- Email: [email protected]
reperfusion via aldose reductase
Ischemic postconditioning during reperfusion attenuates oxidative stress and intestinal mucosal apoptosis induced by intestinal ischemia/ reperfusion via aldose reductase Shi-Hong Wen, MD,a Yi-Hong Ling, MD,b,c Yi Li, MD,a Cai Li, MD,a Jia-Xin Liu, MD,a Yun-Sheng Li, MD,a Xi Yao, MD,a Zhi-Qiu Xia, MD,a and Ke-Xuan Liu, MD, PhD,a Guangzhou, China
Background. We demonstrated previously that ischemic postconditioning (IPo) attenuated intestinal injury induced by intestinal ischemia/reperfusion (I/R), and thereafter employed a proteomic method to identify aldose reductase (AR), a differentially expressed protein in intestinal mucosal tissue, which was downregulated by intestinal I/R and upregulated by IPo. This study aimed to further explore the possible role of AR in intestinal protection conferred by IPo. Methods. Intestinal ischemia was induced by clamping the superior mesenteric artery (SMA) for 60 minutes in male adult rats. Then rats were allocated into 7 groups based on the random number table. The control group involved only sham operation; the control + AR inhibitor epalrestat group underwent sham operation and epalrestat administration; the I/R with and/or without epalrestat groups had SMA clamped for 60 minutes followed by 120 minutes of reperfusion with and/or without epalrestat given before index ischemia; the IPo group underwent 3 cycles of 30 seconds of reperfusion and 30 seconds of re-occlusion imposed immediately on reperfusion; and the epalrestat or vehicle control dimethylsulfoxide + IPo groups had the drugs administrated 10 minutes before ischemia. Results. IPo resulted in significant intestinal protection evidenced as marked decreases in Chiu’s score, reflecting changes intestinal histology, serum diamine oxidase activity, and intestinal mucosal levels of lactic acid, malondialdehyde, and myeloperoxidase, the apoptosis index, and downregulated cleaved caspase-3 protein expression; these changes were accompanied by an increase in superoxide dismutase activity and upregulation of AR protein levels. Epalrestat failed to protect against intestinal I/R insult, but abolished the protective effects of IPo. Conclusion. These findings suggest that IPo attenuates intestinal I/R-induced intestinal injury via ARmediated oxidative defense and apoptosis suppression; AR inhibition reverses the protective effects of IPo. AR seems to be an innate protective factor in this model of intestinal I/R. (Surgery 2013;153:555-64.) From the Department of Anesthesiology,a the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; and the State Key Laboratory of Oncology in South China,b Department of Pathology,c Sun Yat-sen University Cancer Center, Guangzhou, China
INTESTINAL ISCHEMIA/REPERFUSION (I/R) INJURY occurs in a variety of pathophysiologic conditions, for example, shock, severe infection, and certain operative procedures, such as small bowel transplantation, abdominal aortic surgery, and Accepted for publication September 25, 2012. Reprint requests: Ke-Xuan Liu, MD, PhD, Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhongshan 2nd Road, Guangzhou 510080, China. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2012.09.017
cardiopulmonary bypass, and could trigger the development of multiple organ dysfunction.1 The mechanisms of intestinal I/R injury are not understood fully, and effective approaches for its clinical application are still lacking. Murry et al2 first introduced the definition of ischemic preconditioning (IPC), in which repetitive brief periods of ischemia protected the myocardium from a subsequent greater period of ischemic insult. We also demonstrated that IPC protected intestine from intestinal I/R injury in a rat model.3 Clinical events related to intestinal I/R, however, are usually unpredictable, which limits the clinical application of IPC. In contrast, SURGERY 555
556 Wen et al
a clinically more feasible strategy, ischemic postconditioning (IPo), which consists of $1 short cycles of reperfusion followed by $1 short cycles of ischemia, immediately after the conclusion of ischemia and before starting permanent reperfusion, can confer effective organ protection comparable to IPC.4-9 Our previous studies already showed that IPo can decrease the intestinal7 and lung8 injury induced by intestinal I/R, in part by inhibiting oxidative injury, neutrophil filtration, and the proinflammatory response. Further, we identified a differentially expressed protein in the intestinal mucosa via a proteomic method, aldose reductase (AR), which can be downregulated by intestinal I/R and upregulated by IPo.9 This finding indicates that AR might be involved in the intestinal protection conferred by IPo, which needs to be confirmed. AR, a member of the aldo–keto reductase superfamily, is proposed to function as an NADPHdependent oxidoreductase owing to its ability to catalyze the reduction of a broad spectrum of substrates that range from steroid carbonyls to aldo–keto sugars. Investigations also have shown that AR exhibits high affinity for hydrophobic aldehydes, such as those generated during lipid peroxidation.10 Lipid-derived aldehydes are cytotoxic, so the ability of AR to metabolize these aldehydes suggests that this enzyme may be involved in protection against oxidative injury. Given that oxidative stress is among the mechanisms causing intestinal I/R injury7-9,11 and that IPo can confer intestinal protection by suppressing oxidative stress as we documented previously,7 we hypothesized that AR, as an antioxidant enzyme, plays an important role in intestinal protection conferred by IPo. In addition, we7 and others12 demonstrated that apoptosis is the main mode of intestinal mucosal cell death induced by intestinal I/R. Whether IPo confers intestinal protection via inhibiting intestinal mucosal apoptosis or not remains obscure. Because AR can attenuate cell apoptosis by inhibiting oxidative stress in different tissues,13,14 we hypothesized that IPo attenuates oxidative stress and intestinal mucosal cell apoptosis via AR and thereby confers its intestinal protection. This hypothesis was tested in a rat model of intestinal I/R injury. MATERIALS AND METHODS Ethics statement and animal model of intestinal I/R. This study was approved by the Animal Care Committee of Sun Yat-sen University, China, and was performed in accordance with National Institutes of Health guidelines for the use of experimental animals. Fifty-six adult, pathogen-free,
Surgery April 2013
male, Sprague–Dawley rats weighing 220–250 g were housed in individual cages in a temperaturecontrolled room with alternating 12-hour light/ dark cycles and acclimated for 1 week before experiments. All animals were fasted overnight before the experiment but had free access to water ad libitum. Rats were anesthetized with 20% urethane (induction and maintenance, 6 mL/kg intraperitoneally). A 2- to 3-cm midline abdominal incision was made, and the small intestine was exteriorized. The intestinal I/R injury was established by occluding the superior mesenteric artery (SMA) with a microvessel clip for 60 minutes followed by 120 minutes reperfusion. SMA was isolated from the superior mesenteric vein before occlusion. Ischemia was recognized by the existence of a pulseless or pale color of the small intestine. The return of pulses and the reestablishment of the pink color indicated appropriate reperfusion of the intestine. Experimental groups. According to the random number table, the rats were allocated into 1 of 7 groups based on the interventions (n = 8 each): (1) Control group (control), sham surgical preparation including isolation of the SMA without occlusion; (2) I/R group (I/R), SMA occlusion for 60 minutes followed by 120 minutes reperfusion; (3) AR inhibitor (ARI) (epalrestat) + control group (ARI + control); (4) epalrestat + I/R group (ARI + I/R); (5) IPo group (I/R + IPo), immediately at the onset of reperfusion, reflow was initiated with 30 seconds of full SMA flow, followed by 30 seconds of reocclusion, repeated for a total of 3 cycles (3 minutes total intervention); (6) epalrestat group (ARI + I/R + IPo); and (7) dimethylsulfoxide (DMSO) group (DMSO + I/R + IPo), in which DMSO was the solvent of epalrestat and served as vehicle control. Epalrestat (dissolved in DMSO as 10 mg/mL; 10 mg/kg IV) or DMSO (1 mL/kg) was given 10 minutes before SMA occlusion. Our recent study demonstrated that ARI epalrestat can inhibit the activity of AR at a dose of 10 mg/kg.3 Intravenous administration of epalrestat. Before the microvascular clip was placed across the SMA, epalrestat (10 mg/kg body weight [BW]; Wako Co., Osaka, Japan), or vehicle (DMSO, 1 mL/kg BW, Sigma Chemical Co., St Louis, MO) was administered intravenously over a period of 10 minutes through a pump (Harvard Apparatus, Holliston, MA). Animals were then subjected to midline laparotomy and to index ischemia. Preparation of specimens. After the completion of the experiments, blood samples were collected and centrifuged at 3,500 rpm for 15 minutes at 48C, and the supernatant was stored at 808C for
Surgery Volume 153, Number 4
subsequent measurement of diamine oxidase (DAO) activity. A 5- to 10-cm segment of ileum was resected from the terminal ileum and divided into 3 segments. One of the segments was fixed in 10% neutral formaldehyde and paraffin embedded for morphologic analysis, anther was used to evaluate intestinal mucosal epithelial apoptosis, and the third was washed with cold saline and after being scraped off, the intestinal mucosa was dried with filter paper and preserved at 808C for protein extraction. Histologic measurement of intestinal mucosal injury. The segment of small intestine was stained with hematoxylin and eosin. Damage to the intestinal mucosa was evaluated independently by 2 pathologists blind to the study groups. The degree of injury was evaluated using the criteria of Chiu’s score method15 according to changes of the villus and glands of the intestinal mucosa. In brief, mucosal damage was graded from 0 (normal) to 5 (severely damaged) as follows: Grade 0, normal mucosal villi; grade 1, development of subepithelial Gruenhagen’s space at the apex of the villus, often with capillary congestion; grade 2, extension of the subepithelial space with moderate lifting of the epithelial layer from the lamina propria; grade 3, massive epithelial lifting down the sides of villi, possibly with a few denuded tips; grade 4, denuded villi with lamina propria and dilated capillaries exposed, possibly with increased cellularity of lamina propria; and grade 5, digestion and disintegration of the lamina propria, hemorrhage, and ulceration. A minimum of 5 randomly chosen fields from each rat were evaluated and averaged to determine mucosal damage. Detection of lactic acid level in intestinal mucosa. Intestinal mucosal tissues were weighed and made into 10% homogenates. The lactic acid (LD) content in tissues was determined using a chemical assay kit (Nanjing Jiancheng Biochemicals Ltd, Nanjing, China) as described.16 The results were expressed as nmol/mg protein. Detection of DAO activity in serum. To further confirm intestinal injury, serum DAO, a sensitive marker reflecting small intestinal mucosal injury,17 was detected using a chemical assay kit (Nanjing Jiancheng Biochemicals Ltd, Nanjing, China) with an ultraviolet spectrophotometer at the wavelength of 436 nm according to the provided manufacturer’s protocol. Results were expressed as U/L serum. Determination of malondialdehyde level and superoxide dismutase activity in intestinal mucosa. Intestinal mucosal tissues were homogenized on ice with normal saline, frozen in refrigerator at 208C for 5 minutes, and centrifuged for 15 minutes at
