DSS (36–50 kDa) was purchased from MP Biomedicals, LLC (Santa Ana, CA, USA). Berberine was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and TRIzol reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). FastStart Universal SYBR-Green Master (Rox) and PrimeScript Reverse Transcription (RT) Master Mix Perfect Real-Time kits were purchased from Roche Diagnostics GmbH (Mannheim, Germany) and Takara Biotechnology Co., Ltd. (Dalian, China) respectively. Superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) activity assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Primary antibodies for occludin and E-cadherin were from Thermo Fisher Scientific, Inc. and Cell Signaling Technology, Inc. (Danvers, MA, USA), respectively. Primary antibodies for ZO-1 and β-actin were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and Sigma-Aldrich (Merck KGaA), respectively. Mouse monoclonal anti-signal transducer and activator of transcription 3 (STAT3; 124H6) and anti-phosphorylated (p)-STAT-3 (p-Tyr705) antibodies were from Cell Signaling Technology, Inc. Mouse monoclonal anti-cluster of differentiation (CD68) antibody was from Abcam (Cambridge, England).

A total of 40 C57BL/6 J male mice (six weeks old) weighing 18–22 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were kept under an automated 12-h light-dark cycle at a controlled temperature of 22±2°C, relative humidity of 50–60% and had ad libitum access to a standard dry diet and tap water. The animals received humane care and experimental procedures were performed in accordance with the health and care of experimental animals guidelines of the Second Military Medical University (Shanghai, China).

Mice were divided into the following three groups: i) Control group (group 1); ii) DSS-induced colitis group (group 2); and iii) DSS-induced colitis plus berberine-treated group (group 3). Each group consisted of ten mice. Mice in group 1 received drinking water alone and were used as negative controls. Colitis was induced in mice of groups 2 and 3 by administration of DSS (3% w/v, dissolved in drinking water) for six days. Berberine (100 mg/kg, dissolved in distilled water) was administered to group 3 mice via oral gavage once a day for a period of five days after the six days DSS treatment period. Daily assessments of weight loss, stool consistency and fecal blood, as indicators of disease activity index, were measured. The mice in all groups were sacrificed 11 days after initiation of experimental treatments, following the final administration of berberine to group 3 mice. Prior to sacrifice by cervical dislocation, mice were anesthetized with an intraperitoneal injection of 4% (w/v) chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in order to collect blood samples. Colonic tissues were then removed for histopathological examination.

Colon sections were taken from the mice for histological examination. The length of the colon from the anus to appendix was measured by a ruler, then segments of colon were excised, fixed in 10% phosphate-buffered saline (PBS)-buffered formalin at room temperature for 24 h, then embedded in paraffin. Sections measuring 5–10 µm in thickness were cut, placed on glass slides and stained with hematoxylin and eosin (H&E). Histopathological changes were observed under a light microscope and graded semiquantitatively using a histological injury scale, as described by Wirtz et al (16). The scores for histological changes were as follows: 0, no evidence of inflammation; 1, low level inflammation with scattered mononuclear cells (1–2 foci); 2, moderate inflammation with multiple foci of mononuclear cells; 3, high level inflammation with increased vascular density and marked wall thickening; and 4, maximal inflammation with transmural leukocyte infiltration and loss of goblet cells.

The ability of MPO to modulate levels of hydrogen peroxide is considered to be an indicator of MPO activity (17). MPO activity was determined using an MPO activity assay kit, according to a modified method of the manufacturer's protocol, as follows: Freshly excised colon was rinsed with PBS, homogenized in tissue lysis buffer and centrifuged at 12,000 × g and 4°C for 15 min to isolate proteins. Serum was isolated without lysis buffer for later analysis of serum MPO. Blood samples were then centrifuged at 3,500 × g for 20 min at 4°C. Pellets were resuspended in PBS containing 0.5% hexadecyl-trimethylammonium bromide (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), then freeze-thawed three times. MPO activity was determined by measuring the absorbance of the resulting solutions at 460 nm with an automatic microplate reader at 37°C. Protein concentration was then measured quantitatively using a Bicinchoninic Acid-100 Protein Determination kit (Shenneng Bocai Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's protocol. As the degradation of peroxide by MPO results in a color change, MPO activity was defined as the quantity of enzyme degrading 1 µmol/ml peroxide at 37°C, as determined from absorbance measurements, expressed as U/mg of colon protein.

Freshly excised colon was rinsed with PBS, homogenized in tissue lysis buffer and centrifuged at 12,000 × g and 4°C for 15 min. Individual activities of SOD and CAT in tissue lysates and serum were then measured using SOD and CAT activity assay kits, respectively, according to the manufacturer's protocol. Individual activities of SOD and CAT were measured at 450 and 405 nm, respectively, with a microplate fluorometer. Total protein concentration was measured using the Bradford method (Beyotime Institute of Biotechnology, Haimen, China). The concentration of SOD and CAT in colon and serum samples was presented as pg/mg of total protein.

