Just Accepted by Free Radical Research
Role of 15-F2t-isoprostane in Intestinal Injury Induced by Intestinal Ischemia/Reperfusion in Rats Shi-Hong Wen, Yi-Hong Ling, Wei-Feng Liu, Yi-Xin Qiu, Yun-Sheng Li, Yan Wu, Jian-Tong Shen, Zheng-Yuan Xia, Ke-Xuan Liu
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doi: 10.3109/10715762.2014.926010 Abstract 15-F2t-isoprostane is not only a specific marker of lipid peroxidation but also demonstrated to have potent bioactivities and can exert deleterious effects via activating thromboxane A2 receptor (TxA2r). We already demonstrated that lipid peroxidation represents a mechanism of intestinal ischemia/reperfusion (I/R) injury. But no studies have focused on 15-F2t-isoprostane production and its biological actions on postischemic intestine during intestinal I/R. This study was carried to investigate whether the mechanism of endogenous 15-F2t-isoprostane action is involved in the pathogenesis of intestinal ischemia/reperfusion (I/R) and administration of synthetic 15-F2t-isoprostane could exacerbate intestinal insult after intestinal I/R in vivo and in vitro. In comparison with sham control, we reported that endogenous 15-F2t-isoprostane was liberated following intestinal I/R injury in rats, and using the TxA2r antagonist SQ29548 resulted in significant intestinal protection, evidenced by reduced lipid peroxidation, inflammation and alleviated intestinal mucosal microvascular vasoconstriction. Further research found that in vivo administration of synthetic 15-F2t-isoprostane exacerbated intestinal I/R injury by disturbing microvascular perfusion and accumulating anaerobic metabolism. Meanwhile, 15-F2t-isoprostane did not change Hypoxia/ Reoxygenation-induced IEC-6 cell viability but aggravated HUVECs cell death in vitro. Collectively, our results showed that locally produced 15-F2t-isoprostane was in proportion to the severity of oxidative stress-induced intestinal injury and its detrimental effects can be attenuated through TxA2r inactivation. Exogenous 15-F2t-isoprostane exacerbated intestinal I/R injury, which may be contributable to its biological actions on endothelium, rather than intestinal epithelium.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Role of 15-F2t-isoprostane in Intestinal Injury Induced by Intestinal Ischemia/Reperfusion in Rats
Jian-Tong Shen,* Zheng-Yuan Xia,$ Ke-Xuan Liu*
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Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University,
Guangzhou 510080, China; †Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China; §Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou $
Anesthesiology Research Laboratory, Department of Anesthesiology,
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510060, China;
University of Hong Kong, Hong Kong, China
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Corresponding author: Ke-Xuan Liu, Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan 2nd Road, Guangzhou 510080, China. Tel: +86 20-87755766-8273; E-mail: [email protected] The authors contributed equally to this study
Short title: Role of 15-F2t-Isop in intestinal ischemia/reperfusion
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*
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Shi-Hong Wen,*,1 Yi-Hong Ling,†,§ Wei-Feng Liu,* Yi-Xin Qiu,* Yun-Sheng Li,* Yan Wu,*
Abstract
15-F2t-isoprostane is not only a specific marker of lipid peroxidation but also demonstrated to
have potent bioactivities and can exert deleterious effects via activating thromboxane A2 receptor (TxA2r). We already demonstrated that lipid peroxidation represents a mechanism of intestinal ischemia/reperfusion (I/R) injury. But no studies have focused on 15-F2t-isoprostane
production and its biological actions on postischemic intestine during intestinal I/R. This study was carried to investigate whether the mechanism of endogenous 15-F2t-isoprostane 1
action is involved in the pathogenesis of intestinal ischemia/reperfusion (I/R) and administration of synthetic 15-F2t-isoprostane could exacerbate intestinal insult after intestinal I/R in vivo and in vitro. In comparison with sham control, we reported that endogenous
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15-F2t-isoprostane was liberated following intestinal I/R injury in rats, and using the TxA2r
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peroxidation, inflammation and alleviated intestinal mucosal microvascular vasoconstriction. Further research found that in vivo administration of synthetic 15-F2t-isoprostane exacerbated intestinal I/R injury by disturbing microvascular perfusion and accumulating anaerobic
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metabolism. Meanwhile, 15-F2t-isoprostane did not change Hypoxia/Reoxygenation-induced IEC-6 cell viability but aggravated HUVECs cell death in vitro. Collectively, our results showed that locally produced 15-F2t-isoprostane was in proportion to the severity of oxidative
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stress-induced intestinal injury and its detrimental effects can be attenuated through TxA2r inactivation. Exogenous 15-F2t-isoprostane exacerbated intestinal I/R injury, which may be contributable to its biological actions on endothelium, rather than intestinal epithelium.
Key words: 15-F2t-isoprostane; Ischemia/reperfusion injury; SQ29548; Intestine;
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antagonist SQ29548 resulted in significant intestinal protection, evidenced by reduced lipid
Hypoxia/Reoxygenation
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Introduction Major surgery is often associated with ischemia/reperfusion (I/R) injury and subsequent inflammatory response in clinical scenarios. Meanwhile, inflammation itself stimulates the
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generation of free radicals and creates a vicious circle of oxidative stress, which plays a
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systemic inflammatory response syndrome (SIRS), sepsis and multiple organ dysfunction syndrome (MODS) (1).
