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DOI: 10.3748/wjg.v21.i16.4986
World J Gastroenterol 2015 April 28; 21(16): 4986-4996
ISSN 1007-9327 (print) ISSN 2219-2840 (online)
© 2015 Baishideng Publishing Group Inc. All rights reserved.
4986 April 28, 2015
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Moxibustion combined with acupuncture increases tight
junction protein expression in Crohn’s disease patients
Hai-Xia Shang, An-Qi Wang, Chun-Hui Bao, Huan-Gan Wu, Wei-Feng Chen, Lu-Yi Wu, Rong Ji, Ji-Meng Zhao,
Yin Shi
Hai-Xia Shang, An-Qi Wang, Lu-Yi Wu, Rong Ji, Ji-Meng
Zhao, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China
Chun-Hui Bao, Huan-Gan Wu, Yin Shi, Shanghai Institute
of Acupuncture-Moxibustion and Meridian, Shanghai 200030,
China
Wei-Feng C hen, Zhongshan Hospital affiliated to Fudan
University, Shanghai 200032, China
Author contributions: Shang HX, Wang AQ and Bao CH
contributed equally to this work; Shi Y designed the study; Wang
AQ, Bao CH and Wu LY contributed to the patient enrollment;
Bao CH and Wu LY performed the moxibustion and acupuncture
procedure; Chen WX performed enteroscopy and tissue sampling;
Ji R and Shang HX performed the assays; Zhao JM analyzed the
data; Wang AQ and Shang HX drafted the gures and wrote the
manuscript; Wu HG and Shi Y conducted the study and revised
the manuscript.
Supported by National Natural Science Foundation of China,
No. 30772831, No. 81473757; and the National Basic Research
Program of China, 973 Program, No. 2009CB522900.
Ethics approval: This study has been approved by the Ethics
Committee of Yueyang Hospital of Integrated Traditional Chinese
and Western Medicine, Shanghai University of Traditional
Chinese Medicine approved the research project, No. 2009-02.
Clinical trial registration: This study is registered at the Chinese
Clinical Trial Register Center. The registration identification
number is ChiCTR-TRC-10000950.
Infor med consent : A ll s tu dy participants , or their lega l
guardian, provided informed written consent prior to study
enrollment.
Conict-of-interest: All authors stated that there is no conict of
interest related to the manuscript.
Data sharing: Technical appendix, statistical code, and dataset
available from the corresponding author at ysy0636@163.com.
Participants gave informed consent for data sharing.
Open-Access: This article is an open-access article which was
selected by an in-house editor and fully peer-reviewed by external
reviewers. It is distributed in accordance with the Creative
Commons Attribution Non Commercial (CC BY-NC 4.0) license,
which permits others to distribute, remix, adapt, build upon this
work non-commercially, and license their derivative works on
different terms, provided the original work is properly cited and
the use is non-commercial. See: http://creativecommons.org/
licenses/by-nc/4.0/
Correspondence to: Yin Shi, MD, PhD, Shanghai Institute of
Acupuncture-Moxibustion and Meridian, No. 650 South Wanping
Road, Xuhui District, Shanghai 200030,
China. ysy0636@163.com
Telephone: +86-21-64383910
Fax: +86-21-64644238
Received: November 13, 2014
Peer-review started: November 15, 2014
First decision: December 2, 2014
Revised: December 9, 2014
Accepted: February 5, 2015
Article in press: February 5, 2015
Published online: April 28, 2015
Abstract
AIM: To investigate the effect of herb-partitioned
mo xibu s tion c ombi n ed wi t h acup unctu re on t he
expression of intestinal epithelial tight junction (TJ)
proteins.
MET HODS: Sixty patients diagnosed with mild to
moderate Crohn’s disease (CD) were allocated into
the he rb-p art itio ned moxi bustion combine d wit h
acupuncture (HMA) group (
n
= 30) or the mesalazine
(MESA) group (
n
= 30) using a parallel control method.
