2.1. Mice

Male BALB/c mice, aged 8–9 weeks, weighing 20–22 g, were used in this study. All mice were obtained from the Hunan Silaike Jingda Experimental Animal Co. Ltd. (Changsha, China) (Animal Certificate Number: SCXK 2019-0004). The animals were maintained under specific pathogen-free (SPF) conditions (temperature 23 ± 2°C, relative humidity 55 ± 10%, alternating 12 h light/dark cycle). All mice were fed standard food and water ad libitum and acclimatized for 3 days before the start of the experiment. The experimental protocol (permit number: JZ2019-126) was reviewed and approved by Jiangxi University of Traditional Chinese Medicine Animal Care and Use Committee and were performed in accordance with its prescribed guidelines. All mice were randomly divided into four groups: Control group, normal feeding without DSS-induced colitis; DSS group, DSS-induced colitis without drug treatment; DSS + Cur group,DSS-induced colitis treated with curcumin; DSS+5-ASA group, DSS-induced colitis treated with 5-ASA (mesalazine).

2.2. DSS-Induced Experimental Colitis

As described previously [23, 24], experimental colitis was first treated with a 3% (wet/vol) solution of DSS for 7 days, followed by sterile drinking water for 7 days and with 2% (wet/vol) DSS for the last 7 days. Meanwhile, the control mice received only tap water. Curcumin was supplied by GANGRUN Biotechnology (Nanjing, China) (purity >95%, High Performance Liquid Chromatography). Mesalazine (batch number: 130407) was purchased from Sunflower Pharma (Jiamusi, China). The doses of curcumin and mesalazine were based on our previous study [18], in which curcumin was added to 1.5% sodium carboxymethylcellulose. The DSS + Cur group was orally administrated with curcumin (100 mg/kg/day) for 14 consecutive days, and the DSS+5-ASA group was orally administrated with mesalazine (300 mg/kg/day), and the Control and DSS groups were given an equal volume of saline, starting from day 8. Mice were weighed at the same time each day to determine changes in body weight and monitored daily for diarrhoea, blood in the stool, humping, and hair loss [25].

2.3. Histological Evaluation

All mice were weighed before anesthesia, and the entire colons were rapidly collected. The total colon lengths and weights were measured [26], and the colon weight index (CWI), CWI = colon weight/body weight × 100%, was calculated [27, 28]. Distal colon tissue was taken for pathological tissue testing. The tissues were washed with phosphate-buffered saline (PBS, pH = 7.2) and fixed in 4% paraformaldehyde for 24 h at room temperature. After paraffin embedding, 4 μm thick sections were cut to dehydrate by an ethanol gradient and stained with hematoxylin-eosin (H&E) (Solarbio, Beijing, China) for pathological histological analysis. Next, colon injury and inflammation were observed under a microscope (Leica, Wetzlar, Germany). Histological damage was assessed as a combined score of inflammatory cell infiltration (scores 0–3), mucosal damage (scores 0–3), crypt damage (scores 0–4), and regeneration (scores 0–4), using a previously described method [29].

2.4. Flow Cytometry

Briefly, spleen from mice was homogenized. The cell samples were resuspended in RPMI (Roswell Park Memorial Institute) 1640 and lysed with lysing buffer (BD Biosciences, Franklin Lakes, NJ, USA) to clear red blood cells. Then, these cells were incubated with an Fcγ receptor-blocking mAb (CD16/32; BioLegend, San Diego, CA, USA) for 15 minutes at 4°C. Subsequently, for surface antigen detection, the cells were shielded from light and labeled with Percp-Cy5.5 rat anti-mouse CD11b, AF647 rat anti-mouse F4/80, AF488 rat anti-mouse iNOS antibodies, PE rat anti-mouse TIM-1, PE-Cy7 rat anti-mouse TLR4, PE-Cy7 rat anti-mouse CD206, and PE rat anti-mouse CD163. All antibodies were purchased from BD Bioscience (San Jose, CA, USA). The cells were fixed and permeabilized with a Cytofix/Cytoperm Kit (BD Biosciences) prior to the standard surface and intracellular staining procedures. Finally, the single-cell suspensions were incubated for 30 min at 4°C and washed with stain buffer twice before analysis by a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). All data were analyzed with FlowJo 7.6.1 software (TreeStar, San Carlos, CA, USA). Gates were set for the quadrant markers based on negative populations and isotype controls. The numbers in the corners of the FACS dot plots represented the percentage of each cell population within that quadrant as a fraction of the total cell population.

