Male BALB/c mice (7–8 weeks old), weighing 20–22 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and fed in a controlled environment at a temperature of 25±2°C and a 12 h light/dark cycle at 50–70% humidity. Animals were acclimatized for 1 week prior to the initiation of the study and were maintained with ad libitum access to standard laboratory chow and water. All animal procedures were ethically approved by the Institutional Animal Care and Use Committee of Qingdao Municipal Hospital (Qingdao, China).

A total of 80 mice were randomly divided into four groups: Control group, DSS group, curcumin-treated (DSS + Cur) group, and resveratrol-treated (DSS + Res) group, with 20 mice per group. In the control group, mice were fed with a standard diet throughout the course of the experiment (14 days). In the DSS group, the mice received a standard diet for 14 days in addition to DSS (3.5% w/v) during the first 7 days of the experiment (from day 1 to 7). In the DSS + Cur group, the mice received the standard diet supplemented with 50 mg/kg curcumin for 14 days in addition to 3.5% DSS during the first 7 days of the study. In the DSS + Res group, the mice received the standard diet supplemented with 80 mg/kg resveratrol for 14 days in addition to 3.5% DSS during the first 7 days.

The mice were assessed daily for colitis development by monitoring body weight, gross rectal bleeding, stool consistency and survival. Mice were sacrificed by cervical dislocation at day 15 or judged as moribund (inability or unwillingness to walk, inability to reach water or food, palpable hypothermia, or lack of overt response to manipulation) before day 15 and immediately sacrificed, and the colons were removed, length and weight were measured. Scoring systems are used to assess the severity of overall disease (26), and the disease activity index (DAI) was calculated daily for each mouse. In brief, the scoring was as follows: 0, no weight loss, no occult blood in the stools and normal stool consistency; 1, weight loss of 1–5% of total body mass, no occult blood and normal stool consistency; 2, 5–10% weight loss of total body mass, positive for fecal occult blood and loose stools; 3, 10–20% weight loss of total body mass, positive for fecal occult blood and loose stools; and 4, >20% weight loss of total body mass, gross rectal bleeding and diarrhea.

Mice were sacrificed on day 8 or 15 by cervical dislocation, and the length of the colon was measured. Samples for histology were excised from the distal 6–8 cm of the colon, fixed in 10% formalin overnight at room temperature and embedded in paraffin blocks. Paraffin blocks were sliced into sections, 4 µm in thickness, and stained with hematoxylin for 6 min and eosin (cat. no. C0105; Beyotime Biotechnology, Shanghai, China) for 1 min at room temperature.

The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture supernatants of the colon tissues were measured using a Bio-Rad Multiplex bead array instrument and cytokine kits (cat. no. #7050; Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol.

Double immunofluorescence staining for autophagy-related 12 (Atg12), Beclin-1, microtubule-associated protein light chain 3 (LC3)II, phospho-mechanistic target of rapamycin (mTOR) and sirtuin 1 (SIRT1) were performed on the sections. Paraffin sections were deparaffinized with xylene and rehydrated. After endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min at room temperature, the sections were treated with 0.01 mol/l citrate (pH 6.0) in a 500-W microwave oven for 15 min for antigen retrieval. Subsequently, sections were blocked with normal goat serum (cat. no. 16210-064; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h, and incubated with the following primary antibodies overnight at 4°C: Atg12 (cat. no. ab155589), Beclin-1 (cat. no. ab62557), LC3B (cat. no. ab48394), mTOR (phospho S2448; cat. no. ab84400) and SIRT1 (cat. no. ab32441; all 1:1,000 dilution; all from Abcam, Cambridge, MA, USA). Then, Alexa Fluor 594 (red)-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1,000; cat. no. A-11032; Molecular Probes, Eugene, OR, USA) was incubated with the sections for 1 h at room temperature. Cell nuclei were stained with DAPI (1 µg/ml; cat. no. 12131; Roche Diagnostics, Basel, Switzerland) for 5 min at room temperature. Images were obtained using a fluorescence microscope (Leica SP-8; Leica Microsystems GmbH, Wetzlar, Germany).

