Dextran sulfate sodium (molecular weight of 36–50 kDa) was obtained from MP Biomedicals (Irvine, CA, USA). TER was obtained from TCI Chemical Industry Co., Ltd. (Shanghai, China). Rabbit mAb, IκBα, and p65 and mouse p-IκBα monoclonal antibodies and p-p65, NLRP3, caspase-1, ASC, and IL-1β were provided by Cell Signaling Technology Inc. (Beverly, MA, USA). The primary antibodies that were raised against occludin and zonula occludens-1 (ZO-1) were obtained from Santa Cruz (Santa Cruz, CA, USA). β-actin and horseradish peroxidase conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Tianjin Sungene Biotech Co., Ltd. (Tianjin, China). The myeloperoxidase (MPO) determination kit was purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, China). All enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, CA, USA). The Nuclear and Cytoplasmic Protein Extraction Kit was provided by Beyotime Institute of Biotechnology (Jiangsu, China). All other chemicals were of reagent grade.

Male C57BL/6 mice (21–23 g) were provided from the Center of Experimental Animals of Jilin University, China. NLRP3^−/− mice were on C57BL/6 background. The mice were housed in an air-conditioned room with the laboratory temperature maintained at 24 ± 1°C and provided water and food ad libitum. All experimental protocols were guided in accordance with the approval of the Institutional Animal Care and Use Committee of our university under the approved protocol number SCXXK (JI-2016-0003).

Acute colitis was induced by feeding mice with 2.5% (w/v) DSS, which was dissolved in drinking water, continuously for 7 days. The experiment was randomly divided into six groups: control group;DSS group; TER (5, 10, 20 mg/kg) + DSS groups, and TER (20 mg/kg) group. The experimental time lines of the animal model are described in Figure 1A. To assess chemoprevention effect of TER on DSS-induced acute colitis in C57BL/6 mice, the mice were treated with indicated dose of TER 14 days before DSS treatment once per day. Subsequently, to further prove the key role of NLRP3 in the effect of TER, TER was administered intragastrically daily at 20 mg/kg. Wild-type (WT) or NLRP3^−/− mice were randomly assigned to control group, DSS group, and TER (20 mg/kg) + DSS group. The experimental time lines of the animal model are described in Figure 1A.

In all groups, mice weight, stool characters, and blood in stool were recorded daily. The disease activity index (DAI) evaluations were determined by previously established scoring system (20). Briefly, DAI scores were based on the following: changes in body weight, consistency of stool, and hemoccult bleeding. These parameters were assessed on a scale from 0 to 4, with 4 being the most severe. The DAI data are presented as an average score of these parameters taken daily. Animals were sacrificed by cervical dislocation under anesthesia, and the colon was excised from cecum to 1 cm above the anus. The length of colon was detected. For histological analysis, the colon specimens were fixed in 10% formalin and embedded in paraffin. Sections were stained with hematoxylin–eosin (H&E) according to standard protocols. Histological scoring was performed using a previously described method (21).

Myeloperoxidase activity reflects the number and distribution of neutrophils in the tissues. The colon tissues were weighed and homogenized with phosphate-buffered saline (PBS) [1:9 (w/v)]. The supernatants were collected. The activity of MPO was measured according to the manufacturer’s instructions.

The RAW264.7 cells were purchased from China Cell Line Bank (Beijing, China) and cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C with 5% CO₂.

Human intestinal epithelial cells (Caco-2 cells) were cultured with 15% FBS/DMEM-F12 supplied with 1 mM sodium pyruvate and 50 U/mL penicillin–streptomycin. Caco-2 cells were maintained at 37°C in a humidified 5% CO₂ incubator.

Cells were pretreated with or without TER (0.025, 0.05, and 0.1 mM) for 1 h. After that, cells were treated with LPS (1 µg/mL). After 18 h of LPS stimulation, MTT (20 µL of 5 mg/mL) was added to each well for 4 h. The supernatant was removed and dimethyl sulfoxide (150 µL per well) was added. The optical density was tested at 570 nm using a microplate reader.

Cecal bacterial lysates (CBLs) were prepared as described by Dieleman et al. (22). Briefly, the cecal contents in each group were solubilized by vortexing the contents in RPMI-1640 medium and then incubating them with 0.01 M MgCl₂, 10 µg/mL DNase. Then, the contents were homogenized with 0.1-mm glass beads for 3 min. The homogenate was centrifuged at 10,000 × g for 10 min. The supernatant was filtered through a 0.45-µm syringe filter.

