Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, Trypsin-EDTA were obtained from Life Technologies Corporation (Grand Island, NY, USA). A 24-well cell culture insert was purchased from BD Biosciences (Franklin Lakes, NJ, USA). DSS (molecular weight, 40,000 kDa) was purchased from MP Biomedicals (Solon, OH, USA). Antibodies for western blot analysis were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The immunoblot detection system (ECL Plus) and bicinchoninic acid (BCA) protein assay reagent were obtained from Pierce Biotechnology, Inc. (Rockford, IL, USA). Mouse IL-6 or serum amyloid A (SAA) detection ELISA kit was purchased from BioLegend, Inc. (San Diego, CA, USA) or Immunology Consultants Laboratory, Inc. (Portland, OR, USA), respectively. All the other chemicals, unless otherwise stated, were obtained from Sigma Chemicals (St. Louis, MO, USA).

PZH was obtained from and authenticated by the sole manufacturer Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd. (Zhangzhou, China; Chinese FDA approval no. Z35020242). The stock solution of PZH was prepared by dissolving PZH powder in saline to a concentration of 20 mg/ml, followed by sonication for 30 min. The working concentrations for cell-based experiments were made by diluting the stock solution in the cell culture medium.

Seven-week-old male BALB/c mice (weight, 20–22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and were acclimatized for 1 week before the experiment. Animals were housed individually in a room maintained at 22°C under a 12-h day/night cycle. Food and water were given ad libitum throughout the experiments. The animal experiments conducted in this study were approved by the Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine.

Mouse colitis model was established as we described previously (51). Briefly, acute colitis was induced by administration with 3% DSS in the drinking water for 8 days. On the first day of model construction, the animals were randomly divided into three groups (n=5): The normal control group in which mice received neither DSS stimulation nor PZH treatment; the DSS-induced model or PZH-treated group in which mice received DSS stimulation and were given intra-gastric administration with saline or 234 mg/kg, respectively, daily for 12 days. The progression of colitis was monitored in a blinded manner, including measurement of body weight, evaluation of stool consistency, and presence of rectal bleeding tested by guaiac paper. Disease activity index (DAI) was represented as the sum of scores for weight loss, stool consistency and rectal bleeding (Table I).

At the end of the experiment, the animals were anaesthetized with Avertin. Blood was collected via right heart ventricle puncture in lightly heparinized syringes and kept on ice. Sera were separated by 5 min centrifugation at 5,000 × g and stored at −80°C prior to the analysis. The colons were excised and length was measured. One portion of each distal colon was cut and fixed in 10% formalin for histological examination. The remainder of each distal colon was snap-frozen in liquid nitrogen and stored at −80°C for further analysis of the tissue IL-6 level and STAT3 phosphorylation. Proteins in frozen colons were extracted using T-PER Tissue Protein Extraction Reagent kit according to the manufacturer's protocol. Protein concentrations were determined by BCA protein assay kit.

The formalin fixed section of distal colons were processed and stained with hematoxylin and eosin (H&E) and evaluated under light microscopy in a blinded manner by an experienced pathologist.

The level of SAA in the sera and the expression of IL-6 in colonic tissues were measured using ELISA kits according to the manufacturer's protocol. Absorbance was read at 450 nm using a microplate reader (model ELX800; BioTek Instruments, Inc., Winooski, VT, USA). All the samples were analyzed in triplicate.

Equivalent amounts of protein were resolved in 12% Novex Bis-Tris gel electrophoresis (NuPAGE; Life Technologies Corporation). Proteins were then transferred into nitrocellulose membranes in an iBlot Western Blotting system (Invitrogen, Grand Island, NY, USA). The membranes were blocked for 1 h at room temperature with super SuperBlock^® Blocking Buffers (Pierce Biotechnology, Inc.). They were then incubated at 4°C overnight with primary antibodies against p-STAT3 (rabbit, polyclonal, 1:1,000, CST, cat. no. 9131), regular STAT3 (rabbit, polyclonal, 1:1,000, CST, cat. no. 9132)and β-actin (rabbit, polyclonal, 1:2,000, CST, cat. no. 4967) in blocking buffer. After the membranes were washed, they were further incubated with appropriate horseradish anti-rabbit IgG, HRP-linked antibody (1:5,000, CST, cat. no. 7074) for 1 h at room temperature. The membranes were analyzed using enhanced chemiluminescence Plus reagents and scanned with the Storm Scanner (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

Human colon cancer Caco-2 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells (passages 20–40) were grown in DMEM supplemented with 20% (v/v) FBS, 1,000 mg/l of glucose, 50 U/ml penicillin and 50 µg/ml streptomycin in a 37°C humidified incubator with 5% CO₂. Caco-2 cells usually reached confluence 3 days after seeding, and differentiated into enterocyte-like cells 18–20 days post-confluence. To test the effect of PZH on IL-6-induced phosphoralytion of STAT3, we seeded these fully differentiated cells in 24-well biocameral inserts (area, 0.33 µm²; pore size, 0.4 µm insert). On the day of the experiment, the medium was removed and supplemented with 0.5% FBS medium. Differentiated Caco-2 cells (20 days post-confluence) in 24-well biocameral inserts were pre-incubated with 0.5 mg/ml of PZH for 2 h followed by stimulation with 10 ng/ml of IL-6 for 30 min.