There were 2 sets of acupoints used alternately for
HMA treatment. The following points were included in
Set A: ST25 (
Tianshu
), RN6 (
Qihai
), and RN9 (
Shuifen
)
for herb-partitioned moxibustion and ST36 (
Zusanli
),
ST37 (
Shangjuxu
), LI11 (
Quchi
), and LI4 (
Hegu
)
for acupuncture. The points for Set B included BL23
(
Shenshu
) and BL25 (
Dachangshu
) for herb-partitioned
moxibustion and EX-B2 of T6-T1 (
Jiajixue
) for
acupuncture. The patients received the same treatment
6 times a week for 12 consecutive weeks. The MESA
group received 1 g of mesalazine enteric coated tablets
4 times daily for 12 consecutive weeks. Intestinal
Clinical Trials Study
ORIGINAL ARTICLE
tissues were stained and examined to compare the
morphological and ultrastructural changes before and
after the treatment session. Immunohistochemistry
and
in situ
hybridization assays were used to detect
the expression of intestinal epithelial TJ proteins zonula
occludens-1 (ZO-1), occludin, and claudin-1. The mRNA
levels were also evaluated.
RESULTS: After the treatment, both herb-partitioned
moxibustion combined with acupuncture and mesalazine
improved intestinal morphology and ultrastructure of
CD patients; the patients treated with HMA showed
better improvement. HMA significantly increased the
expression of ZO-1 (
P
= 0.000), occludin (
P
= 0.021),
and claudin-1 (
P
= 0.016). MESA signicantly increased
the expression of ZO-1 (
P
= 0.016) and occludin (
P
= 0.026). However, there was no significant increase
in the expression of claudin-1 (
P
= 0.935). There was
no statistically significant difference between the two
groups for the expression of occludin and claudin-1
(
P
> 0.05). The HMA group showed a significant
improvement in ZO-1 expression compared to the
MESA group (2333.34 ± 352.51
vs
2160.38 ± 307.08,
P
= 0.047). HMA signicantly increased the expression
of
ZO-1
mRNA (
P
= 0.000), occludin mRNA (
P
= 0.017),
and claudin-1 mRNA (
P
= 0.017). MESA significantly
increased the expression of ZO-1 mRNA (
P
= 0.000),
occludin mRNA (
P
= 0.042), and claudin-1 mRNA (
P
=
0.041). There was no statistically signicant difference
between the two groups in the expression of occludin
and claudin-1 mRNA (
P
> 0.05). However, the HMA
group showed a signicant improvement in
ZO-1
mRNA
expression compared with the MESA group (2378.17 ±
308.77
vs
2200.56 ± 281.88,
P
= 0.023).
CONCLUSION: HMA can repair intestinal epithelial
barrier lesions and relieve inammation by upregulating
the expression of TJ proteins and their mRNAs.
Key words: Crohn’s disease; Herb-partitioned moxibustion;
Acupuncture; Intestinal epithelial cells; Tight junction
proteins
© The Author(s) 2015. Published by Baishideng Publishing
Group Inc. All rights reserved.
Core tip: Crohn’s disease (CD) is a chronic relapsing
inflammatory condition involving all layers of the
gastrointestinal tract. Although its etiopathogenesis
remains unclear, increased permeability of the intestinal
epithelial barrier is one of the crucial factors in CD
onset. Tight junctions (TJs) within intestinal epithelial
cells form the structural basis of the intestinal epithelial
barrier, and reduced e xpres sion o f TJ pro teins is
positi vely corre lated wi th CD severity. This study
investigated the therapeutic effect of herb-partitioned
moxibustion combined with acupuncture on CD. We
found that this treatment upregulated the expression of
intestinal epithelial TJ proteins and their mRNAs.