Mesenteric lymph node (MLN) was harvested from mice of six experimental groups. Single-cell suspensions were prepared as described by Ruyssers et al. (23). Briefly, approximately 4 × 10⁵ MLN cells and 20 µg/mL CBL were cultured in RPMI-1640 medium with 10% FBS and 50 mg/mL gentamicin at 37°C with 5% CO₂ for 72 h. The culture media was then collected for cytokine analysis and stored at −20°C.

Adipose tissue was removed from excised colons and washed with PBS. Colon tissues (about 50 mg per well) were cultured in RPMI-1640 medium (100 mg/mL of streptomycin and 100 U/mL of penicillin) at 37°C in 5% CO₂. After 24 h, colon supernatants were centrifuged at 12,000 × g at 4°C for 10 min. The RAW264.7 cells were pretreated with or without TER (0.025, 0.05, and 0.1 mM) for 1 h, and then stimulated with 1 µg/mL of LPS for 18 h. The TNF-α, IL-1β, and IL-12 autocrine levels in the colon were detected with ELISA kits according to the manufacturer’s protocol.

Total RNA was extracted from mouse colon samples and cells using the TRIzol reagent as per the manufacturer’s protocol (Invitrogen). The RNA was reverse-transcribed into complementary DNA (cDNA) using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative real-time PCR (qRT-PCR) was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR Green Plus reagent kit (Roche), as described elsewhere (24). The sequences of primers were listed in Table 1. β-actin was used as the reference gene.

Colon samples were homogenized. Total protein was extracted as per the manufacturer’s protocol. Cells were treated with TER (0.025, 0.05, and 0.1 mM) for 1 h. After LPS treatment for 1 h, total cellular protein was extracted. Protein contents were quantified and transferred onto PVDF membrane. Next, the blocked membranes with 5% non-fat milk were incubated overnight at 4°C with primary antibodies against p65 (1:1,000), p-p65 (1:1,000), IκBα (1:1,000), p-IκBα (1:1,000), NLRP3 (1:500), caspase-1 (1:1,000), ASC (1:250), IL-1β (1:1,000), ZO-1 (1:300), occludin (1:300), and β-actin (1:1,000). The secondary antibodies used were goat anti-rabbit or -mouse IgG (1:20,000) and were incubated with membranes at room temperature for 2 h. The blots were tested using a western blotting detection program. β-actin was used as an internal control.

Immunohistochemical stains against ZO-1 and occludin were detected using IHC kit (MaiXin, China). Briefly, paraffin-embedded slides were deparaffinized, rehydrated, and washed in 1% PBS. After that, they were incubated with 3% hydrogen peroxide and blocked with 10% goat serum for 1 h at 37°C. Then, slides were treated with primary antibodies (1:100) overnight at 4°C. Biotinylated secondary anti-rabbit antibodies were added and incubated at room temperature for 1 h. Streptavidin-HRP was added, and after 40 min the sections were achieved using diaminobenzidine as a chromogen and counter-stained with hematoxylin. Images at 200× magnification were examined with a microscope (Olympus, Japan).

Immunofluorescence was performed on paraffin-embedded colonic tissue sections. The sections were deparaffinized, rehydrated, and washed in 1% PBS-Tween. After that, they were treated with 3% hydrogen peroxide, blocked with 10% goat serum, and incubated with ZO-1 and occludin primary antibody in PBS-Tween containing 1% BSA (1:50) for 1 h at 37°C. Slides were washed and incubated for 1 h with species-specific fluorescently labeled secondary antibodies. The slides were stained with DAPI. Cover slips were mounted and analyzed on a confocal laser microscope.

Plasma endotoxin contents were detected by Limulus amebocyte lysate (LAL) assay kit according to manufacturer’s protocol. Briefly, plasma (5 µL) was diluted 1:10 in pyrogen-free water, inactivated at 70°C for 10 min, and incubated with LAL solution at 37°C for 30 min. Addition of reagents led to formation of a magenta derivative that absorbs light at 545 nm.

The mice were previously marked so individual mice could be followed in the whole experiment. In order to collect stool samples, individual mice were put in isolation containers. For bacterial community analysis, stool samples were weighed and stored at −80°C. DNA was extracted using a phenol–chloroform extraction technique with mechanical disruption (bead-beating) based on a previously described protocol (25). For the preparation of a standard for Escherichia coli (E. coli) and Lactobacillus, a clone obtained from mice was used. The sequences of specific bacterial primers were listed in Table 1. Results are expressed as 16SrRNA gene copies per wet weight of feces.

All data are shown as the mean ± SD. One-way analysis of variance (Dunnett’s test) was used where three or more groups of data were compared. All experiments were repeated at least three times. p ≤ 0.05 were considered statistically significant.