Wen et al 557
4,000 rpm. Supernatants were transferred into fresh tubes for the evaluation. The fatty acid peroxidation product malondialdehyde (MDA) and the superoxide dismutase (SOD) activity were measured using chemical assay kits (Nanjing Jiancheng Biochemicals Ltd) as described previosuly.18 The results were expressed as nmol/mg protein and U/mg protein, respectively. Determination of myeloperoxidase (MPO) activity in intestinal mucosa. The intestinal sample was homogenized, and the homogenate was freezethawed twice, and then centrifuged at 13,000 rpm for 5 minutes. The resulting supernatant was assayed spectrophotometrically for myeloperoxidase (MPO) activity as previously described.19 One unit of MPO was defined as the capacity to degrade 1 mmol peroxide per minute at 258C. Results were expressed as U/g intestinal tissue. Detection of intestinal mucosal epithelial apoptosis. The ileal fragments were fixed in 10% neutral formaldehyde and embedded in paraffin. Analysis of apoptosis of intestinal mucosal epithelial cell was carried out by the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP-biotin nick-end labeling (TUNEL) method as described previously.16 Cell death was assessed using an In Situ Detection assay kit (Roche, Indianapolis, IN). TUNEL-positive cells were characterized by dark brown staining of the nucleus and nuclear membrane. Quantitation was performed independently by 2 pathologists who were blind to the study groups by counting the number of positive cells in 5 randomly chosen fields within each slide at an original magnification of 3400. The rate of cell apoptosis (apoptotic index) was expressed as percentage of TUNEL-positive cells using the following formula: Number of TUNEL-positive cell nuclei/the number of total cell nuclei 3 100. Detection of cleaved caspase-3 and AR expressions in intestinal mucosal epithelial cells by Western blotting. To further confirm the existence of apoptosis and explore the role of AR, the protein expressions of cleaved caspase-3 and AR were detected via Western blot. Intestinal mucosal samples were added with RIPA (13 phosphate-buffered saline, 1% NP40, 0.1% SDS, 5 mmol/L EDTA, 0.5% sodium deoxycholate, 1 nmol/L sodium orthovanadate, 1 mmol/L PMSF, and protease inhibitor cocktail) for extraction of total protein. After the centrifugation at 12,000 rpm for 30 minutes, supernatants were collected, and the protein concentration was determined by the Bradford method. We subjected 30 mg of total protein to 12% SDS-polyacrylamide gel electrophoresis, transferred onto 0.22-mm PVDF membranes (Millipore, Bedford, MA), and blocked with 5%
558 Wen et al
nonfat skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST; pH 7.6). Membranes were incubated overnight at 48C with specific antibodies against cleaved Caspase-3 (1:2,000; Cell Signaling Technology, Beverly, MA), AR (1:1,000; Abcam, Cambridge, UK), and actin (1:5,000; Cell Signaling Technology). After being washed with TBST 3 times, the membrane was incubated for 1 hour at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000; Cell Signaling Technology) directed at the primary antibody. Chemiluminescence (ECL plus, Amersham, Buckinghamshire, UK) was used for analyzing levels of protein, and blots were exposed to Hyperfilm ECL (Amersham). Protein levels were quantitated with Image J software (National Institutes of Health, Bethesda, MD). b-Actin was used as internal housekeeping control to calculate relative ratio of optical density with values compared with those of normal controls. Statistical analysis. Data were expressed as mean values ± standard deviation (SD). Biochemical assays for LD, DAO, MDA, and SOD were performed in duplicate or triplicate for each specific sample. Therefore, all the data points are means of numbers that themselves are means of duplicate or triplicate measurements for these parameters. Significance was evaluated using 1-way ANOVA (SPSS 13.0, SPSS Inc, Chicago, IL) followed by the Tukey post hoc procedure for multiple comparisons. Correlation between different variables was assessed by Spearman’s coefficient. A value of P < .05 was considered significant. RESULTS Baseline data. No animal died during the experiments. Body weight and temperature during the experiments did not differ among groups (P > .1; data not shown). Evaluation of intestinal mucosal histopathologic changes. In terms of the morphologic changes, normal villi were observed in the control and ARI + control groups (Fig 1, A and B). By contrast, the I/R group had in severe edema of the villi and infiltration inflammatory cells, and large numbers of intestinal villi were severed and denuded. In addition, the gap between epithelial cells increased significantly, and capillaries and lymph vessels were markedly dilated (Fig 1, C). ARI administration before ischemia without IPo provided no salutary nor aggravating effects on intestinal I/R tissues (Fig 1, D). In contrast, in the IPo (Fig 1, E) and DMSO groups (Fig 1, G), slight edema and necrotic mucosal villi were seen, and the damage was further attenuated as compared with I/R group. In the
Surgery April 2013
ARI + I/R + IPo group (Fig 1, F), edema of the villi, and infiltration of the necrotic epithelial by inflammatory cells were observed, which was comparable to that seen in the I/R and ARI + I/R groups. In parallel with the mucosal morphologic changes, Chiu’s scores in the control and ARI + control groups were less than that in other groups (P < .01), and those in the IPo and DMSO groups were less than that in the I/R, ARI + I/R, and ARI + I/R + IPo groups (P < .05; Fig 1, H). Evaluation of intestinal mucosal injury. LD is a product from glucose metabolism in anaerobic metabolism, and its increase reflects a decrease in tissue perfusion or ischemia and is used to assess intestinal injury.20 In terms of mucosal histopathologic changes, the intestinal levels of LD in the I/ R (4.38 ± 0.75), ARI + I/R (4.13 ± 0.86), and ARI + I/R + IPo (3.76 ± 0.69) groups were greater than those in the control (1.38 ± 0.36), ARI + control (1.25 ± 0.45), and in the IPo (2.82 ± 0.65), and DMSO (2.31 ± 0.50) groups (all P <.05), whereas intestinal levels of LD did not differ between the I/R, ARI + I/R and ARI + I/R + IPo groups (all P > .05). DAO is an enzyme synthesized primarily in gastrointestinal mucosal cells, and the serum level of DAO have been used as an indicator of the integrity and functional mass of the intestinal mucosa.21 The serum DAO activity was increased in I/R (25.83 ± 3.14), ARI + I/R (28.50 ± 4.32) and ARI + I/R + IPo (27.24 ± 4.18) and was decreased in the IPo (17.81 ± 3.45) and the DMSO (18.73 ± 3.12) groups (all P < .05). Evaluation of intestinal lipid peroxidation. Production of MDA is an indicator for lipid peroxidation and development of oxidative stress in intestinal tissue. After 120 minutes of reperfusion, the intestinal MDA levels increased from 6.90 ± 1.70 in the control group and 5.60 ± 2.10 in the ARI + control group to 17.80 ± 3.40 in the I/R group and 19.40 ± 3.80 in the ARI + I/R group (all P <.01), but decreased to 10.10 ± 2.00 in the IPo group and 11.60 ± 3.10 in the DMSO group (all P <.01 versus I/R and ARI + I/R). When given before intestinal ischemia with and/or without postconditioning, epalrestat was still associated with increased MDA levels to 19.40 ± 3.80 and 21.25 ± 2.62 (all P < .01 versus control and ARI + control). In contrast, SOD activity, a reactive oxygen species scavenger, was 108.20 ± 25.10 in the control group and 103.90 ± 13.40 in the ARI + control group and decreased to 48.70 ± 22.20 in the I/R, 39.70 ± 15.20 in the ARI + I/R, and 50.76 ± 6.45 in the ARI + I/R + IPo groups (all P < .01). IPo and DMSO, however, restored the activity of SOD (92.60 ± 10.80 and 82.40 ± 11.50; all P > .05 versus control and ARI + control).
Surgery Volume 153, Number 4
Wen et al 559
Fig 1. Histopathologic changes of small intestinal mucosa under light microscopy (original magnification, 3200; A–G) and the evaluation of intestinal injury with Chiu’s scores (H). In the control and ARI + control groups (A, B), normal intestinal mucosa was seen. In contrast, severe mucosal damage was observed in the I/R and ARI + I/R groups (C, D). Compared with the I/R group, only mild damage in intestinal architecture was seen in the IPo (E) and DMSO (G) groups. The histopathologic change in the ARI + I/R + IPo group (F) was similar to that in the I/R and ARI + I/R groups and could be described as in C. H, Changes in intestinal mucosal Chiu’s scores. Data are expressed as mean values ± SD (n = 8). **P <.01 versus control and ARI + control; #P < .05 versus I/R, ARI + I/R and ARI + I/R + IPo. (Color version of figure is available online.)
Evaluation of neutrophil infiltration in the intestinal mucosa tissues. MPO activity was used to assess neutrophil infiltration in the intestine and also as a sensitive variable reflecting damage to the intestinal mucosal barrier.22 After 120 minutes of reperfusion, MPO activity was 0.65 ± 0.13 in the control and 0.60 ± 0.18 in the ARI + control group and increased to 1.07 ± 0.29 in the I/R, 0.89 ± 0.11 in the ARI + I/R, and 0.94 ± 0.21 in the ARI + I/R + IPo groups (all P < .05), but decreased to 0.47 ± 0.14 in the IPo group (P < .05 versus I/R, ARI + I/R, and ARI + I/R + IPo). Meanwhile, the MPO activity did not changes in the DMSO group (0.65 ± 0.17; P > .05 versus IPo).
Evaluation of intestinal mucosal epithelial apoptosis and cleaved caspase-3 expression. TUNEL-positive epithelial cells at the villus surface demonstrated marked dark brown staining in the nuclei (Fig 2). TUNEL staining in the control and ARI + control groups were barely detectable, and only a few apoptotic cells were present at the tip of the villi (Fig 2, A and B). In contrast, many TUNEL-positive apoptotic cells and intercellular apoptotic fragments from the detached epithelium at the tips to the lower part of the villi were seen in the I/R and ARI + I/R groups (Fig 2, C and D), and the apoptotic index was greater in the I/R and ARI + I/R groups than that in the control and ARI + control groups (P < .01; Fig 2, H).
