Taraxasterol (purity, 98% HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, China). Dextran sodium sulfate (DSS) was purchased from MP Biomedicals, LLC (Santa Ana, CA, USA). The ELISA kits for TNF-α (cat. no. A0119), IL-6 (cat. no. A0107) were purchased from Nanjing Jiancheng Bioengineering Institute (China). All plastic materials were purchased from Falcon Labware (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). RPMI Medium-1640, fetal bovine serum (FBS), and phosphate buffer saline (PBS) were obtained from Gibco (Thermo Fisher Scientific, Inc. Waltham, MA, USA). Penicillin G/streptomycin, MTT, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck KGaA Darmstadt, Germany). Apoptosis Assay kit was purchased from Nanjing KeyGen Biological Co., Ltd. (Nanjing, China). Rabbit anti-human Caspase antibody (cat no. 9661; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-human BAX antibody (cat no. 2774; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-human p53 antibody (cat no. 48818; 1:1,000; Cell Signaling Technology, Inc.) and rabbit anti-human β-actin antibody (cat no. 622101; 1:2,000) were purchased from BioLegend Inc. (San Diego, CA, USA). Human colon cancer epithelial cells (HT-29) were purchased from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China).

Adult male C57BL/6 mice (20–24 g) were purchased from the Laboratory Animal Services Center, The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). All animals were housed in accordance with the National Institutes of Health Guide for Laboratory animals' use. The study was approved by the Animal Ethics Committee of The First Affiliated Hospital of Zhengzhou University. Animals were housed under standard environment condition of temperature at 20–25°C under a 12-h dark/light cycle and allowed free access to sterilized water and standard food.

A total of 60 male C57BL/6 mice were randomly divided into 5 groups (n=12); control group, model group (DSS group), low, medium and high dose (25, 50 and 100 mg/kg/day respectively) taraxasterol groups. Colonic inflammation was established by oral gavage of 5% DSS solution for 5 days. Establishment of a successful model was determined based on thin stools, fecal blood, weight loss and other symptoms and morphological changes. Intestinal inflammation was graded in a blinded fashion, according to Cooper et al (9): 0, no inflammation; 1, mild inflammation of the mucosal intrinsic layer and submucosal layer; 2, severe inflammation of the mucosal intrinsic layer and submucosal layer; 3, mild inflammation of the whole colon wall; and 4, severe inflammation of the whole colon wall.

For taraxasterol treatments, C57BL/6 mice were fed with low, medium or high dose (25, 50 and 100 mg/kg/day) taraxasterol. Mice in the control group were treated with normal saline.

Changes in mucosa and serosal surfaces of colonic tissue were observed via microscope. Fresh colonic tissue was isolated (size ~1×1×0.2 cm). The embedding box was fixed in 4% paraformaldehyde for 24 h. The tissue was embedded in paraffin and stained continuously for 4 µm. Tissue lesions were observed via microscopic observation through hematoxylin and eosin (HE) staining. The criteria was as follows: 0, normal colonic mucosa; 1, crypt defect of 1/3; 2, crypt defect of 2/3; 3, the inherent layer covers the monolayer epithelium with mild inflammatory cell infiltration; and 4, erosion and ulcer in mucosa with plentiful inflammatory cell infiltration.

Paraffin-embedded colonic tissues were continuously sliced into 4 µm thick sections, at 65°C for 2 h. The streptavidin-peroxidase (SP) method was used where sections were dewaxed, hydrated then 0.01 M sodium citrate buffer solution was used for antigen repair for 15 min (1:300) at 95°C. Sections were washed and blocked with goat serum (cat. no. ab7481; Abcam) at room temperature for 20 min. Samples were incubated with primary antibody anti-p53 (1:300; cat no. 48818; Cell Signaling Technology, Inc.) at 4°C overnight. Sections were then washed three times for 5 min using PBS with Tween (PBS-T) then incubated with horseradish peroxidase linked secondary antibody (1:2,000; cat no. 7076; Cell Signaling Technology, Inc.) at 37°C for 1 h. Following washes, SP was added at room temperature for 30 min. DAB staining upon the observation of the slices, and finally the slices were dehydrated, transparent and sealed. Images were taken using an image scanner (Zeiss 125 AxioScan.Z1; Zeiss AG).

