Human colon fibroblast CCD-18Co cells were purchased from The American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), penicillin and streptomycin (100 U/ml) in a humidified 5% CO₂ incubator at 37°C.

A Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) assay was used to evaluate the cell viability, according to the manufacturer's protocols. CCD-18Co cells were seeded into a 96-well plate (5×10³ cells/well) and cultured overnight. Then, CCD-18Co cells were pretreated with ASI (0, 10 or 50 µM) for 24 h, followed by treatment with LPS (0 or 1 µg/ml) for an additional 2 h. After 24 h incubation, 10 µl CCK-8 reagent was added to each well. The absorbance values were measured at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.). LPS was provided by Sigma-Aldrich; Merck KGaA. ASI was purchased from MedChemExpress.

CCD-18Co cells were seeded into a 24-well plate (2×10⁶ cells/well) and cultured overnight. Cells were pretreated with ASI (0, 10 or 50 µM) for 24 h, followed by treatment with LPS (0 or 1 µg/ml) for an additional 2 h. Then, after 24 h incubation, samples of the supernatant were collected from each well to measure TNF-α, IL-1β and IL-6 levels by ELISA. TNF-α (cat. no. H052), IL-1β (cat. no. H002) and IL-6 (cat. no. H007) ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute.

CCD-18Co cells and segments of mouse colon were collected and homogenized using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with protease and phosphatase inhibitors. A Bradford protein assay (Beyotime Institute of Biotechnology) was used to quantify the protein concentration. Equal amounts of proteins (30 µg) in the lysate were then separated via 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.) for 2 h. The PVDF membranes were blocked in TBST containing 5% skim milk at room temperature for 1 h, and then incubated at 4°C with primary antibodies overnight. The primary antibodies were as follows: Rabbit anti-NF-κB transcription factor p65 (p65; 1:1,000; cat. no. ab16502), rabbit anti-phosphorylated NF-κB transcription factor p65 (p-p65; 1:1,000; cat. no. ab86299), rabbit anti-inhibitor of NF-κB (IκB; 1:1,000; cat. no. ab32518), rabbit anti-phosphorylated inhibitor of NF-κB (p-IκB; 1:1,000; cat. no. ab133462), rabbit anti-claudin-1 (1:1,000; cat. no. ab15098), rabbit anti-tight junction protein ZO-1 (ZO-1; 1:1,000; cat. no. ab96587) and anti-β-actin (1:1,000; cat. no. ab8227). The PVDF membranes were washed 3 times with TBST, then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (1:5,000; cat. no. ab7090) at room temperature for 1 h. All antibodies were purchased from Abcam. The protein blots were detected with an enhanced chemiluminescence system (Bio-Rad Laboratories, Inc.) and visualized using a Fluor Chem E imager on a FluorChem E Imaging System 46688 (ProteinSimple). The density of the blots for target proteins were normalized to β-actin.

Male C57BL/6 mice (21–23 g, aged 8–10 weeks) were provided by Shanghai SLAC Laboratory Animal Co., Ltd. The animals were housed at a temperature of 24±1°C, a 12 h light: Dark schedule starting at 08:00, and a humidity of 50–60% for 4 days prior to the initiation of the experiment. The animals were provided with water and food ad libitum.

Following acclimation, the 30 mice were randomly divided into five experimental groups (n=6): Control (water); model group (DSS); DSS + 50 mg/kg ASI (DSS/ASI); DSS + 200 mg/kg ASI (DSS/ASI); and 200 mg/kg ASI treatment (ASI). The mice were orally administered 50 or 200 mg/kg ASI once daily for 3 days (from day 0 to day 3). On day 3, UC was induced in the animals via oral administration of 3% (w/v) DSS (ad libitum; MP Biomedicals) in fresh drinking water for 5 days. From day 8, animals were orally administered 50 or 200 mg/kg ASI alone until the end of the experiment. The body weight of the mice was monitored every other day. The disease activity index (DAI) score was determined as described previously (20). At the end of the experiments, the mice were sacrificed using CO₂ at a displacement rate of 20% of the chamber volume/min (CO₂ flow rate, 2.5 l/min). The length of the colon between the ileocecal junction and the proximal rectum in the mice was measured. The colon tissues were immediately fixed in 4% formalin overnight at room temperature, embedded in paraffin wax for histological analysis and stored at −80°C for other experiments. All experimental procedures were approved by the Ethical Committee of Suzhou Integrated Traditional Chinese and Western Medicine Hospital. The National Institutes of Health Guide for the Care and Use of laboratory animals was followed (21).

Colon specimens from the different groups were embedded in paraffin and fixed with 4% formalin at room temperature for 2 weeks, and sliced at a 5-mm thickness. These fixed tissues were stained via hematoxylin-eosin staining, as described previously (22). A BH22 light microscope (Olympus Corporation; magnification, ×400) was used to observe the histological damage and inflammation of the colon tissues. Severity of histological inflammation was graded as described previously (23,24): Ulceration (0, none; 1, erosion; 2, submucosa ulceration); crypt abscesses (0, none; 1, mild; 2, severe), degree of mononuclear cell infiltration (MNCI) (0, no infiltration; 1, <25%; 2, 25–50%; 3, 50–75%; 4, >75%); segmental distribution of MNCI (0, continuous; 1, mildly segmental; 2, markedly segmental); and eosinophil infiltration (0, none or minimal; 1, mild; 2, severe).