Differentiated Caco-2 cells (20 days post-confluence) in 96-well plates were treated with various concentrations of PZH for 24 h. The cytotoxic effect of PZH on Caco-2 cells was examined by the MTT colorimetric assay. Briefly, MTT (100 µl) (0.5 mg/ml in PBS) was added to each well, and the samples were incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 µl DMSO. The absorbance was measured at 570 nm using a spectrophotometer reader (Model ELX800; BioTek Instruments, Inc., Winooski, VT, USA).

Data were presented as mean ± SD for the indicated number of independently performed experiments. The data were analyzed using the SPSS package for Windows (version 17.0; SPSS, Inc., Chicago, IL, USA). Statistical analysis was carried out using Student's t-test and one-way ANOVA, followed by LSD's test or Dunnett's test. Differences with P<0.05 were considered to be statistically significant.

To determine whether PZH could inhibit the development of UC, we evaluated the clinical manifestations in experimental mice. The DAI was calculated based on the observation of body weight, stool consistency and rectal bleeding. As shown in Fig. 1, upon DSS stimulation mice exhibited apparent manifestations of colitis which was characterized by body weight loss, diarrhea and rectal bleeding. Administration of PZH significantly ameliorated DSS-induced colitis symptoms. The DAI score of normal control, DSS-stimulated model or PZH-treated group was 0.6±0.54, 10.4±1.67 or 4.0±0.71, respectively (P<0.05; Fig. 1A). Moreover, colons were harvested from mice in each group after sacrifice; and length from cecum to anus was measured. We found that PZH treatment profoundly prevented DSS-induced colon shortening. The average colonic length in normal control, model or PZH-treated group was 7.74±0.34, 3.6±0.41 or 6.06±0.38 cm, respectively (P<0.05; Fig. 1B). Taken together, PZH is potent in suppressing the development of UC in vivo.

We next evaluated the histopathological changes of colon tissues through H&E staining. As shown in Fig. 2, the normal control mice displayed normal colonic histology with an intact epithelium, well-defined gland lengths and no leukocyte infiltration in the mucosa, whereas DSS induced severe colonic histological damages such as mucosal ulceration, inflammatory cell infiltration, crypt distortion and hyperplastic epithelium. However, PZH treatment significantly alleviated DSS-induced histopathological changes in colonic mucosa.

It has been shown that the level of the inflammatory marker SAA is increased in UC patients. We therefore performed ELISA assay to examine SAA level in experimental mice. As shown in Fig. 3, PZH treatment significantly inhibited DSS-induced increase of serum level of SAA in UC mice. The SAA level in mice of normal control, model or PZH-treated group was 304.9±62.8, 1,667.9±202.9 or 558.2±92.5 µg/ml, respectively (P<0.05).

To investigate the mechanism of the anti-inflammatory activity of PZH, we evaluated the activation of IL-6/STAT3 pathway in colon tissues of experimental mice. The protein expression of IL-6 in colon tissues was determined by ELISA assay and STAT3 activation was examined by western blot analysis using antibody that recognizes STAT3 phosphorylation at Tyr705. As shown in Fig. 4, the expression of IL-6 and the phosphorylation level of STAT3 in DSS-induced UC mice was significantly increased, as compared to that in normal control mice; which, however, was significantly inhibited by PZH treatment.

By stimulating the differentiated human colorectal carcinoma Caco-2 cells with IL-6, we generated an inflammatory cell model of human intestinal epithelium to examine the in vitro effect of PZH on IL-6/STAT3 pathway. As shown in Fig. 5, IL-6 stimulation resulted in a significant increase of pSTAT3 level in Caco-2 cells. However, PZH treatment profoundly inhibited IL6-induced STAT3 phosphorylation. The levels of non-phosphorylated STAT3 remained unchanged after the treatment with IL-6 and/or PZH.

To exclude the possibility that the in vitro suppressive activity of PZH on STAT3 pathway was due to cytoxicity, we determined its effect on the viability of differentiated Caco-2 cells using MTT assay. As shown in Fig. 6, Caco-2 cell viability was not affected by treatment with PZH, suggesting that the inhibitory effect of PZH on IL-6-induced STAT3 activation in intestinal epithelial cells did not result from its cytotoxic action.