Contributed equally
Curcumin has a therapeutic effect on ulcerative colitis, but the underlying mechanism has yet to be elucidated. The aim of the present study was to clarify the possible mechanisms. Dextran sulfate sodium-induced colitis mice were treated with curcumin via gavage for 7 days. The effects of curcumin on disease activity index (DAI) and pathological changes of colonic tissue in mice were determined. Interleukin (IL)-6, IL-10, IL-17 and IL-23 expression levels were measured by ELISA. Flow cytometry was used to detect the ratio of mouse spleen regulatory T cells (Treg)/Th17 cells, and western blotting was used to measure the nuclear protein hypoxia inducible factor (HIF)-1α level. The results demonstrated that curcumin can significantly reduce DAI and spleen index scores and improve mucosal inflammation. Curcumin could also regulate the re-equilibration of Treg/Th17. IL-10 level in the colon was significantly increased, while inflammatory cytokines IL-6, IL-17 and IL-23 were significantly reduced following curcumin treatment. No significant difference in HIF-1α was observed between the colitis and the curcumin group. It was concluded that oral administration of curcumin can effectively treat experimental colitis by regulating the re-equilibration of Treg/Th17 and that the regulatory mechanism may be closely related to the IL-23/Th17 pathway. The results of the present study provided molecular insight into the mechanism by which curcumin treats ulcerative colitis.
The incidence of ulcerative colitis (UC) has recently increased (
SPF grade BALB/c male mice (6–7 weeks old, weight 22–26 g, n=36; Laboratory Animal Center of Southern Medical University, certificate number: SCXK Guangdong, China) were reared in a clean animal room. The temperature was 22–25°C and the relative humidity was ~55%. The environment was a 12-h light/dark cycle. Water was given as required and mice were given free access to food. DSS (average molecular weight 5,000; FUJIFILM Wako Pure Chemical Corporation) was formulated as 5% solution using sterile distilled water. Curcumin was purchased from Guangzhou QiYun Biotechnology Co., Ltd., (purity ≥98.5%). The mice were fed adaptively for one week and randomly divided into three groups with 12 in each group: Normal group, Colitis control group and Curcumin 100 mg/kg.d treatment group (CUR group). The Normal group was given sterile distilled water for 14 days. The Colitis group and the CUR group were given 5% DSS for 7 days. Once the model was established, they were given distilled water for 7 days. On the 8th day, mice in the Normal group and the Colitis group were given 0.2 ml of 0.5% ethanol per day for 7 days, while mice in the CUR group were given (via gavage) the dose of curcumin 100 mg/kg.d in 0.2 ml of 0.5% ethanol for 7 days. All experiments on animals were approved by the Experimental Animal Ethics Committee of Southern Medical University.
Mouse daily disease activity index (DAI) was recorded according to the Murthy scoring system (
Fresh colon tissue (without cecum) of each experimental group was collected and immediately placed in 10% formaldehyde. After fixing at room temperature for 48 h, the tissues were dehydrated using an ascending alcohol series at room temperature: 30% ethanol for 40 min, 50% ethanol for 40 min, 70% ethanol for 30 min, 80% ethanol for 30 min, 90% ethanol for 30 min, 95% ethanol for 30 min, 95% ethanol for 30 min, 100% ethanol for 20 min and 100% ethanol for 20 min. Subsequently, the tissue sections were cut into 5-µm thick sections and heated at 60°C for 55 min. Then the tissues sections were embedded in paraffin and hematoxylin and eosin (H&E) staining was performed as follows: Hematoxylin for 10 min at room temperature and eosin for 2 min at room temperature All tissue sections were observed under a BX-51 light microscope (Olympus Corporation; magnification, ×200).
The spleen of the mouse was removed from the laminar flow cabinet, and the appropriate amount of spleen tissue was taken. Subsequently, 5 ml mouse lymphocyte separation solution was added to separate the lymphocytes, and the number of lymphocytes was adjusted to 2×106/tube. Samples were prepared according to the manufacturer's protocol of Th17/Treg kit (BD Biosciences). Lymphocytes were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich; Merck KGaA), 1 µg/ml ionomycin (Sigma-Aldrich; Merck KGaA) and appropriate concentration of Monensin (BD Biosciences) for 5 h. Cells were collected and incubated at room temperature for 30 min with 20 µl flow antibody in each tube after staining, fixation, membrane breaking and other steps. The main flow antibodies included mouse CD4-PerCP-Cy5.5, IL-17A-PE, and Alexa Fluor 647-FoxP3. A negative control group was also included. Samples were analyzed by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences) and FACStation software (version FACS101; BD Biosciences).