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

Colon tissue (100 mg) was collected and homogenized in 1000 μL RIPA (Radio Immunoprecipitation Assay) lysis buffer (Cell Signaling Technology, Danvers, MA, USA) and incubated for an hour at 4°C, followed by ultrasonic trituration and centrifugation at 10,000 rpm for 10 min to obtain colon tissue homogenate. The BCA method was used to detect the concentration of total protein and was normalised. The concentrations of cytokines were measured using mouse immunoassay kits (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instruction. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of IL-1β, IL-6, IL-10, IL-33, and CCL-2 by commercial ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA). 100 μL of standard and sample in sequence was added to a 96-well plate and mixed thoroughly before reaction with the corresponding antibody. After terminating the reaction with 100 μL of a stop solution, the optical density (OD) values of these cytokines in each sample were detected using a microplate reader (Thermo, Varioskan Flash, MA, USA).

2.6. Western Blot Analysis

Colon tissue protein samples were prepared in accordance with the protein samples in 2.5. Equal weight of protein per sample was separated using SDS-PAGE gels and transferred to PVDF membranes. These membranes were blocked with 3% BSA for 1 h at room temperature and then incubated with the indicated primary antibody overnight at 4°C, including anti-GAPDH (1 : 1000), AP-1 (1 : 1000), MyD88 (1 : 600), p38MAPK (1 : 600), NF-κBp65 (1 : 1000), TLR2 (1 : 500), and TLR4 (1 : 500) antibodies. The HRP-coupled secondary antibody was then added. Protein detection was performed using the ECL substrate (Thermo, Rockford, IL, USA) before exposure, and photos were taken using the Highly Sensitive Chemiluminescence Imaging system (UVP ChemStudio 515; Analytik Jena, Jena, Germany). The images were quantified using Image-Pro Plus 6.0 software (Media Cybernetic, Bethesda, MD, USA).

2.7. Statistics

All data were presented as mean ± standard error of the mean (SEM). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the least-significant difference (LSD) test to compare the differences between every two groups in GraphPad Prism 7.0 software (San Diego, CA, USA). P < 0.05 was considered as significant difference.

3.1. Curcumin Alleviated DSS-Induced Colitis

In the present study, the body weight (Figure 1(a)) of DSS-induced colitis mice decreased significantly from day 4 to day 21 of the experiment, colonic weight (Figure 1(b)), colonic weight/length (Figure 1(c)) and index of colonic weight (Figure 1(d)) also increased significantly, and colonic length decreased significantly (Figures 1(e) and 1(f)). Meanwhile, colonic histological disorders, loss of intestinal crypts, mucosal ulceration, lymphocytic infiltration, and edema (Figure 1(g)), and significant upregulation of pathological injury scores (Figure 1(h)) were observed in colitis mice. These findings indicated that the chronic colitis was successfully modeled in this study.

After treatment with curcumin and mesalazine, effective reversal of the significant changes in body weight, index of colon weight, colon weight, and colon length and histopathology in colitis mice was achieved. And colonic weight (Figure 1(b)), colonic weight/length (Figure 1(c)), and index of colonic weight (Figure 1(d)) were significantly lower in the DSS + Cur and DSS+5-ASA groups than in the DSS group, and colonic length (Figures 1(e) and 1(f)) was significantly higher in the DSS + Cur group than in the DSS group. In addition, the ulcer formation and inflammatory infiltration (Figure 1(g)) were significantly improved, and the pathological damage scores (Figure 1(h)) were significantly downregulated in colitis mice. Therefore, curcumin can effectively alleviate DSS-induced colonic injury.

