygen species and cytokines in gastroprotection against stress-induced gastric damage by nitric oxide releasing aspirin. International Journal of Colorectal Disease 18 (4), 320–329, 2003. Brzozowski T, Konturek PC, Konturek SJ, Brzozowska I, Pawlik T. Role of prostaglandins in gastroprotection and gastric adaptation. Journal of Physiology and Pharmacology 56 (Suppl 5), 33–55, 2005.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Cellular and molecular mechanisms of 17β-estradiol postconditioning protection against gastric mucosal injury induced by ischemia/reperfusion in rats Dongshu Du a,b, Xiaobo Ma a,b, Jianfu Zhang a,b,⁎, Yongmei Zhang a,b, Xiaoyan Zhou a,b, Yu Li a,b a b
Department of Physiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China Department of Neurobiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China
a r t i c l e
i n f o
Article history: Received 17 June 2009 Accepted 30 October 2009 Keywords: 17β-estradiol Gastric ischemia/reperfusion Apoptosis Proliferation Bcl-2 Bax SOD activity MDA content Rat
a b s t r a c t Aims: To investigate the protective effects of 17β-estradiol postconditioning against ischemia/reperfusion (I–R)induced gastric mucosal injury in rats. Main methods: The animal model of gastric ischemia/reperfusion was established by clamping of the celiac artery for 30 min and reperfusion for 30 min, 1 h, 3 h, 6 h, 12 h or 24 h. 17β-estradiol at doses of 5, 50 or 100 μg/kg (rat) was administered via peripheral veins 2 min before reperfusion. In a subgroup of rats, the estrogen receptor antagonist fulvestrant (Ful, 2 mg/kg) was intravenously injected prior to 17β-estradiol administration. Histological and immunohistochemical methods were employed to assess the gastric mucosal injury index and gastric mucosal cell apoptosis and proliferation. The malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, xanthine oxidase (XOD) activity and hydroxyl free radical (–OH) inhibitory ability were determined by colorimetric assays. Subsequently, the expression of Bcl-2 and Bax in rat gastric mucosa was examined by western blotting. Key findings: 17β-estradiol dose-dependently inhibited gastric I–R (GI–R) injury, and 17β-estradiol (50 μg/kg) markedly attenuated GI–R injury 1 h after reperfusion. 17β-estradiol inhibited gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation in addition to increasing SOD activity and –OH inhibitory ability and decreasing the MDA content and XOD activity. The Bax protein level increased 1 h after GI–R and was markedly reduced by intravenous administration of 17β-estradiol. In contrast, the level of Bcl-2 protein decreased 1 h after GI–R and was restored to normal levels by intravenous administration of 17β-estradiol. These effects of 17β-estradiol were inhibited by pretreatment with fulvestrant. Significance: 17β-estradiol postconditioning should be investigated further as a possible strategy against gastric mucosal injury. © 2009 Elsevier Inc. All rights reserved.