A total of 10 non-repeatable fields were selected at ×100 magnification to analyze the % of positively stained cells and coloring intensity. The intensity was scored as: 0, negative; 1, weak; 2, medium; 3, strong. The % of positive-stained cancer cells was scored as: 0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The intensity number multiplied by extent of staining number produced the final staining score of p53 (range 0–12).

Human colon cancer epithelium HT-29 cells were removed from liquid nitrogen, thawed in a water bath at 37°C, then transferred into a centrifuge tube with RPMI 1640 medium containing 10% fetal bovine serum, and centrifuged at 200 × g for 10 min. The cell pellet was resuspended with complete medium in a 75 ml standard flask, then incubated under standard conditions (37°C, 5% CO₂). For LPS and taraxasterol treatments, either LPS (10 µg/ml) or taraxasterol (2.5, 5 or 10 µg/ml respectively) were added into the medium.

In the logarithmic growth phase, 5,000 cells/well were seeded in 5 wells of a 96-well plate. Following 12 h, 1640 medium with 1% serum containing no taraxasterol was added to the control group and culture medium 1640 with 1% serum containing 2.5, 5 or 10 µg/ml taraxasterol was added to the model group. A total of 100 µl of medium containing MTT (0.5 g/l) was added to each well then samples were incubated for 4 h under standard conditions (37°C, 5% CO₂). The purple formazan crystals were dissolved using dimethyl sulfoxide and viability was subsequently analyzed at a wavelength of 490 nm with a microplate reader (BIO-RAD680; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

HT-29 cells were treated with different concentrations of taraxasterol (2.5, 5 or 10 µg/ml) for 24 h. After digestion, cells were collected and centrifuged at 800 × g for 5 min then the supernatant was collected. For animal experiments, colon tissue was cut into 2–3 mm samples, homogenized with PBS, centrifuged at 12,000 × g for 15 min then the supernatant was collected. Levels of TNF-α and IL-6 in the supernatant were measured according to the instructions of the kit. The experiment was repeated at least three times.

HT-29 cells were treated with different concentrations of taraxasterol (2.5, 5 or 10 µg/ml) for 24 h. Cells were washed with PBS twice, digested and collected by centrifugation. Cells were resuspended with 500 µl binding-buffer, followed by 5 µl Annexin-V/fluorescein isothiocyanate (FITC) and 5 µl propidium iodide (PI) for 5 min. Cells were detected by flow cytometry (BD FACSCanto A) and analyzed using FlowJo v10 (FlowJo LLC). FITC-/PI- were deemed viable cells, FITC-/PI+ were necrotic cells, FITC+/PI- were early apoptotic cells, and FITC+/PI+ were late apoptotic cells. The apoptosis rate was calculated as follows: Apoptosis rate % = (number of early apoptotic cells + number of late apoptotic cells)/total number of cells ×100%. The experiment was repeated at least three times.

Proteins were extracted from cell suspension and tissue homogenates with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). The concentration of proteins were detected via Bicinchoninic acid Protein Assay Kit. Proteins (30 µg) were separated via for SDS-PAGE on a 12% gel for 2 h, then the proteins were transferred for 2 h to a polyvinylidene difluoride membrane. Blocking of the membrane with 5% skim milk was performed at 37°C for 2 h. The membranes were incubated with primary antibodies anti-p53 (cat no. 48818; 1:1,000; Cell Signaling Technology, Inc.), BAX (cat no. 2774; 1:1,000; Cell Signaling Technology, Inc.) and cleaved caspase-3 (cat no. 9661; 1:1,000; Cell Signaling Technology, Inc.) overnight at 4°C. Membranes were washed with PBS for 15 min then incubated with secondary antibody IgG (cat on. 7076; 1:2,000; Cell Signaling Technology, Inc.) for 1.5 h at room temperature. Electrochemiluminescence imaging with gel imaging analysis system (Bio-Rad Laboratories, Inc.) was used to measure protein band density value. The experiment was repeated at least three times with β-actin as the loading control.