560 Wen et al
Surgery April 2013
Fig 2. Changes of the intestinal mucosal cell apoptosis as measured by TUNEL-like staining under light microscopy (original magnification, 3400; A–G) and cleaved caspase-3 expression (I) and protein content (densitometry; J). Apoptotic nuclei are stained dark brown and indicated by arrows. Few apoptotic cells were observed in the distal ileum from control and ARI + control rats (A, B). In the I/R (C), ARI + I/R (D), and ARI + I/R + IPo (F) groups, many TUNELpositive detached epithelial cells were observed. In contrast, some apoptotic epithelia cells were seen in the tissues obtained from the IPo (E) and DMSO (G) groups, and the apoptotic indexes were significantly less than that in the I/R,
Surgery Volume 153, Number 4
Wen et al 561
Fig 3. Changes of intestinal mucosal aldose reductase (AR) protein expression (densitometry). Expression of AR protein in the I/R and ARI + I/R + IPo groups was significantly decreased, whereas IPo and vehicle control DMSO attenuated the downregulation of intestinal AR expression. Data are expressed as mean values ± SD (n = 8). *P < .05 and **P < .01 versus control and ARI + control; #P < .05 versus I/R and ARI + I/R + IPo.
Moreover, the apoptosis and apoptotic index in the ARI + I/R + IPo group were similar to that in the I/R and ARI + I/R groups (P > .05; Fig 2, F and H). As shown in Fig 2, E, G, and H, fewer positive staining cells were seen in ileal sections taken from the IPo and DMSO groups, suggesting that IPo suppressed apoptosis and decreased the apoptotic index compared with the I/R and ARI + I/ R groups (P < .05). Furthermore, such a striking decrease in epithelial apoptosis correlated with less cleaved caspase-3 protein expression in the IPo and DMSO groups in comparison with the I/ R and ARI + I/R + IPo groups (P < .05; Fig 2, I; see densitometry analysis Fig 2, J). Evaluation of intestinal mucosal AR expression. As shown in Fig 3 (densitometry analysis), the intestinal AR expression was decreased in the I/R group (P < .01 versus control and ARI + control). Moreover, when ARI was given before index ischemia, AR expression was also markedly downregulated (P < .01 versus control and ARI + control), and IPo attenuated the downregulation of intestinal AR expression (P < .05 versus I/R and ARI + I/R + IPo). DMSO did not change the AR expression level (P > .05 versus IPo). These data indicate that AR plays possibly a critical role in intestinal protection against I/R injury, and IPo diminishes this source of injury by upregulating AR expression. Correlation analysis. In groups of control, I/R, I/R+ IPo, ARI + I/R + IPo, and DMSO + I/R + IPo (n = 40), negative correlation between AR
Fig 4. Correlations between AR expression and Chiu’s scores (A), and AR expression and malondialdehyde (MDA) levels (B). AR expression was negatively correlated with Chiu’s scores (r = 0.671; P < .001). In addition, AR expression was also strongly inversely correlated with the MDA levels (r = 0.842; P < .001).
expression and Chiu’s scores existed (r = 0.671; P < .001, Fig 4, A). In addition, AR expression was also inversely correlated with MDA levels (r = 0.842; P < .001; Fig 4, B). DISCUSSION The present results showed that IPo attenuated intestinal injury, as evidenced by decreases in Chiu’s score, LD level, and serum DAO activity, as well as an increase in SOD activity and upregulated AR expression in intestinal mucosa, which are in concert with our previous studies.3,7,9 We also found that intestinal I/R significantly decreased AR expression. This result is contradictory with the findings in the rat heart that AR protein expression can be increased when subjected to ischemia or I/R ex vivo or to coronary occlusion or occlusion-reperfusion in situ and that AR upregulation can be abolished by pretreatment with Tiron, a superoxide dismutase mimetic.23 Why
= ARI + I/R and ARI + I/R + IPo groups. H, Changes in intestinal mucosal epithelial apoptosis. Representative Western blots showed increased expression of cleaved caspase-3 in the I/R and ARI + I/R + IPo groups, whereas cleaved caspase3 levels were decreased in intestinal mucosa obtained from the IPo and DMSO groups (I, J). Data are expressed as mean values ± SD (n = 8). *P < .05 and **P < .01 versus control and ARI + control; #P < .05 versus I/R and ARI + I/R + IPo. (Color version of figure is available online.)
562 Wen et al
these results are discordant with the changes of AR expression reported here are not clear, but perhaps seem to be attributable to variations in the animal model/organ, animal strain, and durations of ischemia or reperfusion. Of note, our results showed that the ARI epalrestat alone at the dose used had no toxic effects and the intestinal mucosal tissues in the epalrestat-treated control rats were all intact. Moreover, similar to the finding that epalrestat could not attenuate the myocardial injury owing to I/R in nondiabetic rat hearts,24 we for the first time also found that pretreatment with epalrestat before ischemic insult provided no salutary effects on intestinal I/R tissues, but completely abolished the well-documented protective effect of IPo. These findings demonstrated that AR inhibition resulted in redox imbalance and further exacerbated the oxidative stress-induced lipid peroxidation in the ischemic intestine. Likewise, these results suggest that simultaneously enhanced expressions of AR and endogenous antioxidant enzyme SOD after IPo provided intestinal protection by serving as an important antioxidant. AR not only catalyzes the reduction of the aldehyde form of glucose to sorbitol, but also exhibits a wide range substrate specificity for a variety of aldehydes from endogenous or exogenous origin.25 In addition, extensive studies showed that the AR gene can be induced by oxidants such as aldehydes26,27 and hydrogen peroxide,27,28 and its expression also increased 24 hours after the myocardial IPC.29 Recent studies support the view that upregulation of AR contributed to the detoxification of toxic aldehydes and modulated the survival of the cells exposed to various noxious stimuli.13,27,30 AR inhibition, however, abrogated the infarct-sparing effect of late myocardial preconditioning,29 exacerbated aldehyde toxicity in both ischemic heart23 and rat vascular smooth muscle cell lines,26 and increased vascular cell death and accumulation of toxic aldehydes during inflammation.13 Keith et al31 also found that AR protected against myocardial I/R injury, possibly by decreasing the stress in the endoplasmic reticulum by removing aldehydic products of lipid peroxidation. In this regard, AR is thought to function as an oxidative defense enzyme and may be involved in cellular defense against toxic aldehydes. Interestingly, AR protects the heart from I/R injury only during the late stages of ischemia and during reperfusion.32 Therefore, it is reasonable to postulate that the postconditioning-induced AR activation during the early stages of reperfusion is required for subsequent protection. In addition, negative correlations between AR expression and Chiu’s score as well as between AR expression
Surgery April 2013
and MDA level indicate that AR could be among the major contributors able to ameliorate intestinal I/R injury in this model. As such, we propose that upregulation of AR seems to represent an adaptive response that removes toxic aldehydes (such as MDA) generated in the ischemic intestine and contributes to an endogenous and complex self-defense mechanism, ensuring an increased cellular resistance of intestine against toxic injury. Although the detailed molecular mechanism remains unclarified, extensive data showed that AR catalyzes reduction of phospholipid-derived aldehydes and their glutathione conjugates and regulates the proinflammatory and immunogenic effects of oxidized phospholipids.23,33 This observation supports the concept that the AR level in the intestinal mucosa can be upregulated by IPo, and that the AR-mediated intestinal protective effect of IPo may be related to more efficient removal of toxic lipid peroxidation products triggered by oxidative stress. Several studies demonstrated that an increase in AR activity and expression via the PI3K/Akt/eNOS pathway provides cardioprotection against I/R injury,32,34,35 and ARIs decreased the postischemic recovery in the rat hearts subjected to global ischemia and increased the infarct size when given before ischemia or on reperfusion.32 Meanwhile, activation of the PI3K/Akt-linked pathway is involved in the cardioprotection,36,37 neuroprotection5,38,39 of IPo, and gastric protection40 of genistein postconditioning after I/R injury. Altogether, we proposed that intestinal IPo-mediated upregulation of AR and the subsequent intestinal protection seen in the present study might have been achieved in part via activation of PI3K/Akt pathway. However, the precise mechanisms related to intracellular signaling needs to be further elucidated. In apparent contradiction to these findings and those reported by Calderone et al,24 several studies have concluded that pharmacologic inhibition of AR decrease I/R injury in isolated hearts41,42 and retina,43 and mitigates inflammatory diseases like allergic asthma,44 whereas overexpression of AR promotes atherogenesis in apoE-null mice.45 Different from our research, Ramasamy et al41 showed that nitric oxide synthase 2 inhibitors, not a specific ARI, decreased AR activation and exerted their cardioprotective effects by inhibiting the polyol pathway. The disparities between our findings and the above reports may be related to the inconsistent and uncertain effects of ARIs on different organs or tissues, which could be associated with several reasons, namely (1) differences in organs or tissues as well as animal models (durations of
Wen et al 563
Surgery Volume 153, Number 4
