A total of 70 female BALB/c mice, aged 4–6 weeks and weighing 20–24 g were purchased from the Animal Care Facility of Yangzhou University (Yangzhou, China). Mice were maintained in cages at the Animal Care Facility of Nantong University (Nantong, China) for two weeks at 22–24°C and 49% humidity with a 12/12 h light/dark cycle. Mice were fed a mixed-feed formula and had ad libitum access to distilled drinking water. Drinking water and feed were fresh and changed daily. All animal experiments were approved by the Ethics Committee for Animal Care and Use of the Affiliated Hospital of Nantong University.

Following acclimatization for two weeks, a DSS-induced (MP Biomedicals, Santa Ana, CA, USA) mouse model was established according to the method described by Stevceva et al (27). A total of 70 BALB/c female mice were randomly classified into seven equal groups and raised in separate cages (10 mice/cage). Group A received no treatment and was the blank control; group B, the DSS-induced model, received 0.2 ml normal saline by intraperitoneal injection once a day; group C was intraperitoneally injected with 0.2 ml propylene glycol (20%) as a vehicle control once a day following successful DSS induction; groups D (TL1), E (TL2) and F (TL3) were intraperitoneally injected with TL (Zelang Medical Science and Technology Co., Ltd. Nanjing, China) dissolved in 20% propylene glycol at a daily dose of 0.2, 0.4 and 0.6 mg/kg, respectively, following successful DSS induction; and group G was intraperitoneally injected with dexamethasone dissolved in normal saline at a daily dose of 0.1 mg/kg.

From the first day of DSS induction, the vigor, activity, diet, body weight change, stool characteristics and hematochezia condition of the mice were recorded each day. The DAI was calculated to analyze the effects of TL on UC, as follows: DAI score=(body weight loss score + stool characteristics score + hematochezia score) / 3 (Table I) (28).

Mice were sacrificed by cervical dislocation and the intestinal cavity was exposed via a longitudinal incision along the mesenteric junction. Following removal of the stools, the intestine was washed several times in pre-cooled normal saline, and dried using filter paper. The intestinal wall was spread and fixed with pins for visual inspection of inflammation and ulcer formation. Disease severity was evaluated according to the criteria outlined by Ekstrom (29), as shown in Table II. The intestinal segment with lesions was subsequently cut into three symmetrical sections (4 µm each) using a microtome (CM1950; Leica Microsystems GmbH, Wetzlar, Germany). One section of the intestine was fixed in 4% formaldehyde for pathological analysis with hematoxylin and eosin (HE; Beyotime Institute of Biotechnology, Inc., Haimen, China) staining or paraffin-embedded for immunohistochemical staining; the second section was preserved at −80°C for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis; and the third section was used for protein extraction and western blot analysis.

Following conventional paraffin embedding and segmentation using a microtome, tissue samples with prominent lesions were subjected to pathological analysis via HE staining in order to observe histological alterations. Using blinded analysis, the samples were graded according to the criteria outlined by Boirivant et al (30), as shown in Table III. Samples were independently graded by two pathologists, whom were blinded to the scoring of one another, and the mean of the two histology scores was calculated.

ELISA was performed using an ELISA kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. A standard linear regression curve was obtained by plotting a standard curve of the concentrations of the standard samples and the corresponding optical density (OD) values. The sample concentration was calculated according to the equation of the standard curve.

An immunohistochemical assay was performed according to the manufacturer's protocol (Abcam, Cambridge, MA, USA). IL-1β-positive cells were stained a brown/yellow color, with the background appearing as light blue under the light microscope (Olympus Corporation, Tokyo, Japan). All sections were observed using an optical microscope under identical conditions. Cells with brown or brown/yellow granular particles in the nucleus or cytoplasm were considered as positive for IL-1β expression, whereas those without brown/yellow particles were considered to be IL-1β-negative cells. At ×400 magnification, cells in ≥10 visual fields were counted for each section and the percentage of positive cells was calculated. Cell staining intensity was graded in four tiers as follows: 0, negative staining; 1, weak positive staining; 2, moderate positive staining; and 3, strong positive staining. Five scales were used to grade the number of positive cells: 0, no positive cells; 1, 1–25% positive cells; 2, 26–50% positive cells; 3, 51–75% positive cells; and 4, 76–100% positive cells.