Introduction Gastric ulceration is very prevalent in humans and is usually preceded by burns, sepsis, major surgery, ischemia, trauma and other heterogeneous forms of stress. In recent years, studies on gastric ischemia/reperfusion injury (GI–RI) have revealed that reactive oxygen species (ROS), endothelin, microvascular dysfunction, polymorphonuclear leukocyte infiltration, nitric oxide release, gastric acid secretion and decreased prostaglandin concentrations during reperfusion may play a role in the pathogenesis of gastric mucosal injury induced by I–R (Blebea et al. 1996; Kishimoto et al. 1997; Kitano et al. 1997; Brzozowski et al. 2000; Cabeza et al. 2001; Kim and Hwan 2001;
⁎ Corresponding author. Department of Physiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China. Tel.: +86 516 83262105; fax: +86 516 83262004. E-mail addresses: [email protected], [email protected] (J. Zhang). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.11.001
Tanaka et al. 2001). Mucosal integrity is maintained by the equilibrium between proliferation and apoptosis of the gastric mucosal cells. To better understand the pathogenesis of gastric lesions, it is of great importance to study the imbalance between proliferation and apoptosis. Many researchers have proposed approaches to the protection of the gastric mucosa against GI–RI, such as antacid drugs (Kitano et al. 1997; Cabeza et al. 2001), the endothelin-converting enzyme inhibitor phosphoramidon (Hassan et al. 1997), tetrahydrobiopterin (an anti-ROS drug) (Ishii et al. 2000), (+)-catechin, ghrelin (antioxidant drug) (Rao and Vijayakumar 2007; El Eter et al. 2007), prostaglandin (Brzozowski et al. 2005) and electrical stimulation of the paraventricular nucleus (Zhang et al. 2007). Estrogen is a steroid hormone whose actions involve genomic and non-genomic mechanisms (Acconcia et al. 2005). Previous research has shown that estrogen can induce endothelial progenitor cell proliferation (Zhao et al. 2008) and abrogate murine skeletal muscle cell apoptosis (Vasconsuelo et al. 2008). However, little is known about the systemic
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cellular and molecular mechanisms of 17β-estradiol postconditioning against ischemia/reperfusion (I/R)-induced gastric mucosal injury in rats. In this study, to investigate the cellular and molecular mechanisms of 17β-estradiol in protecting against GI–R injury, the characteristics of gastric mucosal cellular apoptosis and proliferation induced by GI–R were observed. To further examine whether the mitochondrial anti-apoptotic pathway is involved in the effects of 17β-estradiol postconditioning against GI–R injury, variations of Bcl-2 and Bax protein levels were detected by western blotting. Progress in this field may shed new light on the pathology of GI–R injury at both the cellular and molecular levels. Materials and methods Reagents The reagents and detection kits are described as follows: 17βestradiol (ABCR Co., Germany); fulvestrant (Sigma Co., USA); ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., Temecula, CA, USA); mouse anti-proliferative nucleolus monoclonal antibody (proliferative nuclear antigen, PCNA); PowerVisionTM two-step immunohistochemistry detection kit and 3,3′-diaminobenzidine (DAB) (Maixin-Bio Co., Fuzhou, China); substrate solution (Zhongshan Golden Bridge Biotech Co., Beijing, China); mouse anti-Bcl-2, mouse anti-Bax and mouse anti-β-actin antibodies (Zhongshan Golden Bridge Biotech Co., Beijing, China); malondialdehyde (MDA), superoxide dismutase (SOD), xanthine oxidase (XOD) and Hydroxyl Free Radical Detection Kit (Nanjing Jiancheng Bioengineering Institute, China). Animals Adult male Sprague–Dawley (SD) rats (Xuzhou Medical College Experimental Animal Center, Xuzhou, China) weighing 200 ± 15 g were used in all studies (usage certificate NO.: SYXK (su) 20020038).
