A total of 80 female BALB/c mice (age, 4–6 weeks; weight, 20–24 g) were obtained from the Animal Center of the Medical College of Nantong University (Nantong, China). They were raised in the Animal Center of Nantong University (Nantong, China) for 2 weeks at 22–24°C and 49% humidity with a 12-h light/dark cycle and were fed a mixed-feed formula and had ad libitum access to distilled drinking water. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals promulgated by the National Research Council, and supported by the Chinese National Committee for the Use of Experimental Animals for Medical Purposes, Jiangsu Branch (Nanjing, China). The study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University (Nantong, China).

According to the method by Stevceva et al (28) from 2001, a mouse model of UC was established using dextran sulfate sodium (DSS; MP Biomedicals, Santa Ana, CA, USA). A total of 80 female BALB/c mice were stochastically divided into eight groups and housed in separate cages (10 mice per cage). The eight groups were designated as Group A-H. Group A was composed of non-treated sham mice; groups B-H contained DSS-induced model animals. Mice in group B were injected 0.2 ml normal saline intraperitoneally once daily; group C received 0.2 ml 20% propylene glycol as vehicle control daily, also intraperitoneally. Groups D-F were intraperitoneally injected daily with TL (Zelang Medical Science and Technology, Nanjing, China) dissolved in 20% propylene glycol at 0.20 mg/kg (TL1), 0.40 mg/kg (TL2) and 0.60 mg/kg (TL3). Group G was intraperitoneally injected with dexamethasone (Sichuan Guangda Pharmaceutical Co., Ltd., Pengzhou, China) dissolved in normal saline at a daily dose of 0.1 mg/kg, and group H was administered mesalazine (Ipsen Ltd., Slough, UK) dissolved in water by oral gavage at a daily dose of 20 mg/kg.

To assess the severity of colitis, the mice were monitored daily for activity, weight, dietary intake, stool properties and hematochezia. According to the standard by Murano et al (29), the DAI score was calculated as follows: DAI=(score of body weight loss + score of stool properties + score of hematochezia)/3.

At day 8, the mice were sacrificed by cervical dislocation and sectioned along the mesenteric junction to expose the intestinal cavity. After washing and drying, the collected intestinal wall was spread and fixed for visual inspection of inflammation and ulcer formation. The disease status was evaluated using the criteria by Ekström (30).

Colonic tissue specimens were paraffin-embedded and then stained with hematoxylin and eosin (H&E). The level of inflammation was evaluated by two independent pathologists according to a well-established scoring system (31).

Tissue samples were stained according to the manufacturer's protocol using and immunohistochemistry kit (cat. no. ab64261; Abcam, Cambridge, MA, USA), and the stained sections were analyzed by microscopy. To avoid possible technical errors, immunostaining data were evaluated by two independent pathologists. IL-6-positive cells (anti-IL-6 antibody; cat. no. ab7737; Abcam) were stained a brown/yellow color, ten high-power fields were randomly selected in each sample, with at least 500 cells counted. The following scoring system was employed for density evaluation: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. In addition, the extent of staining was recorded as follows: 0, <1%; 1, 1–25%; 2, 26–50%; 3, 51–75%; 4, >75%. IL-6 expression was evaluated by the two scales. The final score was obtained by multiplication of the two scores.

The blood was obtained from the mice following scarification. IL-6 levels in plasma were measured using ELISA kits purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). ELISA was performed according to the manufacturer's instructions. The optical density was measured at a wavelength of 450 nm using a microtiter plate reader (Luwen Biological Technology Co., Ltd., Shanghai, China). The final results were expressed as pg/ml.

Total RNA from the colonic mucosa was extracted according to standard TRIzol RNA isolation instructions (Invitrogen; Thermo Fisher Scientific, Inc.) and evaluated with a spectrophotometer for quantity and purity. The RT reaction was performed for 15 min at 37°C. Forward and reverse primer sequences used to amplify IL-6 and β-actin were synthesized by the Sangon Biotech Co., Ltd. (Shanghai, China). The primers for IL-6 were as follows: Forward, 5′-GGAATTCGTGGAAATGAGAA-3′ and reverse 5′-GCACTAGGAAAGCCGAGTAC-3′. The primers for β-actin were as follows: Forward 5′-TCATCACTATTGGCAACGAGC-3′, and reverse 5′-AACAGTCCGCCTAGAAGCAC-3′. The amplification was performed under the following conditions: 95°C for 10 min (initial denaturation), followed by 45 cycles of 95°C for 10 sec (denaturation), 60°C for 20 sec (annealing) and 20°C for 35 sec (extension). Melting curves were generated at 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec in order to assess the specificity of each reaction. β-actin mRNA was used as an internal control. The mRNA ratio of target gene to β-actin was calculated as the relative expression level of the target gene using the 2-∆∆Cq method (32).