Intestinal I/R is a challenging clinical syndrome arising in critically ill patients and also
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due to certain surgery, such as small bowel transplantation, abdominal aneurysm repair, cardiopulmonary bypass, etc (2). Because of the often nonspecific clinical presentation and the continued difficulty in recognizing the condition before intestinal necrosis occurs, the
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in-hospital mortality rates remained high over the last few decades, ranging between 67 and 80%, and the reported incidence of intestinal I/R injury is increasing (3,4). Previous studies have shown that re-introduction of oxygen to the ischemic intestine leads to the release of proinflammatory factors and bacteria-derived endotoxin, which may
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pivotal role in the pathogenesis of postoperative complications, sometimes leading to
instigate the damage of cellular lipids, proteins and DNA and can overwhelm endogenous antioxidant defenses (5). Furthermore, irreversible membrane and cellular dysfunction secondary to extensive lipid peroxidation and, subsequently, organ injury or failure may ensue. Arachidonic acid is a complex lipid macromolecule of cell membrane, which is pivotal to inflammatory and coagulation cascades. A recent advance in free radical biology has been the discovery of isoprostanes, which are formed by the free radical-catalyzed peroxidation of arachidonic acid independent of cyclo-oxygenase activity. Among these compounds, most
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attention has been focused on the 15-series, which are the most abundant in vivo (6). Of the variety of isoprostanes detected, 15-F2t-isoprostane (15-F2t-Isop, also named isoprostane F2α-III or iPF2α-III, 8-iso-PGF2α) has been found to be the most reliable and reproducible
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marker of oxidative stress (7). And the measurement of 15-F2t-Isop provides a mean to assess
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free radical or I/R injury. Interestingly, next to being accurate markers of oxidant injury and inflammation, 15-F2t-Isop appears to possess important biological effects at pharmacological and physiological concentrations, including constriction of several vascular beds (8,9),
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increased vascular permeability (10), and nociceptor sensitization (11), as well as smooth muscle constriction of the uterine, gastrointestinal and tracheobronchial systems (12). Till now, seldom studies have been focused on the production of 15-F2t-Isop during the process of
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intestinal I/R, and its biological actions on postischemic intestine remain unknown. Evidence from various studies indicates that the effects of 15-F2t-Isop are mediated through the activation of receptors analogous to those for thromboxane A2 (TP receptor) (13-16). And using the specific thromboxane A2 receptor (TxA2r) antagonist SQ29548 can
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the effects of prophylactic and therapeutic interventions, such as antioxidants, to attenuate
largely abrogate most of its biological actions. Although 15-F2t-Isop was known to possess
potent bioactivity under pathophysiological conditions, it remains unclear whether 15-F2t-Isop,
by itself, can be liberated following intestinal I/R and is one of the mediators operating in the complex network of biological messages evoked by intestinal oxidative injury. To this end, we aimed to investigate whether the mechanism of endogenous 15-F2t-Isop action is involved in the pathogenesis of intestinal I/R and administration of synthetic 15-F2t-Isop could exacerbate intestinal insult after intestinal I/R in vivo and in vitro.
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Materials and Methods In vivo Animals and model preparation
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The study was approved by the Committee of Animal Care of Sun Yat-sen University,
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guidelines. Adult pathogen-free male Sprague-Dawley rats weighing 220-240 g were housed in climate-controlled conditions and provided with standard rat chow. All animals were fasted overnight before the experiment but had free access to water ad libitum. Rats were
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anesthetized with 20% urethane (induction and maintenance, 6 ml kg-1, intraperitoneally), tracheostomized and mechanically ventilated with a standard tidal volume ventilation protocol (control mode; tidal volume 8ml/kg; respiratory rate 50 breaths/min and 100% oxygen). An
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arterial catheter was placed into the left carotid artery and connected to a digital data acquisition system (PowerLab/4SP ADI Instruments, Ugo Basile Comerio, VA, Italy) for constant measurement of the mean arterial pressure (MAP). The small intestine was exteriorized by midline laparotomty. The intestinal I/R injury was established by occluding
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China. The experimental animals were cared in accordance with National Institutes of Health
the superior mesenteric artery (SMA) with a microvessel clip for 60 min followed by 120 min reperfusion. SMA was isolated from superior mesenteric vein (SMV) before occlusion. Ischemia was recognized by immediate blanching of the small instine and cecum. The return of pulses and the re-establishment of the pink color were assumed to indicate valid reperfusion of the intestine. During the experiments, normothermia (36-38°C) was maintained with heating pads.
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Experiment 1: To investigate whether locally produced 15-F2t-Isop contributes to the progression of intestinal I/R Experimental groups and drug administration
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The rats were block-randomized assigned to one of four groups based on the
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including isolation of the SMA without occlusion. (2) I/R group (I/R): SMA occlusion for 60 min followed by 120 min reperfusion. (3) TxA2 receptor-specific (TP) antagonist SQ29548 ((1S-(1a,2a(Z),3a,4a))-7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.
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1)hept-2-yl)-5-heptanoic acid, Cayman Chemical, Ann Arbor, MI, 19025) treatment group (I/R-SQ29548). (4) DMSO (Dimethyl Sulfoxide) group (I/R-DMSO): in which DMSO was the solvent of SQ29548 and served as vehicle control. SQ29548 (dissolved in DMSO to
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2μmol/ml; 2μmol/kg) or DMSO (1ml/kg) was given subcutaneously 30 min before intestinal ischemia. The dose of SQ29548 was chosen based on the previous study (15).
Experiment 2: To investigate whether synthetic 15-F2t-Isop exacerbates intestinal insult
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interventions (n=8 each, Fig. 1A): (1) Control group (Control): sham surgical preparation
after intestinal I/R.
