Previous studies have demonstrated that acupuncture is beneficial to patients with Crohn’s disease (CD), but the mechanism underlying its therapeutic effects remains unclear. To identify the mechanism by which acupuncture treats CD, the balance between Th17 and Treg cells was assessed in CD patients. In this study, Ninety-two CD patients were randomly and equally assigned to a treatment group that were treated with herb-partitioned moxibustion and acupuncture or a control group with wheat bran-partitioned moxibustion and superficial acupuncture. The effect of these treatments on Th17 and Treg cells and their related molecular markers in the intestinal mucosa were detected before (week 0) and after (week 12) treatment. The results suggested that the ratio of Th17 and Treg cells was significantly decreased after treatment and that the levels of IL-17 and ROR
Crohn’s disease (CD) is a nonspecific granulomatous inflammatory bowel disease (IBD) with an unknown etiology. The main clinical manifestations are recurrent episodes of abdominal pain, diarrhea, and weight loss and are often complicated with abdominal mass, anal fistula, and intestinal obstruction. The disease is characterized with a long duration and frequent recurrence and cure difficulty. With the disease incidence increasing over years, it has become one of the most common diseases in the intestinal tract [
Acupuncture and moxibustion are an important component of Traditional Chinese Medicine (TCM) with a history of over 4,000 years and have gradually been accepted by many countries worldwide. They are widely used in the clinical treatment of various diseases, especially gastrointestinal diseases, such as Crohn’s disease [
Th17 and Treg cells are two recently discovered subsets of T lymphocytes. Research has shown that an imbalance between Th17 and Treg cells is involved in the development of CD [
From January 2010 to April 2013, patients with Crohn’s disease (CD) were recruited for this study at the following hospitals: the outpatient clinic for inflammatory bowel disease of Shanghai Institute of Acupuncture and Meridian at Shanghai University of TCM, the Endoscopy Center of Zhongshan Hospital at Fudan University, the Shuguang Hospital at Shanghai University of TCM, and the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine at Shanghai University of TCM. The diagnosis of CD was confirmed in all patients by imaging, endoscopic, and histopathological examinations [
Inclusion criteria were as follows: patients with mild to moderate CD (CDAI between 151 and 350), without taking any medication or taking only salicylates and/or prednisone (dose ≤ 15 mg treatment lasting at least one month) and without the use of immunosuppressants or anti-TNF-
Ninety-two patients with active CD were randomly assigned at a 1 : 1 ratio to a treatment group and a control group. Patients in the treatment group were treated with herb-partitioned moxibustion combined with acupuncture: moxibustion was performed on the Tianshu (ST25, bilateral), Qihai (CV6), and Zhongwan (CV12) acupoints; and acupuncture was performed at the Zusanli (ST36), Shangjuxu (ST37), Sanyinjiao (SP6), Taixi (KI3), Gongsun (SP4), and Taichong (LR3) acupoints. For herb-partitioned moxibustion, the herbal cake contained
Ten patients were randomly selected from each group for the present study. At the time of recruitment (week 0) and at the end of treatment (week 12), these patients received colonoscopic examination and intestinal biopsies were taken from the ileocecal region. Four biopsies were taken each time and stored in formaldehyde or liquid nitrogen for future detection.
This clinical trial was approved by the Ethics Committee at the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine at Shanghai University of TCM. All subjects signed the informed consent forms. This trial was registered at the following website: http://clinicaltrials.gov/ (
The tissue biopsies were processed in an automated tissue processing machine and then embedded into paraffin. Then 4
An immunofluorescence double labeling technique was used to determine the ratio of Th17 and Treg cells in the intestinal mucosa. Deparaffinized sections were washed three times in 0.01 M PBS (pH 7.2–7.6) for 5 min each and then heated to 92–98°C for antigen retrieval. Sections were blocked with 10% goat serum for 30 min and then incubated with rabbit anti-human IL-17 and rabbit anti-human FOXP3 (both at 1 : 100, Abcam, U.K.) in a humidified chamber overnight at 4°C. Sections were then incubated with diluted secondary antibodies and mounted with an antifluorescence quenching agent or glycerol: 0.01 M PBS (1 : 1). The sections were photographed under a fluorescent microscope (BX53 Olympus). IL-17 served as the marker for Th17 cells and was labeled with Cy3, and FOXP3 served as the marker for Treg cells and was labeled with FITC. Nuclei were stained by DAPI. Three microscopic fields were randomly chosen from each slide and analyzed with the Image Pro Plus (IPP) analysis system to obtain fluorescence intensity for the positive staining.