Colon samples were homogenized on ice in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing phenylmethylsulfonyl fluoride and protease inhibitor. The protein concentrations of the samples were determined using the Bradford method (using a Bio-Rad Protein assay; Bio-Rad Laboratories, Inc.). Equivalent amounts of protein (50 µg) from each sample were separated on 12% sodium dodecyl sulfate-polyacrylamide gels, and fractioned proteins were transferred onto 0.22 µM nitrocellulose membranes (EMD Millipore, Billerica, MA, USA) at 75 V for 45 min. Subsequently, the membranes were blocked with Tris-buffered saline solution containing 5% nonfat dried milk at room temperature for 1 h, and then incubated with specific antibodies against Atg12 (cat. no. 2011), Beclin-1 (cat. no. 3495), LC3II (cat. no. 4108), mTOR (cat. no. 2972) and SIRT1 (cat. no. 8469; all 1:1,000 dilution; all from Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. Bands were visualized using a chemiluminescence detection system (cat. no. #34095; Pierce; Thermo Fisher Scientific, Inc.) and quantified with Quantity One Software 4.6.7 (Bio-Rad Laboratories, Inc.).

Data analysis was performed using GraphPad Prism software (version 4.03; GraphPad Software, Inc., La Jolla, CA, USA). Parametric data are presented as the mean ± standard error of at least three independent experiments. The statistical significance of any difference in each parameter among the groups was evaluated using one-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Symptomatic parameters, including the survival rate, body weight loss and DAI, caused by colitis in mice were recorded. On day 15, six mice were randomly selected from each group for body weight and DAI analysis. As presented in Fig. 1A, DSS exposure resulted in a 50% mortality rate at day 14, which was similar to the data reported in a previous study (27). However, the survival rate of the DSS-treated mice was improved in the curcumin (66.7%) and resveratrol (83.3%)-treated groups. In the DSS-treated group, a loss of body weight was recorded from day 4 onwards following the administration of DSS (4%) and this loss remained significantly higher than the normal mice until day 8 (83.85±2.36%) (P<0.05). The administration of curcumin or resveratrol significantly reduced the body weight loss in mice with colitis compared with the DSS-treated mice (P<0.05; Fig. 1B). Another common feature of the DSS-induced model of colitis is an increase in the DAI (28). Curcumin or resveratrol treatment significantly ameliorated diarrhea and rectal bleeding compared with the DSS alone-treated group (P<0.05; Fig. 1C).

Colon tissues were collected and measured at days 7 and 14, in order to study the effect of curcumin or resveratrol on the inflammation-induced decrease in colon length, a morphological parameter used to assess the degree of inflammation in the DSS-induced colitis mouse model. The colon length of the mice exposed to DSS was significantly shorter compared with that of the control mice (P<0.05; Fig. 2). The colon of mice treated with DSS and curcumin, or DSS and resveratrol exhibit a statistically significant increase in colon length when compared with the DSS group and almost completely reverted to a normal length (P<0.05).

Histological analysis of the distal colon tissue revealed that the treatment of mice with DSS resulted in the destruction of colonic architecture with ulcerations, crypt dilation, goblet cell depletion, a disrupted epithelial layer and intense infiltration of the inflammatory cells compared with the normal morphology of colon tissue (Fig. 2B). By contrast, colon tissues from the curcumin or resveratrol-treated mice exhibited predominantly intact colon histology, with a preserved epithelial layer and crypt structure, and reduced numbers of infiltrating cells (Fig. 2B).

Colonic TNF-α and IL-6 levels were significantly elevated in the DSS group compared with the controls (P<0.05; Fig. 3). Curcumin or resveratrol administration prevented significant increases in TNF-α and IL-6 expression levels at day 7 (P<0.05). Similar effects of curcumin or resveratrol treatment were observed at day 14.

Immunofluorescence staining for components of cellular autophagy signaling pathways, including Atg12, Beclin-1 and LC3II, was performed on colon tissues. As presented in Fig. 4A, a marked increase in the expression of the autophagy-associated proteins Atg12, Beclin-1 and LC3II was observed in the DSS-treated mice when compared with the control mice (P<0.05). LC3I expression also appeared to increase. Treatment with curcumin or resveratrol significantly reversed the changes in the levels of autophagy-associated genes when compared with the DSS-treated mice (P<0.05). To further confirm this observation, the LC3II, Atg12 and Beclin-1 expression, and the LC3II/I ratio were assessed using western blot analysis (Fig. 4B). The expression levels of Atg12, Beclin-1 and LC3II were significantly increased by exposure to DSS (P<0.05), and curcumin or resveratrol treatment significantly reduced their expression.

Furthermore, the present study intended to investigate whether the inhibition of autophagy was mediated via mTOR and SIRT1 activation. As determined by immunofluorescence staining and western blot analysis, a substantial decrease in the protein expression levels of phospho-mTOR and SIRT1 were observed in the DSS group compared with the normal mice (Fig. 5A and B). Additionally, phospho-mTOR and SIRT1 demonstrated a higher expression in the curcumin or resveratrol-treated groups compared with the DSS alone treated group.