Adult male BALB/c mice were obtained from the Laboratory Animal Center of the Fourth Military Medical University, Xi’an, China and housed under standard conditions. They were kept in cages at a fairly constant room temperature and were exposed to a 12/12 h light/dark cycle. Each animal was fasted overnight prior to surgery, but had access to water ad libitum. All animal experiments were performed in accordance with the Prevention of Cruelty to Animals Act 1986, under the guidelines of the National Health and Medical Research Council for the Care and Use of Animals for Experimental Purposes in China.

The mice were randomly divided into 3 groups (n=13 per group): i) Sham group: all the surgical steps were performed; however, intestinal I/R was not induced. The animals were kept under anesthesia for the duration of the intestinal I/R procedure. ii) I/R group: intestinal I/R was induced and this group served as the control for the osthole-treated group. iii) Osthole group: a single dose of osthole (50 mg/kg daily for 3 days) was injected via the tail vein. Osthole (>98% purity) was a gift from Dr W. Liu (Department of Neurology, Xijing Hospital, Fourth Military Medical University, Xi’an, China). The mice in each group were subdivided into 3 subgroups. The first subgroup was used for histological examination and immunofluorescence staining (n=4 per group); the second subgroup was used for detecting MPO, GSH, SOD, MDA and NO levels (n=6 per group); and the third subgroup was used for western blot analysis (n=3 per group).

Following an acclimation period of at least 3 days, the mice were prepared for surgery under deep sodium pentobarbital anesthesia. All procedures were performed with the animals breathing spontaneously and body temperature was maintained at 37°C using a water-circulating heating pad. The animals were subjected to I/R as previously described (22). Briefly, a midline laparotomy was performed. The superior mesenteric artery was identified and isolated, and the blood supply to the intestine was interrupted for 1 h by occlusion of the superior mesenteric artery using a microvascular clamp. Ischemia was confirmed by the loss of pulsation of the mesenteric artery and its branches or a pale color of the intestine. Following the ischemic phase, the clamp was removed to allow the restoration of the blood flow (reperfusion) for 6 h, which was confirmed by the return of the pulses, re-establishment of the pink color and enhanced intestinal peristalsis. Once reperfusion was secured, the wound was sutured and the animal was allowed to awaken from the anesthesia. The mice were administered an overdose of pentobarbital prior to euthanasia.

For isolation of the intestinal segments, tissue from the entire intestine was cut into 6 equal segments, of which a section of 1–1.5 cm at the middle of each segment was collected for histological analysis. Formalin-fixed and paraffin-embedded tissue sections were cut (4-μm-thick) and stained with hematoxylin and eosin for histological examination. The above procedure was performed by a physician.

Intestinal sections from each animal were scored for mucosal damage as previously described (23). Briefly, a score of 0 was assigned to a normal villus; villi with tip distortion were scored as 1; villi lacking goblet cells and containing Guggenheims’ spaces were scored as 2; villi with patchy disruption of the epithelial cells were scored as 3; villi with exposed but intact lamina propria and epithelial cell sloughing were assigned a score of 4; villi in which the lamina propria was exuding were scored as 5; and finally, villi displaying hemorrhage or denudation were scored as 6. All histological analyses were performed by a pathologist in a blinded manner. Five random high-power fields (×200) were examined per sample.

The intestinal samples were weighed and homogenized in a solution prepared from the assay kit (Jiancheng Institute of Biotechnology, Nanjing, China), and homogenates were obtained and used for MPO assay. The reaction was initiated by adding hydrogen peroxide in the medium and 3,3,5,5-tetramethylbenzidine was used as an oxidizable dye to produce yellow color compounds. The optical density was recorded at 460 nm and the results were expressed as U/g wet tissue.

The intestinal samples were homogenized in 0.05 mol/l phosphate buffer. The homogenates were centrifuged at 4,000 rpm for 20 min at 4°C and the MDA, GSH and SOD content in the supernatant was measured using the corresponding kits (Jiancheng Institute of Biotechnology).

SOD activity and MDA levels were measured using the xanthine oxidase and thiobarbituric acid method, respectively. SOD activity was measured at 500 nm. One SOD activity unit was defined as the enzyme amount causing 50% inhibition in 1 ml reaction solution/mg tissue protein and the result was expressed as U/mg protein. MDA was determined at 532 nm and the results were expressed as nmol/mg protein. GSH levels were measured through a reaction using dithiobisnitrobenzoic acid. GSH activity was determined at 420 nm and the results were expressed as mg/g protein.

NO synthesis can contribute to nitrite accumulation. The concentration of nitrite in the intestinal samples was measured following a reaction with Griess reagent (sulfanilamide 1%, naphthylethylene diamine 0.01%, H₃PO₄ 5%) to reflect the production of NO at 550 nm. The results were expressed as μmol/g protein. Detailed procedures for the above measurements were performed according to the instructions of kit. The protein content was determined using a kit based on the Bradford assay and bovine serum albumin was used as a standard.

Total protein lysates from frozen tissues were prepared in ice-cold RIPA buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS and protease inhibitor cocktails). Nuclear extracts were prepared using a Nuclear Protein Extraction kit (Beyotime Biotechnology, Shanghai, China). Protein samples were immunoblotted with antibodies to IκB, p-IκB and NF-κB-p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and the protein-antibody immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents (Pierce Biotechnology, Rockford, IL, USA). For detection, an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) was used. Signals were detected and quantified using the LAS-3000 image analyzer (FujiFilm, Tokyo, Japan).