Total RNA was extracted from tissues using Trizol (Invitrogen, U.S.) and the RNA content and quality were determined with Nanodrop. Then 4 mg of total RNA was reverse-transcribed into cDNA with a reverse transcription kit (Thermo, U.S.), and 100 ng cDNA was used as template for the real-time PCR reaction. Primers were added at 200 nM into the reactions, and SYBR Green was used for detection. Real-time PCR was run in an ABI7300 instrument (running ABI Prism 7300 SDS Software). The relative expression levels of IL-17, ROR
Primer sequences used in RT-PCR.
Gene name | Primer | Sequence |
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IL-17 | Forward | 5′-TGAAGGCAGGAATCACAATC-3′ |
Reverse | 5′-CGGTTATGGATGTTCAGGTT-3′ | |
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ROR |
Forward | 5′-ATGGAGCTCTGCCAGAATGA-3′ |
Reverse | 5′-TGCGGTTGTCAGCATTGTAG-3′ | |
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FOXP3 | Forward | 5′-CAGCGTGGTTTTTCTTCTCGGTATA-3′ |
Reverse | 5′-TGGTGAAGTGGACTGACAGAAAAG-3′ |
The Envision method was used for immunohistochemistry. Sections were deparaffinized, rehydrated, antigen-retrieved with sodium citrate buffer, and blocked with 20% normal goat serum in a humidified chamber at 37°C for 30 min and then incubated with primary antibodies (mouse anti FOXP3, 1 : 100, Abcam; rabbit anti-IL-17 and rabbit anti-ROR
Statistical analysis was performed using the SPSS13.0 software package. Quantitative data with normal distribution and homogeneous variances are represented as mean ± standard deviation (
After the treatments, the CDAI scores of patients in the treatment group (
Before treatment, the intestinal biopsies from both groups showed pathological characteristics typical of Crohn’s disease. A large number of giant, multinucleated cells were present in granulomas, the centers of which contained necrotic cells and infiltrated inflammatory cells. The mucosa epithelium was damaged or missing, and the lamina propria was infiltrated by many lymphocytes. Also visible were structures resembling lymphoid follicles. The intestinal glands were damaged and disorganized, containing necrotic epithelial cells, infiltrated inflammatory cells, and multinucleated giant cells. After the moxibustion and acupuncture treatment, the intestinal mucosal epithelium was intact, the intestinal glands were reorganized, and less inflammatory cells infiltrated. After control treatment, the mucosal epithelium also became intact, but inflammatory cell infiltration was still visible and the glands were less organized than the treatment group (Figure
HE staining images of the intestinal mucosa from both groups of patients (a) before and (b) after treatment from patients in the treatment group and (c) before and (d) after treatment from patients in the control group (200x).
Before the treatments, a large number of IL-17 + T cells in the intestinal mucosa infiltrated in both groups, and the IL-17 + T cells were mainly located in the lamina propria. After treatment, there were significantly more FOXP3 + Treg cells in both cases but significantly fewer IL-17 + T cells. Treg cells were localized mainly in the cortex and lamina propria. In both groups of patients, the ratio of Th17/Treg was significantly lower after treatment (
Th17 and Treg cells in the intestinal mucosa. Th17 (red) and Treg (green) cells were detected by immunofluorescence (a) before and (b) after treatment from patients in the treatment group and (c) before and (d) after treatment from patients in the control group (200x).