Total RNA from each mouse colon was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). The extracted RNA was treated with DNAse I (Roche Diagnostics, Basel, Switzerland). Following measurement of concentration, the total RNA was stored at −80°C. Total RNA was reverse transcribed to cDNA using a SYBR Green Supermix kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China). Forward and reverse primer sequences used to amplify IL-1β and β-actin were synthesized by the Sangon Biotech Co., Ltd. (Shanghai, China). The primers for IL-1β (607 bp) were as follows: Forward, 5′-AGCTGACCCTAAACAGATGA3′, and reverse, 5′-GATCTACACTCTCCAGCTGTAGC-3′. The primers for β-actin (446 bp) were as follows: Forward, 5′-GAGACCTTCAACACCCCAGC-3′, and reverse, 5′-CCACAGGATTCCATACCCAA-3′. The mRNA expression levels of IL-1β were analyzed by RT-qPCR using the SYBR Green Supermix kit, according to the manufacturer's protocol. PCR was performed using a fluorescence quantitative PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.), as follows: 95°C pre-degeneration for 2 min, 95°C degeneration for 15 sec, annealing at 58°C for 25 sec, and extension at 72°C for 35 sec for 45 cycles. The fluorescent readout was obtained at 72°C during each cycle. In order to test the specificity of each reaction, melting curve analysis was performed, as follows: 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. The ratio of target gene to β-actin mRNA was used to evaluate the expression levels of the target gene. The mRNA expression levels of the target gene were normalized to those of β-actin using the 2^−ΔΔCq method.

IL-1β protein expression was measured by western blot analysis. Total protein (20 µl) was extracted from intestinal tissue samples and protein concentration was measured by UV spectrophotometry (Shanghai Mapada Instruments Co., Ltd., Shanghai, China). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto a nitrocellulose membrane (GE Healthcare Bio-Sciences, Pittsburg, PA, USA). Following blocking for 4 h in 5% Tris-buffered saline with Tween-20 (TBST), the membrane was incubated with anti-mouse IL-1β primary antibody (1:2,500; cat. no. ab200478; Abcam) at 4°C overnight. Subsequently, the membrane was washed five times in TBST for 10 min and incubated with secondary antibody (1:5,000; cat. no. A28175; Pierce Biotechnology; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. Bands were visualized by enhanced chemiluminescence. Data were processed using ImageJ version 1.36b (National Institutes of Health, Bethesda, MA, USA), and the normalized expression level of IL-1β was measured as the gray scale ratio of IL-1β to β-actin bands.

Windows Excel 2010 software (Microsoft Corp., Redmond, WA, USA) was utilized to form a database of experimental results. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Numerical data was presented as the mean ± standard deviation and analyzed by analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Characteristics including vigor, body weight, activity, hair color, diet, stool property and hematochezia were assessed from the first day of DSS induction and DAI scores were calculated to assess the effect of TL on UC. Mice in the blank control group exhibited shiny hair, increased body weight, normal vigor and activity levels, and their diet and stool were normal. From the second or third day of receiving normal saline or propylene glycol injection, DSS-induced mice were anorexic, with decreased activity, piloerection, darkened hair color, weight loss and altered stool properties, including loose and soft stools. Hematochezia or occult fecal blood were observed on the fourth day following treatment, accompanies by prominent body weight loss and reduced activity. One mouse in the normal saline group succumbed to the treatment after six days. Mice in the TL2, TL3 and dexamethasone treatment groups exhibited improved diet and body weight, as compared with the normal saline or propylene glycol treatment groups. In addition, the vigor and activity of mice were improved, hair shine and color returned, stool properties returned to normal and hematochezia and occult fecal blood were alleviated (Fig. 1). In summary, The DAI scores of the normal saline injection and propylene glycol injection groups were significantly elevated as compared with the blank control group (P<0.05); conversely, the scores for the TL and dexamethasone treatment groups were significantly decreased (P<0.05). The results suggest that treatment with TL and dexamethasone may alleviate the symptoms of mouse colon ulcerative colitis.

Mice in each group were scored according to the scale outlined in Table II. The results are presented in Table IV. The gross morphology scores of the normal saline injection and propylene glycol injection groups were significantly elevated as compared with the blank control group (P<0.01), and the scores significantly decreased in the TL and dexamethasone treatment groups (P<0.01). No hyperemia, edema, erosion, or ulcer formations were detected in the colonic mucosa of mice in the blank control group. Three mice exhibited mild hyperemia of the colonic mucosa in the distal colon. Colonic lesions were found in the anus of mice in the normal saline and propylene glycol treatment groups, which were gradually aggravated resulting in moderate to severe mucosal hyperemia and edema, multiple sites of erosion and superficial ulceration. Mild hyperemia and edema were detected in the proximal colon. Conversely, mice that received TL or dexamethasone treatment demonstrated markedly reduced erosion and ulceration, and edema of the colonic mucosa was mild or moderate. Hyperemia and edema in the proximal colon were not obvious. The results suggest that treatment with TL and dexamethasone may reduce gross morphology and scoring.

