The TNF-α (cat. no. H052), IL-1β (cat. no. H002), IL-6 (cat. no. H007), IL-10 (cat. no. H009), D-lactic acid (D-LA; cat. no. A019-2) and intestinal-type fatty acid-binding protein (I-FABP; cat. no. H266) enzyme-linked immunosorbent assay (ELISA) kits specific for mouse cytokines were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies to Nrf2 (cat. no. sc-722), NF-κB (cat. no. sc-71675) and phosphorylated inhibitor of NF-κB (p-IκBα; cat. no. sc-101713) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies directed against β-actin (cat. no. 4970) and lamin B1 (cat. no. 13435) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). IRDye 800CW secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE, USA), Brusatol (cat. no. SML1868) and all-trans retinoic acid (ATRA; cat. no. R2625), specific antagonists of Nrf2 (12,13), were purchased from Sigma-Aldrich, Merck KGaA (Darmstadt, Germany). t-Butylhydroquinone (t-BHQ; cat. no. 112976), a specific activator of Nrf2 (12), was also purchased from Sigma-Aldrich, Merck KGaA. All of the chemicals used were of the highest grade commercially available.

This study was approved by the Animal Care Committee of Wuhan University (Wuhan, China) and protocols were in accordance with the National Institutes of Health (NIH) guidelines for the care and use of experimental animals (NIH publication no. 80-23). This study was performed at the animal center of Renmin Hospital of Wuhan University (Wuhan, China). A total of 64 adult male C57BL/6J mice (Hunan Slac JD Laboratory Animal Co., Ltd., Hunan, China; age, 8–10 weeks; weight, 25±3 g) were housed in individual cages (4 mice/cage) in a climate-controlled room (23±1°C; relative humidity 60±5%) with a 12-h light/dark cycle and free access to food and water. The mice were allowed to acclimatize to the environment for 2 weeks prior to the experiments. All of the animals were fasted for 12 h prior to the experiments but had free access to water.

All mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). Surgery was performed after the loss of blink and withdrawal reflexes. The mice were then placed in the supine position and allowed to breathe spontaneously. The IIR model was established by superior mesenteric artery (SMA) occlusion (12). In brief, after laparotomy, the SMA was isolated and temporarily occluded with a microvascular clip. The mice were subjected to ischemia (45 min) followed by 120 min of reperfusion by gently removing the clip. After 120 min of reperfusion, the mice were euthanized and the intestinal tissues and blood were collected and processed for further analysis.

After surgical preparation, the animals were randomly allocated into 8 groups as follows (n=8 in each group): i) S group: Sham surgical preparation with isolation of the SMA but without occlusion; ii) IIR group: SMA occlusion for 45 min followed by 120 min of reperfusion; iii) A+S group: Sham surgery plus ATRA treatment; iv) A+IIR group: IIR procedure plus ATRA treatment; v) B+S group: Sham surgery plus brusatol treatment; vi) B+IIR group: IIR procedure plus brusatol treatment; vii) T+S group: Sham surgery plus t-BHQ treatment; viii) T+IIR group: IIR procedure plus t-BHQ treatment. For ATRA treatment, the animals received ATRA [2 mg/ml dissolved in 1% dimethyl sulfoxide (DMSO); 10 ml/kg intraperitoneally per day] for two weeks prior to the experiment (13). Brusatol was diluted with 1% DMSO to 0.5 mg/ml and 4 ml/kg was injected intraperitoneally once every 2 days for 10 days prior to the experiment (14). t-BHQ was diluted with 1% DMSO and 16.7 mg/kg was administered intraperitoneally 3 times/day (every 8 h) for 3 days prior to the experiment, as described previously (12).

After reperfusion, 1 cm of small intestine without adipose tissue was biopsied from the same site from each animal at the distal end of the ileum and fixed in 4% formaldehyde. Sections (4-µm) were prepared from the paraffin-embedded tissue and assessed by hematoxylin and eosin (H&E) staining (hematoxylin staining for 10–30 sec and eosin staining 1–3 min at 23±1°C) and light microscopic examination (original magnification, ×200; Olympus BX50; Olympus Optical, Tokyo, Japan). Intestinal mucosal damage was evaluated in at least 2 different sections of each specimen using the improved Chiu et al scoring method (15), with blinding to the experimental groups, using a 5-point grading scale according to the changes in the villi and the glands of the intestinal mucosa: 0, normal mucosa; 1, development of subepithelial Gruenhagen's space at the tip of a villus; 2, extension of the space with moderate epithelial lifting; 3, massive epithelial lifting with a few denuded villi; 4, denuded villi with exposed capillaries; and 5, disintegration of the lamina propria, ulceration and hemorrhage.