Intestinal epithelial permeability in vivo was determined according to a previously described method (18). Briefly, mice were fasted overnight and fluorescein isothiocyanate (FITC)-dextran solution (4 kDa, 600 mg/kg) was delivered via gavage the following day. Mice were then anesthetized with 4% (w/v) chloral hydrate (intraperitoneal injection) 4 h after FITC-dextran administration. Abdominal cavities were exposed with surgical instruments and blood was harvested via cardiac puncture with a 1-ml injector. Blood samples were then centrifuged at 3,500 × g and 4°C for 20 min. Serum levels of FITC were measured at 480 and 520 nm using a microplate fluorometer.

Immunoblotting to detect colonic proteins was performed according to a previously described method (19). Briefly, colonic tissues were removed and washed in PBS. Whole tissue was then cut into pieces and homogenized in five volumes of ice-cold radioimmunoprecipitation assay buffer (Kangcheng Pharmaceutical Co., Ltd., Guangzhou, China) containing 1 µl protease and phosphatase inhibitors. The mixture was then centrifuged at 14,000 × g and 4°C for 15 min, and protein concentration of the resulting supernatants was determined by a Bradford assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Equal quantities of protein were separated by a 10% (w/v) SDS-PAGE gel (20 µg/lane) and transferred to nitrocellulose membranes. The membranes were blocked for 3 h at room temperature with 5% blocking reagent, then incubated overnight at 4°C with anti-STAT3 antibody (1:1,000), anti-p-STAT3 antibody (1:1,000), anti-occludin antibody (1:2,000), anti-E-cadherin antibody (1:2,000), anti-ZO-1 antibody (1:1,000) and β-actin antibody (1:10,000), according to the manufacturer's instructions. After washing with PBS-Tween 20, membranes were incubated with Cyanine™-conjugated affinipure donkey anti-rabbit immunoglobulin (Ig)-G [heavy (H) and light (L) chains; cat. no. 712-167-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA] and HiLyte 488™-conjugated affinipure donkey anti-goat IgG (H+L chains; cat. no. 705-167-003; Jackson Immunoresearch Laboratories, Inc.) at room temperature for 1 h. Protein bands were visualized and quantified with an Odyssey Infrared Imaging 3.0 system (LI-COR Biosciences, Lincoln, NE, USA). β-actin was used as a protein loading control. Immunoblotting experiments were repeated at least three times.

Total RNA was extracted from tissue homogenates of mouse colon with TRIzol reagent, according to the manufacturer's protocol. RNA was reverse transcribed into cDNA with a PrimeScript Reverse Transcription (RT) Master Mix Perfect Real-Time kit, according to the manufacturer's protocol. Resulting cDNA was then used as a template for qPCR. qPCR was performed using an ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA). The primer sequences were as follows: GAPDH, forward, 5′-GTATGACTCCACTCACGGCAAA-3′ and reverse, 5′-GGTCTCGCTCCTGGAAGATG-3′; interleukin (IL)-1β, forward, 5′-CTCACAAGCAGAGCACAAGC-3′ and reverse, 5′-CAGTCCAGCCCATACTTTAGG-3′; IL-6, forward, 5′-CGGAGAGGAGACTTCACAGAG-3′ and reverse, 5′-CATTTCCACGATTTCCCAGA-3′; tumor necrosis factor (TNF)-α, forward, CATTTCCACGATTTCCCAGA-3′ and reverse, 5′-GGAAAGCCCATTTGAGTCCT-3′. GAPDH was used as an internal control and RNA products were quantified by the DDCq method (20). RT-qPCR experiments were performed at least three times.

An immunofluorescence assay to detect macrophage infiltration in mouse colonic mucosa was performed according to a previously described method (21). Briefly, after colons were excised and rinsed with PBS, colon tissues were fixed in 10% PBS-buffered formalin at room temperature for 24 h, then embedded in paraffin, cut into 5 µm sections and mounted on glass slides. Sections were subsequently deparaffinized in xylene and rehydrated in a graded series of ethanol solutions (50 75, 85, 95 and 100%, 3 min/solution). Sections were then blocked with 5% bovine serum albumin (BSA; Beyotime Institute of Biotechnology) in Tris-buffered saline (TBS) for 90 min at room temperature, then immunostained overnight at 4°C with anti-CD68 antibody (1:100) and 5% BSA in TBS. Following immunostaining, sections were washed three times with TBS, then incubated with Alexa Fluor 488-conjugated secondary antibody (1:200; cat. no. 715-547-003; Jackson Immunoresearch Laboratories, USA) in TBS for 2 h at room temperature in the dark. Sections were then mounted with mounting medium containing 5 mg/ml DAPI (Beyotime Institute of Biotechnology, Shanghai, China) for nuclear counterstaining and visualized under an Olympus IX71 inverted fluorescent microscope (Olympus Corporation, Tokyo, Japan).

Quantified data are presented as the mean ± standard deviation. Differences between two groups were determined with the Student's t-test. Statistical analysis was performed using SPSS 11.0 software (SPSS, Inc., Chicago, IL, USA) and P<0.05 was considered to indicate a statistically significant difference.

