Four-week-old male ICR mice were purchased from Orient-Bio, Inc. (Seongnam, Republic of Korea). They were acclimated for 1 week with tap water and basal diet under the conventional housing conditions of humidity (50 ± 10%), temperature (24 ± 2°C), and light (12/12 h of light/dark cycle). All studies were approved by the Institutional Animal Care and Use Committee of Seoul National University.

DSS (MW of 36,000–50,000) was obtained from MP Biomedicals, LLC (Solon, OH, United States). PE was obtained from Amino Up Chemical Co., Ltd. (Sapporo, Japan). Groups of six mice were treated for 2 weeks in accordance with the experimental design illustrated in Figure 1A. Acute colitis was induced by DSS (3%, w/v) given in double distilled drinking water for seven consecutive days. Mice in the PE group received PE (20 or 100 mg/kg) 1 week before and during the DSS administration which was dissolved in double distilled water (DDW) once daily by oral gavage. Mice in control and DSS alone groups were also treated with DDW in the same way to get exposed to an equal stress level. In another experiment, PE was given alone at a daily oral dose of 20 or 100 mg/kg for 2 weeks. At the end of the treatment, mice were sacrificed by cervical dislocation. Their colon tissues were taken out and washed with phosphate-buffered saline (PBS). The tissues were separated into several parts for extraction of protein and mRNA. The distal part was fixed in 10% buffered formalin for immunohistochemical examination, and other parts were frozen in liquid nitrogen immediately and kept at -70°C until use.

Since the beginning of the DSS treatment, the body weight of every mice was measured daily. Rectal bleeding and stool consistency were observed and graded from 0 to 4, depending on the severity of blood and diarrhea (Fukata et al., 2005; Lavi et al., 2010). Disease Activity Index (DAI) was graded as the sum of scores of rectal bleeding and stool consistency. Colorectal parts were photographed, and their length was measured.

Mouse colon tissues were homogenized in ice-cold lysis buffer [150 mM NaCl, 0.5% triton-X 100, 50 mM Tris–HCl (pH 7.4), 20 mM ethylene glycol tetra acetic acid, 1 mM phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free cocktail tablet] followed by periodical vortex mixing for 30 min at 0°C. Lysates were centrifuged at 18,000 g for 15 min at 4°C. The supernatants were collected and stored at -70°C till use.

For Western blot analysis, the total protein concentration was quantified by using the bicinchoninic acid protein assay kit (Pierce; Rockford, IL, United States). Lysates (30–50 μg protein) were mixed and boiled in a sodium dodecyl sulfate (SDS) sample buffer for 5 min. The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Gelman Laboratory; MI, United States). The blots were blocked in 5% fat-free dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membranes were incubated for 12–24 h at 4°C with dilutions of primary antibodies against α-tubulin, actin (Sigma Aldrich, St. Louis, MO, United States), laminB, STAT3, phosphorylated STAT3 (P-STAT3), p65, IκBα, P-IκBα, COX-2, iNOS, CXC chemokine receptor 2 (CXCR2), heme oxygenase-1 (HO-1; Stressgen Biotechnologies Co., San Diego, CA, United States), NF-E2-related factor 2 (Nrf2) and interferon regulatory factor 3 (IRF3; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States). The membranes were washed, followed by incubation with 1:4000 dilution of respective horseradish peroxidase (HRP)-conjugated secondary antibody (rabbit, mouse, or goat; Zymed Laboratories; San Francisco, CA, United States) for 2 h, and washed again with TBST. Protein expression was visualized with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech; Buckinghamshire, United Kingdom) and LAS-4000 image reader (Fujifilm; Tokyo, Japan) according to the manufacturer’s instructions.

Cytosolic extracts were obtained from lysates dissolved in hypotonic buffer A [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.8), 1.5 mM MgCl₂, 10 mM KCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, and 0.1 mM PMSF] with 0.1% Nonidet P-40 by homogenization and vortex mixing at 10 min intervals for 3 h. After centrifugation at 18,000 g for 15 min, the supernatants (the cytosolic extracts) were collected and stored at -70°C until use. Precipitated pellets were suspended in buffer C [20 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 20% glycerol]. After centrifugation at 18,000 g for 15 min, the supernatants (the nuclear extracts) were collected and stored at -70°C until use.