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Experimental protocol The animals were randomized into five groups (n = 6): sham (with the same surgical procedures except clamping the celiac artery); GI–R (reperfusion for 1 h after 30 min of ischemia); 17β-E2 (17β-estradiol + GI–R); vehicle (DMSO + GI–R); and 17β-E2 + Ful (17β-estradiol + GI–R + fulvestrant). Each group was housed in wire mesh cages at room temperature and in a 12/12 hours day/ night cycle. The project protocol was approved by Xuzhou Medical College Animal Care and Use Committee. Prior to the experiment, all rats were fasted for 24 h and allowed access to tap water ad libitum. The animals were anesthetized under sodium pentobarbital (40 mg/kg, i.p.). Their abdomens were incised along the midline, and the celiac artery and its adjacent tissues were carefully isolated. The animals were included into different protocols as illustrated in Fig. 1. The celiac artery was clamped with a small non-traumatic vascular clamp for 30 min to induce gastric ischemia and then released for 30 min, 1 h, 3 h, 6 h, 12 h, or 24 h to allow reperfusion. Following reperfusion, the rats were sacrificed and the stomachs were immediately removed. 17β-estradiol (dissolved in DMSO) was intravenously injected in doses of 5 μg/kg, 50 μg/kg and 100 μg/kg 2 min before reperfusion in the postconditioning group. Fulvestrant (2 mg/kg, estrogen receptor antagonist) (Tissier et al. 2007) was intravenously injected 5 min before injection of 17β-estradiol. In the 17β-estradiol preconditioning group, 17β-estradiol was intravenously injected at a dose of 50 μg/kg before ischemia. Measurement of gastric mucosal injury index According to Zhang et al. (2007), the murine stomach was incised along the greater curvature and flushed with ice-cold PBS (0.1 mol/l). The index is based on a cumulative-length scale on which an individual lesion limited to the mucosal epithelium (including pinpoint erosions, ulcers, and hemorrhagic spots) is scored according to its length as follows: 1, ≤1 mm; 2, N1 mm and ≤2 mm; 3, N2 mm and ≤3 mm. For lesions N1 mm in width, the lesion score was
Fig. 1. Experimental protocol. CAO, celiac artery occlusion; CAR, celiac artery reperfusion; vehicle and genistein were administrated 2 min prior to reperfusion; fulvestrant was administered before injection of 17β-estradiol (17β-E2).
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doubled. The sum total of the scores of all lesions represents the gastric mucosal injury index. To avoid bias, the index was determined by a researcher who was blind to the treatments. Observation of gastric mucosal pathological changes by light microscopy Following gastric injury index measurements, two pieces of tissue from the gastric mucosa were immersed in 4% formaldehyde solution at 4 °C for paraffin tissue slicing. The resulting slices (5 μm) were inconsecutively pasted to polylysine-treated slides, which were then stained with hematoxylin–eosin. Gastric mucosal injury and repair in different groups were observed and photographed under a light microscope. Immunohistochemical assay of gastric mucosal apoptosis and proliferation following GI–R For immunohistochemistry, gastric mucosal cell apoptosis was visualized with ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., Temecula, CA, USA). PCNA was used as a marker for gastric mucosal cell proliferation, and the immunohistochemical staining was performed with PowerVisionTM two-step immunohistochemistry detection kit (Zhongshan Golden Bridge Biotech Co., Beijing, China). The sections were stained with 3,3′-diaminobenzidine (DAB), counterstained using hematoxylin and then analyzed under an inverted research microscope (Olympus, Model IX71, Japan). Cells with brown granules visible in the cytoplasm or nucleus were considered positive. The number of positive cells per section was counted in ten random high-power (× 40) or lower-power (× 10) fields, and the percentage of positive cells (positive cells/total cells×100%) was calculated. Three inconsecutive sections were selected from each specimen and those indexes were averaged. Measurement of malondialdehyde content and antioxidant enzyme activity As an index of lipid peroxidation, MDA was determined by the thiobarbituric acid reaction method. Briefly, the gastric mucosa was excised and subsequently homogenized in saline at 4 °C. The homogenate was centrifuged at 3000 g for 10 min, and the supernatant was retained. The protein concentrations were determined by Coomassie brilliant blue protein assay. The level of lipid peroxidation was indicated by thiobarbituric acid-reactive substances and was determined spectrophotometrically at 532 nm.
Fig. 2. Dose-dependent effects of 17β-E2 injection (i.v.) on gastric ischemia/reperfusion (GI–R) injury in rats. Before GI–R, vehicle or 17β-E2 (5 μg/kg, 50 μg/kg, 100 μg/kg) was intravenously injected. GI/R injury was induced by 30 min ischemia and 1 h reperfusion. Mean ± SD; n = 6; ⁎P b 0.05 compared with the GI/R group.
Fig. 3. Effects of 17β-E2 on GI/R injury at different time points of reperfusion in rats. Vehicle or 17β-E2 (50 µg/kg rat) was intravenously injected 2 min before reperfusion. GI/R injury was induced by 30 min ischemia and reperfusion for 30 min, 1 h, 3 h, 6 h, 12 h, 24 h. Mean ± SD; n = 6; ⁎ P b 0.05 compared with the Vehicle group at corresponding time points.