Staining was performed on slides of 4-μm-thick sections from intestinal tissue. Following deparaffinization and dehydration, the slides were immersed in 10 mM sodium citrate buffer containing 0.05% Tween-20 (pH 6.0) for 5 min at 55°C for antigen retrieval. The slides were then permeabilized with 10 μg/ml proteinase K in 10 mM Tris/HCl (pH 7.6) for 15 min and incubated with 5% BSA in a humidified chamber for 30 min to block non-specific binding. The slides were incubated with antibody against NF-κB-p65 (1:200 in PBS containing 1% BSA; Santa Cruz Biotechnology, Inc.) in a humidified chamber overnight at 4°C, and with fluorescent-fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G (1:500 in PBS) at room temperature for 1 h. The nuclei were counterstained with Hoechst 33258 (1:5,000; 10 min). Fluorescence imaging was assessed under a fluorescence microscope (Fluorescence E-1000 microscopy, Nikon, Japan) and subjected to identical exposure times. Instead of the primary antibody, PBS was used for the negative controls.

Data were analyzed using SPSS v 11.5 software for Windows (SPSS Inc, Chicago, IL, USA). All values are presented as the means ± standard error of the mean (SEM). Differences between groups were compared using analysis of variance with the Bonferroni correction for multiple comparisons. Values of P<0.05 were considered to indicate statistically significant differences.

In order to determine the effect of osthole on intestinal I/R injury, the mice were administered various doses of osthole (5, 10, 25, 50 mg/kg) for 3 days prior to the induction of intestinal I/R injury. The results revealed that osthole preconditioning (25 mg/kg and 50 mg/kg) prior to intestinal I/R injury significantly improved the survival rate at 24 h following reperfusion as compared to the vehicle-treated mice (Fig. 1).

To further investigate the effect of osthole on intestinal I/R injury, the morphology of the intestinal epithelium and villi was examined by immunohistochemistry. In the sham group, the results of histopathological examination of the intestinal epithelium and villi were normal. Mucosal necrosis and bleeding were observed in the intestinal mucosa and submucosa in the I/R group which also showed a marked reduction in villus height and destruction of epithelial cells within the villi. In the osthole group, mice pre-treated with osthole (50 mg/kg) demonstrated intestinal architecture with almost normal histological features. Although the median intestinal injury score of the osthole group was higher than the sham group (P<0.001), it was significantly lower than that of the I/R group (P<0.01) (Fig. 2B). Taken together, these data suggest that osthole exerts protective effects against intestinal I/R injury.

To elucidate the underlying mechanisms of action of osthole in protecting against intestinal I/R injury, the antioxidant and oxidant products were evaluated. The levels of SOD, an enzyme occurring naturally in the body which protects cells by cleaning up free radicals, were significantly reduced in the I/R group compared with the sham group (P<0.05). Following pre-treatment with osthole, SOD activity was significantly improved compared with the I/R group (P<0.01) (Fig. 3A). The levels of MDA, an important marker of lipoperoxidation associated with oxidative stress, were decreased following pre-treatment with osthole (P<0.05 vs. I/R group) (Fig. 3B). Following I/R injury, the levels of GSH, a common antioxidant, were slightly decreased; however, following pre-treatment with osthole, these levels increased. However, the difference was not statistically significant among the different groups (Fig. 3C). These results demonstrate that osthole relieves oxidative stress through the upregulation of SOD and the downregulation of MDA levels following intestinal I/R injury.

Tissue MPO activity is a hallmark of intestinal I/R injury which reflects the infiltration of neutrophils. In order to examine the effect of osthole on inflammation, the activity of MPO was measured. Compared to the sham group, a significant increase in MPO activity was observed in the I/R group, which was alleviated in the osthole-treated group (P<0.01) (Fig. 4).

The overproduction of NO following intestinal I/R injury has been reported to be closely associated with oxidative damage (11). NO synthesis can contribute to nitrite accumulation. The concentration of nitrite in the intestinal samples was measured to reflect the production of NO. As shown in Fig. 5, pre-treatment with osthole significantly inhibited the elevation of NO levels in the I/R group (P<0.01).

It has been reported that NO produced by the iNOS gene is induced during inflammation (24). NF-κB is an important nuclear transcription factor that regulates iNOS gene expression by binding the iNOS promoter region and plays a central role in inflammation through its ability to induce the transcription of pro-inflammatory genes (25). The data mentioned above suggest that osthole reduces neutrophil infiltration and inhibits the NO levels induced by intestinal I/R injury. Thus, we hypothesized that osthole protects against intestinal I/R injury by inhibiting the activation of the NF-κB signaling pathway. To confirm our hypothesis, we examined the effects of osthole on NF-κB nuclear translocation. In the sham group, NF-κB was localized in the cytoplasm and translocated to the nucleus in the I/R group. Pre-treatment with osthole inhibited the nuclear translocation of NF-κB compared with the I/R group (Fig. 6). In order to further confirm these results, western blot analysis was performed to examine the nuclear expression of NF-κB and the phosphorylation state of IκBα. Consistent with the immunofluorescence staining results, we observed that the nuclear expression of NF-κB was markedly increased in the I/R group, while pre-treatment with osthole inhibited the nuclear expression of NF-κB (P<0.01 vs. I/R group). We also found that phosphorylated IκBα was increased in the I/R group. In the osthole-treated group, phosphorylated IκBα was significantly inhibited (P<0.01 vs. I/R group) (Fig. 7).