EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: The purpose of this study was to evaluate the effect of intravenous injection of osthole on intestinal ischemia-reperfusion injury and parameters of oxidative stress.

Materials and Methods: In 45 Kunming male mice, treatment included sham surgery (15 mice); intestinal ischemia-reperfusion injury (clamping of the superior mesenteric artery, 2 h; clamp release, 1 h; 15 mice); or osthole treatment before and after ischemia-reperfusion injury (15 mice). Evaluation included histopathology, determination of intestinal wet/dry weight ratio, and measurement of levels of diamine oxidase, superoxide dismutase, malon-dialdehyde, interleukin 1β, tumor necrosis factor α, and interleukin 2. Intestinal barrier permeability was evaluated with Evans blue test.

Results: The mean wet-to-dry weight ratio, Evans blue content, and Chiu score were significantly greater in the ischemia-reperfusion than in the sham group and lower in the osthole-treated than the ischemia-reperfusion group. The mean serum diamine oxidase, malondialdehyde, interleukin 1β, and tumor necrosis factor α levels were significantly greater in the ischemia-reperfusion than in the sham group and lower in the osthole-treated than in the ischemia-reperfusion group. The mean superoxide dismutase activity and interleukin 2 levels were lower in the ischemia-reperfusion than in the sham group and greater in the osthole-treated than in the ischemia-reperfusion group.

Conclusions: Treatment with osthole may protect against oxidative stress and tissue damage from intestinal ischemia-reperfusion injury.

Key words : Cytokines, Inflammation, Intestine, Oxidative stress, Superoxide dismutase

Intestinal ischemia-reperfusion is a serious and complex disease with varied causes. Intestinal ischemia may include acute mesenteric ischemia, chronic mesenteric ischemia, or ischemic colitis.¹ Subclinical progression of the disease and continued difficulty in diagnosis may occur before the development of intestinal necrosis. Intestinal ischemia-reperfusion injury may be caused by cardiovascular surgery including thoracoabdominal aortic aneurysm repair, cardiopulmonary bypass, and small bowel transplant.^2-4 Intestinal ischemia may increase the rapid progression of tissue necrosis and may cause serious metabolic disorders, reactive oxygen species production, bacterial translocation, release of proinflammatory cytokines (interleukin 1β [IL-1β] and tumor necrosis factor α [TNF-α]), cell damage from inflammation, and multiple organ dysfunction syndrome.⁵

Natural products for treatment may have advantages such as low toxicity and a wide range of sources.⁶ Astragalus membranaceus, green tea, and Nigella sativa may cause antioxidative effects on the intestinal mucosa of rats after ischemia-reperfusion.^7-9 Osthole (7-methoxy-8-[3-methylpent-2-enyl]coumarin, C₁₅H₁₆O₃; 244.39 Da) is a plant-derived natural product from the fruit of the herb Cnidium monnieri., a well-known traditional Chinese medicine, which has therapeutic effects on several skin diseases. Osthole has several biologic functions including antihepatic, antiosteoporotic, antiallergic, antiseizure, anti-inflammatory, and antiproliferative functions.^10-15 The purpose of the present study was to evaluate the effect and mechanism of action of intravenous injection of osthole on intestinal ischemia-reperfusion injury.

Materials and Methods

Chemicals
Osthole (> 98% purity) was purchased (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China), dissolved in dimethylformamide, and mixed with polysorbate 80 (Tween 80 [< 20%], Sigma-Aldrich, St. Louis, MO, USA) and physiological saline (dimethylforma-mide:polysorbate 80:physiological saline = 1:1:8). The osthole solution was injected intravenously (20 mg/kg). All the experiments on animals were performed according to the ethical guidelines for animal testing of Anhui Medical University.

Animals
Kunming male mice (weight, 27-31 g) were purchased (Laboratory Animal Center, Anhui Medical University, Hefei, China). Mice were housed in animal facilities with constant temperature (24°C ± 2°C), a 12-hour light/ dark cycle, and fasting for 24 hours before experiments.