A total of 50 mg of mouse colon tissue was collected and an appropriate amount of pre-cooled saline was added. Following homogenization with a glass homogenizer on ice, it was centrifuged at low temperature, and the supernatant was collected for quantification of the protein with Nanodrop 2000 (Thermo Fisher Scientific, Inc.). The concentrations of IL-6 (cat. no. DKW12-2060; Dakewe Biotech Co., Ltd.), IL-10 (DKW12-2100; Dakewe Biotech Co., Ltd.), IL-17 (DKW12-2170; Dakewe Biotech Co., Ltd.), and IL-23 (cat. no. M2300; R&D Systems, Inc.) in the intestine of mice were measured in strict accordance with the ELISA kit instructions.
A total of 100 mg of colon tissue was taken, and the nuclear protein was extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit (Pierce; Thermo Fisher Scientific, Inc.), protease and phosphatase inhibitors were added. Protein quantification was performed by using Nanodrop 2000 quantitative analyzer (Thermo Fisher Scientific, Inc.) and the BCA method. An appropriate amount of loading buffer was added to adjust to the same concentration. Then 10% protein electrophoresis separation gel and 5% aminated gel were prepared, and 30 µg nuclear protein was sampled. After the electrical conversion, the PVDF membrane was removed and blocked in 5% skimmed milk powder/TBST solution (0.05% Tween-20) for 1 h at room temperature. The samples were separated by electrophoresis, followed by membrane transfer, blocking, primary and secondary antibodies incubation, ECL imaging (Thermo Fisher Scientific, Inc.), and analysis. The primary antibodies were anti-mouse HIF-1α (cat. no. 36169S; 1:200; Cell Signaling Technology, Inc.) and Lamin (cat. no. ABP0099; 1:1,500 dilution; Abbkine Scientific Co., Ltd.), and the incubation was at 4°C overnight. The secondary antibodies for horseradish peroxidase labeled goat anti mouse (cat. no. 79233; 1:1,500 dilution, OriGene Technologies, Inc.) incubated for 1 h at room temperature. Quantity One software (v1-D; Bio-Rad Laboratories, Inc.) was used to calculate the relative grayscale value with Lamin as the reference protein. SPSS v13.0 statistical software (SPSS, Inc.) was used for statistical analysis.
The experimental data were all expressed as mean ± standard deviation. All experimental data were analyzed using SPSS v13.0 statistical software (SPSS, Inc.). One-way analysis of variance was used to analyze the differences between groups. S-N-K (Student-Newman-Keuls, q test) was selected to compare the mean of multiple samples. P<0.05 was considered to indicate a statistically significant difference.
No animal died during the experiment. The Normal group score was 0. Colitis group and CUR group demonstrated umbelliferous bloody stool, weight loss, and activity decreased in the first 7 d after administration of 5% DSS. As shown in
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The present study observed increasing DAI and SI and severe mucosa erosion in colitis mice, which is similar to the symptoms of humans UC. After 7 days of curcumin treatment in experimental colitis mice, DAI and SI were significantly decreased, and colonic mucosal inflammation was significantly improved, suggesting that curcumin has a good therapeutic effect on mice with colitis. Previous studies have achieved similar therapeutic effects on colitis mice (
It was demonstrated that the CUR group had more significant improvement in DAI and SI compared with the colitis group. The ratio of Th17 cells increased significantly in splenic lymphocytes of colitis mice, suggesting that there was a serious imbalance in Treg/Th17 ratio in colitis. DSS-induced colitis mice experienced a disruption in Treg/Th17 balance, which led to intestinal immune disorders, one of the key factors for the formation and progression of colitis. It was thus speculated that that the spleen serves as an important immune organ and that DSS can induce immunological disorders in colitis mice and cause compensatory enlargement of the spleen. The present study further confirmed that there was a significant Treg/Th17 imbalance in the splenic lymphocytes of mice with colitis, which is consistent with human UC. Th17-related cytokines IL-6, IL-17 and IL-23 in mice with colitis was also increased, while Treg-related cytokine IL-10 was significantly decreased.