Shang HX, Wang AQ, Bao CH, Wu HG, Chen WF, Wu LY, Ji
R, Zhao JM, Shi Y. Moxibustion combined with acupuncture
increases tight junction protein expression in Crohn’s disease
patients. World J Gastroenterol 2015; 21(16): 4986-4996
Available from: URL: http://www.wjgnet.com/1007-9327/full/
v21/i16/4986.htm DOI: http://dx.doi.org/10.3748/wjg.v21.
i16.4986
INTRODUCTION
Crohn’s disease (CD) is a chronic relapsing inammatory
condition involving all layers of the gastrointestinal (GI)
tract and can affect any part of the GI tract from the
mouth to anus[1]. Clinical epidemiological studies have
shown the prevalence of CD in mainland China is 1.4
cases per 100000 person-years, and the incidence has
increased steadily and rapidly in recent years[2].
The clinical management of CD involves conventional
medical treatments including non-steroidal anti-infla-
mmatory drugs, such as 5-aminosalicylate (5-ASA),
corticosteroids and immunosuppressive drugs, such
as thiopurines[3]. However, the European Crohn’s and
Colitis Organization has recognized the efficiency
of 5-ASA as “limited”[4] in improving patients’ CD
Activity Index (CDAI). The inappropriate use of large
amounts of corticosteroids and insufficient amounts
of thiopurines is common among Asian physicians[5]
and results in drug resistance and adverse effects.
One new treatment approach in Western countries is
biologic therapy, such as anti-tumor necrosis factor
(anti-TNF) agents or iniximab[6]. However, in Asia, the
use of biologics is limited by economic burdens due to
the strict coverage range of medical insurance.
Complementary and alternative medicines (CAMs),
such as Chinese medicine, flourish in Asia and
especially in East Asia. In China, more than half of
CD patients receive concomitant traditional Chinese
therapies[7] including Chinese materia medica
decoction, acupuncture and moxibustion. The efcacy
and safety of these therapies have been frequently
questioned because of the indiscriminate use of
CAMs in patients with CD. As a result, many attempts
have been made to obtain concrete evidence of their
therapeutic effect and to investigate their possible
mechanisms. Recent studies[8] have shown that apart
from the undeniable placebo effect, acupuncture and
moxibustion offer additional therapeutic benefits in
patients with mild to moderately active CD. Compared
with the conventional 5-ASA treatment, moxibustion
and acupuncture have a significant advantage in
improving quality-of-life ratings and CDAI scores
of patients with mild to moderate CD[9]. In further
clinical studies, we found that herb-partitioned
moxibustion combined with acupuncture can inhibit
intestinal epithelial cell apoptosis by decreasing
the overexpression of intestinal mucosa tumor
Shang HX
et al
. Moxibustion increases tight junction protein expression
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necrosis factor alpha (TNF-α), tumor necrosis factor
1 (TNFR1), and tumor necrosis factor 2 (TNFR2)[10].
The treatments also reduce intestinal inflammation
by increasing hemoglobin (HGB) counts and by
decreasing C-reactive protein levels and erythrocyte
sedimentation rates in CD patients[11].
Previous studies have suggested that increased
intestinal permeability may appear precede clinical
manifestations[12] and could be used to predict clinical
relapses[13] of CD. Animal experiments have been
performed to understand the pathological basis
underlying the occurrence of CD and to investigate
the cause of increased intestinal permeability from
two aspects. One aspect is excessive epithelial cell
apoptosis[14,15]. The other aspect is intestinal mucosal
epithelial barrier dysfunction[16]. The main function
of the intestinal epithelial barrier is to maintain
intestinal permeability, and the barrier depends on the
dynamically changing formation of tight junctions (TJs)
reacting to varied extracellular stimuli[17]. Through
protein-protein interactions, the cytoplasmic protein
zonula occludens-1 (ZO-1) and the transmembrane
proteins claudin-1 and occludin are able to modulate
and associate with different forms of multimolecular
complexes to regulate the formation of TJs[18]. In
previous study, we observed decreased intestinal
permeability and reduced expression of TJ proteins
ZO-1, claudin-1 and occludin in a TNBS-induced
CD rat model. However, treatment with herb-
partitioned moxibustion and acupuncture increased
the expression of TJ proteins ZO-1, claudin-1 and
occludin. Additionally, the inammatory reaction in the
intestinal mucosa was improved based on histological
observation[19].