Experimental protocol
The mice were anesthetized with 1.5% sodium pentobarbital solution (0.15 g/kg, intraperitoneal). The intestinal ischemia-reperfusion injury model was established by superior mesenteric artery occlusion with intestinal ischemia (2 h) and reperfusion (1 h).¹⁶ Mice were allocated to 3 experimental groups (15 mice per group): (1) a sham group that underwent sham surgery with isolation of the superior mesenteric artery without occlusion; (2) an intestinal ischemia-reperfusion group that was subjected to intestinal ischemia and reperfusion after the superior mesenteric artery had been isolated and occluded; (3) ischemia-reperfusion plus osthole treatment group (osthole group) subjected to intravenous injection of osthole solution 10 minutes before intestinal ischemia and immediately after reperfusion (total, 2 injections). At the end of the experiment, blood samples were collected in polypropylene tubes from mice by eyeball extraction and centrifuged (3000 rpm for 10 minutes). Serum samples were stored at -80°C until analysis.

Intestinal edema
The intestinal wet-to-dry weight ratios were used as a measurement of intestinal edema. A 4-cm ileal segment located 5 cm from the terminal ileum was weighed immediately after reperfusion and placed in a drying oven at 110°C for 24 hours. After drying, the specimen was reweighed, and the ratio of the weight before and after drying was calculated. The presence of intestinal edema was confirmed by an increase in this ratio.

Intestinal barrier permeability
Evans blue dye, which strongly binds to albumin, is a marker of protein extravasation in models of intestinal injury.¹⁷ In each group, mice were injected with 0.2% Evans blue dye (20 mg/kg) into the tail vein 15 minutes before the superior mesenteric artery clamp was released and killed by cervical dislocation. A standard curve was prepared by dissolving Evans blue in distilled water at various concentrations (range, 0.25-20 μg/mL) and measuring optical density with a microplate reader at 620 nm.

After reperfusion, saline (37°C) was perfused via the abdominal aorta for 3 minutes (0.8 mL/g body weight) to eliminate residual blood and Evans blue dye, cut from the abdominal vein, and operated as described previously. A 4-cm ileal segment was cut. The wet ileal segment was weighed, placed into formamide solution (1 g/L) for 48 hours at 45°C, and centrifuged (3500 rpm for 10 minutes). The optical density of the extraction solution was measuring with a microplate reader at 620 nm. The ileal segment was placed in a drying oven at 180°C for 12 hours and reweighed. The Evans blue content was calculated per intestinal dry weight (Evans blue content = Evans blue concentration × 3 mL/dry weight).

Histopathology
The ileum specimens were fixed in 10% formalin for 48 hours, embedded in paraffin, and cut into sections (5 μm). Slides were stained with hematoxylin-eosin and examined under a light microscope. Intestinal mucosal lesions were scored on a scale (Chiu score) from 0 to 5 as previously described: grade 0, normal mucosal villi; grade 1, development of subepithelial Gruenhagen space, usually at the apex of the villus, often with capillary congestion; grade 2, extension of the subepithelial space with moderate raising of the epithelial layer from the lamina propria; grade 3, massive epithelial raising with a few denuded villi; grade 4, denuded villi with exposed dilated capillaries; and grade 5, digestion and disintegration of lamina propria, hemorrhage, and ulceration.18

Assays
The total superoxide dismutase activity was measured at 550 nm. The supernatant was assayed using the xanthine oxidase-cytochrome C method.¹⁹ The amount of superoxide dismutase activity required to inhibit 50% cytochrome C reduction at 25°C was defined as 1 unit, and data were expressed as units/μL.

Malondialdehyde concentration in the super-natant was determined after incubation at 95°C and pH 3.4 with thiobarbituric acid as an indicator of lipid peroxidation, as previously described.²⁰ The pink color produced by these reactions was identified at 532 nm to determine the malondialdehyde content.