Relative to the untreated control group (group 1), DSS-induced colitis mice (groups 2 and 3) exhibited marked weight loss five to seven days after initiation of a six days DSS treatment (Fig. 1A). After day 8, group 2 and 3 mice exhibited recovery of body weight, however body weight recovery was significantly greater on days 8, 9 and 11 for group 3 mice administered with berberine (day 7), relative to berberine-untreated group 2 mice (P<0.05; Fig. 1A).

Colon length was also measured as an indicator of colitis. Relative to that of the control group, colon length was significantly decreased in group 2 DSS-colitis mice (P<0.01). In turn, relative to that of group 2 mice, colon length was significantly increased in group 3 DSS-colitis mice administered with berberine (P<0.01; Fig. 1B).

Histological and morphological characteristics of the colons were subsequently examined by H&E staining. In the control group, colon tissue exhibited normal crypt morphology and goblet cell count, and absence of mucosal thickening and ulcerations (Fig. 1C). By contrast, colons of group 2 DSS-colitis mice exhibited superficial ulcerations, loss of goblet cells, neutrophil infiltration and goblet cell damage. Thus, group 2 colon tissue received a significantly higher histological damage score than that of the control group (P<0.01; Fig. 1C). In turn, relative to the colon tissue of group 2 mice, colons of berberine-treated group 3 colitis mice displayed reduced neutrophil infiltration, minimal goblet cell loss and thus a significantly lower histological damage score (P<0.01; Fig. 1C).

Intestinal epithelial tight junctions serve a key role in protecting against inflammation, and disrupted tight junctions are a main cause of intestinal barrier dysfunction and inflammation (22). Therefore, the present study evaluated the effects of berberine on intestinal permeability in vivo using FITC-dextran. It was observed that the level of FITC-dextran in the serum of group 2 DSS-colitis mice was significantly higher than that in control mice (P<0.01; Fig. 2A). In turn, the elevated serum levels of FITC-dextran in DSS-colitis mice were significantly reduced by berberine treatment in group 3 mice (P<0.01; Fig. 2A).

To verify the involvement of epithelial tight junctions, the levels of tight junction-associated protein expression, namely ZO-1, E-cadherin and occludin, were determined by western blotting (Fig. 2B). Relative to control mice, significant decreases in the levels of colonic ZO-1, E-cadherin and occludin and were identified in group 2 DSS-colitis mice (P<0.05, P<0.01 and P<0.05, respectively; Fig. 2C-E). In turn, the reduced levels of ZO-1, E-cadherin and occluding expression in DSS-colitis mice were significantly increased by berberine treatment in group 3 mice (P<0.05, P<0.01 and P<0.05, respectively; Fig. 2C-E).

Levels of the proinflammatory cytokines, IL-1β, IL-6 and TNF-α, in the colon of DSS-colitis mice were measured at the mRNA level by RT-qPCR. It was observed that relative to control mice, levels of IL-1β, IL-6 and TNF-α mRNA in group 2 DSS-colitis mice were significantly increased (P<0.05, P<0.05 and P<0.01, respectively; Fig. 2A-C). In turn, the elevated levels of IL-1β, IL-6 and TNF-α mRNA in DSS-colitis mice were significantly decreased by berberine treatment in group 3 mice (all P<0.05; Fig. 3A-C).

STAT3 is involved in colonic inflammation and is activated by a number of cytokines and growth factors, including as IL-6 and IL-17 (23,24). Therefore, the effect of berberine on the phosphorylation of STAT3 in DSS-colitis mice was evaluated by western blot analysis (Fig. 3D). It was demonstrated that the levels of p-STAT3 in colonic tissue of DSS-colitis mice were significantly increased relative to control mice (P<0.01; Fig. 3D). In turn, the elevated levels of p-STAT3 in DSS-colitis mice were significantly decreased by berberine treatment in group 3 mice (P<0.05; Fig. 3D).

MPO, SOD and CAT serve key roles in inflammation and oxidative stress (25). Therefore, their levels in the serum and colonic tissue of DSS-colitis mice were determined by enzyme activity assays (Fig. 4). Relative to control mice, it was observed that the levels of MPO in serum and colonic tissue of DSS-colitis mice were significantly higher (both P<0.01; Fig. 4A and D). In turn, the elevated serum and colon levels of MPO in DSS-colitis mice were significantly decreased by berberine treatment in group 3 mice.

By contrast, significant decreases in the serum and colon levels of SOD (both P<0.01; Fig. 4B and E) and CAT (P<0.05 and P<0.01, respectively; Fig. 4C and F) were identified in group 2 DSS-colitis mice, relative to control mice. In turn, significant increases in the serum and colon levels of SOD (both P<0.05; Fig. 4B and E) and CAT (P<0.05 and P<0.01, respectively; Fig. 4C and F) were observed following berberine treatment in group 3 colitis mice, relative to group 2 mice.

CD68 may be used as a marker of macrophages (and monocytes) in the colon of mice. Therefore, the present study measured CD68 expression in colonic tissue of DSS-colitis mice in order to determine the level of macrophage infiltration. It was observed that CD68 expression was markedly increased in DSS-induced mice relative to control mice, and that subsequent berberine treatment reduced the elevated expression of CD68 in DSS-colitis mice (Fig. 5).