To investigate the protective effect of TER on UC, we established a model of DSS-induced mice colitis by feeding C57BL/6 mice with drinking water containing 2.5% DSS for 7 days. The mice treated with only DSS had a significant increase in DAI scores compared with control group. The DAI scores were lower in the DSS-induced mice that received TER administration vs that in the group treated with only DSS (Figure 1B). In addition, DSS typically caused colonic shortening, whereas such change was significantly improved in TER groups (Figure 1C). The severity of colonic inflammation was further evaluated by histopathological analysis (Figures 1D,E). The DSS group existed distortion of crypts, loss of goblet cells, and severe mucosal damage. However, administration of TER to DSS-induced UC mice could obviously improve the pathological changes in a dose-dependent manner. Only TER (20 mg/kg) did not generate a histopathological change in the colons of mice. Moreover, TER at 10 and 20 mg/kg significantly reduced the level of DSS-induced hyperactivated MPO (Figure 1F).

The anti-inflammatory activity of TER was further confirmed by measuring TNF-α, IL-1β, and IL-12. The results showed that the levels of TNF-α, IL-1β, and IL-12 were significantly increased after DSS challenge. However, the increase of these cytokines was dramatically reduced by TER treatment in colon explants (Figure 2A). In addition, to investigate the effect of TER on host-dependent immune responses, we investigated the cytokine responses of the MLN cells to CBL. The results showed that TER could significantly inhibit the elevated expression of these cytokines in a dose-dependent manner (Figure 2B). In addition, we measured the mRNA levels of TNF-α, IL-1β, and IL-12 in colon tissues as well. Treatment with TER markedly decreased TNF-α, IL-1β, and IL-12 levels (Figure 2C).

NF-κB plays a pivotal role in regulating cytokines. To detect whether the suppression of inflammation by TER is mediated by NF-κB pathway, NF-κB p65 and IκB phosphorylation levels were determined. The resulted showed that the phosphorylation of p65 and IκBα was significantly increased in DSS group, but reversed in TER treatment groups (Figures 2D,E). Furthermore, emerging evidence suggests the pivotal role of NLRP3 inflammasome in the developments and pathogenesis of IBD (6). In our study, we have demonstrated that TER could decrease IL-1β production. Consequently, we further investigated whether TER could inhibit NLRP3 inflammasome activation in DSS-induced colitis mice. The results showed that the levels of NLRP3, ASC, Caspase-1, and IL-1β were remarkably enhanced after DSS challenge. The administration of TER (5, 10, and 20 mg/kg) inhibited the expression of production of NLRP3, ASC, Caspase-1, and IL-1β in a dose-dependent manner (Figures 2F,G).

Tumor necrosis factor-α, IL-1β, and IL-12 are three major cytokines in UC. As described above, TER suppressed the secretion of TNF-α, IL-1β, and IL-12 in vivo (Figures 2A–C). Subsequently, we confirmed the anti-inflammatory effect of TER in vitro. First, we detected the potential cytotoxicity of TER on RAW264.7 cells. The result showed that TER (0.025, 0.05, and 0.1 mM) had no toxic effect on RAW264.7 cells (Figure 3A). Thus, we detected the anti-inflammatory effects of TER on LPS-induced RAW264.7 cells with the concentration of 0.025, 0.05, and 0.1 mM. As expected, the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-12) was increased quickly in LPS-induced RAW264.7 cells. The concentration of TNF-α, IL-1β, and IL-12 was significantly reduced in TER treatment groups compared with LPS group (Figure 3B). To further test the effect of TER, mRNA expression levels of pro-inflammatory cytokines were evaluated by qRT-PCR. As shown in Figure 3C, gene expression levels of TNF-α, IL-1β, and IL-12 were also significantly increased by LPS stimulation compared with the control and clearly reduced by treatment with TER. These results demonstrated that TER inhibited the transcription and final secretion of TNF-α, IL-1β, and IL-12.

To test whether the anti-inflammatory effect of TER is mediated through NF-κB pathway, we measured the expression of NF-κB protein by western blotting. Our results showed that LPS significantly induced NF-κB activation in RAW264.7 cells. However, TER markedly inhibited LPS-induced phosphorylation of p65 and IκBα (Figures 3D,E). Furthermore, our in vivo study has also demonstrated that TER decreased IL-1β production (Figures 2A–C,F) and suppressed NLRP3 inflammasome activation (Figures 2F,G). To elucidate its anti-inflammatory mechanisms, we further explored whether TER could influence NLRP3 inflammasome activation in vitro. As shown in Figures 3F,G, TER significantly inhibited activated NLRP3 inflammasome-related protein levels and caspase-1 activity.