Therefore, we hypothesized that herb-partitioned
moxibustion combined with acupuncture could
increase intestinal permeability by upregulating
the expression of TJ proteins ZO-1, claudin-1 and
occludin in CD patients. In this study, we examine the
mechanism of herb-partitioned moxibustion combined
with acupuncture in CD patients and try to verify the
feasibility of acupuncture and moxibustion methods for
treating mild to moderate CD.
MATERIALS AND METHODS
Patients and group allocation
The study was performed from July 2009 through
March 2010 in the CD clinic of the Shanghai Research
Institute of Acupuncture within the Moxibustion and
Meridian Endoscopy Center of Zhongshan Hospital
affiliated to Fudan University. Ethics approval was
obtained from the Chinese Clinical Trial Register
Center (registration number: ChiCTR-TRC-10000950).
All patients provided written informed consent prior
to the beginning of the trial. Patients with CD were
recruited in accordance with Jinan diagnostic criteria
(revised by the National Conference on Inflammatory
Bowel Disease in 2007[20]). Patients with mild to
moderate disease and CDAI scores between 150 and
450 were included. The patients were not treated
with other relevant pharmacological therapies and
signed an informed consent. The exclusion criteria
included the following: pregnant or lactating patients,
psychotic patients, and patients with severe heart,
brain, liver, kidney, or hematopoietic system diseases
and other severe diseases. This study was conducted
as a controlled trial with 2 parallel treatment groups.
Patients from the Shanghai Research Institute of
Acupuncture-Moxibustion and Meridian were enrolled
in the herb-partitioned moxibustion combined with
acupuncture group (HMA group, n = 30). The patients
from the endoscopy center of Zhongshan Hospital
affiliated with Fudan University were included in the
mesalazine group (MESA group, n = 30).
Treatments
The patients in the HMA group received herb-parti-
tioned moxibustion combined with acupuncture. There
were 2 sets of acupoints used for treatment. The
points in Set A included the following: ST25 (Tianshu),
RN6 (Qihai), and RN9 (Shuifen) for herb-partitioned
moxibustion and ST36 (Zusanli), ST37 (Shangjuxu),
LI11 (Quchi), and LI4 (Hegu) for acupuncture. The
points in Set B were BL23 (Shenshu) and BL25
(Dachangshu) for herb-partitioned moxibustion
and EX-B2 of T6 - T1 (Jiajixue) for acupuncture.
These acupoints were located based on the national
GB-12346-90 acupoint standard. The procedure used
involved placing moxa cones (1.7 cm in height and 1.8
g in weight; Hanyi, Henan, China) on a herbal cake (2.3
cm in diameter and 0.5 cm in length). The herbal cake
consisted of 3 g of Shaoxing wine and 2.5 g of herbal
powder [medicinal formula: Aconite preparata (radix),
Cinnamomi (cortex), etc.]. Four sets of moxa cones
and herbal cakes were used for each treatment. The
skin was cleaned with a tincture of iodine and alcohol,
and then sterile single-use acupuncture needles (Φ 0.30
mm × 40 mm specication, Huatuo, Suzhou, China)
were inserted between 20 mm and 40 mm into the
acupoints. Acupuncture was performed by the same
qualied and skilled physician. When two moxa cones
burned out, the moxa cones, herbal cake and needles
were removed. The treatment was applied once per
day, 6 times per week for 12 consecutive weeks. The
MESA group received mesalazine enteric coated tablets
four times a day for 12 consecutive weeks.
Tissue sampling
On a voluntary basis and after signing an informed
consent form, intestinal mucosa tissue samples from
10 patients of each group were removed via painless
enteroscopy before and after the treatment session.