Experimental groups and drug administration In a separate set of experiments (shown in Fig. 1B), the rats were divided into the
following four groups (n=8 each): (1) Control + DMSO group (Control + DMSO): sham surgical preparation including isolation of the SMA without occlusion. (2) Control + 15-F2t-Isop group (Control +15-F2t-Isop). (3) I/R + DMSO group (I/R + DMSO). (4) I/R +
15-F2t-Isop group (I/R + 15-F2t-Isop). DMSO was the solvent of 15-F2t-Isop (Cayman
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Chemical, Ann Arbor, MI, 16350) and served as vehicle control. 1μg/kg/min 15-F2t-Isop was administered intravenously over a period of 10 min through a pump (Harvard Apparatus, Holliston, MA) after 60 min of sham operation or immediately at the onset of reperfusion.
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Preparation of specimens
At the end of the experiments, blood samples were collected from the heart of rat and centrifuged at 3,500 rpm for 15 minutes at 4 , the supernatant was used for subsequent
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measurements of 15-F2t-Isoprostane, endothelin-1 (ET-1) and thromboxane B2 (TxB2, the stable metabolite of thromboxane A2) concentrations and diamine oxidase (DAO) activity. And then, a segment of 10cm intestine was cut 5 cm away from the ileocecal valve, and was
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divided into two segments. The segments are, respectively, fixed in 10% neutral formaldehyde and paraffin embedded for morphological analysis, washed with cold saline and after being scraped off, intestinal mucosa was dried with filter paper and preserved at -80
for
determination of lactic acid (LD), malondialdehyde (MDA), superoxide dismutase (SOD) and
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The dose of 15-F2t-Isop infusion was chosen based on the previous studies (13, 17).
myeloperoxidase (MPO).
Plasma 15-F2t-Isop assays
Enzyme immunoassay (EIA) of free 15-F2t-Isop was performed with a commercially
available kit (Cayman Chemical, Ann Arbor, MI) according to the methods provided by the manufacturer. The values of the unknown were expressed as pg/ml 15-F2t-Isop of plasma.
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Histopathology measurement of intestinal mucosa The segment of small intestine was stained with hematoxylin-eosin for histological assessment. Two independent pathologists blinded to treatment group evaluated histological
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damage of specimens and assigned a grade based on the Chiu’s classification of intestinal
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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
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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,
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hemorrhage, and ulceration. A minimum of five randomly chosen fields from each rat were evaluated and averaged to determine mucosal damage.
Detection of LD level in intestinal mucosa and plasma DAO activity
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injury (18). In brief, mucosal damage was graded from 0 (normal) to 5 (severely damaged) as
Intestinal mucosal tissues were weighed and made into 10% homogenate. The LD content
in tissues was determined as previously described (19).The results were expressed as nmol/mg protein. In order to further confirm intestinal injury, DAO, a sensitive marker reflecting small intestinal mucosal injury (20), was detected using a chemical assay kit according to the provided protocol of the manufacturer. The above results were expressed as nmol/mg protein and U/L plasma, respectively.
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Detection of MDA level and SOD activity in intestinal mucosa Intestinal mucosal tissues were homogenized on ice with normal saline, frozen in refrigerator at -20 ℃ for 5 min and centrifuged for 15 min at 4 000 rpm. Supernatants were
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transferred into fresh tubes for the evaluation. The fatty-acid peroxidation product MDA and
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expressed as nmol/mg protein and U/mg protein, respectively.
Detection of MPO activity in intestinal mucosa
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Intestinal mucosal samples were homogenized and the homogenate was freeze-thawed twice, and then centrifuged at 13,000 rpm for 5 min. The resulting supernatant was assayed spectrophotometrically for MPO activity as previously described (22). One unit of MPO was
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defined as the capacity to degrade 1 μmol peroxide per minute at 25 ℃. Results were expressed as U/g intestinal tissue.
Plasma ET-1 and TxB2 assays
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the SOD activity were measured as previously described (21). The above results were
ET-1 and TxB2 levels in plasma were performed by EIA kits (R&D Systems,
Minneapolis, USA) according to the manufacturer’s instruction. They were determined using a standard curve generated from known concentrations of ET-1 and TxB2. All measurements were performed in duplicate, and the intra- and interassay variability were < 10%. The values of the unknown were all expressed as pg/ml of plasma.
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In vitro IEC-6 cells and HUVEC cells culture Intestinal epithelial cells (IEC-6, Catalog No. RL-1592) were obtained from American
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Type Culture Collection (ATCC, Manassas, VA, USA) and were culture in Dulbecco’s
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4.5g/L D-glucose and 1% penicillin/ streptomycin antibiotics (Gibco, Invitrogen Ltd, Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were used at
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passages 2–12 to ensure genetic stability. HUVECs were cultured in RMPI-1640 medium containing 10% v/v FBS and 1% penicillin/streptomycin antibiotics (Gibco, Invitrogen Ltd, Shanghai, China) The above two cells were maintained under standard cell culture conditions with a humid environment of 21% O2 and 5% CO2. They were used soon after
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at 37
reaching 80% confluence.
Hypoxia/Reoxygenation injury of cultured IEC-6 and HUVECs
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modified Eagle’s medium (DMEM), supplemented with 10% v/v fetal bovine serum (FBS),
Hypoxia/Reoxygenation (H/R) injury of cells was induced employing a glucose oxidase
(2 U/ml, Sigma-Aldrich, G6125) and catalase (120 U/ml, Sigma-Aldrich, C1345) based two-enzyme system (GOX/CAT system) first described by Baunman et al. in 2008 (23). In
this enzymatic model, depletion of oxygen is achieved rapidly within minutes, representing the situation of intestinal ischemia in vivo. Briefly, cells were grown under normoxic conditions up to a confluence of 80%. Hypoxia was induced by exchanging the normoxic medium to hypoxic medium. After a 1 h hypoxia period, medium was changed back to
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normoxic medium and cells were cultured under the described conditions for an additional 2 h (Reoxygenation). Control experiments were performed by omitting hypoxia and incubating
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the cells for 3 h under normoxic conditions.