Serum diamine oxidase was measured with an automatic biochemical analyzer and a diamine oxidase assay. The reaction mixture was incubated for 5 minutes at 37°C and serum diamine oxidase samples were added at 37°C for 10 minutes. Hydrochloric acid was added and absorbance was read at 340 nm.²¹

Serum levels of IL-1β, interleukin 2 (IL-2), and TNF-α were measured with enzyme-linked immuno-sorbent assay kits according to the instructions from the manufacturer. The serum samples were added to 96-well plates that were coated with antibodies against IL-1β, IL-2, and TNF-α. Stop solution was added to the wells and absorbance was read at 450 nm.²²

Statistical analyses
Data were reported as mean ± standard derivation (SD). For all statistical tests, multiple comparisons were performed by 1-way analysis of variance with the Tukey–Kramer test. The least squares method was used for linear correlation between selected variables. The significance level was defined by P ≤ .05 for all tests.

The mean wet-to-dry weight ratio was significantly greater in the ischemia-reperfusion than in the sham group (P ≤ (.05) and lower in the osthole than in the ischemia-reperfusion group (P ≤ .05) (Figure 1). The mean Evans blue content of intestinal tissue was significantly greater in the ischemia-reperfusion than it was in the sham group (P ≤ .01) and lower in the osthole than in the ischemia-reperfusion group (P ≤ .01) (Figure 2). The serum diamine oxidase level was significantly greater in the ischemia-reperfusion than it was in the sham group (P ≤ .01) and lower in the osthole than it was in the ischemia-reperfusion group (P ≤ .05) (Figure 3).

Histopathologic examination showed that small intestinal epithelium and villi were normal in sham mice, but mice in the ischemia-reperfusion group had loss of villi, villous atrophy and fragmentation, villous edema, vascular congestion, hemorrhage, and mucosal damage (higher Chiu score; mostly grades 1-4 and some mice with grade 5) (P ≤ .01). Mice treated with osthole had preserved structure of the intestinal mucosa (lower Chiu score in osthole than ischemia-reperfusion group; frequently grade 0 and significantly less grades 1-3) (P ≤ .01) (Figure 4).

The mean superoxide dismutase activity was lower in ischemia-reperfusion than in the sham group and greater in the osthole than it was in the ischemia-reperfusion group (Figure 5). Mean serum malondialdehyde level was greater in the ischemia-reperfusion than it was in the sham group, and lower in the osthole than it was in the ischemia-reperfusion group (P ≤ .05) (Figure 5).

The mean serum IL-1β and TNF-α levels were greater in the ischemia-reperfusion than they were in the sham group and lower in the osthole than they were in the ischemia-reperfusion group (Figure 6). The mean serum IL-2 level was lower in the ischemia-reperfusion than it was in the sham group and greater in the osthole than it was in the ischemia-reperfusion group (Figure 6).

The mechanisms of ischemia-reperfusion injury are complex and several treatment modalities have been used to limit intestine reperfusion injury in animals.²³ The pathogenesis and prevention of intestinal ischemia-reperfusion injury have been studied.²⁴ Ischemia causes hypoxia, increased vascular permeability, mucosal epithelial edema, and apoptosis. Intestinal mucosa normally functions as a barrier, but ischemia causes increased intestinal permeability, bacterial translocation, and bacterial sepsis.²⁵ Therefore, intestinal ischemia-reperfusion injury damages the intestinal barrier function and may cause multiple organ dysfunction syndrome.

In the intestinal ischemia-reperfusion experiments, indices used to evaluate the injury included edema (increase in wet-to-dry intestinal weight ratio); histopathologic scores for the degree of oxidative stress caused by lipid peroxidation and protein oxidation; and Evans blue content and serum diamine oxidase content as measures of intestinal barrier permeability.^26,27 The intestinal mucosal surface layer, with tightly arranged epithelial cells, is an important component of the intestinal barrier.²⁸ When intestinal mucosal damage occurs, including cell necrosis and shedding, the mucous membrane barrier is damaged, gut permeability is increased, and intestinal diamine oxidase is released into the bloodstream. Consistent with previous studies, the present study showed that ischemia (2 h) and reperfusion (1 h) caused marked intestinal injury with increased intestinal water and Evans blue content, increased serum diamine oxidase content, and marked intestinal histopathologic changes. However, intravenous osthole injection prevented intestinal injury in this mouse model of ischemia-reperfusion injury, confirmed with a decrease in intestinal water and Evans blue content, decrease in serum diamine oxidase content, and improvement of intestinal histopathologic changes.