SOD activity and –OH inhibitory ability were determined spectrophotometrically at 550 nm by the xanthine/xanthine oxidase and the fenton reaction method, respectively. SOD activity and –OH inhibitory ability are expressed in Units/mg of protein. XOD was determined spectrophotometrically at 530 nm using a commercial XOD kit, and XOD activity is expressed in Units/g of protein. Western blotting Tissue samples were homogenized in ice-cold buffer A (mmol/l) [3(N-morpholino) propanesulfonic acid (MOPS) 50; MgCl2 0.5; KCl 10; sucrose 320; dithiothreitol (DTT) 1; Na3VO4 1; EDTA 1; EGTA 1; benzamidine 1; phenylmethylsulfonyl fluoride (PMSF) 1; aprotinin 10; leupeptin 10; and pepstain A 10 mg/l at pH 7.4]. The homogenate was centrifuged at 8000 g for 15 min at 4 °C, and the supernatant containing the cytoplasmic components was retained. Samples that had been stored at −80 °C were thawed. Protein concentrations were determined by Coomassie brilliant blue protein assay. Samples were mixed with loading buffer and boiled for 5 min. The proteins were isolated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The blots were incubated with 4% bovine serum albumin in TBST (10 mmol/l Tris, pH 7.5; 150 mmol/l NaCl, 0.05% Tween-20) at room temperature for 3 h and probed with primary antibodies at 4 °C overnight. Membranes were rinsed and incubated with secondary antibody for 2 h and were detected with an NBT/BCIP assay kit. At the end of immunoblotting, bands were scanned and analyzed by Image J software.
Fig. 4. Reversal of the protective effect of 17β-E2 by the estrogen receptor antagonist fulvestrant. Before to intravenous injection of vehicle or 17β-E2 (50 µg/kg rat), fulvestrant (2 mg/kg rat) was first injected. Summarized data are reported as mean ± SD; n = 6; # P b 0.05 compared with the Vehicle group; ⁎P b 0.05 compared with the 17β-E2 group.
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gastric mucosa, whereas in the sham group no lesions were observed. Following I–R, the gastric mucosal injury index was 126.4 ± 18.15. When 17β-estradiol at three doses (5, 50, or 100 μg/kg) was administered subsequent to ischemia, the gastric mucosal injury index decreased to 112.4 ± 25.95, 82.0 ± 27.97 (P b 0.05 versus I–R group) and 53.0 ± 26.83 (P b 0.01 versus I–R group). The results indicate that 50 μg/kg of 17β-estradiol was the optimal protective dose, which was applied to the following experiments. 17β-estradiol significantly improved gastric mucosal integrity against GI–R. Effects of 17β-estradiol on GI–R injury in rats at different time points The effects of 17β-estradiol on GI–R injury were determined at various time points after reperfusion. As Fig. 3 shows, gastric mucosal lesions were evident at 30 min, peaked at 1 h, and thereafter lessened at 3 h, 6 h and 12 h and were restored to normal at 24 h. 17β-estradiol significantly attenuated the injury at 1 h and 3 h (*P b 0.05) with a greater attenuation at 1 h. Thus, GI–R injury 1 h after reperfusion was observed. Fig. 5. Protective effects of 17β-E2 postconditioning on GI/R injury compared with 17β-E2 preconditioning. 17β-E2 (50 µg/kg rat) was intravenously injected 2 min before reperfusion in the postconditioning group while 17β-E2 (50 µg/kg rat) was intravenously injected before ischemia. ⁎P b 0.05 compared with the Vehicle group. #P N 0.05 compared with the 17β-E2 preconditioning group.