Morphological observation
Pieces of intestinal mucosa tissues (0.5 cm3 for each)
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Shang HX
et al
. Moxibustion increases tight junction protein expression
In situ hybridization
The expression levels of ZO-1, occludin and claudin-1
mRNAs were detected by in situ hybridization.
Digoxigenin-labelled RNA probes were generated
with a DIG RNA labeling kit (Boehringer Mannheim,
Mannheim, Germany) and a relevant plasmid (provided
by JRDUN Biotechnology Co., Ltd., Shanghai, China)
according to the manufacturer’s protocol. The tissues
were dewaxed and hydrated. The sections were
heated at 98 ℃ for 20 min in EDTA solution (0.01 mol/L,
pH 8.0) and pre-treated with the following solutions:
2 µg/mL proteinase K in TE at 37 ℃ for 8 min; 0.1
mol/L glycine in PBS for 10 min; a graded ethanol
series (each concentration for 1 min); and 0.2 × SSC
and 50% formamide to pre-hybridize at 37 ℃ for 30
min. Pre-treated sections were covered with 20-30
mL of hybridization buffer (1 µg/mL RNA probe) and
incubated at 48 ℃ for 8-12 h under a coverslip. The
slides were washed twice at 45 ℃ for 5 min in 2 × SSC
and twice at 37 ℃ for 5 min in 1 ×, 0.5 ×, 0.1 × SSC.
The samples were then blocked in 10% serum. The
sections were then incubated at 37 ℃ for 30 min in
mouse McAb anti-Dig IgG (cat#84-0146, lot#305359,
Invitrogen). The slides were washed three times in
PBS and further incubated at 37 ℃ for 40 min with
goat anti-Mouse IgG-HRP antibody (cat#84-01450,
lot#651053A, Invitrogen). The sections were washed
three times in PBS for 3 min each. The staining detection
was performed using 0.04% DAB and 0.03% H2O2
according to the manufacturer’s recommendations.
The tissues were dehydrated and mounted after
color development. We performed semi-quantitative
analyses of the results, and the positive expression
area index (positive area/total area × OD) values of
ZO-1, occludin and claudin-1 mRNA were calculated in
3 high-power optical elds.
Statistical analysis
All measurement data are presented as the mean
± SD. All statistical analyses were performed using
SPSS 16.0 (SPSS Inc. Illinois, United States). The
comparison of sex and severity from the baseline
data were analyzed using the
χ
2 test. The changes
in age, duration of disease from the baseline data
and between group differences after treatment were
compared using two independent sample t-tests.
A paired-samples t-test was used for within group
comparisons. All two-sided P values < 0.05 were
considered statistically signicant.
RESULTS
Baseline subject characteristics
Table 1 presents the baseline characteristics of the two
groups by age, sex, duration of disease, and severity
of disease. There were no differences between the
HMA group and the MESA group before the procedure.
were collected from CD patients. The intestinal mucosa
tissues were fixed in 10% neutral-buffered formalin,
embedded in paraffin and then sectioned into 4-µm-
thick tissue sections. The tissue sections were then
stained with hematoxylin and eosin, dehydrated in
95%, 90% and 70% ethanol and cleared in xylene.
The stained sections were mounted in Permount
or Histoclad and observed using an Olympus DP73
microscope (Olympus, Tokyo, Japan).
Ultrastructural observation
The intestinal mucosa tissues were cut into 1-mm3
strips, xed for 4 h at 4 ℃ in 5% glutaraldehyde and
then washed 3 times in 0.1 mol/L phosphate buffered
saline (PBS). The tissues were then postxed for 2 h
at 4 ℃ in 2% osmium tetroxide and dehydrated in a
graded ethanol series. The tissues were embedded in
Epon 812 and then cut into ultrathin sections (75 nm)
and stained with uranyl acetate and lead citrate. The
sections were viewed in a HITACHI H-600 electron
microscope at 80 kV (HITACHI, Tokyo, Japan).
