A total of 32 Sprague-Dawley (SD) rats (male, 6–8-weeks old, mean weight, ~200 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Animals were kept in standard cages (5 rats/cage) under specific pathogen-free conditions in the Laboratory Animal Research Center of Wenzhou Medical University. Rats were given free access to autoclaved tap water and to a standard diet, maintained under controlled conditions of light (12-h light-dark cycle), temperature (22–24°C) and humidity (45–55%). All procedures were ethically approved according to the Guide for the Care and Use of the Administration Committee of Experimental Animals of Wenzhou Medical University (Wenzhou, China).

In accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care International, rats were maintained under specific pathogen-free conditions and studied according to protocols approved by the Wenzhou Medical University Animal Care and Use Committee (approval no. wydw2013-0071).

Rats were weighed and anesthetized by intraperitoneal injection of ketamine/xylazine solution (80 ml/10 g body weight). TNBS (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was dissolved in alcohol (50:50 vol/vol). TNBS solution (100 mg/kg body weight) was administered intrarectally via 3.5 F-catheter to rats maintained for 60 sec in a vertical position. The catheter was inserted into the colon 8 cm proximal to the anus. The normal control group received 0.9% saline intrarectally (38).

SD rats were randomly assigned to four groups (n=8/group): Normal control group, TNBS-treated group, sulfasalazine (SASP)-treated group and TGP-treated group. SASP (Shanghai Sunve Pharmaceutical Co., Ltd., Shanghai, China) and TGP (Ningbo Liwah Pharmaceutical Co., Ltd., Ningbo, China) were respectively administered at the doses of 100 mg/kg/day in the SASP-treated and TGP-treated group by gastric gavage daily from day 1 (24 h after the induction of colitis) for 7 days prior sacrifice. Normal control group and TNBS-treated group were treated with an equal volume of 0.9% saline alone intragastrically.

The clinical progression of colitis was monitored daily for body weight, diarrhea and hemafecia (3). Loss of body weight was calculated as the percentage difference relative to initial body weight. Diarrhea was scored as follows: 0, Normal; 2, loose stools; and 4, diarrhea that remained adhesive to the anus. Bleeding scores were assessed as follows: 0, Negative hemoccult; 2, positive hemoccult; and 4, obvious bleeding (39).

Rats were sacrificed 7 days after drug treatment. The colon was removed and opened longitudinally. The macroscopic damage was measured by a blinded observer with the following score system (40,41): 0, Normal; 1, hyperemia, edema, no ulcer; 2, hyperemia, edema, small linear ulcers or petechiae; 3, hyperemia, edema, wide ulcers, necrosis, or adhesions; 4, hyperemia, edema, megacolon, stenosis, or perforation. For histological analysis, the colonic fragments (0.5 cm) were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned (4 µm thickness) and stained with 10% hematoxylin for 2 min and 0.5% eosin for 1 min at room temperature. The pathological sections were observed under a light microscope by two blinded pathologists and the microscopic damage was scored as follows (42): 0, No evidence of inflammation; 1, low level of inflammation with scattered infiltrating mononuclear cells (1–2 foci); 2, moderate inflammation with multiple foci; 3, high level of inflammation with increased vascular density and marked wall thickening; 4, maximal severity of inflammation with transmural leukocyte infiltration and loss of goblet cells.

The colon specimens were fixed with 4% paraformaldehyde, embedded with paraffin, and sectioned at 4 µm for immunohistochemical staining for IL-17, IL-6, ROR-γt, tumor necrosis factor-α (TNF-α), IL-10, TNF-β and Foxp3. After incubation with xylene and descending concentrations of ethanol, antigens were retrieved by citrate buffer for 15 min at 100°C. Then endogenous peroxidases were removed in 3% hydrogen peroxidase for 15 min at room temperature followed by 5% goat serum for 1 h at 37°C for blocking. Subsequently, sections were incubated with polyclonal rabbit anti-rat antibodies IL-17 (cat. no. ab79056; 1:300), ROR-γt (cat. no. ab78007; 1:50), TNF-α (cat. no. ab6671; 1:250), IL-10 (cat. no. ab192271; 1:1,000), TNF-β (cat. no. ab92486; 1:100) and monoclonal mouse anti-rat IL-6 (cat. no. ab9324; 1:250), Foxp3 (cat. no. ab22510; 1:50; Abcam, Cambridge, MA, USA) in an optimum concentration overnight at 4°C and then incubated with horseradish peroxidase-conjugated secondary antibody (cat. nos. PV.6001 and PV.6002; OriGene Technologies, Inc., Beijing, China) for 30 min at 37°C. Antibody bindings were counterstained with hematoxylin, dehydrated with ascending concentrations of ethanol, cleared in xylene and mounted. A negative control was performed according to the same procedure. Images were acquired with a biological imaging microscope (BX53; Olympus Corporation, Tokyo, Japan).