Statistical analysis Data are presented as mean ± SD (n = 6). Statistical analyses were performed with SPSS13.0 software and multiple-group analyses were conducted by the one-way analysis of variance (ANOVA). In all cases, P b 0.05 was considered statistically significant. Results
Effect of the estrogen receptor antagonist fulvestrant on the protective effect of 17β-estradiol To ascertain whether the estrogen receptor antagonist fulvestrant could reverse the protective effect of 17β-estradiol against GI–R injury, fulvestrant was injected via the femoral vein prior to 17β-estradiol administration (i.v.). As shown in Fig. 4, fulvestrant completely blocked the protective effect of 17β-estradiol against GI–R injury (*P b 0.05). These results indicate that the protective effect of 17βestradiol might be mediated by the estrogen receptor. Effects of 17β-estradiol postconditioning compared with 17β-estradiol preconditioning
Effects of different doses of 17β-estradiol on GI–R injury in rats Fig. 2 shows the total area of the erosions expressed as a morphological index of gastric injury. GI–R caused erosions in the
In order to further ascertain the protective effects of 17β-estradiol postconditioning against GI–R injury, a 17β-estradiol preconditioning group was added as a comparison. As Fig. 5 shows, 17β-estradiol
Fig. 6. Photomicrographs showing the gastric mucosa after GI/R and 17β-E2 treatment. Sham: normal gastric mucosa; GI/R: GI/R alone; Vehicle: vehicle injection + GI–R; 17β-E2: 17β-estradiol (50 μg/kg rat) injection + GI–R; 17β-E2 + Ful: fulvestrant (2 mg/kg) injection prior to 17β-E2 + GI/R. Scale bar, 100 μm.
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postconditioning prevented GI–R injury, and this effect of 17β-estradiol postconditioning was similar to 17β-estradiol preconditioning (*P N 0.05).
against GI–R, but this effect was blocked by estrogen receptor antagonist fulvestrant (Fig. 6 17β-E2 + Ful).
Effects of 17β-estradiol postconditioning on pathological changes of the gastric mucosa
Immunohistochemical assay of quantitative variations of apoptotic cells in gastric mucosa
Fig. 6 shows light microscopic photographs of the gastric mucosa. The control sample (sham-operated) obtained prior to the ischemic period showed a normal appearance (Fig. 6, Sham). Lesions, widespread hemorrhage with a loss of surface epithelium and infiltration of leukocytes in the deeper mucosal strata of the stomach were observed microscopically in the GI–R group. As shown in Fig. 6, 17β-estradiol lessened these lesions and protected gastric mucosa
As shown in Fig. 7A, apoptotic cells (with brown staining in nuclei) were predominantly distributed in the strata of the gastric glands and the epithelium. In the Sham group, apoptotic cells were scarce. As shown in Fig. 7B, in the GI–R group the percentage of apoptotic cells significantly increased (P b 0.01). There was no significant difference between the gastric I/R group and the vehicle group (P N 0.05). Intravenous administration of estradiol markedly
Fig. 7. Effect of 17β-E2 on GI/R-induced apoptosis of gastric mucosal cells in rats. A: The apoptotic-like cells were probed with anti-M30 Cyto DEATH antibody and counterstained with hematoxylin in the rat gastric mucosa. Sham: normal gastric mucosa; GI/R: GI/R alone; Vehicle: vehicle injection + GI/R; 17β-E2: 17β-E2 (50 μg/kg rat) injection + GI–R; 17β-E2 + Ful: fulvestrant (2 mg/kg rat) injection prior to 17β-E2 + GI–R. Scale bar, 25 μm. B: The percentage of apoptotic cells was calculated by counting the cells in 10 microscopic fields (×400). Sham: normal gastric mucosa; GI/R: reperfusion for 60 min following 30 min ischemia; Vehicle: vehicle injection + GI/R; 17β-E2: 17β-E2 injection + GI/R; 17β-E2 + Ful: fulvestrant injection before 17β-E2 + GI/R. ⁎P b 0.01 compared with the Vehicle group; #P b 0.01 compared with the 17β-E2 group.