Malondialdehyde is an important marker of oxidative damage and a naturally occurring product of lipid peroxidation. The concentration of malondialdehyde is directly proportional to cell damage caused by free radicals.²⁹ Conversely, superoxide dismutase is an important enzyme for eliminating reactive oxygen species. Therefore, malondialdehyde and superoxide dismutase levels are markers of oxidative stress. The intestine may be the greatest source of oxidants compared with other organs.³⁰ The generation of reactive oxygen species and reactive oxygen species-mediated damage may be important in intestinal ischemia-reperfusion injury. Many oxygen-free radicals are generated during ischemia and reperfusion, causing excessive superoxide dismutase consumption.³¹ During intestinal ischemia, adenosine triphosphate in cells may be converted to xanthine, and ischemia may promote the conversion of xanthine dehydrogenase oxidase. After reperfusion, oxygen delivery and use are increased. The production of xanthine by hypoxanthine-xanthine oxidase and release of reactive oxygen-free radicals may cause oxidative tissue damage. Intestinal cells may develop additional oxidative stress injury from xanthine oxidase, which is abundant in intestinal tissue.³² Consistent with previous studies, we observed that oxidative stress occurs in intestinal ischemia-reperfusion injury in mice, manifested by a significant increase in serum malondialdehyde and decrease in serum superoxide dismutase. Osthole decreased oxidative stress, indicating potential antioxidant effects of osthole, as reported previously.³³

During ischemia-reperfusion, the ischemic tissue and remote organs release inflammatory cytokines such as TNF-α and IL-1β, inflammatory cells accumulate, and intestinal inflammatory damage occurs.³⁴ The cytokine IL-1β can induce and promote damage by inflammatory mediators, edema formation, and various pathologic processes. In addition, IL-1β promotes the expression of adhesion molecules in the blood vessel endothelium and facilitates neutrophil binding. The inflammatory cytokine TNF-α up-regulates the expression of other inflammatory cytokines such as IL-1β and activates neutrophils, stimulates production of platelet activating factor and endothelin, and causes platelet thrombus formation and blood vessel contraction.³⁵ In addition, TNF-α provides rapid defense for the host against infection but may cause death and may aggravate the injury to the intestines after reperfusion.

The cytokine IL-2 is important for defense against infection and tumors. It is produced by CD4+ cells and may increase the activity of T cells and NK cells for immune function.³⁶ The present study showed an increase of serum IL-1β and TNF-α level, and decrease of serum IL-2 level, after intestinal ischemia-reperfusion injury. Therapies combined with osthole may suppress the production of IL-1β and TNF-α and promote the production of IL-2.

Oxidative stress induced by free radicals may be the primary cause of tissue injury and may occur in inflammation.³⁷ During reperfusion, oxygen is reintroduced to the hypoxic intestine, and oxygen reacts with hypoxanthine and xanthine oxidase to generate reactive oxygen species (superoxide anion and hydrogen peroxide) that are detrimental. In addition, the reactive oxygen species are signal transduction molecules that are involved in several signaling cascades that affect numerous cellular processes including inflammation and apoptosis.³⁸ Therefore, oxidative stress may contribute to the inflammatory response after intestinal ischemia-reperfusion injury.

In summary, marked oxidative stress may occur in intestinal ischemia-reperfusion in mice, and may participate in intestinal injury. Treatment with osthole prevented pathologic changes. The protective effect most likely is due to the attenuating effect of osthole on oxidative stress. However, more studies are required to evaluate the protective effects of osthole on intestinal ischemia in other experimental models and clinical trials.

Volume: 12 Issue: 3 June 2014

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