ischemia or reperfusion), (2) the specificity and selectivity of ARIs in different tissues or organs, (3) because glucose is not a preferred substrate for AR rather than aldehydes,10 the importance of the polyol pathway may be limited under normoglycemic conditions because only a minor part of nonphosphorylatyed glucose enters the polyol pathway, and (4) different posttranslational modifications of AR,23,32 which modulate both substrate and inhibitor binding. The current study is the first to find that IPo can attenuate intestinal mucosal cell apoptosis. Because oxidative stress and inflammatory reaction in the gastrointestinal tract induce apoptosis,11,46 and IPo can partly abrogate the above pathophysiologic phenomenon induced by intestinal I/R, it is likely that IPo suppressed the intestinal mucosal apoptosis seen in the present study. Work in vasculitis showed that AR inhibition increased the lipid peroxidation products and led to increased cell apoptosis.13 Moreover, Kang et al found that activation of PI3K and p38 MAPK by curcumin upregulated the expression of AR gene, inhibition of enzyme activity, or gene silencing of AR markedly augmented the glucose oxidase-mediated apoptosis,47 and that overexpression of AR suppressed the increase in intracellular ROS and attenuated the apoptotic cell death induced by ultraviolet B radiation.14 In our study, pretreatment with ARI completely abolished the effect of IPo on apoptosis, indicating that apoptosis suppression by AR plays, at least in part, an important role in the intestinal protection conferred by IPo. The current study focused on elucidating the role of AR by employing its specific inhibitor. Whether AR is specific for the intestinal IPo effect is not clear, and further studies utilizing AR gene knock-out models or employing the RNA interference methodologies are needed to confirm the role of AR in intestinal IPo against I/R injury. Moreover, the possibility of off-target effects by epalrestat should be considered, and whether epalrestat can inhibit other enzymes involved in providing protection against redox stress deserves further investigations. Nevertheless, the findings from the present study provide new clues for understanding the molecular mechanisms of intestinal I/R injury and the mechanisms whereby IPo induces beneficial effects. S.-H.W. and Y.-H.L. contributed equally to this study. This work was financially supported by grants from National Natural Science Foundation of China (Grant Numbers: 81270456, 81171847, to Ke-Xuan Liu), partly by ‘‘the Fundamental Research Funds for the Central Universities’’ in China and China Education program
for New Century Excellent Talents of Ministry of Education (NCET-10-0853, to Ke-Xuan Liu), partly by a grant from Natural Science Foundation of Guangdong Province (no. S2011020002332, to Ke-Xuan Liu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Professor Wanlong Lei (Department of Anatomy and Brain Research, Sun Yat-sen University, Guangdong, China) and Professor Weikang Wu (Department of Pathophysiology, Sun Yat-sen University, Guangdong, China) for their technical assistance. REFERENCES 1. Mallick IH, Yang W, Winslet MC, Seifalian AM. Ischemia-reperfusion injury of the intestine and protective strategies against injury. Dig Dis Sci 2004;49:1359-77. 2. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36. 3. Liu KX, Li C, Li YS, Yuan BL, Xu M, Xia Z, et al. Proteomic analysis of intestinal ischemia/reperfusion injury and ischemic preconditioning in rats reveals the protective role of aldose reductase. Proteomics 2010;10:4463-75. 4. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:579-88. 5. Rehni AK, Singh N. Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked behavioral deficits in mice. Pharmacol Rep 2007;59:192-8. 6. Jiang B, Liu X, Chen H, Liu D, Kuang Y, Xing B, et al. Ischemic postconditioning attenuates renal ischemic/reperfusion injury in mongrel dogs. Urology 2010;76:1519.e1-7. 7. Liu KX, Li YS, Huang WQ, Chen SQ, Wang ZX, Liu JX, et al. Immediate postconditioning during reperfusion attenuates intestinal injury. Intensive Care Med 2009;35:933-42. 8. Liu KX, Li YS, Huang WQ, Li C, Liu JX, Li Y. Immediate but not delayed postconditioning during reperfusion attenuates acute lung injury induced by intestinal ischemia/reperfusion in rats: comparison with ischemic preconditioning. J Surg Res 2009;157:e55-62. 9. Li YS, Wang ZX, Li C, Xu M, Li Y, Huang WQ, et al. Proteomics of ischemia/reperfusion injury in rat intestine with and without ischemic postconditioning. J Surg Res 2010; 164:e173-80. 10. Srivastava S, Watowich SJ, Petrash JM, Srivastava SK, Bhatnagar A. Structural and kinetic determinants of aldehyde reduction by aldose reductase. Biochemistry 1999;38:42-54. 11. Liu KX, Chen SQ, Huang WQ, Li YS, Irwin MG, Xia Z. Propofol pretreatment reduces ceramide production and attenuates intestinal mucosal apoptosis induced by intestinal ischemia/reperfusion in rats. Anesth Analg 2008;107: 1884-91. 12. 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. 13. Rittner HL, Hafner V, Klimiuk PA, Szweda LI, Goronzy JJ, Weyand CM. Aldose reductase functions as a detoxification
564 Wen et al
14.
15.
16.
17.
18.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
system for lipid peroxidation products in vasculitis. J Clin Invest 1999;103:1007-13. Kang ES, Iwata K, Ikami K, Ham SA, Kim HJ, Chang KC, et al. Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation. Free Radic Biol Med 2010;50:680-8. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg 1970;101:478-83. Liu KX, Rinne T, He W, Wang F, Xia Z. Propofol attenuates intestinal mucosa injury induced by intestinal ischemiareperfusion in the rat. Can J Anaesth 2007;54:366-74. Tsunooka N, Maeyama K, Nakagawa H, Doi T, Horiuchi A, Miyauchi K, et al. Localization and changes of diamine oxidase during cardiopulmonary bypass in rabbits. J Surg Res 2006;131:58-63. Liu KX, He W, Rinne T, Liu Y, Zhao MQ, Wu WK. The effect of Ginkgo biloba extract (EGb 761) pretreatment on intestinal epithelial apoptosis induced by intestinal ischemia/reperfusion in rats: role of ceramide. Am J Chin Med 2007;35:805-19. Cuzzocrea S, Mazzon E, Esposito E, Muia C, Abdelrahman M, Di Paola R, et al. Glycogen synthase kinase-3 beta inhibition attenuates the development of ischaemia/reperfusion injury of the gut. Intensive Care Med 2007;33:880-93. Vejchapipat P, Williams SR, Spitz L, Pierro A. Intestinal metabolism after ischemia-reperfusion. J Pediatr Surg 2000;35:759-64. Wolvekamp MC, de Bruin RW. Diamine oxidase: an overview of historical, biochemical and functional aspects. Dig Dis 1994;12:2-14. Naito Y, Takagi T, Uchiyama K, Handa O, Tomatsuri N, Imamoto E, et al. Suppression of intestinal ischemia-reperfusion injury by a specific peroxisome proliferator-activated receptorgamma ligand, pioglitazone, in rats. Redox Rep 2002;7:294-9. Kaiserova K, Srivastava S, Hoetker JD, Awe SO, Tang XL, Cai J, et al. Redox activation of aldose reductase in the ischemic heart. J Biol Chem 2006;281:15110-20. Calderone V, Testai L, Martelli A, La Motta C, Sartini S, Da Settimo F, et al. Anti-ischaemic activity of an antioxidant aldose reductase inhibitor on diabetic and non-diabetic rat hearts. J Pharm Pharmacol 2010;62:107-13. Yabe-Nishimura C. Aldose reductase in glucose toxicity: a potential target for the prevention of diabetic complications. Pharmacol Rev 1998;50:21-33. Ruef J, Liu SQ, Bode C, Tocchi M, Srivastava S, Runge MS, et al. Involvement of aldose reductase in vascular smooth muscle cell growth and lesion formation after arterial injury. Arterioscler Thromb Vasc Biol 2000;20:1745-52. Spycher SE, Tabataba-Vakili S, O’Donnell VB, Palomba L, Azzi A. Aldose reductase induction: a novel response to oxidative stress of smooth muscle cells. FASEB J 1997;11:181-8. Keightley JA, Shang L, Kinter M. Proteomic analysis of oxidative stress-resistant cells: a specific role for aldose reductase overexpression in cytoprotection. Mol Cell Proteomics 2004;3:167-75. Shinmura K, Bolli R, Liu SQ, Tang XL, Kodani E, Xuan YT, et al. Aldose reductase is an obligatory mediator of the late phase of ischemic preconditioning. Circ Res 2002;91:240-6. Chang KC, Paek KS, Kim HJ, Lee YS, Yabe-Nishimura C, Seo HG. Substrate-induced up-regulation of aldose reductase by methylglyoxal, a reactive oxoaldehyde elevated in diabetes. Mol Pharmacol 2002;61:1184-91. Keith RJ, Haberzettl P, Vladykovskaya E, Hill BG, Kaiserova K, Barski O, et al. Aldose reductase decreases endoplasmic reticulum stress in ischemic hearts. Chem Biol Interact 2009;178:242-9.
Surgery April 2013
32. Kaiserova K, Tang XL, Srivastava S, Bhatnagar A. Role of nitric oxide in regulating aldose reductase activation in the ischemic heart. J Biol Chem 2008;283:9101-12. 33. Srivastava S, Spite M, Trent JO, West MB, Ahmed Y, Bhatnagar A. Aldose reductase-catalyzed reduction of aldehyde phospholipids. J Biol Chem 2004;279:53395-406. 34. Bell RM, Yellon DM. Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. J Mol Cell Cardiol 2003;35:185-93. 35. Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation 2001;104:330-5. 36. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of ‘‘modified reperfusion’’ protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res 2004;95:230-2. 37. Chiari PC, Bienengraeber MW, Pagel PS, Krolikowski JG, Kersten JR, Warltier DC. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits. Anesthesiology 2005;102:102-9. 38. Gao X, Zhang H, Takahashi T, Hsieh J, Liao J, Steinberg GK, et al. The Akt signaling pathway contributes to postconditioning’s protection against stroke; the protection is associated with the MAPK and PKC pathways. J Neurochem 2008;105:943-55. 39. Jiang X, Ai C, Shi E, Nakajima Y, Ma H. Neuroprotection against spinal cord ischemia-reperfusion injury induced by different ischemic postconditioning methods: roles of phosphatidylinositol 3-kinase-Akt and extracellular signalregulated kinase. Anesthesiology 2009;111:1197-205. 40. Du DS, Ma XB, Zhang JF, Zhou XY, Li Y, Zhang YM, et al. The protective effect of capsaicin receptor-mediated genistein postconditioning on gastric ischemia-reperfusion injury in rats. Did Dis Sci 2010;55:3070-7. 41. Ramasamy R, Hwang YC, Liu Y, Son NH, Ma N, Parkinson J, et al. Metabolic and functional protection by selective inhibition of nitric oxide synthase 2 during ischemia-reperfusion in isolated perfused hearts. Circulation 2004;109:1668-73. 42. Hwang YC, Shaw S, Kaneko M, Redd H, Marrero MB, Ramasamy R. Aldose reductase pathway mediates JAK-STAT signaling: a novel axis in myocardial ischemic injury. FASEB J 2005;19:795-7. 43. Obrosova IG, Maksimchyk Y, Pacher P, Agardh E, Smith ML, EI-Remessy AB, et al. Evaluation of the aldose reductase inhibitor fidarestat on ischemia-reperfusion injury in rat retina. Int J Mol Med 2010;26:135-42. 44. Yadav UC, Ramana KV, Aguilera-Aguirre L, Boldogh I, Boulares HA, Srivastava SK. Inhibition of aldose reductase prevents experimental allergic airway inflammation in mice. PLoS One 2009;4:e6535. 45. Vikramadithyan RK, Hu Y, Noh HL, Liang CP, Hallam K, Tall AR, et al. Human aldose reductase expression accelerates diabetic atherosclerosis in transgenic mice. J Clin Invest 2005;115:2434-43. 46. Calvino-Fernandez M, Benito-Martinez S, Parra-Cid T. Oxidative stress by Helicobacter pylori causes apoptosis through mitochondrial pathway in gastric epithelial cells. Apoptosis 2008;13:1267-80. 47. Kang ES, Woo IS, Kim HJ, Eun SY, Paek KS, Kim HJ, et al. Up-regulation of aldose reductase expression mediated by phosphatidylinositol 3-kinase/Akt and Nrf2 is involved in the protective effect of curcumin against oxidative damage. Free Radic Biol Med 2007;43:535-45.