Immunohistochemical assay
The expression levels of occludin, claudin-1, and ZO-1
were detected by immunohistochemical assays. The
sections were dewaxed, hydrated, and then pretreated
in a microwave (antigen retrieval). The endogenous
peroxidase activity was inhibited with 0.3% H2O2. The
nonspecic binding was blocked with 20% normal goat
serum. All sections were incubated with ZO-1 (Rabbit
anti-ZO-1 polyclonal antibody 1:50, Invitrogen, New
York, United States), occludin (Rabbit anti-Occludin
polyclonal antibody 1:100, Invitrogen, New York,
United States) and claudin-1 antibodies (Mouse anti-
Claudin-1 monoclonal antibody 1:100, Invitrogen,
New York, United States) for 2 h at 37 ℃. The samples
were washed and then incubated for 30 min at room
temperature with appropriate preabsorbed biotinylated
secondary antibody. The antigen was visualized using the
streptavidin-peroxidase method (JRDUN Biotechnology
Co., Ltd., Shanghai, China), and 3,3-diaminobenzidine
(DAB) (Liquid DAB-Plus Substrate Kit, JRDUN
Biotechnology Co., Ltd., Shanghai, China) was used as
a chromogen. The slides were washed in distilled water
and counterstained with Mayer’s hematoxylin before
they were dehydrated and mounted. The primary
antibody was replaced with PBS for a negative control.
A semi-quantitative analysis of the staining results was
conducted using the IMS medical image quantitative
analysis system (JRDUN Biotechnology Co., Ltd.,
Shanghai, China). Positive results for ZO-1, occludin and
claudin-1 were brown or yellow particles stained among
intestinal epithelium. The positive area and the optical
density (OD) values in 3 high-power optical fields (×
200) of every slice were measured. The immune positive
area index (positive area/total area × OD) values of
ZO-1, occludin and claudin-1 were calculated in every
high-power optical eld.
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Shang HX
et al
. Moxibustion increases tight junction protein expression
Intestinal morphological observations
Figure 1 shows the morphological observations of
intestinal mucosa tissues from both groups before
and after treatment. Figure 1A shows that in the HMA
group before treatment, the mucosal epithelium was
seriously damaged. The intestinal glands were rare,
and there were ulcerations and obvious submucosal
hyperemia and edema. Tissue damage was observed
in the mucosa, submucosa and muscular layer,
and there was substantial eosinophil and inflamma-
tory cell infiltration in the intestinal mucosa and
submucosa. Figure 1B shows that in the HMA group
after treatment, there was only a small amount of
hyperemia and inflammatory cell infiltration in the
intestinal mucosa and submucosa. The intestinal
glands were arranged in an orderly manner. Figure
1C shows that in the MESA group before treatment,
the mucosal epithelium was also seriously damaged.
There was obvious hyperemia and edema, and
eosinophils and inammatory cells had inltrated into
the intestinal mucosa and submucosa. There was
also ulceration and tissue damage in the mucosa,
submucosa and muscular layer. Figure 1D shows that
in the MESA group after treatment, there was less
severe hyperemia and edema in intestinal mucosa and
submucosa. Additionally, some of the intestinal glands
were restored but some were poorly organized.
Intestinal ultrastructural observations
Figure 2 presents ultrastructural images of the
intestinal mucosa tissues from both groups before
and after treatment. Figure 2A shows that in the HMA
group before treatment, the connections between
the epithelial cells were loose and that there was
a significant broadening of intercellular spaces.
Furthermore, the cell membranes were partly injured,
and intestinal epithelial cells contained a small number
of bubbles inside of the cytoplasm. Figure 2B shows
that after HMA treatment the connections between
the epithelial cells were relatively tight and that the
intercellular spaces between cells were not broadened.
A small amount of particle secretion was observed,
and a few villi could be observed on the cell surfaces.