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decreased the percentage of apoptotic gastric mucosal cells (Fig. 7B, 17β-E2) while intravenous administration of the estradiol receptor antagonist fulvestrant increased the apoptotic percentage as compared to the 17β-E2 group (P b 0.01) (Fig. 7B). Immunohistochemical assay of quantitative variations of the proliferative cells in the gastric mucosa As shown in Fig. 8A, the PCNA-positive cells (i.e., proliferative cells) were predominantly distributed in the neck region of the gastric glands. In the Sham group, the PCNA-positive cells were abundant. As shown in Fig. 8B, in the GI–R groups where animals were subjected to 30 min ischemia prior to the 1 h reperfusion, the percentage of PCNApositive cells decreased rapidly. The percentage of PCNA-positive cells was significantly higher in the 17β-E2 group than in the I–R group
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(P b 0.01), but the proliferation percentage was markedly decreased in the 17β-E2 + Ful group compared with the 17β-E2 group (P b 0.01). Effects of 17β-estradiol on SOD, XOD activity, –OH inhibitory ability and MDA content in gastric mucosa with I/R-induced injury MDA and XOD are regarded as indexes to mucosal injuries from ROS. Scarcity of MDA and low activity of XOD were detected in normal mucosa. One hour after GI–R, the MDA content and the XOD activity significantly increased (P b 0.05) but were attenuated by 17β-estradiol administration via the peripheral vein (P b 0.05). Conversely, fulvestrant (2 mg/kg) pretreatment increased the MDA content and XOD ability in the gastric mucosa of the 17β-E2 group. The enzymatic activity of SOD is an index to the antioxidative properties of cells. As Table 1 shows, the SOD activity of gastric
Fig. 8. Effect of 17β-E2 on I/R-induced proliferation of gastric mucosal cells in rats. A: Proliferative-positive cells (brown) were detected with mouse anti-PCNA antibody followed by hematoxylin staining in the gastric mucosa (×400). Sham: normal gastric mucosa; GI/R: GI/R alone; Vehicle: vehicle injection + GI/R; 17β-E2: 17β-E2 (50 μg/kg rat) injection + GI–R; 17β-E2 + Ful: fulvestrant (2 mg/kg rat) injection prior to 17β-E2 + GI/R. Scale bar, 25 μm. B: The percentage of proliferative cells was calculated by the cell counts in 10 microscopic fields (× 400). ⁎P b 0.01 compared with the Vehicle group; #P b 0.01 compared with the 17β-E2 group.
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Table 1 Effects of 17β-E2 on SOD, XOD activity, –OH inhibitory ability and MDA content in gastric mucosa with GI/R-induced injuries.
Sham GI–R 17β-E2 17β-E2 + Ful Vehicle
SOD (U/mg prot)
–OH (U/mg prot)
XOD (U/g prot)
MDA (nmol/mg prot)
176.35 ± 6.71 120.03 ± 4.12 161.87 ± 5.89⁎⁎ 131.01 ± 4.92# 122.83 ± 7.98
267.06 ± 20.32 93.89 ± 12.01 189.34 ± 10.78⁎⁎ 123.81 ± 13.36## 104.98 ± 9.43
22.39 ± 3.05 31.67 ± 2.12 25.31 ± 1.27⁎ 29.31 ± 2.32# 30.82 ± 4.36
3.54 ± 0.83 6.09 ± 1.08 4.54 ± 0.53⁎ 5.79 ± 1.11# 5.98 ± 0.92
⁎P b 0.05, ⁎⁎P b 0.01, the 17β-E2 group compared with the GI/R group; #P b 0.05, ##P b 0.01, the 17β-E2 group compared with the 17β-E2 + Ful group.