Total protein of colon specimens was extracted using a radioimmunoprecipitation lysis buffer (Solarbio Science & Technology Co., Ltd., Beijing, China) and phenylmethylsulfonyl fluoride. The protein concentration was analyzed using a BCA kit (Tiangen Biotech Co., Ltd., Beijing, China). Equal amounts of protein (50 µg per lane) were separated on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk for 90 min at room temperature, the membranes were incubated overnight at 4°C with anti-IL-17 (cat. no. ab79056; polyclonal, rabbit anti-rat; 1:1,000), anti-IL-6 (cat. no. ab9324; monoclonal, mouse anti-rat; 1:2,500), anti-ROR-γt (cat. no. ab78007; polyclonal, rabbit anti-rat; 1:1,000), anti-TNF-α (cat. no. ab6671; polyclonal, rabbit anti-rat; 1:1,000), anti-IL-10 (cat. no. ab192271; polyclonal, rabbit anti-rat; 1:1,000), anti-TNF-β (cat. no. ab92486; polyclonal, rabbit anti-rat; 1:1,000) and anti-Foxp3 (cat. no. ab22510; monoclonal, mouse anti-rat; 1:1,000; all from Abcam, Cambridge, MA, USA) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. BS60630; polyclonal, rabbit anti-rat; 1:1,000; Bioworld Technology, Inc., St. Louis Park, MN, USA) and washed three times with TBST. Then the membranes were incubated with secondary antibodies-conjugated to horseradish peroxidase (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature followed by washing three times. Immunoreactive bands were visualized by an enhanced chemiluminescence kit (Bio-Rad Laboratories, Hercules, CA, USA).

Total RNA was extracted from the colon tissue, spleen mononuclear cells and mesenteric lymph node (MLN) mononuclear cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. RNA (1 µg total) was reverse-transcribed into cDNA. Reverse transcription was carried out by RevertAid First Strand cDNA Synthesis kit (cat. no. K1622; Thermo Scientific Inc.). The temperature conditions for the reverse transcription were as follows: Initial cycle at 25°C for 5 min, followed by 42°C for 60 min and 72°C for 10 min, allowed to cool to 4°C. The mRNA expression of several genes was quantified with SYBR-Green Real-time PCR Master Mix Plus (Thermo Fisher Scientific, Inc.) by an ABI 7500 Sequence-Detection system (Thermo Fisher Scientific, Inc.). The PCR cycling conditions were as follows: An initial denaturation and activation at 95°C for10 min, followed by 40 amplification cycles of 95°C for 15 sec and 60°C for 60 sec. The primer sequences are presented in Table I. GAPDH was used as reference gene. Relative gene expression levels were calculated by the 2^−∆∆Cq method (43).

Peripheral blood of rats was drawn into EDTA-anticoagulant tubes, then centrifuged for 15 min at 3,000 × g at 4°C. The plasma was collected and stored at −80°C until tested. ELISA kits were used to assess the plasma concentrations of IL-17 (cat. no. 10353-09R), IL-6 (cat. no. 10752-09R), TNF-α (cat. no. 10917-09R), IL-10 (cat. no. 10726-09R) and TNF-β (cat. no. 10973-09R; all from Shanghai Boyun Biotech Co., Ltd., Shanghai China) in accordance with the manufacturer's protocols.

The spleen and MLN were removed from rats 7 days after drug treatment and isolated mononuclear cells. In order to analyze Th17 cells, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (1 µg/ml) in the presence of Brefeldin A (10 µg/ml) and monensin (1.4 µg/ml) (Hangzhou MultiSciences Biotech Co., Ltd., Hangzhou China) at 37°C and 5% CO₂ for 4 h. Cells were washed with PBS and surface-labeled with fluorescein isothiocyanate-(FITC-) conjugated anti-CD4 (0.5%; cat. no. 85-11-0041-81; eBioscience; Thermo Fisher Scientific, Inc.) for 40 min at 4°C. The cells were subsequently fixed and permeabilized by fixation/permeabilization buffer (BD Biosciences, San Jose, CA, USA) and labeled with phycoerythrin-(PE-) conjugated anti-IL-17 (1%; cat. no. 85-12-7177-81; eBioscience; Thermo Fisher Scientific, Inc.) for 40 min at 4°C. For analysis of Treg cells, without PMA and ionomycin stimulation, surface staining was performed with FITC-conjugated anti-CD4 (0.5%; cat. no. 85-11-0041-81; eBioscience; Thermo Fisher Scientific, Inc.) and allophycocyanin-conjugated anti-CD25 (1.5%; cat. no. 85-17-0390-82; eBioscience; Thermo Fisher Scientific, Inc.) for 40 min at 4°C. Afterwards, cells were fixed and permeabilized, and intracellular staining was performed with PE-conjugated anti-Foxp3 (1.25%; cat. no. 85-12-5773-82; eBioscience; Thermo Fisher Scientific, Inc.) for 40 min at 4°C. Appropriate isotype controls were used in the experiments. The stained cells were detected by a FACSCalibur flow cytometer (BD Biosciences) and the data were analyzed with FlowJo version 7.6.1 (FlowJo LLC, Ashland, OR, USA).