The colon tissues were freeze-thawed in liquid nitrogen and levigated. The NO concentration in the colon tissues was detected by Griess assay (Sigma Aldrich; Merck KGaA) as previously described (2). The data are presented as the mean (nitrite) in mM/g colon tissue.

The TNF-α (cat. no. H052), IL-1β (cat. no. H002) and IL-6 (cat. no. H007) content in the colon tissues of the mice was measured with ELISA kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol.

The infiltration of neutrophils in the colon samples was assessed via myeloperoxidase (MPO) activity. Colon tissues were first homogenized in PBS (w/v 1/9), then the MPO activity assay kit (Nanjing Jiancheng Bioengineering Institute) was used to detect the MPO activity according to the manufacturer's protocol. The results are presented as U/g wet tissue.

Each group was analyzed in at least three independent experiments and all data are presented as the mean ± standard deviation. SPSS 17.0 software (SPSS, Inc.) was used for all statistical analyses. Comparisons among multiple groups were performed with one-way analysis of variance followed by Dunnett's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The potential cytotoxic effects of ASI and/or LPS on CCD-18Co cells were initially assessed using a CCK-8 assay. As indicated in Fig. 1A, neither ASI nor LPS affected the cell viability of CCD-18Co cells. A previous study indicated that ASI exhibited gastro-protective effects against acute gastric lesions in rats by alleviating inflammation (25). The anti-inflammatory action of ASI in LPS-treated CCD-18Co cells was subsequently investigated. As demonstrated in Fig. 1B-D, compared with control treatment, LPS induced a significant increase in TNF-α, IL-β and IL-6 levels in cells. By contrast, LPS-induced TNF-α, IL-β and IL-6 upregulation were significantly reversed by 50 µM ASI. These data demonstrated that ASI may decrease the production of TNF-α, IL-β and IL-6 in LPS-stimulated CCD-18Co cells in vitro.

It has been suggested that NF-κB serves as a key factor in the mediation of inflammation, and phosphorylated NF-κB p65 (p-p65) serves a vital role in the activation of NF-κB (26). Therefore, the present study investigated whether ASI exerted anti-inflammatory effects on CCD-18Co cells via the inhibition of NF-κB activation. As indicated in Fig. 2, the expression of p-p65 and p-IκB in cells was significantly increased by LPS. By contrast, the LPS-induced increases in p-p65 and p-IκB protein levels were notably reduced by 50 µM ASI. These data demonstrated that ASI inhibited the phosphorylation of NF-κB p65 and IκB in LPS-treated CCD-18Co cells in vitro.

In order to confirm the anti-inflammatory effects of ASI in vivo, a DSS-induced mouse UC model was established. As indicated in Fig. 3A, compared with the control group, the body weights of the mice were markedly decreased in the DSS group. However, ASI (200 mg/kg) notably prevented body weight loss during the progression of experimental UC in mice (Fig. 3A). In addition, H&E staining was used to observe the infiltration of inflammatory cells in sections of mouse colon segments. As indicated in Fig. 3B, colons in the control group were healthy, exhibiting intact surface epithelia and submucosa. However, DSS resulted in distortion of the crypt epithelium, and ulceration and damage to the surface epithelium. Compared with the DSS group, colons in the ASI (200 mg/kg) group exhibited well-preserved intact surface epithelia and cryptal glands (Fig. 3B). The histological damage scores in the ASI (200 mg/kg) group were significantly decreased compared with the DSS group (Fig. 3C). In addition, 200 mg/kg ASI significantly decreased the DAI during the progression of experimental UC in mice, which reflects the health status of the mice (Fig. 3D). Furthermore, ASI (200 mg/kg) significantly alleviated colon shortening caused by DSS in mice (Fig. 3E). All these data demonstrated that ASI attenuated UC induced by DSS in vivo.

Next, the concentrations of TNF-α, IL-1β and IL-6 in colon tissues were detected in vivo. As indicated in Fig. 4A-C, the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in the colon tissues were significantly upregulated in the DSS group, compared with the control group (P<0.01). However, ASI (200 mg/kg) treatment markedly suppressed the DSS-induced production of these cytokines (Fig. 4A-C). Similar to the pro-inflammatory cytokines, the results for NO and the MPO activity assay indicated that the levels of NO and MPO in the colon tissues were significantly upregulated in the DSS group. Nevertheless, ASI (200 mg/kg) treatment alleviated the DSS-induced production of NO and MPO in the colon (Fig. 4D and E). These data demonstrated that ASI may alleviate inflammatory responses in the colon in the DSS-induced UC mouse model.

It has been demonstrated that UC is associated with increased intestinal penetrability and decreased TJ protein expression, including that of ZO-1 and claudin-1 (27). As presented in Fig. 5, the expression levels of claudin-1 and ZO-1 were markedly decreased in the DSS group. However, DSS-induced claudin-1 and ZO-1 downregulation was notably reversed by 200 mg/kg ASI (Fig. 5). These data demonstrated that ASI increased the expression levels of TJ proteins in colonic tissues.