Female BALB/c mice, aged 6–8 weeks and weighing 20 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., (Shanghai, China) and maintained at 22°C with a 12-h light/dark cycle and ad libitum access to water and a standard rodent diet. Mice were randomly divided into four groups: Normal control, DSS-treated, 5-FU-treated and DSS + 5-FU-treated groups (n=20 per group). Colitis was induced in the mice with 4% DSS (molecular weight, 36–50 kDa; MP Biomedicals, LLC, Santa Ana, CA, USA) dissolved in tap water administered ad libitum (days 1–7). Control mice were administered tap water ad libitum (days 1–7). 5-FU (Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally at a dosage of 15 mg/kg body weight once daily for the first 3 days of DSS treatment. Control mice received phosphate-buffered saline (PBS; Sangon Biotech Co., Ltd., Shanghai, China) via intra-peritoneal injection once daily for the first three days, at the same volume of 5-FU. Half the mice from each group were sacrificed via intraperitoneal injection of 4% pentobarbital on day 3 and the other half on day 7. Colon tissue was excised and fixed in 4% paraformaldehyde (Sangon Biotech Co., Ltd.), or frozen in liquid nitrogen and stored at −80°C. The Animal Welfare Committee of Shanghai East Hospital approved all experimental procedures.

Multiple clinical parameters were measured throughout the present study, including the percentage of initial weight, stool consistency and fecal bleeding. These were recorded and graded for severity on a scale between 0 (less severe) and 3 (most severe), as previously described (13). In order to calculate the disease activity score, each parameter was then rescored on a scale between 0 and 3. Final scoring was as follows: Percentage of initial weight (0, >99%; 1, 92–99%; 2, 85–91%; and 3, <85%); stool consistency (0, normal; 1, slightly soft; 2, loose; and 3, liquid); bleeding on hemoccult test (0, negative; 1, faint blue; 2, blue; 3, red) (Sangon Biotech Co., Ltd.). The mean of these parameters represented the disease activity score.

Inflammation-induced reduction in colon length was used as a marker for the severity of acute DSS-induced inflammation of the colon (14). Half the mice from each group were sacrificed on day 3 and the other half on day 7. For all mice, the entire colon was removed and measured following sacrifice. The extracted colon tissue was spread on a plastic sheet, fixed with 4% paraformaldehyde and embedded in a paraffin block. Sections of paraffin-embedded tissue (4-µm-thick) were subjected to hematoxylin and eosin (H&E; Beyotime Institute of Biotechnology, Shanghai, China) staining for the evaluation of colitis severity. Histological sections were scored for severity based on the following parameters (ranging between 0 and 3): Crypt damage; leucocyte infiltration; and submucosal edema and haemorrhage. The score was further multiplied by the extent of involvement (×1, <10%; ×2, 10–25%; ×3, >25%). A value of four was added to the histological score if there was evidence of transmural involvement (15). The scoring was graded in a blinded manner by two independent investigators.

Total RNA was extracted from the colon tissue of mice with induced colitis using Tripure Isolation Reagent (Roche Diagnostics, Basel, Switzerland) and RNA quality was assessed by the A260/A280 ratio using a BioPhotometer (Eppendorf, Hamburg, Germany). Total RNA (1 µg) was reverse transcribed using a Prime Script RT Reagent kit with gDNA Eraser (Perfect Real Time; Takara Bio, Inc., Otsu, Shiga, Japan). RT-qPCR was conducted using the SYBR Green Master Mix kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The relative quantity of target mRNA was determined using the quantification cycle (Cq) method (16) by normalizing target mRNA Cq values to those for β-actin. The primer sequences used were as follows: TNF-α, F 5′-GTCGTAGCAAACCACCAAGTG-3′ and R 5′-CAGATTTGTGTTGGTCCTTC-3′; interleukin-1β (IL-1β), F 5′-AGGCTGCTCTGGGATTC-3′ and R 5′-GCCACAACAACTGACGC-3′; interferon γ (IFN-γ), F 5′-TGTAGTGAGGAACAAGCCAGAG-3′ and R 5′-TACATTTGCCGAAGAGCC-3′; IL-10, F 5′-ATGCTGCCTGCTCTTACTGAC-3′ and R 5′-CGGTTAGCAGTATGTTGTCCAG-3′; and β-actin, F 5′-GTCCACCTTCCAGCAGATGT-3′ and R 5′-AGGGAGACCAAAGCCTTCAT-3′. RT-qPCR was performed on an Applied Biosystems Step One Real-Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Neutrophil infiltration in the colon was monitored by measuring the MPO activity (17). Briefly, segments of colon were homogenized in PBS at 50 mg/ml (50 mmol/l, pH 6.0) with 0.5% hexadecyltrimethylammonium bromide (Sangon Biotech Co., Ltd.). Samples were frozen and thawed three times, and then centrifuged at 30,000 × g for 20 min for 4°C. The supernatants were diluted 1:30 with assay buffer consisting of PBS, 0.167 mg/ml o-dianisidine (Sigma-Aldrich) and 0.0005% H₂O₂ (Sangon Biotech Co., Ltd.). The colorimetric reaction was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). MPO activity was calculated as follows: MPO activity = (A450 × 13.5) / tissue weight (g), where A450 is the change in the absorbance at a wavelength of 450 nm between 1 and 3 min after the initiation of the reaction. The coefficient 13.5 was determined empirically for 1 U MPO activity to represent the amount of enzyme that will reduce 1 mol peroxide/min.