Background. We demonstrated previously that ischemic postconditioning (IPo) attenuated intestinal injury induced by intestinal ischemia/reperfusion (I/R), and thereafter employed a proteomic method to identify aldose reductase (AR), a differentially expressed protein in intestinal mucosal tissue, which was downregulated by intestinal I/R and upregulated by IPo. This study aimed to further explore the possible role of AR in intestinal protection conferred by IPo. Methods. Intestinal ischemia was induced by clamping the superior mesenteric artery (SMA) for 60 minutes in male adult rats. Then rats were allocated into 7 groups based on the random number table. The control group involved only sham operation; the control + AR inhibitor epalrestat group underwent sham operation and epalrestat administration; the I/R with and/or without epalrestat groups had SMA clamped for 60 minutes followed by 120 minutes of reperfusion with and/or without epalrestat given before index ischemia; the IPo group underwent 3 cycles of 30 seconds of reperfusion and 30 seconds of re-occlusion imposed immediately on reperfusion; and the epalrestat or vehicle control dimethylsulfoxide + IPo groups had the drugs administrated 10 minutes before ischemia. Results. IPo resulted in significant intestinal protection evidenced as marked decreases in Chiu’s score, reflecting changes intestinal histology, serum diamine oxidase activity, and intestinal mucosal levels of lactic acid, malondialdehyde, and myeloperoxidase, the apoptosis index, and downregulated cleaved caspase-3 protein expression; these changes were accompanied by an increase in superoxide dismutase activity and upregulation of AR protein levels. Epalrestat failed to protect against intestinal I/R insult, but abolished the protective effects of IPo. Conclusion. These findings suggest that IPo attenuates intestinal I/R-induced intestinal injury via ARmediated oxidative defense and apoptosis suppression; AR inhibition reverses the protective effects of IPo. AR seems to be an innate protective factor in this model of intestinal I/R. (Surgery 2013;153:555-64.) From the Department of Anesthesiology,a the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; and the State Key Laboratory of Oncology in South China,b Department of Pathology,c Sun Yat-sen University Cancer Center, Guangzhou, China
INTESTINAL ISCHEMIA/REPERFUSION (I/R) INJURY occurs in a variety of pathophysiologic conditions, for example, shock, severe infection, and certain operative procedures, such as small bowel transplantation, abdominal aortic surgery, and Accepted for publication September 25, 2012. Reprint requests: Ke-Xuan Liu, MD, PhD, Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhongshan 2nd Road, Guangzhou 510080, China. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2012.09.017
cardiopulmonary bypass, and could trigger the development of multiple organ dysfunction.1 The mechanisms of intestinal I/R injury are not understood fully, and effective approaches for its clinical application are still lacking. Murry et al2 first introduced the definition of ischemic preconditioning (IPC), in which repetitive brief periods of ischemia protected the myocardium from a subsequent greater period of ischemic insult. We also demonstrated that IPC protected intestine from intestinal I/R injury in a rat model.3 Clinical events related to intestinal I/R, however, are usually unpredictable, which limits the clinical application of IPC. In contrast, SURGERY 555
556 Wen et al
a clinically more feasible strategy, ischemic postconditioning (IPo), which consists of $1 short cycles of reperfusion followed by $1 short cycles of ischemia, immediately after the conclusion of ischemia and before starting permanent reperfusion, can confer effective organ protection comparable to IPC.4-9 Our previous studies already showed that IPo can decrease the intestinal7 and lung8 injury induced by intestinal I/R, in part by inhibiting oxidative injury, neutrophil filtration, and the proinflammatory response. Further, we identified a differentially expressed protein in the intestinal mucosa via a proteomic method, aldose reductase (AR), which can be downregulated by intestinal I/R and upregulated by IPo.9 This finding indicates that AR might be involved in the intestinal protection conferred by IPo, which needs to be confirmed. AR, a member of the aldo–keto reductase superfamily, is proposed to function as an NADPHdependent oxidoreductase owing to its ability to catalyze the reduction of a broad spectrum of substrates that range from steroid carbonyls to aldo–keto sugars. Investigations also have shown that AR exhibits high affinity for hydrophobic aldehydes, such as those generated during lipid peroxidation.10 Lipid-derived aldehydes are cytotoxic, so the ability of AR to metabolize these aldehydes suggests that this enzyme may be involved in protection against oxidative injury. Given that oxidative stress is among the mechanisms causing intestinal I/R injury7-9,11 and that IPo can confer intestinal protection by suppressing oxidative stress as we documented previously,7 we hypothesized that AR, as an antioxidant enzyme, plays an important role in intestinal protection conferred by IPo. In addition, we7 and others12 demonstrated that apoptosis is the main mode of intestinal mucosal cell death induced by intestinal I/R. Whether IPo confers intestinal protection via inhibiting intestinal mucosal apoptosis or not remains obscure. Because AR can attenuate cell apoptosis by inhibiting oxidative stress in different tissues,13,14 we hypothesized that IPo attenuates oxidative stress and intestinal mucosal cell apoptosis via AR and thereby confers its intestinal protection. This hypothesis was tested in a rat model of intestinal I/R injury. MATERIALS AND METHODS Ethics statement and animal model of intestinal I/R. This study was approved by the Animal Care Committee of Sun Yat-sen University, China, and was performed in accordance with National Institutes of Health guidelines for the use of experimental animals. Fifty-six adult, pathogen-free,
Surgery April 2013
male, Sprague–Dawley rats weighing 220–250 g were housed in individual cages in a temperaturecontrolled room with alternating 12-hour light/ dark cycles and acclimated for 1 week before experiments. All animals were fasted overnight before the experiment but had free access to water ad libitum. Rats were anesthetized with 20% urethane (induction and maintenance, 6 mL/kg intraperitoneally). A 2- to 3-cm midline abdominal incision was made, and the small intestine was exteriorized. The intestinal I/R injury was established by occluding the superior mesenteric artery (SMA) with a microvessel clip for 60 minutes followed by 120 minutes reperfusion. SMA was isolated from the superior mesenteric vein before occlusion. Ischemia was recognized by the existence of a pulseless or pale color of the small intestine. The return of pulses and the reestablishment of the pink color indicated appropriate reperfusion of the intestine. Experimental groups. According to the random number table, the rats were allocated into 1 of 7 groups based on the interventions (n = 8 each): (1) Control group (control), sham surgical preparation including isolation of the SMA without occlusion; (2) I/R group (I/R), SMA occlusion for 60 minutes followed by 120 minutes reperfusion; (3) AR inhibitor (ARI) (epalrestat) + control group (ARI + control); (4) epalrestat + I/R group (ARI + I/R); (5) IPo group (I/R + IPo), immediately at the onset of reperfusion, reflow was initiated with 30 seconds of full SMA flow, followed by 30 seconds of reocclusion, repeated for a total of 3 cycles (3 minutes total intervention); (6) epalrestat group (ARI + I/R + IPo); and (7) dimethylsulfoxide (DMSO) group (DMSO + I/R + IPo), in which DMSO was the solvent of epalrestat and served as vehicle control. Epalrestat (dissolved in DMSO as 10 mg/mL; 10 mg/kg IV) or DMSO (1 mL/kg) was given 10 minutes before SMA occlusion. Our recent study demonstrated that ARI epalrestat can inhibit the activity of AR at a dose of 10 mg/kg.3 Intravenous administration of epalrestat. Before the microvascular clip was placed across the SMA, epalrestat (10 mg/kg body weight [BW]; Wako Co., Osaka, Japan), or vehicle (DMSO, 1 mL/kg BW, Sigma Chemical Co., St Louis, MO) was administered intravenously over a period of 10 minutes through a pump (Harvard Apparatus, Holliston, MA). Animals were then subjected to midline laparotomy and to index ischemia. Preparation of specimens. After the completion of the experiments, blood samples were collected and centrifuged at 3,500 rpm for 15 minutes at 48C, and the supernatant was stored at 808C for
Surgery Volume 153, Number 4
subsequent measurement of diamine oxidase (DAO) activity. A 5- to 10-cm segment of ileum was resected from the terminal ileum and divided into 3 segments. One of the segments was fixed in 10% neutral formaldehyde and paraffin embedded for morphologic analysis, anther was used to evaluate intestinal mucosal epithelial apoptosis, and the third was washed with cold saline and after being scraped off, the intestinal mucosa was dried with filter paper and preserved at 808C for protein extraction. Histologic measurement of intestinal mucosal injury. The segment of small intestine was stained with hematoxylin and eosin. Damage to the intestinal mucosa was evaluated independently by 2 pathologists blind to the study groups. The degree of injury was evaluated using the criteria of Chiu’s score method15 according to changes of the villus and glands of the intestinal mucosa. In brief, mucosal damage was graded from 0 (normal) to 5 (severely damaged) as follows: Grade 0, normal mucosal villi; grade 1, development of subepithelial Gruenhagen’s space at the apex of the villus, often with capillary congestion; grade 2, extension of the subepithelial space with moderate lifting of the epithelial layer from the lamina propria; grade 3, massive epithelial lifting down the sides of villi, possibly with a few denuded tips; grade 4, denuded villi with lamina propria and dilated capillaries exposed, possibly with increased cellularity of lamina propria; and grade 5, digestion and disintegration of the lamina propria, hemorrhage, and ulceration. A minimum of 5 randomly chosen fields from each rat were evaluated and averaged to determine mucosal damage. Detection of lactic acid level in intestinal mucosa. Intestinal mucosal tissues were weighed and made into 10% homogenates. The lactic acid (LD) content in tissues was determined using a chemical assay kit (Nanjing Jiancheng Biochemicals Ltd, Nanjing, China) as described.16 The results were expressed as nmol/mg protein. Detection of DAO activity in serum. To further confirm intestinal injury, serum DAO, a sensitive marker reflecting small intestinal mucosal injury,17 was detected using a chemical assay kit (Nanjing Jiancheng Biochemicals Ltd, Nanjing, China) with an ultraviolet spectrophotometer at the wavelength of 436 nm according to the provided manufacturer’s protocol. Results were expressed as U/L serum. Determination of malondialdehyde level and superoxide dismutase activity in intestinal mucosa. Intestinal mucosal tissues were homogenized on ice with normal saline, frozen in refrigerator at 208C for 5 minutes, and centrifuged for 15 minutes at