DNA binding activity of NF-κB was measured with electrophoretic mobility shift assay (EMSA). In brief, T4 polynucleotide kinase transferred ³²P labeled γ-phosphate from ATP to NF-κB oligonucleotide. After purification with a G-50 micro column (GE Healthcare LS; Buckinghamshire, United Kingdom), the γ-³²P labeled probe was mixed with 10 μg of nuclear extracts and incubation buffer [10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% glycerol, and 0.1 mg/ml sonicated salmon sperm DNA]. All the samples were mixed with 2 μl of 0.1% bromophenol blue loading dye after 50 min incubation and separated on 6% non-denatured polyacrylamide gel in a cold room. Finally, gels were dried and exposed to X-ray films (Agfa Healthcare; Mortsel, Belgium).

The dissected colon tissues were prepared for immunohistochemical analysis to measure the expression of COX-2, P-STAT3, and Nrf2. Four-micrometer sections of 10% formalin-fixed and paraffin-embedded tissues were cut on silanized glass slides and deparaffinized three times with xylene and rehydrated through graded alcohol bath. The deparaffinized sections were heated by using microwave and boiled twice for 6 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. To diminish non-specific staining, each section was treated with 3% hydrogen peroxide and 4% peptone casein blocking solution for 15 min. For the detection of respective protein expression, slides were incubated with COX-2 (Cayman Chemical; Ann Arbor, MI, United States), P-STAT3 (Cell Signaling Technology, Inc.; Beverly, MA, United States) and Nrf2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States) antibodies at room temperature for 40 min in Tris-buffered saline containing 0.05% Tween 20, and then developed using respective HRP-conjugated secondary antibodies (rabbit, mouse, or goat) EnVision^TM System (Dako; Glostrup, Denmark). The peroxidase-binding sites were detected by staining with 3,3′-diaminobenzidine tetrahydrochloride (Dako; Glostrup, Denmark). Finally, counterstaining was performed by using Mayer’s hematoxylin.

The CCD841CoN cells were obtained from the American Type Culture Collection (Manassas, VA, United States) and maintained in MEM containing 10% fetal bovine serum and an antibiotic–antimycotic mixture (Gibco BRL; Grand Island, NY, United States) at 37°C with 5% CO₂ and 95% air. The CCD841CoN cells were treated with PE (10 mg/ml) 1 h before stimulation with 10 ng/ml of tumor necrosis factor-α (TNF-α; R&D Inc.; Minneapolis, MN, United States) dissolved in 0.1% bovine serum albumin in PBS. After TNF-α treatment, CCD841CoN cells were incubated for 1 h and prepped. The cells in the apical slide were harvested and centrifuged at 18,000 g for 5 min at 4°C. The pellets were suspended in the cell lysis buffer (Cell Signaling Technology; Beverly, MA, United States). After centrifugation at 18,000 g for 15 min, the supernatants were collected and stored at -70°C until use.

Total RNA was isolated from CCD841CoN cells by using TRIzol^® (Invitrogen; Carlsbad, CA, United States) according to manufacturer’s protocol. One microgram of total RNA reverse transcribed with MLV reverse transcriptase at 42°C for 50 min and at 72°C for 15 min. PCR was conducted according to the standard procedures. The primer sequences used were (forward and reverse, respectively): cox-2, 5′-GCTGAGCCATACAGCAAATCC-3′ and 5′-GGGAGTCGGGCAATCATCAG-3′; il-6, 5′-GTGTGAAAGCAGCAAAGAGGC-3′ and 5′-CTGGAGGTACTCTAGGTATAC-3′; il-8, 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ and 5′-TCTCAGCCCTCTTCAAAAACTTCT-3′; gapdh, 5′-AAGGTCGGAGTCAACGGA-3′ and 5′-GCAGTGAGGGTCTCTCTC-3′; interferon (ifn)-α, 5′-AGTGAGCTGACCCAGCAGAT-3′ and 5′-AGACAGCCTTGCAGGTCATT-3′; ifn-β, 5′-CCCTATGGAGATTGACGGAGA-3′ and 5′-ACCCAGTGCTGGAGAAATTG-3′; actin, 5′-AGAGCATAGCCCTCGTAGAT-3′ and 5′-CCCAGAGCAAGAGAGGTATC-3′. Amplified products were analyzed on 2% agarose gel electrophoresis, stained with SYBR^® Green (Invitrogen; Carlsbad, CA, United States) and photographed using fluorescence in LAS-4000.

All values presented are the mean ± standard error (SE) of at least three independent experiments. Statistical significance was determined by the One-way analysis of variance (ANOVA) with Tukey’s post hoc test, and p < 0.05 was considered to be statistically significant. The bar-graphs were analyzed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, United States).