mucosa in the Sham group remained at a high level while it was markedly decreased in the GI–R group. In contrast to the GI–R group, 17β-estradiol administration via the peripheral vein significantly enhanced SOD activity (P b 0.05). Fulvestrant pretreatment attenuated SOD activity as compared to the 17β-E2 group. The pattern of the variations of –OH inhibitory ability in gastric mucosa was similar to that of the SOD activity in each group. Effects of 17β-estradiol on the expression of Bcl-2 and Bax in gastric mucosa with I/R-induced injuries As shown in Fig. 9, Bcl-2 and Bax were expressed in normal gastric mucosa in rats. Compared with the Vehicle group, the expression of Bcl-2 increased in the 17β-estradiol group (P b 0.01) whereas the expression of Bax decreased (P b 0.05). In addition, in the 17β-E2 + Ful group the expression of Bcl-2 decreased (P b 0.01), while the expression of Bax increased (P b 0.05) in comparison to the 17β-E2 group. Discussion The present study demonstrates that administration of pharmacological doses of estradiol via the peripheral vein attenuates GI–R injury in a dose and time-dependent manner. This effect was supported by the evidence that the estrogen receptor antagonist fulvestrant reversed the effect of estradiol. Fulvestrant, a new type of estrogen receptor antagonist with no agonist effects, blocks and causes degradation of the estrogen receptor, culminating in complete abrogation of estrogen-sensitive gene transcription (Dowsett et al. 2005). In addition, it can competitively bind to the estrogen receptor with a much greater affinity than tamoxifen (McKeage et al. 2004). It is well known that gastric mucosal cells have a strong capacity to self-restore. Our previous study (Qiao et al. 2006) demonstrated that gastric mucosal injury occurs primarily in the early stages of reperfusion, and repair begins quickly during reperfusion. Ischemia for 30 min alone caused only slight gastric injury; reperfusion led to increased injury that peaked after 1 h and then gradually declined to the basal level after 72 h of reperfusion. In addition, the effect of 17β-estradiol was gradually attenuated during reperfusion. Therefore, 17β-estradiol only plays a role in protecting gastric injury at the 1 h and 3 h time points. All of these results support the notion that 17β-estradiol can protect gastric mucosal integrity from GI–R, which has been implicated in regulating the genesis of gastric mucosal injury in gastric response to noxious visceral stimuli (Filaretova et al. 1998) and in stressinduced gastric ulceration (Zhang and Zheng 1997). Our present study demonstrates that the protective effect of estradiol on GI–R injury might be mediated by the estrogen receptor. Estrogen receptors, which are expressed in the gastric epithelium (Campbell-Thompson et al. 2001) in normal rats and can be regulated by estradiol (Murphy et al. 2009), mediate estrogen protection against gastric acid-induced duodenal injury (Smith et al. 2008). Therefore,
Fig. 9. Effects of 17β-E2 on the expression of Bcl-2 and Bax in rat gastric mucosa. The western immunoblot from cellular protein extracts was probed with antibodies to Bcl-2 and Bax. Quantitative assessment, values are means ± SD of optical density of Bcl-2 and Bax bands from western immunoblots (n = 6). A: Bcl-2 expression; B: Bax expression. ⁎P b 0.05 compared with the Vehicle group; #P b 0.05 compared with the 17β-E2 group.