Wen et al 557
4,000 rpm. Supernatants were transferred into fresh tubes for the evaluation. The fatty acid peroxidation product malondialdehyde (MDA) and the superoxide dismutase (SOD) activity were measured using chemical assay kits (Nanjing Jiancheng Biochemicals Ltd) as described previosuly.18 The results were expressed as nmol/mg protein and U/mg protein, respectively. Determination of myeloperoxidase (MPO) activity in intestinal mucosa. The intestinal sample was homogenized, and the homogenate was freezethawed twice, and then centrifuged at 13,000 rpm for 5 minutes. The resulting supernatant was assayed spectrophotometrically for myeloperoxidase (MPO) activity as previously described.19 One unit of MPO was defined as the capacity to degrade 1 mmol peroxide per minute at 258C. Results were expressed as U/g intestinal tissue. Detection of intestinal mucosal epithelial apoptosis. The ileal fragments were fixed in 10% neutral formaldehyde and embedded in paraffin. Analysis of apoptosis of intestinal mucosal epithelial cell was carried out by the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP-biotin nick-end labeling (TUNEL) method as described previously.16 Cell death was assessed using an In Situ Detection assay kit (Roche, Indianapolis, IN). TUNEL-positive cells were characterized by dark brown staining of the nucleus and nuclear membrane. Quantitation was performed independently by 2 pathologists who were blind to the study groups by counting the number of positive cells in 5 randomly chosen fields within each slide at an original magnification of 3400. The rate of cell apoptosis (apoptotic index) was expressed as percentage of TUNEL-positive cells using the following formula: Number of TUNEL-positive cell nuclei/the number of total cell nuclei 3 100. Detection of cleaved caspase-3 and AR expressions in intestinal mucosal epithelial cells by Western blotting. To further confirm the existence of apoptosis and explore the role of AR, the protein expressions of cleaved caspase-3 and AR were detected via Western blot. Intestinal mucosal samples were added with RIPA (13 phosphate-buffered saline, 1% NP40, 0.1% SDS, 5 mmol/L EDTA, 0.5% sodium deoxycholate, 1 nmol/L sodium orthovanadate, 1 mmol/L PMSF, and protease inhibitor cocktail) for extraction of total protein. After the centrifugation at 12,000 rpm for 30 minutes, supernatants were collected, and the protein concentration was determined by the Bradford method. We subjected 30 mg of total protein to 12% SDS-polyacrylamide gel electrophoresis, transferred onto 0.22-mm PVDF membranes (Millipore, Bedford, MA), and blocked with 5%
558 Wen et al
nonfat skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST; pH 7.6). Membranes were incubated overnight at 48C with specific antibodies against cleaved Caspase-3 (1:2,000; Cell Signaling Technology, Beverly, MA), AR (1:1,000; Abcam, Cambridge, UK), and actin (1:5,000; Cell Signaling Technology). After being washed with TBST 3 times, the membrane was incubated for 1 hour at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000; Cell Signaling Technology) directed at the primary antibody. Chemiluminescence (ECL plus, Amersham, Buckinghamshire, UK) was used for analyzing levels of protein, and blots were exposed to Hyperfilm ECL (Amersham). Protein levels were quantitated with Image J software (National Institutes of Health, Bethesda, MD). b-Actin was used as internal housekeeping control to calculate relative ratio of optical density with values compared with those of normal controls. Statistical analysis. Data were expressed as mean values ± standard deviation (SD). Biochemical assays for LD, DAO, MDA, and SOD were performed in duplicate or triplicate for each specific sample. Therefore, all the data points are means of numbers that themselves are means of duplicate or triplicate measurements for these parameters. Significance was evaluated using 1-way ANOVA (SPSS 13.0, SPSS Inc, Chicago, IL) followed by the Tukey post hoc procedure for multiple comparisons. Correlation between different variables was assessed by Spearman’s coefficient. A value of P < .05 was considered significant. RESULTS Baseline data. No animal died during the experiments. Body weight and temperature during the experiments did not differ among groups (P > .1; data not shown). Evaluation of intestinal mucosal histopathologic changes. In terms of the morphologic changes, normal villi were observed in the control and ARI + control groups (Fig 1, A and B). By contrast, the I/R group had in severe edema of the villi and infiltration inflammatory cells, and large numbers of intestinal villi were severed and denuded. In addition, the gap between epithelial cells increased significantly, and capillaries and lymph vessels were markedly dilated (Fig 1, C). ARI administration before ischemia without IPo provided no salutary nor aggravating effects on intestinal I/R tissues (Fig 1, D). In contrast, in the IPo (Fig 1, E) and DMSO groups (Fig 1, G), slight edema and necrotic mucosal villi were seen, and the damage was further attenuated as compared with I/R group. In the
Surgery April 2013
ARI + I/R + IPo group (Fig 1, F), edema of the villi, and infiltration of the necrotic epithelial by inflammatory cells were observed, which was comparable to that seen in the I/R and ARI + I/R groups. In parallel with the mucosal morphologic changes, Chiu’s scores in the control and ARI + control groups were less than that in other groups (P < .01), and those in the IPo and DMSO groups were less than that in the I/R, ARI + I/R, and ARI + I/R + IPo groups (P < .05; Fig 1, H). Evaluation of intestinal mucosal injury. LD is a product from glucose metabolism in anaerobic metabolism, and its increase reflects a decrease in tissue perfusion or ischemia and is used to assess intestinal injury.20 In terms of mucosal histopathologic changes, the intestinal levels of LD in the I/ R (4.38 ± 0.75), ARI + I/R (4.13 ± 0.86), and ARI + I/R + IPo (3.76 ± 0.69) groups were greater than those in the control (1.38 ± 0.36), ARI + control (1.25 ± 0.45), and in the IPo (2.82 ± 0.65), and DMSO (2.31 ± 0.50) groups (all P <.05), whereas intestinal levels of LD did not differ between the I/R, ARI + I/R and ARI + I/R + IPo groups (all P > .05). DAO is an enzyme synthesized primarily in gastrointestinal mucosal cells, and the serum level of DAO have been used as an indicator of the integrity and functional mass of the intestinal mucosa.21 The serum DAO activity was increased in I/R (25.83 ± 3.14), ARI + I/R (28.50 ± 4.32) and ARI + I/R + IPo (27.24 ± 4.18) and was decreased in the IPo (17.81 ± 3.45) and the DMSO (18.73 ± 3.12) groups (all P < .05). Evaluation of intestinal lipid peroxidation. Production of MDA is an indicator for lipid peroxidation and development of oxidative stress in intestinal tissue. After 120 minutes of reperfusion, the intestinal MDA levels increased from 6.90 ± 1.70 in the control group and 5.60 ± 2.10 in the ARI + control group to 17.80 ± 3.40 in the I/R group and 19.40 ± 3.80 in the ARI + I/R group (all P <.01), but decreased to 10.10 ± 2.00 in the IPo group and 11.60 ± 3.10 in the DMSO group (all P <.01 versus I/R and ARI + I/R). When given before intestinal ischemia with and/or without postconditioning, epalrestat was still associated with increased MDA levels to 19.40 ± 3.80 and 21.25 ± 2.62 (all P < .01 versus control and ARI + control). In contrast, SOD activity, a reactive oxygen species scavenger, was 108.20 ± 25.10 in the control group and 103.90 ± 13.40 in the ARI + control group and decreased to 48.70 ± 22.20 in the I/R, 39.70 ± 15.20 in the ARI + I/R, and 50.76 ± 6.45 in the ARI + I/R + IPo groups (all P < .01). IPo and DMSO, however, restored the activity of SOD (92.60 ± 10.80 and 82.40 ± 11.50; all P > .05 versus control and ARI + control).
Surgery Volume 153, Number 4
Wen et al 559
Fig 1. Histopathologic changes of small intestinal mucosa under light microscopy (original magnification, 3200; A–G) and the evaluation of intestinal injury with Chiu’s scores (H). In the control and ARI + control groups (A, B), normal intestinal mucosa was seen. In contrast, severe mucosal damage was observed in the I/R and ARI + I/R groups (C, D). Compared with the I/R group, only mild damage in intestinal architecture was seen in the IPo (E) and DMSO (G) groups. The histopathologic change in the ARI + I/R + IPo group (F) was similar to that in the I/R and ARI + I/R groups and could be described as in C. H, Changes in intestinal mucosal Chiu’s scores. Data are expressed as mean values ± SD (n = 8). **P <.01 versus control and ARI + control; #P < .05 versus I/R, ARI + I/R and ARI + I/R + IPo. (Color version of figure is available online.)
Evaluation of neutrophil infiltration in the intestinal mucosa tissues. MPO activity was used to assess neutrophil infiltration in the intestine and also as a sensitive variable reflecting damage to the intestinal mucosal barrier.22 After 120 minutes of reperfusion, MPO activity was 0.65 ± 0.13 in the control and 0.60 ± 0.18 in the ARI + control group and increased to 1.07 ± 0.29 in the I/R, 0.89 ± 0.11 in the ARI + I/R, and 0.94 ± 0.21 in the ARI + I/R + IPo groups (all P < .05), but decreased to 0.47 ± 0.14 in the IPo group (P < .05 versus I/R, ARI + I/R, and ARI + I/R + IPo). Meanwhile, the MPO activity did not changes in the DMSO group (0.65 ± 0.17; P > .05 versus IPo).
Evaluation of intestinal mucosal epithelial apoptosis and cleaved caspase-3 expression. TUNEL-positive epithelial cells at the villus surface demonstrated marked dark brown staining in the nuclei (Fig 2). TUNEL staining in the control and ARI + control groups were barely detectable, and only a few apoptotic cells were present at the tip of the villi (Fig 2, A and B). In contrast, many TUNEL-positive apoptotic cells and intercellular apoptotic fragments from the detached epithelium at the tips to the lower part of the villi were seen in the I/R and ARI + I/R groups (Fig 2, C and D), and the apoptotic index was greater in the I/R and ARI + I/R groups than that in the control and ARI + control groups (P < .01; Fig 2, H).