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For dose-response assays and to determine whether the administration of synthetic 15-F2t-Isop leads to the damage of IEC-6 and HUVECs under the normal condition, cells were incubated with different concentrations (10-10 to 10-5 mol/L) of 15-F2t-Isop or vehicle for
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24 h (Fig. 2A), subsequently, to test whether 15-F2t-Isop (10-10 to 10-5 mol/L) or vehicle administrated immediately at the time of reoxygenation exacerbates IEC-6 and HUVECs H/R injury (Fig. 2B). DMSO was the solvent of 15-F2t-Isop and at the concentrations used was
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without any effect on cell viability.
Cell viability assessment
Cell viability was determined using MTT assay. Briefly, 1×105 cells were plated into
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Study groups and experimental protocol
each well in 96-well tissue culture plates. After H/R or 15-F2t-Isop incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide, 5 mg/ml in Phosphate buffered saline, Sigma) was added to each well and incubated at 37
for 4h. After careful
removal of the medium, 0.15ml buffered DMSO was added to each well. The absorbance was recorded on microplate reader at the wavelength of 560nm. All experiments were repeated a minimum of three times.
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Statistical analysis Data were expressed as mean ± standard deviation (SD). Biochemical markers were performed in duplicate or triplicate for each specific sample. Therefore, all the data points are
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means of duplicate or triplicate measurements for these parameters. One-way ANOVA and the
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test failed, Kruskal-Wallis test was performed. Correlation between different variables was assessed by Pearson or Spearman coefficient (SPSS 13.0，SPSS Inc., Chicago, USA). A
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P 0.05 vs. the I/R + DMSO
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groups)
2. 15-F2t-Isop is liberated following intestinal I/R injury As shown in Fig. 4, locally produced plasma 15-F2t-Isop were significantly increased 2 h
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during the experiments did not differ among groups (data not shown, P > 0.1). As Fig. 3A
after reperfusion in the I/R and I/R-DMSO groups when compared with the Control group (all P 0.05 vs. the I/R and I/R-DMSO groups).
3. Effect of SQ29548 on attenuating intestinal I/R injury Representative mucosal morphological changes are presented in Figs. 5A-D. In the
Control group (Fig. 5A), normal villi were seen as expected. By contrast, I/R and I/R-DMSO
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groups (Figs. 5B and 5D) animals resulted in severe edema of mucosal villi and infiltration of necrotic epithelial and inflammatory cells, and intestinal glands showed evidence of severe injury. However, in the I/R-SQ29548 group (Fig. 5C), slight edema could be seen in the
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intestinal villi, and some intestinal villi were severed, and the gap between epithelial cells
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I/R-DMSO groups were significantly higher than that in the Control group (all P < 0.01, Fig. 5E). However, Chiu’s score in I/R-SQ29548 group was markedly lower when compared with the I/R and I/R-DMSO groups (P < 0.05). A weak but statistically significant positive
Chiu’s scores.
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correlation (r = 0.57, P < 0.01; Fig. 5F) was noted between concentrations of 15-F2t-Isop and
LD and DAO are used as markers of intestinal cellular injury described in our previous
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researches (24). As shown in Fig. 6, intestinal mucosal LD level (A) and DAO activity (B) in the I/R and I/R-DMSO groups were significantly higher than that in the Control group (all P < 0.05). However, LD level and DAO activity were markedly reduced by SQ29548 (all P < 0.05 vs. the I/R and I/R-DMSO groups).
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increased slightly. Consistent with histological changes, Chiu’s scores in the I/R and
4. SQ29548 reduces lipid peroxidation and inflammation in intestinal I/R injury At 2 h after reperfusion, rats pretreated with SQ29548 had significantly decreased
concentrations of MDA (A) and MPO (C) activity whereas SOD (B) activity in intestinal mucosa homogenates was increased (all P < 0.05 vs. the I/R and I/R-DMSO groups, Fig. 7).
5. SQ29548 alleviates intestinal mucosal microvascular vasoconstriction
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Plasma ET-1 levels in the I/R and I/R-DMSO groups were significantly higher than that in the Control group (P < 0.05). However, SQ29548 pretreatment markedly reduced the production of ET-1 (P < 0.05 vs. the I/R and I/R-DMSO groups, Fig. 8A). After 2 h
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reperfusion, TxB2 concentrations in the I/R, DMSO and I/R-SQ29548 groups were
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differences in TxB2 levels among the I/R, I/R-DMSO and I/R-SQ29548 groups (P > 0.05, Fig. 8B). Meanwhile, plasma 15-F2t-Isop concentrations correlated significantly with ET-1 and
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TxB2 levels (r=0.74 and r=0.78, both P 0.05). Meanwhile, intestinal mucosal LD levels and plasma DAO activity in the I/R+DMSO
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and I/R+15-F2t-Isop groups were significantly increased after 2 h reperfusion (P < 0.05 vs. the Control+DMSO and Control+15-F2t-IsoP groups, Figs. 9F and 9G). What’s more, LD levels in the I/R+15-F2t-Isop group were markedly higher than that in the I/R+DMSO group (P0.05, Fig. 9G).