Figure 2C shows that in the MESA group before
treatment, the connections between the epithelial
cells were quite loose and that there was significant
broadening of intercellular spaces. Particle secretion
was detected. Figure 2D shows that after MESA
treatment the connections between the epithelial
cells were tight. However, there was broadening of
some intercellular spaces, and particle secretion was
detected.
Expression of TJ proteins occludin, claudin-1 and ZO-1
After treatment, the HMA group showed signicantly
increased expression of occludin (P = 0.021), claudin-1
(P = 0.016), and ZO-1 (P = 0.000) (Figure 3A, B and
E). The MESA group showed a signicant increase in
the expression of occludin (P = 0.026) and ZO-1 (P =
0.016). However, there was no signicant increase in
the expression of claudin-1 (P = 0.935) (Figure 3C-E).
There was no statistical difference for the expression
of occludin (P = 0.512) and claudin-1 (P = 0.055)
between groups. The HMA group showed a signicant
improvement in ZO-1 expression compared to the
MESA group (2333.34 ± 352.51 vs 2160.38 ± 307.08,
P = 0.047) (Figure 3B, D and E).
Expression of TJ proteins occludin, claudin-1 and ZO-1
mRNAs
The HMA group showed significant increases in the
expression of ZO-1 mRNA (P = 0.000), occludin mRNA
(P = 0.017), and claudin-1 mRNA (P = 0.017) (Figure
4A, B and E). The MESA group showed significant
increases in the expression of ZO-1 mRNA (P = 0.000),
occludin mRNA (P = 0.042), and claudin-1 mRNA
(P = 0.041) (Figure 4C-E). There was no difference
between groups in the expression of occludin mRNA
and claudin-1 mRNA (P = 0.748, P = 0.388). The HMA
group showed a significant increase in ZO-1 mRNA
expression compared to the MESA group (2378.17 ±
308.77 vs 2200.56 ± 281.88, P = 0.023) (Figure 4B,
D and E).
DISCUSSION
The dominant symptom of CD is “leak-ux diarrhea”
due to epithelial barrier dysfunction, which results in
increased epithelial permeability and a continuous
loss of solutes[21]. Previous studies have shown that
increases in intestinal permeability not only act as
an etiological factor in CD[22] but also precede clinical
relapses in CD and are an indicator of subclinical
disease[13,23]. However, increased intestinal permeability
has also presented in first-degree relatives of CD
patients in the absence of clinical symptoms[24,25]. This
result suggests that increased intestinal permeability
might be one of several pathogenic factors in CD.
In recent decades, new findings have revealed the
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Table 1 Baseline characteristics
HMA
n
= 30
MESA
n
= 30
P
value
Age (yr)
Min 22 18
Max 56 65
mean ± SD 31.77 ±
8.77
36.93 ± 13.25 0.080
Sex (M/F) 23/7 19/11 0.260
Duration of disease (yr)
Min 1 1
Max 12 18
mean ± SD 6.33 ± 3.63 5.67 ± 4.08 0.507
Severity of disease (mild/moderate) 22/8 18/12 0.273
HMA: Herb-partitioned moxibustion combined with acupuncture group;
MESA: Mesalazine group.
Shang HX
et al
. Moxibustion increases tight junction protein expression
major factors underlying CD etiopathogenesis. These
factors include the following: excessive bacterial
translocation caused by intestinal epithelial barrier
dysfunction[26], infection resulting in dysfunction of
immunotolerance and aggressive immune response
to bacteria[27,28], significant loss of complexity in
species of the Firmicutes and the Bacteroidetes
phyla and increased Enterobacteriaceae, particularly
Escherichia coli species[29,30]. The epithelial barrier is
the primary defense against exogenous pathogens.
Thus, maintaining and repairing the epithelial barrier is
crucial for the treatment and prevention of CD.