The body weight of mice in the DSS group became decreased after the day 3 of DSS treatment compared to the control group. However, oral administration of PE attenuated the loss of body weight (Figure 1B). Following the DSS treatment, the DAI score was increased after the day 3. DSS-treated mice exhibited serious symptoms, such as diarrhea and bloody stool. PE treatment reduced the severity of the symptoms (Figure 1C). In addition, shrinkage and thickening of colon occurred as a consequence of colitis caused by DSS (Figure 2A). Oral administration of PE ameliorated the pathological symptoms of colitis. Inhibitory effects of PE against DSS-induced inflammatory events in mouse colon were also verified by histological, immunohistochemical, and immunoblot analyses. Colorectal tissues from DSS-treated mice showed collapse and expansion of epithelium which were accompanied by infiltration of inflammatory cells (Figure 2B) and overexpression of COX-2, iNOS, and cyclin D1 (Figures 3A,B). Thus, PE protected the epithelium from DSS-induced colitis as evidenced by attenuation of morphological and biochemical alterations related to inflammatory colonic tissue damage.

NF-κB is a transcription factor regulating expression of COX-2 and iNOS. It is activated in IBD (Atreya et al., 2008), resulting in disorders of the immune system, elevation of cell survival, and reduction of apoptosis (Aggarwal, 2004). NF-κB activation and translocation into nucleus are dependent on phosphorylation and degradation of IκBα. DSS administration stimulated phosphorylation and subsequent degradation of IκBα, thereby augmenting nuclear translocation of p65, a functionally active subunit of NF-κB. Treatment with PE (100 mg/kg) inhibited the phosphorylation and degradation of IκBα and nuclear migration of p65 (Figure 4A). Likewise, the DSS-induced DNA binding activity of NF-κB in colonic mucosa was reduced by the PE administration (Figure 4B).

STAT3 is mainly activated via the interleukin (IL)-6-gp130-JAK axis, leading to phosphorylation on Tyr705. After dimerization, it translocates into nucleus and regulates transcription of target genes by binding to their promoters. The level of P-STAT3 in the colonic tissue was increased by DSS administration (Figure 5A), but the phosphorylation and nuclear translocation of STAT3 were significantly inhibited by PE (100 mg/kg) (Figures 5A,B).

TNF-α is a pleiotropic inflammatory cytokine and a central regulator of inflammation. TNF-α activates NF-κB and MAPK signaling (Bendtzen et al., 2009). CCD841CoN human colon epithelial cells were stimulated with TNF-α (10 ng/ml). Phosphorylation of IκBα and STAT3 was increased in 15 min and 1 h, respectively after TNF-α treatment (Figure 6A). IL-6 plays a role not only in acute inflammation, but also in chronic inflammation by changing the nature of leukocytes and modulating some immune cells, such as T- and B-cells. IL-8 is involved in promotion of neutrophil infiltration. TNF-α treatment induced the expression of genes encoding both proteins at 1 h (Figure 6B). CXCR2 is a receptor for inflammatory chemokines, such as IL-8. Treatment with PE (10 mg/ml) 1 h prior to TNF-α reduced the expression levels of CXCR2 and some representative mediators involved in inflammatory signaling, such as iNOS, P-IκBα, and P-STAT3 (Figure 6C).

Toll-like receptor 4 (TLR4) is an upstream signaling molecule involved in NF-kB activation and pathogenesis of IBD. TLR4 expression has been shown to be upregulated in the colonic mucosa of DSS-induced colitis (Zhang et al., 2014). One of the key mediators of TLR4 signaling is IRF3 that encodes type I interferon (IFN), such as IFN-α and IFN-β. We noticed that there was robust accumulation of IRF3 and expression of ifn-α and ifn-β in the colonic mucosa of DSS-treated mice (Figure 7). PE administration suppressed the expression of IRF3 (Figure 7A) and its target genes (Figure 7B).

DSS administration caused not only inflammation, but also oxidative mucosal damage as revealed by increased accumulation of 4-HNE-modified protein (Figure 8A). PE administration attenuated the DSS-induced oxidative stress. Nrf2 is a transcription factor encoded by the NFE2L2 gene and regulates the expression of many antioxidant enzymes and other stress-responsive proteins, including HO-1 (Moi et al., 1994). PE enhanced the expression of HO-1 as well as Nrf2 (Figure 8B). The colonic accumulation of Nrf2 was further verified by immunohistochemical analysis (Figure 8C). The nuclear localization of Nrf2 was also enhanced by PE (Figure 8D).