560 Wen et al
Surgery April 2013
Fig 2. Changes of the intestinal mucosal cell apoptosis as measured by TUNEL-like staining under light microscopy (original magnification, 3400; A–G) and cleaved caspase-3 expression (I) and protein content (densitometry; J). Apoptotic nuclei are stained dark brown and indicated by arrows. Few apoptotic cells were observed in the distal ileum from control and ARI + control rats (A, B). In the I/R (C), ARI + I/R (D), and ARI + I/R + IPo (F) groups, many TUNELpositive detached epithelial cells were observed. In contrast, some apoptotic epithelia cells were seen in the tissues obtained from the IPo (E) and DMSO (G) groups, and the apoptotic indexes were significantly less than that in the I/R,
Surgery Volume 153, Number 4
Wen et al 561
Fig 3. Changes of intestinal mucosal aldose reductase (AR) protein expression (densitometry). Expression of AR protein in the I/R and ARI + I/R + IPo groups was significantly decreased, whereas IPo and vehicle control DMSO attenuated the downregulation of intestinal AR expression. Data are expressed as mean values ± SD (n = 8). *P < .05 and **P < .01 versus control and ARI + control; #P < .05 versus I/R and ARI + I/R + IPo.
Moreover, the apoptosis and apoptotic index in the ARI + I/R + IPo group were similar to that in the I/R and ARI + I/R groups (P > .05; Fig 2, F and H). As shown in Fig 2, E, G, and H, fewer positive staining cells were seen in ileal sections taken from the IPo and DMSO groups, suggesting that IPo suppressed apoptosis and decreased the apoptotic index compared with the I/R and ARI + I/ R groups (P < .05). Furthermore, such a striking decrease in epithelial apoptosis correlated with less cleaved caspase-3 protein expression in the IPo and DMSO groups in comparison with the I/ R and ARI + I/R + IPo groups (P < .05; Fig 2, I; see densitometry analysis Fig 2, J). Evaluation of intestinal mucosal AR expression. As shown in Fig 3 (densitometry analysis), the intestinal AR expression was decreased in the I/R group (P < .01 versus control and ARI + control). Moreover, when ARI was given before index ischemia, AR expression was also markedly downregulated (P < .01 versus control and ARI + control), and IPo attenuated the downregulation of intestinal AR expression (P < .05 versus I/R and ARI + I/R + IPo). DMSO did not change the AR expression level (P > .05 versus IPo). These data indicate that AR plays possibly a critical role in intestinal protection against I/R injury, and IPo diminishes this source of injury by upregulating AR expression. Correlation analysis. In groups of control, I/R, I/R+ IPo, ARI + I/R + IPo, and DMSO + I/R + IPo (n = 40), negative correlation between AR
Fig 4. Correlations between AR expression and Chiu’s scores (A), and AR expression and malondialdehyde (MDA) levels (B). AR expression was negatively correlated with Chiu’s scores (r = 0.671; P < .001). In addition, AR expression was also strongly inversely correlated with the MDA levels (r = 0.842; P < .001).
expression and Chiu’s scores existed (r = 0.671; P < .001, Fig 4, A). In addition, AR expression was also inversely correlated with MDA levels (r = 0.842; P < .001; Fig 4, B). DISCUSSION The present results showed that IPo attenuated intestinal injury, as evidenced by decreases in Chiu’s score, LD level, and serum DAO activity, as well as an increase in SOD activity and upregulated AR expression in intestinal mucosa, which are in concert with our previous studies.3,7,9 We also found that intestinal I/R significantly decreased AR expression. This result is contradictory with the findings in the rat heart that AR protein expression can be increased when subjected to ischemia or I/R ex vivo or to coronary occlusion or occlusion-reperfusion in situ and that AR upregulation can be abolished by pretreatment with Tiron, a superoxide dismutase mimetic.23 Why
= ARI + I/R and ARI + I/R + IPo groups. H, Changes in intestinal mucosal epithelial apoptosis. Representative Western blots showed increased expression of cleaved caspase-3 in the I/R and ARI + I/R + IPo groups, whereas cleaved caspase3 levels were decreased in intestinal mucosa obtained from the IPo and DMSO groups (I, J). Data are expressed as mean values ± SD (n = 8). *P < .05 and **P < .01 versus control and ARI + control; #P < .05 versus I/R and ARI + I/R + IPo. (Color version of figure is available online.)
562 Wen et al
these results are discordant with the changes of AR expression reported here are not clear, but perhaps seem to be attributable to variations in the animal model/organ, animal strain, and durations of ischemia or reperfusion. Of note, our results showed that the ARI epalrestat alone at the dose used had no toxic effects and the intestinal mucosal tissues in the epalrestat-treated control rats were all intact. Moreover, similar to the finding that epalrestat could not attenuate the myocardial injury owing to I/R in nondiabetic rat hearts,24 we for the first time also found that pretreatment with epalrestat before ischemic insult provided no salutary effects on intestinal I/R tissues, but completely abolished the well-documented protective effect of IPo. These findings demonstrated that AR inhibition resulted in redox imbalance and further exacerbated the oxidative stress-induced lipid peroxidation in the ischemic intestine. Likewise, these results suggest that simultaneously enhanced expressions of AR and endogenous antioxidant enzyme SOD after IPo provided intestinal protection by serving as an important antioxidant. AR not only catalyzes the reduction of the aldehyde form of glucose to sorbitol, but also exhibits a wide range substrate specificity for a variety of aldehydes from endogenous or exogenous origin.25 In addition, extensive studies showed that the AR gene can be induced by oxidants such as aldehydes26,27 and hydrogen peroxide,27,28 and its expression also increased 24 hours after the myocardial IPC.29 Recent studies support the view that upregulation of AR contributed to the detoxification of toxic aldehydes and modulated the survival of the cells exposed to various noxious stimuli.13,27,30 AR inhibition, however, abrogated the infarct-sparing effect of late myocardial preconditioning,29 exacerbated aldehyde toxicity in both ischemic heart23 and rat vascular smooth muscle cell lines,26 and increased vascular cell death and accumulation of toxic aldehydes during inflammation.13 Keith et al31 also found that AR protected against myocardial I/R injury, possibly by decreasing the stress in the endoplasmic reticulum by removing aldehydic products of lipid peroxidation. In this regard, AR is thought to function as an oxidative defense enzyme and may be involved in cellular defense against toxic aldehydes. Interestingly, AR protects the heart from I/R injury only during the late stages of ischemia and during reperfusion.32 Therefore, it is reasonable to postulate that the postconditioning-induced AR activation during the early stages of reperfusion is required for subsequent protection. In addition, negative correlations between AR expression and Chiu’s score as well as between AR expression
Surgery April 2013
and MDA level indicate that AR could be among the major contributors able to ameliorate intestinal I/R injury in this model. As such, we propose that upregulation of AR seems to represent an adaptive response that removes toxic aldehydes (such as MDA) generated in the ischemic intestine and contributes to an endogenous and complex self-defense mechanism, ensuring an increased cellular resistance of intestine against toxic injury. Although the detailed molecular mechanism remains unclarified, extensive data showed that AR catalyzes reduction of phospholipid-derived aldehydes and their glutathione conjugates and regulates the proinflammatory and immunogenic effects of oxidized phospholipids.23,33 This observation supports the concept that the AR level in the intestinal mucosa can be upregulated by IPo, and that the AR-mediated intestinal protective effect of IPo may be related to more efficient removal of toxic lipid peroxidation products triggered by oxidative stress. Several studies demonstrated that an increase in AR activity and expression via the PI3K/Akt/eNOS pathway provides cardioprotection against I/R injury,32,34,35 and ARIs decreased the postischemic recovery in the rat hearts subjected to global ischemia and increased the infarct size when given before ischemia or on reperfusion.32 Meanwhile, activation of the PI3K/Akt-linked pathway is involved in the cardioprotection,36,37 neuroprotection5,38,39 of IPo, and gastric protection40 of genistein postconditioning after I/R injury. Altogether, we proposed that intestinal IPo-mediated upregulation of AR and the subsequent intestinal protection seen in the present study might have been achieved in part via activation of PI3K/Akt pathway. However, the precise mechanisms related to intracellular signaling needs to be further elucidated. In apparent contradiction to these findings and those reported by Calderone et al,24 several studies have concluded that pharmacologic inhibition of AR decrease I/R injury in isolated hearts41,42 and retina,43 and mitigates inflammatory diseases like allergic asthma,44 whereas overexpression of AR promotes atherogenesis in apoE-null mice.45 Different from our research, Ramasamy et al41 showed that nitric oxide synthase 2 inhibitors, not a specific ARI, decreased AR activation and exerted their cardioprotective effects by inhibiting the polyol pathway. The disparities between our findings and the above reports may be related to the inconsistent and uncertain effects of ARIs on different organs or tissues, which could be associated with several reasons, namely (1) differences in organs or tissues as well as animal models (durations of
Wen et al 563
Surgery Volume 153, Number 4
ischemia or reperfusion), (2) the specificity and selectivity of ARIs in different tissues or organs, (3) because glucose is not a preferred substrate for AR rather than aldehydes,10 the importance of the polyol pathway may be limited under normoglycemic conditions because only a minor part of nonphosphorylatyed glucose enters the polyol pathway, and (4) different posttranslational modifications of AR,23,32 which modulate both substrate and inhibitor binding. The current study is the first to find that IPo can attenuate intestinal mucosal cell apoptosis. Because oxidative stress and inflammatory reaction in the gastrointestinal tract induce apoptosis,11,46 and IPo can partly abrogate the above pathophysiologic phenomenon induced by intestinal I/R, it is likely that IPo suppressed the intestinal mucosal apoptosis seen in the present study. Work in vasculitis showed that AR inhibition increased the lipid peroxidation products and led to increased cell apoptosis.13 Moreover, Kang et al found that activation of PI3K and p38 MAPK by curcumin upregulated the expression of AR gene, inhibition of enzyme activity, or gene silencing of AR markedly augmented the glucose oxidase-mediated apoptosis,47 and that overexpression of AR suppressed the increase in intracellular ROS and attenuated the apoptotic cell death induced by ultraviolet B radiation.14 In our study, pretreatment with ARI completely abolished the effect of IPo on apoptosis, indicating that apoptosis suppression by AR plays, at least in part, an important role in the intestinal protection conferred by IPo. The current study focused on elucidating the role of AR by employing its specific inhibitor. Whether AR is specific for the intestinal IPo effect is not clear, and further studies utilizing AR gene knock-out models or employing the RNA interference methodologies are needed to confirm the role of AR in intestinal IPo against I/R injury. Moreover, the possibility of off-target effects by epalrestat should be considered, and whether epalrestat can inhibit other enzymes involved in providing protection against redox stress deserves further investigations. Nevertheless, the findings from the present study provide new clues for understanding the molecular mechanisms of intestinal I/R injury and the mechanisms whereby IPo induces beneficial effects. S.-H.W. and Y.-H.L. contributed equally to this study. This work was financially supported by grants from National Natural Science Foundation of China (Grant Numbers: 81270456, 81171847, to Ke-Xuan Liu), partly by ‘‘the Fundamental Research Funds for the Central Universities’’ in China and China Education program
for New Century Excellent Talents of Ministry of Education (NCET-10-0853, to Ke-Xuan Liu), partly by a grant from Natural Science Foundation of Guangdong Province (no. S2011020002332, to Ke-Xuan Liu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Professor Wanlong Lei (Department of Anatomy and Brain Research, Sun Yat-sen University, Guangdong, China) and Professor Weikang Wu (Department of Pathophysiology, Sun Yat-sen University, Guangdong, China) for their technical assistance. REFERENCES 1. Mallick IH, Yang W, Winslet MC, Seifalian AM. Ischemia-reperfusion injury of the intestine and protective strategies against injury. Dig Dis Sci 2004;49:1359-77. 2. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36. 3. Liu KX, Li C, Li YS, Yuan BL, Xu M, Xia Z, et al. Proteomic analysis of intestinal ischemia/reperfusion injury and ischemic preconditioning in rats reveals the protective role of aldose reductase. Proteomics 2010;10:4463-75. 4. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:579-88. 5. Rehni AK, Singh N. Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked behavioral deficits in mice. Pharmacol Rep 2007;59:192-8. 6. Jiang B, Liu X, Chen H, Liu D, Kuang Y, Xing B, et al. Ischemic postconditioning attenuates renal ischemic/reperfusion injury in mongrel dogs. Urology 2010;76:1519.e1-7. 7. Liu KX, Li YS, Huang WQ, Chen SQ, Wang ZX, Liu JX, et al. Immediate postconditioning during reperfusion attenuates intestinal injury. Intensive Care Med 2009;35:933-42. 8. Liu KX, Li YS, Huang WQ, Li C, Liu JX, Li Y. Immediate but not delayed postconditioning during reperfusion attenuates acute lung injury induced by intestinal ischemia/reperfusion in rats: comparison with ischemic preconditioning. J Surg Res 2009;157:e55-62. 9. Li YS, Wang ZX, Li C, Xu M, Li Y, Huang WQ, et al. Proteomics of ischemia/reperfusion injury in rat intestine with and without ischemic postconditioning. J Surg Res 2010; 164:e173-80. 10. Srivastava S, Watowich SJ, Petrash JM, Srivastava SK, Bhatnagar A. Structural and kinetic determinants of aldehyde reduction by aldose reductase. Biochemistry 1999;38:42-54. 11. Liu KX, Chen SQ, Huang WQ, Li YS, Irwin MG, Xia Z. Propofol pretreatment reduces ceramide production and attenuates intestinal mucosal apoptosis induced by intestinal ischemia/reperfusion in rats. Anesth Analg 2008;107: 1884-91. 12. 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. 13. Rittner HL, Hafner V, Klimiuk PA, Szweda LI, Goronzy JJ, Weyand CM. Aldose reductase functions as a detoxification
564 Wen et al
14.