Cell viability decreased significantly after 1 h of hypoxia and 2 h of reoxygenation in
IEC-6 and HUVECs. In contrast, after administration of synthetic 15-F2t-Isop at 10-5 mol/L immediately at the time of reoxygenation, HUVECs cell death was significantly increased when compared with the H/R group (P
Role of 15-F2t-isoprostane in Intestinal Injury Induced by Intestinal Ischemia/Reperfusion in Rats Shi-Hong Wen, Yi-Hong Ling, Wei-Feng Liu, Yi-Xin Qiu, Yun-Sheng Li, Yan Wu, Jian-Tong Shen, Zheng-Yuan Xia, Ke-Xuan Liu
Free Radic Res Downloaded from informahealthcare.com by Washington University Library on 05/27/14 For personal use only.
doi: 10.3109/10715762.2014.926010 Abstract 15-F2t-isoprostane is not only a specific marker of lipid peroxidation but also demonstrated to have potent bioactivities and can exert deleterious effects via activating thromboxane A2 receptor (TxA2r). We already demonstrated that lipid peroxidation represents a mechanism of intestinal ischemia/reperfusion (I/R) injury. But no studies have focused on 15-F2t-isoprostane production and its biological actions on postischemic intestine during intestinal I/R. This study was carried to investigate whether the mechanism of endogenous 15-F2t-isoprostane action is involved in the pathogenesis of intestinal ischemia/reperfusion (I/R) and administration of synthetic 15-F2t-isoprostane could exacerbate intestinal insult after intestinal I/R in vivo and in vitro. In comparison with sham control, we reported that endogenous 15-F2t-isoprostane was liberated following intestinal I/R injury in rats, and using the TxA2r antagonist SQ29548 resulted in significant intestinal protection, evidenced by reduced lipid peroxidation, inflammation and alleviated intestinal mucosal microvascular vasoconstriction. Further research found that in vivo administration of synthetic 15-F2t-isoprostane exacerbated intestinal I/R injury by disturbing microvascular perfusion and accumulating anaerobic metabolism. Meanwhile, 15-F2t-isoprostane did not change Hypoxia/ Reoxygenation-induced IEC-6 cell viability but aggravated HUVECs cell death in vitro. Collectively, our results showed that locally produced 15-F2t-isoprostane was in proportion to the severity of oxidative stress-induced intestinal injury and its detrimental effects can be attenuated through TxA2r inactivation. Exogenous 15-F2t-isoprostane exacerbated intestinal I/R injury, which may be contributable to its biological actions on endothelium, rather than intestinal epithelium.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Role of 15-F2t-isoprostane in Intestinal Injury Induced by Intestinal Ischemia/Reperfusion in Rats
Jian-Tong Shen,* Zheng-Yuan Xia,$ Ke-Xuan Liu*
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Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University,
Guangzhou 510080, China; †Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China; §Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou $
Anesthesiology Research Laboratory, Department of Anesthesiology,
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510060, China;
University of Hong Kong, Hong Kong, China
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Corresponding author: Ke-Xuan Liu, Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan 2nd Road, Guangzhou 510080, China. Tel: +86 20-87755766-8273; E-mail: [email protected] The authors contributed equally to this study
Short title: Role of 15-F2t-Isop in intestinal ischemia/reperfusion
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Shi-Hong Wen,*,1 Yi-Hong Ling,†,§ Wei-Feng Liu,* Yi-Xin Qiu,* Yun-Sheng Li,* Yan Wu,*
Abstract
15-F2t-isoprostane is not only a specific marker of lipid peroxidation but also demonstrated to
have potent bioactivities and can exert deleterious effects via activating thromboxane A2 receptor (TxA2r). We already demonstrated that lipid peroxidation represents a mechanism of intestinal ischemia/reperfusion (I/R) injury. But no studies have focused on 15-F2t-isoprostane
production and its biological actions on postischemic intestine during intestinal I/R. This study was carried to investigate whether the mechanism of endogenous 15-F2t-isoprostane 1
action is involved in the pathogenesis of intestinal ischemia/reperfusion (I/R) and administration of synthetic 15-F2t-isoprostane could exacerbate intestinal insult after intestinal I/R in vivo and in vitro. In comparison with sham control, we reported that endogenous
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15-F2t-isoprostane was liberated following intestinal I/R injury in rats, and using the TxA2r
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peroxidation, inflammation and alleviated intestinal mucosal microvascular vasoconstriction. Further research found that in vivo administration of synthetic 15-F2t-isoprostane exacerbated intestinal I/R injury by disturbing microvascular perfusion and accumulating anaerobic
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metabolism. Meanwhile, 15-F2t-isoprostane did not change Hypoxia/Reoxygenation-induced IEC-6 cell viability but aggravated HUVECs cell death in vitro. Collectively, our results showed that locally produced 15-F2t-isoprostane was in proportion to the severity of oxidative
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stress-induced intestinal injury and its detrimental effects can be attenuated through TxA2r inactivation. Exogenous 15-F2t-isoprostane exacerbated intestinal I/R injury, which may be contributable to its biological actions on endothelium, rather than intestinal epithelium.
Key words: 15-F2t-isoprostane; Ischemia/reperfusion injury; SQ29548; Intestine;
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antagonist SQ29548 resulted in significant intestinal protection, evidenced by reduced lipid
Hypoxia/Reoxygenation
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Introduction Major surgery is often associated with ischemia/reperfusion (I/R) injury and subsequent inflammatory response in clinical scenarios. Meanwhile, inflammation itself stimulates the
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generation of free radicals and creates a vicious circle of oxidative stress, which plays a
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systemic inflammatory response syndrome (SIRS), sepsis and multiple organ dysfunction syndrome (MODS) (1).