For immunohistochemical staining, slides were deparaffinized in xylene (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), rehydrated and immersed in 1.5% H₂O₂ in PBS for 30 min. Slides were then incubated for 1 h with horseradish peroxidase-conjugated avidin-biotin complex. Subsequently, slides were incubated overnight at 4°C with rat anti-mouse CD4 (14-9766; eBioscience, Inc., San Diego, CA, USA) or rabbit anti-mouse MPO (ab9535; Abcam, Cambridge, UK) primary antibodies diluted 1:50 in PBS containing 5% bovine serum albumin (BSA; Sangon Biotech Co., Ltd.) and 10% goat serum (Thermo Fisher Scientific, Inc.). Following washing three times with PBS for 5 min, biotinylated secondary goat anti-rabbit (1:100; A0277; Beyotime Institute of Biotechnology) or rabbit anti-rat (1:100; ab6733; Abcam) polyclonal antibodies were added to the sections and incubated at room temperature for 1 h. Streptavidin-horseradish peroxidase was then added and, following a 40-min incubation, the sections were stained with 3,3′-diaminobenzidine from a ChemMate and EnVision detection kit (Gene Tech Biotechnology Co., Ltd., Shanghai, China) and counterstained with hematoxylin. CD4⁺ T cells were counted in a blinded manner in 10 intercrypt spaces per mouse using an Olympus BX41-32P02-FLB3 microscope (Olympus Corporation, Tokyo, Japan).

Colon tissue was collected, immediately placed in liquid nitrogen and pulverized with a mortar. Tissue was homogenized using lysis buffer (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1 mM EDTA; 1% (m/v) NP-40; 0.2 mM phenylmethylsulfonyl fluoride; 0.1 mM NaF; and 1.0 mM dithiothreitol). Whole cell extracts were prepared by lysing the cells in cold radioimmunoprecipitation assay buffer containing a mixture of proteasome inhibitors (Beyotime Institute of Biotechnology). Lysates were then centrifuged at 15,100 × g for 20 min at 48°C, and the supernatant was collected. The homogenate was centrifuged at 48°C for 15 min at 13,000 × g, and the supernatant was then collected. The concentration of proteins was detected using a BCA assay with a Varioskan Lux Multimode Microplate Reader (Thermo Fisher Scientific, Inc.). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 80 V for 2 h at room temperature and then transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA), which were blocked with 1% BSA in PBS for 1 h at 37°C. The blots were incubated with specific primary antibodies overnight at 48°C, followed by an IRDye 800-conjugated secondary anti-body for 1 h at 37°C. Protein expression was detected using the Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE, USA). All blots were stripped and reprobed with polyclonal β-actin antibody to ascertain equal loading of proteins. Densitometric analysis was performed using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Briefly, the linear range was detected and the background was subtracted prior to normalization to β-actin, which was considered to be 1. Once normalized values were determined for each replicate, the respective means, P-values and fold changes were calculated.