Tissue edema was detected by the wet/dry weight ratio of the biopsied gut segments. At the end of the experiments, 1 cm of small intestine without adipose tissue was taken from the same site in each animal, weighed and then placed in a drying oven at 80°C for 24 h. After this drying procedure the specimens were reweighed, and the ratio of the weight prior to and after drying was calculated.

D-LA, I-FABP, IL-1β, IL-6, IL-10 and TNF-α levels in the intestinal mucosa and in the serum were measured following the standard procedures of the ELISA kits.

Apoptosis in the intestinal sections was examined after TUNEL staining with the Click-iT TUNEL Alexa Fluor 488 Imaging assay (cat. no. C10245; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The 4-µm paraffin-embedded sections were deparaffinized in xylene and double diluted water. The sections were then treated with proteinase K for 20 min at room temperature and subsequently incubated with a mixture of fluorescent labeling solution and TdT enzyme for 1 h in a humidified atmosphere. After washing with phosphate-buffered saline (PBS) and drying, the sections were incubated with DNase I for 10 min in a humidified atmosphere at room temperature. The fluorescein isothiocyanate-labeled TUNEL-positive cells were imaged using fluorescence microscopy. DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) was used to stain the nuclei. The average number of apoptotic cells was calculated from five random fields with Image-Pro Plus software (version 6.0; Media Cybernetics, Rockville, MD, USA).

The 5-µm paraffin-embedded sections were stained using the streptavidin-biotin complex immunohistochemistry technique for Nrf2, NF-κB and p-IκBα detection. A positive signal was visualized by a 3,3′-diaminobenzidine color reaction. The nuclei were stained with hematoxylin. Brown staining in the cytoplasm and the nucleus was considered an indicator of protein expression. Two different sections of each specimen were examined (original magnification, ×400; Olympus BX50; Olympus Optical). The results were semi-quantitatively evaluated with Image-Pro^® Plus version 6.0 according to the optical density values of protein expression. For this purpose, five fields per slide were randomly selected by the viewer for evaluation.

Endochylema and cellular nuclear proteins were extracted from frozen intestinal tissues with a nuclear extract kit (cat. no. P0028; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. Protein concentration was determined using a BCA assay. Equal amounts (100 µg per lane) of protein were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 v for 3 h. After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (cat. no. 88520; Thermo Fisher Scientific, Inc.) at 200 mA for 2 h. The membranes were incubated overnight at 4°C with rabbit anti-mouse polyclonal antibodies to Nrf2 (1:200 dilution), NF-κB (1:1,000 dilution), p-IκB-α (1:1,000 dilution), β-actin (1:2,000 dilution) and lamin B1 (1:200 dilution). After washing for three times with Tris-buffered saline containing Tween-20, the membranes were incubated with the corresponding goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) for 1 h at room temperature. The intensity of the bands was detected using an Odyssey two-color infrared laser imaging system and densitometry was performed using Odyssey 1.0 software (both LI-COR Biosciences).

Values are expressed as the mean ± standard deviation. GraphPad Prism 5.0 statistical software (GraphPad Software Inc., La Jolla, CA, USA) was used to manage the data and calculate the results. A statistical evaluation of the data was performed by one-way or a two-way analysis of variance, followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the underlying mechanisms of the effect of Nrf2 on IIR-induced injury, animals were pretreated with Nrf2 antagonists and an Nrf2 activator prior to reperfusion-induced intestinal injury. H&E staining indicated that IIR induced villous edema, inflammatory cell infiltration and capillary congestion, and markedly increased the gap between epithelial cells (Fig. 1A). These effects were dramatically aggravated after administration of an Nrf2 antagonist (ATRA or Brusatol) (Fig. 1). In addition, IIR-induced intestinal injury was significantly attenuated after treatment with the Nrf2 activator t-BHQ (Fig. 1). Chiu's scoring produced similar results to those of H&E staining.

Next, intestinal permeability damage was assessed by determining the intestinal wet/dry weight ratios (Fig. 2A). The intestinal wet/dry weight ratios were significantly higher in the IIR group than in the S group (P=0.0085). Compared with the IIR group, the intestinal wet/dry weight ratio was significantly decreased in the group pretreated with the Nrf2 activator t-BHQ (P=0.021). Furthermore, the serum levels of D-LA (Fig. 2B) and I-FABP (Fig. 2C) were assessed as biomarkers for the integrity of the intestinal epithelium. Serum levels of D-LA and I-FABP were markedly increased in the IIR group compared with those in the S group (P=0.0083 and 0.00009, respectively) and were significantly decreased in the group pretreated with t-BHQ treatment (P=0.015 or 0.0003, respectively, compared with the IIR group). However, the Nrf2 antagonists had no significant effect on serum D-LA or I-FABP.