Intestinal I/R is a challenging clinical syndrome arising in critically ill patients and also
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due to certain surgery, such as small bowel transplantation, abdominal aneurysm repair, cardiopulmonary bypass, etc (2). Because of the often nonspecific clinical presentation and the continued difficulty in recognizing the condition before intestinal necrosis occurs, the
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in-hospital mortality rates remained high over the last few decades, ranging between 67 and 80%, and the reported incidence of intestinal I/R injury is increasing (3,4). Previous studies have shown that re-introduction of oxygen to the ischemic intestine leads to the release of proinflammatory factors and bacteria-derived endotoxin, which may
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pivotal role in the pathogenesis of postoperative complications, sometimes leading to
instigate the damage of cellular lipids, proteins and DNA and can overwhelm endogenous antioxidant defenses (5). Furthermore, irreversible membrane and cellular dysfunction secondary to extensive lipid peroxidation and, subsequently, organ injury or failure may ensue. Arachidonic acid is a complex lipid macromolecule of cell membrane, which is pivotal to inflammatory and coagulation cascades. A recent advance in free radical biology has been the discovery of isoprostanes, which are formed by the free radical-catalyzed peroxidation of arachidonic acid independent of cyclo-oxygenase activity. Among these compounds, most
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attention has been focused on the 15-series, which are the most abundant in vivo (6). Of the variety of isoprostanes detected, 15-F2t-isoprostane (15-F2t-Isop, also named isoprostane F2α-III or iPF2α-III, 8-iso-PGF2α) has been found to be the most reliable and reproducible
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marker of oxidative stress (7). And the measurement of 15-F2t-Isop provides a mean to assess
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free radical or I/R injury. Interestingly, next to being accurate markers of oxidant injury and inflammation, 15-F2t-Isop appears to possess important biological effects at pharmacological and physiological concentrations, including constriction of several vascular beds (8,9),
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increased vascular permeability (10), and nociceptor sensitization (11), as well as smooth muscle constriction of the uterine, gastrointestinal and tracheobronchial systems (12). Till now, seldom studies have been focused on the production of 15-F2t-Isop during the process of
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intestinal I/R, and its biological actions on postischemic intestine remain unknown. Evidence from various studies indicates that the effects of 15-F2t-Isop are mediated through the activation of receptors analogous to those for thromboxane A2 (TP receptor) (13-16). And using the specific thromboxane A2 receptor (TxA2r) antagonist SQ29548 can
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the effects of prophylactic and therapeutic interventions, such as antioxidants, to attenuate
largely abrogate most of its biological actions. Although 15-F2t-Isop was known to possess
potent bioactivity under pathophysiological conditions, it remains unclear whether 15-F2t-Isop,
by itself, can be liberated following intestinal I/R and is one of the mediators operating in the complex network of biological messages evoked by intestinal oxidative injury. To this end, we aimed to investigate whether the mechanism of endogenous 15-F2t-Isop action is involved in the pathogenesis of intestinal I/R and administration of synthetic 15-F2t-Isop could exacerbate intestinal insult after intestinal I/R in vivo and in vitro.
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Materials and Methods In vivo Animals and model preparation
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The study was approved by the Committee of Animal Care of Sun Yat-sen University,
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guidelines. Adult pathogen-free male Sprague-Dawley rats weighing 220-240 g were housed in climate-controlled conditions and provided with standard rat chow. All animals were fasted overnight before the experiment but had free access to water ad libitum. Rats were
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anesthetized with 20% urethane (induction and maintenance, 6 ml kg-1, intraperitoneally), tracheostomized and mechanically ventilated with a standard tidal volume ventilation protocol (control mode; tidal volume 8ml/kg; respiratory rate 50 breaths/min and 100% oxygen). An
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arterial catheter was placed into the left carotid artery and connected to a digital data acquisition system (PowerLab/4SP ADI Instruments, Ugo Basile Comerio, VA, Italy) for constant measurement of the mean arterial pressure (MAP). The small intestine was exteriorized by midline laparotomty. The intestinal I/R injury was established by occluding
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China. The experimental animals were cared in accordance with National Institutes of Health
the superior mesenteric artery (SMA) with a microvessel clip for 60 min followed by 120 min reperfusion. SMA was isolated from superior mesenteric vein (SMV) before occlusion. Ischemia was recognized by immediate blanching of the small instine and cecum. The return of pulses and the re-establishment of the pink color were assumed to indicate valid reperfusion of the intestine. During the experiments, normothermia (36-38°C) was maintained with heating pads.
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Experiment 1: To investigate whether locally produced 15-F2t-Isop contributes to the progression of intestinal I/R Experimental groups and drug administration
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The rats were block-randomized assigned to one of four groups based on the
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including isolation of the SMA without occlusion. (2) I/R group (I/R): SMA occlusion for 60 min followed by 120 min reperfusion. (3) TxA2 receptor-specific (TP) antagonist SQ29548 ((1S-(1a,2a(Z),3a,4a))-7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.
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1)hept-2-yl)-5-heptanoic acid, Cayman Chemical, Ann Arbor, MI, 19025) treatment group (I/R-SQ29548). (4) DMSO (Dimethyl Sulfoxide) group (I/R-DMSO): in which DMSO was the solvent of SQ29548 and served as vehicle control. SQ29548 (dissolved in DMSO to
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2μmol/ml; 2μmol/kg) or DMSO (1ml/kg) was given subcutaneously 30 min before intestinal ischemia. The dose of SQ29548 was chosen based on the previous study (15).
Experiment 2: To investigate whether synthetic 15-F2t-Isop exacerbates intestinal insult
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interventions (n=8 each, Fig. 1A): (1) Control group (Control): sham surgical preparation
after intestinal I/R.