Data are expressed as the mean ± standard error of the mean. Statistical significance of differences between treatment and control groups was determined by Student's t-test. Data were analyzed with one-way analysis of variance, followed by Student's t-test for experiments involving 2 groups or Dunnett's t-test for experiments involving >2 groups. Statistical analyses were performed using SPSS 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of 5-FU on the severity of acute DSS-induced colitis was assessed. Mice were treated with 5-FU or PBS with the administration of 4% DSS in the drinking water. 5-FU + DSS-treated mice experienced significantly less weight loss (P<0.05; Fig. 1A), bleeding (P<0.05; Fig. 1B) and loss of stool consistency (P<0.05; Fig. 1C) compared with the DSS-treated mice. These clinical markers suggest reduced DSS-induced inflammatory changes in the 5-FU-treated mice. Consequently, the disease activity score was significantly lower in the 5-FU + DSS-treated mice compared with the DSS group (P<0.05), particularly at day 7 (P<0.01; Fig. 1D).

In accordance with the clinical data, gross and histological parameters, including crypt damage, leucocyte infiltration, colon length and histological score, favored the 5-FU + DSS-treated mice, suggesting less severe disease compared with the DSS-treated mice. Accordingly, the mean length of colon in the 5-FU + DSS-treated mice (8.35±1.23 cm) was significantly longer than in the control group (6.48±0.89 cm; P<0.05; Fig. 2A), suggesting decreased inflammation. In addition, the mean histological score from colon tissue in the 5-FU group was significantly lower than the DSS group (P<0.05; Fig. 2B). Fig. 2C demonstrates that 5-FU treatment prevented the abnormal architecture change in the colon that occurred following DSS treatment. The 5-FU control group did not indicate a distorted colon architecture (Fig. 2C). Furthermore, the dose of 15 mg/kg 5-FU resulted in no significant histological or biochemical damage to the liver and kidney of the mice compared with the DSS-treated group (Fig. 3).

Oral administration of DSS is toxic to the colonic epithelium and triggers inflammation by disrupting the compartmentalization of commensal bacteria in the gut with high levels of proinflammatory cytokines, such as TNF-α, IL-1β and IFN-γ (18,19). The release of cytokines is considered to be an indicator of the inflammatory response. Thus, the mRNA expression levels of TNF-α, IL-1β and IFN-γ in the colon tissue were detected using RT-qPCR. As demonstrated in Fig. 4, treatment with DSS increased the release of TNF-α, IL-1β and IFN-γ in mice compared with the normal control group. Notably, in mice treated with 5-FU, the DSS-induced secretion of TNF-α (P<0.05), IL-1β (P<0.01) and IFN-γ (P<0.05) were significantly reduced, indicating that 5-FU may inhibit inflammation in intestinal epithelial cells. The expression levels of the anti-inflammatory cytokine IL-10 did not significantly change.

Another trait of colitis is the invasion of immune cells to the intestinal mucosal with increased expression levels of MPO and CD4 (20). There was a marked increase in lymphocytic infiltration and loss of glandular architecture in the DSS-treated group compared with the 5-FU + DSS-treated group (Fig. 5A). Induction of colitis by DSS resulted in an increased MPO activity in the colon tissue compared with the normal control group (Fig. 5B). Additionally, administration of 5-FU to DSS-treated mice significantly reduced the MPO activity compared with the DSS-treated group (P<0.01; Fig. 5C). Similarly, 5-FU + DSS-treated mice demonstrated significantly decreased CD4⁺ T cell infiltration compared with the DSS-treated group (P<0.01; Fig. 5B).

NF-κB is a critical transcription factor in the inflammatory response. It functions as a pro-inflammatory factor and participates in the pathophysiology of intestinal inflammatory diseases (21). Previous studies detected activated NF-κB in colonic mucosal tissue sections of patients with IBD compared with healthy subjects (22,23). Furthermore, NF-κB was identified as one of the primary factors governing the formation of the molecular network leading to various cellular functions associated with IBD (24). For example, numerous NF-κB-dependent pro-inflammatory mediators, such as IL-1β, TNF-α, IL-12p40 and IL-23p19, are elevated in patients with IBD and, thus, represent therapeutic targets (25). Additionally, previous studies demonstrated that 5-FU inhibits the activation of NF-κB and its subsequent nuclear translocation (26,27). To investigate the mechanism of 5-FU's anti-inflammatory activity, the effect of 5-FU on the activation of the NF-κB pathway in intestinal epithelial cells was investigated. Changes in the expression levels of phosphorylated (p)NF-κB-p65 in the intestinal mucosa of DSS-treated mice were evaluated by western blotting. As demonstrated in Fig. 6, pNF-κB-p65 was upregulated in DSS-treated mice compared with the normal control group. 5-FU treatment significantly reduced this effect compared with the DSS-treated group (P<0.05).