The ratio of Th17/Treg cells in the intestinal mucosa of both groups of patients before and after treatment. Comparison within groups,
IL-17 proteins were mainly found in the lamina propria. IL-17 levels were significantly lower after treatment in both groups (
Expression of IL-17 in the intestinal mucosa (a) before and (b) after treatment from patients in the treatment group and (c) before and (d) after treatment from patients in the control group (200x).
Quantitation of IL-17 mRNA and protein levels in the intestinal mucosa. (a) Levels of IL-17 protein in the intestinal mucosa. (b) Levels of IL-17 mRNA in the intestinal mucosa. Comparison within groups,
IL-17 mRNA levels were significantly lower after treatment in both groups (
Positive staining for ROR
Expression of ROR
Quantitation of ROR
In the treatment group, ROR
FOXP3 protein was mainly detected in the epithelium and lamina propria. In the treatment group, FOXP3 protein levels were significantly higher after the treatment (
Expression of FOXP3 in the intestinal mucosa (a) before and (b) after treatment from patients in the treatment group and (c) before and (d) after treatment from patients in the control group (200x).
Quantitation of FOXP3 mRNA and protein levels in the intestinal mucosa. (a) Levels of FOXP3 protein in the intestinal mucosa. (b) Levels of FOXP3 mRNA in the intestinal mucosa. Comparison within groups,
In the treatment group, FOXP3 mRNA levels were significantly higher after the treatment (
It has demonstrated that moxibustion and acupuncture are effective and safe in treating CD, but their therapeutic mechanism has not been fully elucidated. The results of the present study showed that moxibustion and acupuncture inhibited the protein and mRNA expression of IL-17 and ROR
The subjects of this study were from a previous randomized, controlled clinical trial [
An imbalance between Th17 and Treg cells constituted a key step in the disruption of intestinal homeostasis and is one of the major contributors to the development and progression of CD [
Moreover, studies have suggested that a reduction in the number or function of Treg cells might be a major cause of the pathogenesis of Crohn’s disease [
The present study showed that moxibustion and acupuncture can regulate and restore the balance between Th17 and Treg cells in intestinal mucosa of patients with CD. The moxibustion and acupuncture treatment reduced the number of Th17 cells and inhibited the expression of Th17-related molecules IL-17 and ROR
Previous animal studies of our research group have also indicated that moxibustion and acupuncture can reduce intestinal inflammation in CD rats and promote the recovery of intestinal function. It is reported that moxibustion can decrease the levels of TNF-a and TNFR1, which are abnormally induced in CD rat colons, inhibit excessive apoptosis of colonic epithelial cells, and restore the colonic epithelial barrier [
One of the limitations of this study is that the sample size is small. These results should be further validated with a larger sample size. Although the patients were followed up for 12 weeks after the treatment to assess their CDAI scores, no intestinal mucosa biopsies were taken to evaluate the long-term effect of moxibustion and acupuncture on the balance between Th17 and Treg cells. Future studies should combine clinical evaluation with microscopic and molecular studies of the intestinal mucosa to further elucidate the long-term efficacy of moxibustion and acupuncture.
In conclusion, this study shows that moxibustion and acupuncture can reduce the number of Th17 cells and downregulate the expression of Th17-related molecules IL-17 and ROR
The authors declare that there is no conflict of interests regarding the publication of this paper.
Chen Zhao, Chunhui Bao, and Jing Li contributed equally to this work.
The authors thank Dr. Weifeng Chen at Zhongshan Hospital affiliated with Fudan University and Dr. Xin Guan at Shuguang Hospital affiliated with Shanghai University of TCM for their assistant in recruiting patients and thank Dr. Congqi Dai at Fudan University for the advice and paper revision. This work was supported by National Basic Research Program of China (973 program, no. 2009CB522900), The Ministry of Education Program for New Century Excellent Talents (no. NCET-13-0907), Shanghai Municipal Health Bureau (no. 20124028), and Shanghai top clinical medical center of Acupuncture, Moxibustion and Tuina.