Next, the changes in inflammatory cytokine expression in the intestine and serum were investigated. The levels of tissue IL-1β, IL-6 and TNF-α in the IIR group were significantly higher than those in the S group (P=0.021, 0.0076 and 0.033, respectively) (Fig. 3A–C). However, the tissue levels of IL-10 were markedly reduced in the IIR group compared with those in the S group (P=0.044) (Fig. 3D). In addition, pretreatment with ATRA or brusatol significantly aggravated the IIR-induced increases in IL-1β, IL-6 and TNF-α levels, while further reducing IL-10 levels. The increases in the levels of IL-1β, IL-6 and TNF-α, and the decrease of IL-10 induced by IIR were inhibited by pretreatment with t-BHQ (P=0.032, 0.017, 0.026 and 0.023, respectively) (Fig. 3). The changes in the serum levels of inflammatory cytokines were consistent with those in the intestinal tissue (Fig. 4).

To further investigate the effects of Nrf2 on apoptosis after IIR, the intestine was examined by TUNEL staining (Fig. 5). The amount of TUNEL-positive intestinal cells increased significantly after IIR (P=0.017) (Fig. 5A and B). Pretreatment with ATRA and brusatol aggravated IIR-induced apoptosis in epithelial cells. Conversely, the IIR-induced apoptosis of intestinal epithelial cells was inhibited by pretreatment with Nrf2 activator t-BHQ (P= 0.008).

The expression of apoptosis-associated proteins in the intestine was then examined. It was observed that Bax and cleaved caspase-3 were significantly increased after IIR treatment (P=0.032 and 0.046, respectively) (Fig. 5C, D and F), which was markedly exacerbated by pretreatment with Nrf2 antagonists ATRA (P=0.037 and 0.041, respectively) and brusatol (P=0.040 and 0.035, respectively) (Fig. 5C, D and F). The expression of Bcl-2 in the IIR group was markedly decreased compared with that in the S group (P=0.028), and further significant decreases were observed in the A+IIR and B+IIR groups pretreated with the Nrf2 antagonists (P=0.029 and 0.033, respectively, vs. IIR group) (Fig. 5C and E). In addition, pretreatment with t-BHQ significantly inhibited the IIR-induced increases in the expression of Bax (P=0.041) (Fig. 5C and D) and the levels of cleaved caspase-3 (P=0.037) (Fig. 5C and F), as well as the decrease in the expression of Bcl-2 (P=0.022) (Fig. 5C and E).

The Nrf2/ARE pathway is deeply involved in the protection of organs from IIR injury. The present study evaluated the expression of Nrf2 and inflammation-associated proteins in the intestine by immunohistochemical staining and western blot analysis. Positive immunohistochemical staining was indicated by yellow-brown-stained granules. In the IIR group, protein expression was mainly identified in the cytoplasm and the nuclei of intestinal tissue cells. In the group pretreated with t-BHQ, cellular staining for Nrf2 became lighter after IIR. In the IIR group, a large proportion of the cytoplasm and nuclei of the intestinal tissue cells appeared brownish-yellow or dark brown, and a large proportion of cytoplasm and nuclei of the intestinal tissue cells remained brownish-yellow or dark brown, indicating Nrf2 expression in the group pretreated with t-BHQ prior to IIR (Fig. 6A). After IIR induction, the cytoplasm and the nuclei of the tissues exhibited Nrf2 and NF-κB expression in the epithelial lamina propria according to immunohistochemical staining (Fig. 6A). Quantification of the staining revealed that the expression of Nrf2 (P=0.034) (Fig. 6B) and NF-κB (P=0.029) (Fig. 6C) was significantly increased after IIR. Pretreatment with ATRA did not affect the expression of Nrf2 in the cytoplasm and the nuclei of intestinal cells compared with those in the IIR group, while it remained significantly higher than that in the S group (P=0.009) (Fig. 6A and B). However, pretreatment with brusatol abolished the IIR-induced expression with no significant difference compared with that in the S group (P=0.098) (Fig. 6A and B). The levels of NF-κB and p-IKBα in the IIR group were much higher than those in the group pretreated with the Nrf2 antagonist ATRA prior to IIR (P=0.025 and 0.022, respectively) (Fig. 6A, C and D). In the group pretreated with t-BHQ, the accumulation of Nrf2 in the nuclei was significantly increased compared with that in the IIR group (P=0.0043) (Fig. 6A and B), while the accumulation of NF-κB in the nuclei and the levels of p-IκBα in the cytoplasm decreased significantly compared with those in the IIR group (P=0.014 and 0.028, respectively) (Fig. 6A, C and D).

Furthermore, western blot analysis of the protein expression of nuclear Nrf2 and NF-κB as well as the cytoplasmic levels of p-IKBα provided similar results to those obtained by immunohistochemistry (Fig. 7).