Tissue samples were homogenized in homogenization buffer and centrifuged at 13,000 × g at 4°C for 20 min to collect the supernatants. The supernatants were subjected to BCA protein assay to determine the protein concentrations of the samples. The total tissue protein was extracted and its concentration was measured by UV spectrophotometry (Shanghai Mapada Instruments Co., Ltd., Shanghai, China). Equal amounts of total protein (20 µl) were separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk for 2 h, the membrane was incubated overnight with anti-IL-6 (1:2,500; cat. no. ab7737; Abcam) and anti-β-actin antibodies (1:2,500; cat. no. ab8226; Abcam) at 4°C. This was followed by incubation with horseradish peroxidase-conjugated secondary anti-mouse antibody (1:5,000; cat. no. A32733; Pierce; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Band intensities were measured with ImageJ version 1.36b (National Institutes of Health, Bethesda, MA, USA) and normalized to β-actin levels. Experiments were performed three times.

Values are expressed as the mean ± standard error of the mean. SPSS 21.0 (International Business Machines Corporation, Armonk, NY, USA) was employed for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

To determine DAI scores, factors such as activity, body weight, fur color, grooming, diet, stool consistency and hematochezia were taken into account. Mice with no intervention had a normal body weight, shiny fur as well as a regular dietary intake and stool. However, mice treated with DSS (colitis models) were anorexic and the body weight started to decrease at days 2–3 after injection with normal saline or propylene glycol; their hair became darkened and the stool properties were gradually altered. On the fourth day of treatment, hematochezia or fecal occult blood was observed, along with marked body weight loss. However, after treatment with TL (0.40 or 0.60 mg/kg), dexamethasone or mesalazine, the mice showed increased body weight and dietary intake; indeed, their activity was improved, shiny hair was regained, hematochezia was alleviated and their stool gradually regained its normal consistency (Fig. 1). These findings supported the previously suggested notion that TL has a therapeutic benefit in UC.

Next, the present study assessed the morphological alterations in the intestinal mucosa of the mice. In the blank control group, no hyperemia, edema or erosion was found. However, after treatment with DSS, obvious erosion and superficial ulceration were observed. The lesions were continuous and developed from the anus; the rectum and distal colon were mostly affected, with severe mucosal hyperemia and edema, while the proximal colon showed mild hyperemia and edema. After treatment with TL (0.40 or 0.60 mg/kg), dexamethasone or mesalazine, the colonic mucosal injury was alleviated; the rectum and distal colon mucosae had mild hyperemia and edema, and no hyperemia or edema was observed in the proximal colon. The associated scores are shown in Table I.

For histological observation, H&E-stained colonic mucosa specimens were prepared. The colonic mucosa was intact, without erosion or ulceration; the glands had normal morphology and were well arranged in sham control mice. After treatment with DSS, lesion, erosion and ulceration areas were observed in the colonic mucosa, with massive infiltration of lymphocytes and mononuclear cells as well as a certain number of neutrophils in the mucosa and submucosal. Lymphoid follicles were formed occasionally. The glands were deformed and disordered. However, after treatment with TL (0.40 or 0.60 mg/kg), dexamethasone or mesalazine, the degree of inflammation, erosion and ulceration in the colonic mucosa was significantly alleviated compared with that in the saline- and propylene glycol-treated groups (Table II).

Next, the serum levels of IL-6 in mice were analyzed using ELISA. Serum IL-6 levels in the normal saline- and propylene glycol-treated groups were significantly higher than the blank control groups, indicating a significant increase of IL-6 induced by DSS compared with that in the control group (P<0.05). Serum IL-6 levels in the TL (0.40 and 0.60 mg/kg), dexamethasone and mesalazine treatment groups were significantly reduced compared with those in the normal saline and propylene glycol-treated groups (P<0.05; Table III).

IL-6 mRNA was barely detected in the colonic mucosa of blank control mice; however, after treatment with DSS, IL-6 gene expression levels were significantly increased (P<0.05). Of note, treatment with TL (0.40 or 0.60 mg/kg), dexamethasone or mesalazine significantly decreased colitis-induced IL-6 mRNA levels (P<0.05). However, there was no significant difference in IL-6 mRNA expression levels among these abovementioned treatment groups E-H (P>0.05; Table IV).

As presented in Fig. 2 and Table V, immunohistological analysis revealed an upregulation of IL-6 in the colonic mucosa tissue samples from UC mice in the vehicle-treated groups, compared with those in the blank control (P<0.05). However, TL (0.40 and 0.60 mg/kg), dexamethasone and mesalazine treatment alleviated this UC-associated upregulation of IL-6 (P<0.05). However, there was no significant difference in IL-6 expression levels among the TL (0.40 and 0.60 mg/kg), dexamethasone and mesalazine treatment groups (P>0.05).

To further assess the expression pattern of IL-6 in colonic mucosa tissue samples, IL-6 protein levels were evaluated by western blot analysis. IL-6 protein was weakly expressed in the colonic mucosa tissues of blank control mice; however, significantly higher IL-6 protein levels were found after induction of UC with DSS, and treatment with dexamethasone, mesalazine and TL (0.40 or 0.60 mg/kg) significantly attenuated these effects (Fig. 3).