15.
16.
17.
18.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
system for lipid peroxidation products in vasculitis. J Clin Invest 1999;103:1007-13. Kang ES, Iwata K, Ikami K, Ham SA, Kim HJ, Chang KC, et al. Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation. Free Radic Biol Med 2010;50:680-8. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg 1970;101:478-83. Liu KX, Rinne T, He W, Wang F, Xia Z. Propofol attenuates intestinal mucosa injury induced by intestinal ischemiareperfusion in the rat. Can J Anaesth 2007;54:366-74. Tsunooka N, Maeyama K, Nakagawa H, Doi T, Horiuchi A, Miyauchi K, et al. Localization and changes of diamine oxidase during cardiopulmonary bypass in rabbits. J Surg Res 2006;131:58-63. Liu KX, He W, Rinne T, Liu Y, Zhao MQ, Wu WK. The effect of Ginkgo biloba extract (EGb 761) pretreatment on intestinal epithelial apoptosis induced by intestinal ischemia/reperfusion in rats: role of ceramide. Am J Chin Med 2007;35:805-19. Cuzzocrea S, Mazzon E, Esposito E, Muia C, Abdelrahman M, Di Paola R, et al. Glycogen synthase kinase-3 beta inhibition attenuates the development of ischaemia/reperfusion injury of the gut. Intensive Care Med 2007;33:880-93. Vejchapipat P, Williams SR, Spitz L, Pierro A. Intestinal metabolism after ischemia-reperfusion. J Pediatr Surg 2000;35:759-64. Wolvekamp MC, de Bruin RW. Diamine oxidase: an overview of historical, biochemical and functional aspects. Dig Dis 1994;12:2-14. Naito Y, Takagi T, Uchiyama K, Handa O, Tomatsuri N, Imamoto E, et al. Suppression of intestinal ischemia-reperfusion injury by a specific peroxisome proliferator-activated receptorgamma ligand, pioglitazone, in rats. Redox Rep 2002;7:294-9. Kaiserova K, Srivastava S, Hoetker JD, Awe SO, Tang XL, Cai J, et al. Redox activation of aldose reductase in the ischemic heart. J Biol Chem 2006;281:15110-20. Calderone V, Testai L, Martelli A, La Motta C, Sartini S, Da Settimo F, et al. Anti-ischaemic activity of an antioxidant aldose reductase inhibitor on diabetic and non-diabetic rat hearts. J Pharm Pharmacol 2010;62:107-13. Yabe-Nishimura C. Aldose reductase in glucose toxicity: a potential target for the prevention of diabetic complications. Pharmacol Rev 1998;50:21-33. Ruef J, Liu SQ, Bode C, Tocchi M, Srivastava S, Runge MS, et al. Involvement of aldose reductase in vascular smooth muscle cell growth and lesion formation after arterial injury. Arterioscler Thromb Vasc Biol 2000;20:1745-52. Spycher SE, Tabataba-Vakili S, O’Donnell VB, Palomba L, Azzi A. Aldose reductase induction: a novel response to oxidative stress of smooth muscle cells. FASEB J 1997;11:181-8. Keightley JA, Shang L, Kinter M. Proteomic analysis of oxidative stress-resistant cells: a specific role for aldose reductase overexpression in cytoprotection. Mol Cell Proteomics 2004;3:167-75. Shinmura K, Bolli R, Liu SQ, Tang XL, Kodani E, Xuan YT, et al. Aldose reductase is an obligatory mediator of the late phase of ischemic preconditioning. Circ Res 2002;91:240-6. Chang KC, Paek KS, Kim HJ, Lee YS, Yabe-Nishimura C, Seo HG. Substrate-induced up-regulation of aldose reductase by methylglyoxal, a reactive oxoaldehyde elevated in diabetes. Mol Pharmacol 2002;61:1184-91. Keith RJ, Haberzettl P, Vladykovskaya E, Hill BG, Kaiserova K, Barski O, et al. Aldose reductase decreases endoplasmic reticulum stress in ischemic hearts. Chem Biol Interact 2009;178:242-9.
Surgery April 2013
32. Kaiserova K, Tang XL, Srivastava S, Bhatnagar A. Role of nitric oxide in regulating aldose reductase activation in the ischemic heart. J Biol Chem 2008;283:9101-12. 33. Srivastava S, Spite M, Trent JO, West MB, Ahmed Y, Bhatnagar A. Aldose reductase-catalyzed reduction of aldehyde phospholipids. J Biol Chem 2004;279:53395-406. 34. Bell RM, Yellon DM. Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. J Mol Cell Cardiol 2003;35:185-93. 35. Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation 2001;104:330-5. 36. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of ‘‘modified reperfusion’’ protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res 2004;95:230-2. 37. Chiari PC, Bienengraeber MW, Pagel PS, Krolikowski JG, Kersten JR, Warltier DC. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits. Anesthesiology 2005;102:102-9. 38. Gao X, Zhang H, Takahashi T, Hsieh J, Liao J, Steinberg GK, et al. The Akt signaling pathway contributes to postconditioning’s protection against stroke; the protection is associated with the MAPK and PKC pathways. J Neurochem 2008;105:943-55. 39. Jiang X, Ai C, Shi E, Nakajima Y, Ma H. Neuroprotection against spinal cord ischemia-reperfusion injury induced by different ischemic postconditioning methods: roles of phosphatidylinositol 3-kinase-Akt and extracellular signalregulated kinase. Anesthesiology 2009;111:1197-205. 40. Du DS, Ma XB, Zhang JF, Zhou XY, Li Y, Zhang YM, et al. The protective effect of capsaicin receptor-mediated genistein postconditioning on gastric ischemia-reperfusion injury in rats. Did Dis Sci 2010;55:3070-7. 41. Ramasamy R, Hwang YC, Liu Y, Son NH, Ma N, Parkinson J, et al. Metabolic and functional protection by selective inhibition of nitric oxide synthase 2 during ischemia-reperfusion in isolated perfused hearts. Circulation 2004;109:1668-73. 42. Hwang YC, Shaw S, Kaneko M, Redd H, Marrero MB, Ramasamy R. Aldose reductase pathway mediates JAK-STAT signaling: a novel axis in myocardial ischemic injury. FASEB J 2005;19:795-7. 43. Obrosova IG, Maksimchyk Y, Pacher P, Agardh E, Smith ML, EI-Remessy AB, et al. Evaluation of the aldose reductase inhibitor fidarestat on ischemia-reperfusion injury in rat retina. Int J Mol Med 2010;26:135-42. 44. Yadav UC, Ramana KV, Aguilera-Aguirre L, Boldogh I, Boulares HA, Srivastava SK. Inhibition of aldose reductase prevents experimental allergic airway inflammation in mice. PLoS One 2009;4:e6535. 45. Vikramadithyan RK, Hu Y, Noh HL, Liang CP, Hallam K, Tall AR, et al. Human aldose reductase expression accelerates diabetic atherosclerosis in transgenic mice. J Clin Invest 2005;115:2434-43. 46. Calvino-Fernandez M, Benito-Martinez S, Parra-Cid T. Oxidative stress by Helicobacter pylori causes apoptosis through mitochondrial pathway in gastric epithelial cells. Apoptosis 2008;13:1267-80. 47. Kang ES, Woo IS, Kim HJ, Eun SY, Paek KS, Kim HJ, et al. Up-regulation of aldose reductase expression mediated by phosphatidylinositol 3-kinase/Akt and Nrf2 is involved in the protective effect of curcumin against oxidative damage. Free Radic Biol Med 2007;43:535-45.