Experimental groups and drug administration In a separate set of experiments (shown in Fig. 1B), the rats were divided into the
following four groups (n=8 each): (1) Control + DMSO group (Control + DMSO): sham surgical preparation including isolation of the SMA without occlusion. (2) Control + 15-F2t-Isop group (Control +15-F2t-Isop). (3) I/R + DMSO group (I/R + DMSO). (4) I/R +
15-F2t-Isop group (I/R + 15-F2t-Isop). DMSO was the solvent of 15-F2t-Isop (Cayman
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Chemical, Ann Arbor, MI, 16350) and served as vehicle control. 1μg/kg/min 15-F2t-Isop was administered intravenously over a period of 10 min through a pump (Harvard Apparatus, Holliston, MA) after 60 min of sham operation or immediately at the onset of reperfusion.
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Preparation of specimens
At the end of the experiments, blood samples were collected from the heart of rat and centrifuged at 3,500 rpm for 15 minutes at 4 , the supernatant was used for subsequent
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measurements of 15-F2t-Isoprostane, endothelin-1 (ET-1) and thromboxane B2 (TxB2, the stable metabolite of thromboxane A2) concentrations and diamine oxidase (DAO) activity. And then, a segment of 10cm intestine was cut 5 cm away from the ileocecal valve, and was
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divided into two segments. The segments are, respectively, fixed in 10% neutral formaldehyde and paraffin embedded for morphological analysis, washed with cold saline and after being scraped off, intestinal mucosa was dried with filter paper and preserved at -80
for
determination of lactic acid (LD), malondialdehyde (MDA), superoxide dismutase (SOD) and
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The dose of 15-F2t-Isop infusion was chosen based on the previous studies (13, 17).
myeloperoxidase (MPO).
Plasma 15-F2t-Isop assays
Enzyme immunoassay (EIA) of free 15-F2t-Isop was performed with a commercially
available kit (Cayman Chemical, Ann Arbor, MI) according to the methods provided by the manufacturer. The values of the unknown were expressed as pg/ml 15-F2t-Isop of plasma.
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Histopathology measurement of intestinal mucosa The segment of small intestine was stained with hematoxylin-eosin for histological assessment. Two independent pathologists blinded to treatment group evaluated histological
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damage of specimens and assigned a grade based on the Chiu’s classification of intestinal
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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
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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,
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hemorrhage, and ulceration. A minimum of five randomly chosen fields from each rat were evaluated and averaged to determine mucosal damage.
Detection of LD level in intestinal mucosa and plasma DAO activity
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injury (18). In brief, mucosal damage was graded from 0 (normal) to 5 (severely damaged) as
Intestinal mucosal tissues were weighed and made into 10% homogenate. The LD content
in tissues was determined as previously described (19).The results were expressed as nmol/mg protein. In order to further confirm intestinal injury, DAO, a sensitive marker reflecting small intestinal mucosal injury (20), was detected using a chemical assay kit according to the provided protocol of the manufacturer. The above results were expressed as nmol/mg protein and U/L plasma, respectively.
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Detection of MDA level and SOD activity in intestinal mucosa Intestinal mucosal tissues were homogenized on ice with normal saline, frozen in refrigerator at -20 ℃ for 5 min and centrifuged for 15 min at 4 000 rpm. Supernatants were
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transferred into fresh tubes for the evaluation. The fatty-acid peroxidation product MDA and
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expressed as nmol/mg protein and U/mg protein, respectively.
Detection of MPO activity in intestinal mucosa
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Intestinal mucosal samples were homogenized and the homogenate was freeze-thawed twice, and then centrifuged at 13,000 rpm for 5 min. The resulting supernatant was assayed spectrophotometrically for MPO activity as previously described (22). One unit of MPO was
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defined as the capacity to degrade 1 μmol peroxide per minute at 25 ℃. Results were expressed as U/g intestinal tissue.
Plasma ET-1 and TxB2 assays
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the SOD activity were measured as previously described (21). The above results were
ET-1 and TxB2 levels in plasma were performed by EIA kits (R&D Systems,
Minneapolis, USA) according to the manufacturer’s instruction. They were determined using a standard curve generated from known concentrations of ET-1 and TxB2. All measurements were performed in duplicate, and the intra- and interassay variability were < 10%. The values of the unknown were all expressed as pg/ml of plasma.
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In vitro IEC-6 cells and HUVEC cells culture Intestinal epithelial cells (IEC-6, Catalog No. RL-1592) were obtained from American
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Type Culture Collection (ATCC, Manassas, VA, USA) and were culture in Dulbecco’s
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4.5g/L D-glucose and 1% penicillin/ streptomycin antibiotics (Gibco, Invitrogen Ltd, Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were used at
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passages 2–12 to ensure genetic stability. HUVECs were cultured in RMPI-1640 medium containing 10% v/v FBS and 1% penicillin/streptomycin antibiotics (Gibco, Invitrogen Ltd, Shanghai, China) The above two cells were maintained under standard cell culture conditions with a humid environment of 21% O2 and 5% CO2. They were used soon after
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at 37
reaching 80% confluence.
Hypoxia/Reoxygenation injury of cultured IEC-6 and HUVECs
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modified Eagle’s medium (DMEM), supplemented with 10% v/v fetal bovine serum (FBS),
Hypoxia/Reoxygenation (H/R) injury of cells was induced employing a glucose oxidase
(2 U/ml, Sigma-Aldrich, G6125) and catalase (120 U/ml, Sigma-Aldrich, C1345) based two-enzyme system (GOX/CAT system) first described by Baunman et al. in 2008 (23). In
this enzymatic model, depletion of oxygen is achieved rapidly within minutes, representing the situation of intestinal ischemia in vivo. Briefly, cells were grown under normoxic conditions up to a confluence of 80%. Hypoxia was induced by exchanging the normoxic medium to hypoxic medium. After a 1 h hypoxia period, medium was changed back to
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normoxic medium and cells were cultured under the described conditions for an additional 2 h (Reoxygenation). Control experiments were performed by omitting hypoxia and incubating
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the cells for 3 h under normoxic conditions.