3.2. Curcumin Regulated the Expression of Inflammatory Cytokines in Colon Tissues

In this study, the levels of the proinflammatory cytokines CCL-2 (Figure 2(a)), IL-1β (Figure 2(b)), and IL-6 (Figure 2(c)) were remarkably increased in the DSS group than in the Control group (Figures 2(a), 2(b), and 2(c)), and the anti-inflammatory cytokines IL-33 (Figure 2(d)) and IL-10 (Figure 2(e)) in the DSS group were dramatically decreased. Importantly, the concentrations of CCL-2 (Figure 2(a)), IL-1β (Figure 2(b)), and IL-6 (Figure 2(c)) were significantly downregulated after DSS-induced colitis mice were treated with curcumin; IL-33 (Figure 2(d)) and IL-10 (Figure 2(e)) were significantly upregulated. Altogether, curcumin effectively alleviated DSS-induced colonic mucosal damage by modulating the release of inflammatory cytokines.

3.3. Curcumin Inhibited the Activation of Macrophages in Colitis Mice

In our study, the percentage of CD11b⁺F4/80⁺ (Figure 3(d)) macrophages was significantly increased in the spleen of colitis mice, whereas the percentage of CD11b⁺F4/80⁺ (Figure 3(d)) macrophages was significantly decreased after curcumin administration treatment. Activation of macrophages results in the expression of TIM-1, the expression level of which is positively correlated with the status of macrophage activation [30]. We found a significant increase in the percentage of CD11b⁺F4/80⁺TIM-1⁺ macrophages (Figure 3(e)) in colitis mice, whereas curcumin administration treatment resulted in a significant decrease in the percentage of CD11b⁺F4/80⁺TIM-1⁺ macrophages (Figure 3(e)). Interestingly, the percentage of CD11b⁺F4/80⁺TLR4⁺ macrophages (Figure 3(f)) in colitis mice increased significantly, whereas curcumin administration treatment resulted in a significant decrease in the percentage of CD11b⁺F4/80⁺TLR4⁺ macrophages (Figure 3(f)). These studies suggested that curcumin inhibited macrophage activation and thus interfered with the process of experimental colitis, possibly in close association with TLR4 signaling pathway.

3.4. Curcumin Suppressed the Activation of TLRs Signaling Pathway in Colitis Mice

Naturally, the effects of curcumin on the regulation of TLRs signaling pathways in colitis mice were further investigated by Western blot analysis, including TLRs signaling molecules TLR2 and TLR4 and the downstream proteins MyD88, NF-κBp65, p38MAPK, and AP-1. In our study, the expression levels of TLR2 and TLR4 and those of the downstream proteins NF-κBp65, p38MAPK, and AP-1 (Figure 4) were significantly higher in DSS group than in the Control group. These results indicated that the TLRs signaling pathway was activated in DSS-induced colitis mice. After curcumin treatment, the protein levels of TLR2, TLR4, MyD88, NF-κBp65, p38MAPK, and AP-1 (Figure 4) in the colonic tissues of colitis mice were significantly reduced. Thus, these data suggested that curcumin inhibited the activation of TLRs signaling pathway in colitis mice.

3.5. Curcumin Regulated the Polarization Balance of M1/M2 Macrophage in Colitis Mice

Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization and dysbiosis contributes to IBD pathogenesis [31]. Secretion of iNOS by proinflammatory M1 macrophages is typical [32], and the surface markers for anti-inflammatory M2 macrophages are CD206 and CD163 [33]. In this study, the percentage of M1 macrophage CD11b⁺F4/80⁺iNOS⁺ (Figure 5(e)) was significantly increased in DSS-induced colitis mice, and those of M2 macrophage CD11b⁺F4/80⁺CD206⁺ (Figure 5(f)) and CD11b⁺F4/80⁺CD163⁺ (Figure 5(g)) cells were significantly downregulated. It suggested that DSS-induced colitis disrupted the differentiation balance of M1/M2 macrophage. After administration of Cur, the percentage of CD11b⁺F4/80⁺iNOS⁺ (Figure 5(e)) macrophages in colitis mice was significantly downregulated, and those of M2 macrophage CD11b⁺F4/80⁺CD206⁺ (Figure 5(f)) and CD11b⁺F4/80⁺CD163⁺ (Figure 5(g)) macrophages were significantly upregulated. These results suggested that curcumin effectively regulated the balance of M1/M2 macrophage polarization in colitis mice.