estradiol might regulate GI–R injury mediated by the estrogen receptor. However, estrogen inhibition of ischemia-induced apoptosis may be mediated by the Fas pathway (Jia et al. 2009). In addition, estrogen also limits ischemic cell death by modulating caspase-12mediated apoptotic pathways following middle cerebral artery occlusion (Crosby et al. 2007). Another finding of the present study is that estradiol significantly inhibited I/R-induced apoptosis of gastric mucosal cells and promoted gastric mucosal cell proliferation, which is consistent with previous reports (Drago et al. 1999; Gunal et al. 2003) that estrogen can ameliorate acetic acid-induced colon injuries. However, some reports (Luo et al. 2005) have indicated that 17β-estradiol exacerbates stomach injuries induced by cysteamine administration. Previous relevant studies (Kwiecien et al. 2002; Brzozowski et al. 2003; Tarnawski 2005; Li et al. 2007; Bao et al. 2008) have demonstrated
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that SOD, MDA, ROS and XOD are crucial in the protection against organ injuries, which was in accord with one of our above findings that estradiol had an antioxidative effect against GI–R injury. In order to ascertain the molecular mechanism of estradiol attenuating GI–R injury via the inhibition of apoptosis, the protein expression of Bcl-2 and Bax was determined. Our study indicates that the expression of Bcl-2 protein decreased 1 h after GI–R and was restored to normal levels by 17β-estradiol administration; in contrast, the level of Bax protein increased 1 h after GI–R and was markedly reduced by 17β-estradiol administration. Therefore, the protective effect of estradiol might be mediated by the upregulation of Bcl-2 and the downregulation of Bax. Another important innovation of the present study is the novel research method. Pharmacological postconditioning was employed in gastric ischemic–reperfusion injury, which avoided not only ischemic preconditioning, but also ischemic postconditioning, that might cause mechanical injury to the organism. The research methods of previous relevant studies (Dembiński et al. 2003; Narin et al. 2006; Bulbul et al. 2008; Liu et al. 2009) have focused on ischemic preconditioning, ischemic postconditioning and pharmacological preconditioning. A recent study (Wang et al. 2008) demonstrated that stem cells can inhibit cellular necrosis and apoptosis induced by reperfusion of ischemic kidneys in rats. However, in this report the stem cells were difficult to obtain. In summary, the present project demonstrates that administration of 17β-estradiol, which helped to restore and maintain the structural integrity of I–R gastric mucosa, attenuated GI–R injury. This protective effect might be mediated by the inhibition of gastric mucosal cell apoptosis and the promotion of gastric mucosal cell proliferation. Not only does our study provide insights into the mechanism of gastric mucosal tissue repair; it also provides information that could potentially guide development of a new therapeutic strategy. Conclusion 17β-estradiol exhibited a gastroprotective effect against ischemia/ reperfusion-induced injury, and these protective effects of 17β-estradiol are mediated by the estrogen receptor. Acknowledgements This project was supported by grants from the National Natural Science Foundation of China (No. 30570671) and the Foundation of Education Ministry of Jiangsu Province (No. 02KJD310008). The authors are grateful for this financial support. References Acconcia F, Totta P, Ogawa S, Cardillo I, Inoue S, Leone S, Trentalance A, Muramatsu M, Marino M. Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling. Journal of Cellular Physiology 203 (1), 193–201, 2005. Bao L, Yao XS, Tsi D, Yau CC, Chia CS, Nagai H, Kurihara H. Protective effects of bilberry (Vaccinium myrtillus L.) extract on KBrO3-induced kidney damage in mice. Journal of Agricultural and Food Chemistry 56 (2), 420–425, 2008. Blebea J, Bacik B, Strothman G, Myatt L. Decreased nitric oxide production following extremity ischemia and reperfusion. American Journal of Surgery 172 (2), 158–161, 1996. Brzozowski T, Konturek PC, Konturek SJ, Drozdowicz D, Kwiecien S, Pajdo R, Bielanski W, Hahn EG. Role of gastric acid secretion in progression of acute gastric erosions induced by ischemia/reperfusion into gastric ulcers. European Journal of Pharmacology 398 (1), 147–158, 2000. Brzozowski T, Konturek PC, Konturek SJ, Kwiecien S, Sliwowski Z, Pajdo R, Duda A, Ptak A, Hahn EG. Implications of reactive oxygen species and cytokines in gastroprotection against stress-induced gastric damage by nitric oxide releasing aspirin. International Journal of Colorectal Disease 18 (4), 320–329, 2003. Brzozowski T, Konturek PC, Konturek SJ, Brzozowska I, Pawlik T. Role of prostaglandins in gastroprotection and gastric adaptation. Journal of Physiology and Pharmacology 56 (Suppl 5), 33–55, 2005.
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