Statistical analysis was performed using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). All results were presented as mean ± standard error of the mean (SEM). Comparisons were performed using one-way analysis of variance followed by Bonferroni's post hoc test. P<0.05 was considered to indicate significant difference.

To obtain the optimal concentration of taraxasterol and LPS, HT-29 cells were treated with LPS or taraxasterol at different concentrations, and the cell viability was detected via MTT assay. The results demonstrated that 10 µg/ml LPS decreased the cell viability slightly, while the taraxasterol concentrations used in the experiments did not affect the survival of HT-29 cells compared with the control group (Fig. 1).

Next, the effect of taraxasterol on the expression of inflammation factors was investigated. To this effect, HT-29 cells were treated with LPS to induce an inflammatory response. LPS stimulation significantly increased TNF-α and IL-6 levels in the supernatant (Fig. 2A and B). Taraxasterol treatment decreased the LPS-induced TNF-α and IL-6 expression in a dose-dependent manner which suggested that taraxasterol exerted a significant anti-inflammatory effect.

Since taraxasterol could reduce the expression of inflammatory factors, the effect of taraxasterol on apoptosis activity was investigated. HT-29 cells were treated with LPS or taraxasterol, flow cytometry was used to detect the apoptosis rates. The results demonstrated that LPS induced apoptosis in HT-29 cells. In comparison, taraxasterol significantly decreased apoptosis in HT-29 cells (Fig 3A and B). Next, the expression levels of apoptosis-related proteins were detected via western blotting. The results demonstrated that expression of p53, BAX and cleaved caspase-3 were significantly higher in the LPS-treated group compared with the control group (Fig. 3C-F). Taraxasterol significantly decreased p53, BAX and cleaved caspase-3 protein levels in a dose dependent manner (Fig. 3C-F), which suggests that taraxasterol could reduce LPS-induced apoptosis in vitro.

Animal experiments were used to identify the effects of taraxasterol on UC in vivo, after DSS was used to establish a UC mouse model. The structure of the control group colonic tissue was distinct with smooth intact mucosa and no erosion of the tissue or inflammatory cell infiltration (Fig. 4A). In the UC model group, mucosal erosion, granulation tissue hyperplasia and glandular enlargement was observed in Fig. 4A (indicated by the red arrows). In the taraxasterol groups, there was no obvious erosion in the colonic mucosa of mice with minimal inflammatory cell infiltration (Fig. 4A). Colonic pathological changes in the 100 mg/kg taraxasterol dosage group were significantly reduced compared with the 50 mg/kg or 25 mg/kg taraxasterol treatment groups (Fig. 4A). The histopathological scores of the model group and taraxasterol treated groups were significantly higher compared with the control group (Table I; P<0.05). The histopathological score of the 100 mg/kg group (1.5±0.2) was significantly lower compared with the 25 mg/kg group (2.7±0.3; P<0.05).

Immunohistochemical assay was used to determine p53 protein levels in colon tissues. The results demonstrated that positive p53 staining (brown) was mainly located within nuclei (Fig. 4B). Expression of p53 in colonic mucosa was higher in the UC model groups and the taraxasterol treated groups compared to the control groups (Table I; P<0.01). Treatment with 100 mg/kg taraxasterol significantly decreased p53 levels compared with the UC model group (Fig. 4B). There was no significant difference in IHC score between 100 mg/kg and 50 mg/kg taraxasterol treatment groups, however both had lower IHC scores than the 25 mg/kg treatment groups (Table I; P<0.01).

The expression levels of TNF-α and IL-6 in the supernatant of colonic mucosa tissues was determined. TNF-α and IL-6 expression levels were lower in taraxasterol-treated groups compared with UC model groups (Table I; P<0.01). The levels of TNF-α and IL-6 in the taraxasterol-treated groups were significantly higher compared with the control group (Table I; P<0.05). In taraxasterol-treated groups, the levels of TNF-α and IL-6 in the 100 mg/kg treatment group were significantly lower than those in the 25 mg/kg treatment group (Table I; P<0.05). These results suggested that taraxasterol significantly decreased the expression of TNF-α and IL-6 in vivo.