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For dose-response assays and to determine whether the administration of synthetic 15-F2t-Isop leads to the damage of IEC-6 and HUVECs under the normal condition, cells were incubated with different concentrations (10-10 to 10-5 mol/L) of 15-F2t-Isop or vehicle for
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24 h (Fig. 2A), subsequently, to test whether 15-F2t-Isop (10-10 to 10-5 mol/L) or vehicle administrated immediately at the time of reoxygenation exacerbates IEC-6 and HUVECs H/R injury (Fig. 2B). DMSO was the solvent of 15-F2t-Isop and at the concentrations used was
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without any effect on cell viability.
Cell viability assessment
Cell viability was determined using MTT assay. Briefly, 1×105 cells were plated into
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Study groups and experimental protocol
each well in 96-well tissue culture plates. After H/R or 15-F2t-Isop incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide, 5 mg/ml in Phosphate buffered saline, Sigma) was added to each well and incubated at 37
for 4h. After careful
removal of the medium, 0.15ml buffered DMSO was added to each well. The absorbance was recorded on microplate reader at the wavelength of 560nm. All experiments were repeated a minimum of three times.
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Statistical analysis Data were expressed as mean ± standard deviation (SD). Biochemical markers were performed in duplicate or triplicate for each specific sample. Therefore, all the data points are
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means of duplicate or triplicate measurements for these parameters. One-way ANOVA and the
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test failed, Kruskal-Wallis test was performed. Correlation between different variables was assessed by Pearson or Spearman coefficient (SPSS 13.0，SPSS Inc., Chicago, USA). A
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P 0.05 vs. the I/R + DMSO
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groups)
2. 15-F2t-Isop is liberated following intestinal I/R injury As shown in Fig. 4, locally produced plasma 15-F2t-Isop were significantly increased 2 h
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during the experiments did not differ among groups (data not shown, P > 0.1). As Fig. 3A
after reperfusion in the I/R and I/R-DMSO groups when compared with the Control group (all P 0.05 vs. the I/R and I/R-DMSO groups).
3. Effect of SQ29548 on attenuating intestinal I/R injury Representative mucosal morphological changes are presented in Figs. 5A-D. In the
Control group (Fig. 5A), normal villi were seen as expected. By contrast, I/R and I/R-DMSO
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groups (Figs. 5B and 5D) animals resulted in severe edema of mucosal villi and infiltration of necrotic epithelial and inflammatory cells, and intestinal glands showed evidence of severe injury. However, in the I/R-SQ29548 group (Fig. 5C), slight edema could be seen in the
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intestinal villi, and some intestinal villi were severed, and the gap between epithelial cells
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I/R-DMSO groups were significantly higher than that in the Control group (all P < 0.01, Fig. 5E). However, Chiu’s score in I/R-SQ29548 group was markedly lower when compared with the I/R and I/R-DMSO groups (P < 0.05). A weak but statistically significant positive
Chiu’s scores.
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correlation (r = 0.57, P < 0.01; Fig. 5F) was noted between concentrations of 15-F2t-Isop and
LD and DAO are used as markers of intestinal cellular injury described in our previous
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researches (24). As shown in Fig. 6, intestinal mucosal LD level (A) and DAO activity (B) in the I/R and I/R-DMSO groups were significantly higher than that in the Control group (all P < 0.05). However, LD level and DAO activity were markedly reduced by SQ29548 (all P < 0.05 vs. the I/R and I/R-DMSO groups).
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increased slightly. Consistent with histological changes, Chiu’s scores in the I/R and
4. SQ29548 reduces lipid peroxidation and inflammation in intestinal I/R injury At 2 h after reperfusion, rats pretreated with SQ29548 had significantly decreased
concentrations of MDA (A) and MPO (C) activity whereas SOD (B) activity in intestinal mucosa homogenates was increased (all P < 0.05 vs. the I/R and I/R-DMSO groups, Fig. 7).
5. SQ29548 alleviates intestinal mucosal microvascular vasoconstriction
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Plasma ET-1 levels in the I/R and I/R-DMSO groups were significantly higher than that in the Control group (P < 0.05). However, SQ29548 pretreatment markedly reduced the production of ET-1 (P < 0.05 vs. the I/R and I/R-DMSO groups, Fig. 8A). After 2 h
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reperfusion, TxB2 concentrations in the I/R, DMSO and I/R-SQ29548 groups were
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differences in TxB2 levels among the I/R, I/R-DMSO and I/R-SQ29548 groups (P > 0.05, Fig. 8B). Meanwhile, plasma 15-F2t-Isop concentrations correlated significantly with ET-1 and
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TxB2 levels (r=0.74 and r=0.78, both P 0.05). Meanwhile, intestinal mucosal LD levels and plasma DAO activity in the I/R+DMSO
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and I/R+15-F2t-Isop groups were significantly increased after 2 h reperfusion (P < 0.05 vs. the Control+DMSO and Control+15-F2t-IsoP groups, Figs. 9F and 9G). What’s more, LD levels in the I/R+15-F2t-Isop group were markedly higher than that in the I/R+DMSO group (P0.05, Fig. 9G).
Cell viability decreased significantly after 1 h of hypoxia and 2 h of reoxygenation in
IEC-6 and HUVECs. In contrast, after administration of synthetic 15-F2t-Isop at 10-5 mol/L immediately at the time of reoxygenation, HUVECs cell death was significantly increased when compared with the H/R group (P