Statistical analysis was performed using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as mean ± standard error and one-way analysis of variance was used for multiple comparisons, followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the potential therapeutic effect of TGP in the TNBS-induced colitis, the present study tested the clinical symptoms and macroscopic scores in the aforementioned groups. As expected, the rats acquired severe symptoms characterized by weight loss, diarrhea and bleeding following TNBS instillation compared with the normal control group. However, TGP and SASP treatment rapidly reversed weight loss and decreased bleeding and diarrhea scores compared with the TNBS group (Fig. 1A-C). The present study observed marked macroscopic change accompanied by hyperemia, edema, ulcers, necrosis and adhesion in TNBS-induced group, whereas the colons from TGP or SASP group exhibited no or a slight macroscopic damage (Fig. 1D). Consistently, the macroscopic scores of TNBS-induced rats were significantly increased compared with the control group and the TGP and SASP-treated groups (Fig. 1E). Histopathologically, the colonic tissues of TNBS-induced rats exhibited a reduced number of goblet cells, loss of crypts, damage of crypts, infiltration by inflammatory cells and extensive destruction of the mucosal layer compared with the control group (Fig. 2A). However, histological signs of inflammatory activity were distinctly reduced following TGP administration in TNBS-rats, similar to the SASP-treated group (Fig. 2). Therefore, treatment of TGP is effective in modulating the development of acute TNBS-induced colitis and the efficacy of TGP was similar to SASP.

In order to investigate the effects of TGP on acute inflammation, plasma and colonic tissue samples were collected at 7 days and the production of signature cytokines and transcription factors was measured by ELISA, western blotting and immunohistochemistry. The current results revealed that Th17-associated pro-inflammatory cytokines and transcription factors in colonic tissues of TNBS-induced rats, such as IL-17, IL-6, TNF-α and ROR-γt, were significantly reduced following administration of TGP and SASP treatment (Figs. 3 and 4). The TGP and SASP-treated groups also had higher levels of IL-10, TNF-β and Foxp3 in colonic tissues compared with the untreated TNBS and control groups (Figs. 3 and 4). Similarly, TGP administration downregulated the expression of IL-17, TNF-α, IL-6 and upregulated the level of IL-10 and TNF-β in plasma of TNBS-induced rats (Fig. 5). These findings indicate that TGP inhibits Th17 responses; however, may promote Treg responses in TNBS-induced colitis and there was no significant difference between treatment with TGP or SASP.

To determine whether TGP may adjust the mRNA expression of transcription factors and cytokines in Th17 and Treg cells, we tested the expression levels using RT-qPCR. TGP administration in TNBS-rats led to lower mRNA expression levels of TNF-α, IL-6, IL-17 and ROR-γt in the colonic tissues, MLN and splenic lymphocytes (Figs. 6A-D, 7A-D and 8A-D). In contrast, the mRNA expression of IL-10, TNF-β and Foxp3 increased in TNBS-rats treated with TGP (Figs. 6E-G, 7E-G and 8E-G).

Previous studies have revealed that Th17 and Treg cells play a distinct role in the control and development of IBD and the balance between Th17 and Treg cells proliferation levels is a critical factor in designing therapies for IBD (44,45). Therefore, the present study hypothesized that TGP may differentially contribute to the development of Th17 and Treg cells. In order to determine this, flow cytometry was used to quantify the frequencies of Th17 and Treg cells separated from spleen and MLNs of all groups. As presented in Fig. 9C and D, the percentage of CD4⁺IL17⁺(Th17) cells was markedly increased in the TNBS-induced rats, whereas the administration of TGP markedly reduced the percentage, as well as the treatment with SASP. Additionally, the present study revealed that TGP treatment increased the level of CD4⁺CD25⁺Foxp3⁺ (Treg) cells (Fig. 9A and B). Therefore, these findings indicated that TGP may be able to ameliorate colitis, which was associated with an increased number of Treg cells and a reduction of Th17 cells among spleen and MLNs.