Mice in each group were scored according to the scale outlined in Table III. HE staining demonstrated good colonic mucosa integrity in mice of the blank control group, with well-arranged lamina propria glands of normal morphology. Conversely, colonic mucosal lesion, loss, erosion and ulceration were detected in mice in the normal saline and propylene glycol treatment groups, with extensive lymphocyte and mononuclear cell infiltration and minimal neutrophil infiltration into the mucosa and submucosa. Lymphoid follicles were occasionally formed, accompanied by lamina propria gland deformation and disordered arrangement. The severity of inflammatory cell infiltration, ulceration, and erosion was milder in mice that received TL or dexamethasone treatment, as compared with the normal saline or propylene glycol treatment groups (Table V). The histological scores of the normal saline injection and propylene glycol injection groups were significantly elevated as compared with the blank control group (P<0.01). The scores significantly decreased in the TL and dexamethasone treatment groups (P<0.01). The results suggest that treatment with TL and dexamethasone may reduce ulceration and erosion.

Serum levels of IL-1β in the blank control, normal saline and propylene glycol treatment groups were 42.44±8.26, 96.39±10.10 and 89.53±9.88 pg/ml, respectively. Serum IL-1β levels were significantly increased in mice in the normal saline and propylene glycol treatment groups, as compared with those in the blank control group (P<0.01). Serum IL-1β levels were 69.69±7.36, 61.75±7.10 and 59.26±6.22 pg/ml for mice in the TL2, TL3 and dexamethasone treatment groups, respectively, which was significantly decreased, as compared with the normal saline and propylene glycol treatment groups (P<0.01). However, the serum level of IL-1β was 84.23±8.52 pg/ml in mice that received TL1 treatment, which was not significantly different from the normal saline and propylene glycol treatment groups. No significant differences in serum IL-1β levels were detected between the TL2, TL3 and dexamethasone treatment groups. The results suggest that treatment with TL and dexamethasone may decrease the serum levels of IL-1β.

Fig. 2 shows the immunohistological images and Table VI outlines the comparison of immunohistological scores among the groups. As compared with the weak expression of IL-1β detected in the colonic mucosa of mice in the blank control group, IL-1β expression levels were significantly increased the normal saline and propylene glycol groups (P<0.01). Furthermore, IL-1β expression levels were significantly decreased in the TL2, TL3 and dexamethasone treatment groups, as compared with the normal saline and propylene glycol groups (P<0.01). No significant differences were detected among the TL2, TL3 and dexamethasone treatment group. The results suggest that treatment with TL and dexamethasone may downregulate the expression of IL-1β in colon tissue.

As compared with the weak IL-1β mRNA expression detected in the colonic mucosal tissue of mice in the blank control group, IL-1β mRNA expression was significantly increased in the normal saline and propylene glycol treatment groups (P<0.01). Furthermore, as compared with the blank control group, IL-1β mRNA expression levels were markedly increased in the TL2, TL3 and dexamethasone treatment groups, and were significantly reduced, as compared with the normal saline and propylene glycol treatment groups (P<0.01). No significant differences were detected among the TL2, TL3 and dexamethasone treatment groups (Fig. 3). These results suggest that treatment with TL and dexamethasone may inhibit IL-1β mRNA expression in colon tissue.

IL-1β protein expression was detected by western blot analysis. Fig. 4 shows the western blot images of IL-1β and β-actin in each group. As compared with the weak IL-1β protein expression levels detected in the colonic mucosa tissue of mice in the blank control group, IL-1β protein expression levels were significantly upregulated in the normal saline and propylene glycol treatment groups (P<0.01). Furthermore, as compared with the blank control group, IL-1β protein expression levels were markedly increased in the TL2, TL3 and dexamethasone treatment groups, and were significantly decreased, as compared with the normal saline and propylene glycol treatment groups (P<0.01). No significant differences were detected among the TL2, TL3 and dexamethasone treatment groups. The results suggest that treatment with TL and dexamethasone may suppress IL-1β expression in colon tissue.