The DSS-induced mice colitis model was used to further elucidate the key role of NLRP3 inflammasome in TER protective effects by comparing WT mice and NLRP3^−/− mice. Consistent with the results from colitis experiment in C57BL/6 mice, administration of TER (20 mg/kg) showed a significant improvement on DSS-induced colitis of WT mice (Figures 4A–D). Results from Figures 4E,F also showed that DSS-increased MPO activity, and IL-1β level in the colon were significantly reduced by TER treatment in WT mice. However, we found that NLRP3^−/− mice were significantly protected from DSS-induced colitis, showing a less severity of symptoms, including reduced DAI scores and colon shortening compared with WT mice (Figures 4A–D). MPO activity and IL-1β level in colon of NLRP3^−/− mice also showed a significant inhibitory effect compared with those in the WT mice in response to DSS. Furthermore, histopathological analysis also revealed a significant decrease of mucosal damage, distortion of crypts, and loss of goblet cells. These results showed that mice deficient for NLRP3 were less sensitive to DSS-induced acute colitis. Consistent with the above results from colitis experiment in WT, TER (20 mg/kg) decreased the MPO activity in NLRP3^−/− mice (Figure 4E). However, we are surprising that TER did not protective effects on DSS-treated colitis in NLRP3^−/− mice characterized by DAI scores, colon shortening, pathological changes, and IL-1β secretion (Figures 4A–D,F). These results may suggest that NLRP3 inflammasome play an important role or at least play a partial role in TER-mediated colitis alleviation.

We further compared the activity of NLRP3 inflammasome in colon samples of WT and NLRP3^−/− mice by western blotting. As shown in Figures 5A,B, there is an undetectable level of NLRP3 protein in colon tissues of NLRP3^−/− mice. In addition, IL-1β level did not differ significantly between WT and NLRP3^−/− mice. However, high levels of caspase-1, ASC, and IL-1β were observed in the colon of WT mice receiving DSS, but not in those of NLRP3^−/− mice. Furthermore, DSS-increased caspase-1, ASC, and IL-1β levels in the colon were significantly decreased by TER treatment in WT mice, but did not in NLRP3^−/− mice. Our findings indicated that the NLRP3 inflammasome may play a critical role on DSS-induced colitis and its inhibition contributes to the anti-inflammatory effect of TER.

To analyze whether TER could re-balance the composition of the commensal microbiota, representative bacteria in the feces of TER-treated mice were investigated by qRT-PCR. The copy numbers of E. coli were significantly reduced with TER treatments (10 and 20 mg/kg) compared with DSS group (Figure 6B). Furthermore, the levels of Lactobacillus were decreased by DSS compared with control group. However, TER (20 mg/kg) significantly increased the DSS-induced reduction of Lactobacillus levels (Figure 6C). LPS is a component of Gram-negative bacterial cells. We further detected the plasmatic LPS concentration as well. The result showed that plasmatic LPS concentration was significantly higher in DSS group than that in control group. However, the oral administration of TER suppressed plasmatic LPS level in a dose-dependent manner (Figure 6A).

Epithelial TJ proteins are an especially important aspect of the mechanical barrier, preventing harmful substances from breaching the mucosa, maintaining cellular integrity and permeability, and thus ensuring a relatively stable internal environment. ZO-1 and occludin are important epithelial TJ proteins. Thus, we detected the effect of TER on epithelial TJ proteins (ZO-1 and occludin). As shown in Figures 7A,B, ZO-1 and occludin positive signals were obviously decreased in the colon after DSS administration by immunohistochemical staining compared with the control group, while these changes were significantly increased through pretreatment with TER. IF analysis further demonstrated that ZO-1 and occludin were localized at the surface of the colonic epithelium in control group and TER (20 mg/kg) group. In DSS-treated mice, a substantial loss of staining intensity of ZO-1 and occludin was observed, which was significantly prevented by administration of TER (Figures 7C,D).

To extend our observations, we also detected the effect of TER on ZO-1 and occludin in vitro. First, we investigated the potential cytotoxicity of TER on Caco-2 cells. As shown in Figure 7E, TER (0.025, 0.05, and 0.1 mM) had no toxic effect on Caco-2 cells. We then detected the effect of TER on TJ proteins and mRNA expression. The results showed that the expression levels of ZO-1 and occludin were significantly downregulated in LPS-induced Caco-2 cells, and the TER-administered significantly increased their expressions in protein (Figures 7F,G) and mRNA (Figures 7H) levels compared with the LPS group, suggesting the importance of TER for restoring the integrity of the TJ networks.