It was speculated that there were multiple mechanisms that could mediate the therapeutic effect of curcumin on colitis. First, the imbalance of Treg/Th17 occurs in a variety of inflammatory and autoimmune diseases. The present study confirmed that curcumin primarily regulates Treg/Th17 rebalance by downregulating CD4+IL-17+Th17 cells (
The present study demonstrated that the anti-inflammatory cytokine IL-10 in the CUR treatment group was elevated, and the proinflammatory cytokines IL-6, IL-17, and IL-23 were decreased. Therefore, maintaining the re-equilibration of cytokines serves an important role in curcumin treatment of IBD. IL-10 plays a negative regulatory role in cellular immunity. It can inhibit the antigen presentation and downregulate the transcription and secretion of IL-1β, IL-6 and TNF-α and other inflammatory factors in T cells and macrophages. Ultimately, it inhibits T cell-mediated immune response and thus improves UC intestinal inflammation (
Curcumin is an all-natural compound extracted from plants. It has many biological activities including anti-inflammatory, anti-infective and immune-regulating, and it protects the intestinal mucosa and repairs the function of intestinal tissue (
Curcumin itself has anti-inflammatory and anti-infective effects. By inhibiting production of leukocyte eicosanoid and related inflammatory cytokines, Treg/Th17 disorder is balanced. Through negative feedback regulation of its own immune and anti-inflammatory system, it further prevented the inflammatory cascade amplification, which serves a crucial role in the recovery of UC. At the same time, curcumin can reduce and inhibit the exudation of neutrophils and macrophages, regulate intestinal immune disorders, reduce intestinal endothelial cell swelling and increase permeability, which further reduces intestinal inflammation (
Therefore, the present study suggested that the mechanism of curcumin in treating IBD may be through downregulating the proportion of Th17 cells to regulate the re-equilibration of Treg/Th17 and Th17-related cytokines. Curcumin can significantly decrease the DAI and SI of the mice with colitis. It can regulate the re-equilibration of Treg/Th17 by inhibiting the IL-23/Th17 pathway, downregulating IL-6, IL-17, IL-23 and upregulating anti-inflammatory cytokine IL-10. Curcumin, as a good all-natural drug for the treatment of IBD, possesses good prospects in clinical application.
The authors would like to thank Dr De-Feng Li, Dr Yao Jun and Dr Wang Lisheng (all Department of Gastroenterology, Jinan University of Second Clinical Medical Sciences, Shenzen Municipal People's Hospital, Shenzhen, Guangdong) for providing project funding support and revising the manuscript.
The study was supported by Three Engineering Training Funds in Shenzhen (grant no. SYLY201718) and Technical Research and Development Project of Shenzhen (grant nos. JCYJ20170307100538697, JCYJ20150403101028164, JCYJ20170307100911479 and JCYJ20160429174927286).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
CW designed the study and wrote the manuscript. JW and DL were laboratory personnel; these authors participated in part of the experimental process, guided part of the experimental process design and data analysis, and modified the paper. FX, BW, ML, ZY and HJ performed the histological assessments and evaluations. TL, QT, YL, DZ and ZX performed the flow cytometry and proteomics experiments. LW and JY were responsible for designing the project, guiding the experimental process, revising the paper and providing scientific research funding support. All authors read and approved the final manuscript.
All experiments on animals were approved by the Experimental Animal Ethics Committee of Southern Medical University, China.
Not applicable.
The authors declare that they have no competing interests.
Effects of CUR on disease activity index and mean SI in mice. (A) Disease activity index. (B) Mean SI in the (B-a) normal group, (B-b) colitis control group and (B-c) CUR-treated group. (C) Analysis of mean SI. aP<0.05 vs. normal group and bP<0.05 vs. colitis control group. CUR, curcumin; SI, spleen index.
Effects of CUR on histological score of distal colon in mice. (A) Histological examination of colon (magnification, ×100) in the (A-a) normal group, (A-b) colitis control group and (A-c) CUR-treated group. (B) Analysis of histological score aP<0.05 vs. normal group and bP<0.05 vs. colitis control group. CUR, curcumin.
Effects of CUR on Treg/Th17 in the spleen. The ratio of Treg/Th17 in spleen in normal group, colitis control group and CUR-treated group was (A) determined by flow cytometry and (B) quantified. aP<0.05 vs. normal group and bP<0.05 vs. colitis control group. CUR, curcumin; CD4, cluster of differentiation 4; Foxp3, forkhead box P3; IL, interleukin; Treg, regulatory T cells.
Effects of CUR on cytokine protein levels. Cytokine protein levels in normal group, colitis control group and CUR-treated group. aP<0.05 vs. normal group and bP<0.05 vs. colitis control group. CUR, curcumin; IL, interleukin.
Effects of curcumin on HIF-1α protein expression. (A) HIF-1α protein expression in the (a) normal group, (b) colitis control group and (c) CUR-treated group. (B) Protein expression was semi-quantified. aP<0.05 vs. normal group and bP>0.05 vs. colitis control group. CUR, curcumin; HIF, hypoxia inducible factor.
Murthy scoring system.
Score | Weight loss rate | Stool viscosity | Invisible/visible bloody stools |
---|---|---|---|
0 | (−) | Normal (Granular, shaped) | Normal |
1 | 1–5% | ||
2 | 6–10% | Soft (paste, no adhesion to the anus) | Occult blood (+) |
3 | 11–15% | ||
4 | >15% | Diarrhea (water sample, adhesion to the anus) | Bloody stools |