The epithelial barrier is a single layer of epithelial
cells that lines the entire digestive tract. A TJ seals the
intercellular space between adjacent epithelial cells[31],
thus, serve as the major determinant of epithelial
permeability[32]. The crucial feature of TJs is a fibril-
like protein structure called TJ strands. The strands
are connected with each other to create a continuous
network. The TJ strands act as a diffusion barrier to
regulate the transport of ions, macromolecules and
immune cells in the paracellular pathway[33]. A TJ
is a membrane-associated multimolecular complex
composed of three transmembrane protein families[34].
The protein families consist of the claudin family[35], the
junctional adhesion molecule (JAM) protein family[36]
and the TJ-associated Marvel domain proteins (TAMPs)
family, which includes occludin, tricellulin and Marvel
D3[37]. Claudins are responsible for the charge[38] and
size-selectivity[39,40] of the TJ barrier. The JAM proteins
and TAMPs are mainly responsible for the stabilization
of TJs and the regulation of epithelial permeability[41,42].
ZO-1 is a member of the membrane-associated
guanylate kinases family. ZO-1 is a multi-domain
scaffolding protein with an important role in the
assembly of the TJ barrier and in the maintenance
of the cytoskeleton[32] because it establishes a
connection between the TJ barrier and perijunctional
actomyosin[43].
Previous studies have shown that the expression
of TJ proteins occludin, claudin-1, and ZO-1 were
significantly decreased in the lamina propria in both
active and chronic CD patients. The decrease in ZO-1
leads to increased intestinal epithelial permeability
and TJ barrier dysfunction[44,45]. In this study, we
evaluated occludin, claudin-1, and ZO-1 as indicators
of intestinal epithelial barrier dysfunction during CD
pathologic processes. We also examined the impact of
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DCBA
Figure 1 Morphological observation of patient intestinal mucosa tissue from two groups before and after treatment. A: HMA group before treatment; B: HMA
group after treatment; C: MESA group before treatment; D: MESA group after treatment. Magnication × 200 (A-D). HMA: Herb-partitioned moxibustion combined with
acupuncture group; MESA: Mesalazine group.
Figure 2 Ultrastructural observation of patient intestinal mucosa tissue from two groups before and after treatment. A: HMA group before treatment; B: HMA
group after treatment; C: MESA group before treatment; D: MESA group after treatment. Magnication × 10000 (A-D). HMA: Herb-partitioned moxibustion combined
with acupuncture group; MESA: Mesalazine group.
DCBA
Shang HX
et al
. Moxibustion increases tight junction protein expression
HMA therapy on the intestinal epithelium. We chose
mesalazine treatment for the control group patients
because it is one of the most commonly used 5-ASA
therapies used for treating mild to moderate CD. We
compared HMA with mesalazine treatment to evaluate
the possible application of HMA in CD management.
In this study, we evaluated the morphology and
ultrastructure of intestinal mucosa tissue before and
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3000
2000
1000
0
3000
2000
1000
0
4000
3000
2000
1000
0
Claudin-1 expression
P
= 0.055
P
= 0.016
P
= 0.935
HMA MESA
Occludin expression
P
= 0.512
P
= 0.021
P
= 0.026
HMA MESA
ZO-1 expression
P
= 0.047
P
= 0.000
P
= 0.016
HMA MESA
Before After Before After Before After
D
C
BA
Claudin-1 Occludin ZO-1
Figure 3 Expression of protein zonula occludens-1, occludin and claudin-1 in two groups before and after the treatment session. A: HMA group before
treatment; B: HMA group after treatment; C: MESA group before treatment; D: MESA group after treatment; E: Bar graphs of the expression of protein zonula
occludens-1, occludin and claudin-1 in HMA group and MESA group before and after the treatment session. HMA: Herb-partitioned moxibustion combined with
acupuncture group; MESA: Mesalazine group; ZO-1: Zonula occludens-1.
E
Shang HX
et al
. Moxibustion increases tight junction protein expression
after treatment. Before treatment, the morphological
observations using light microscopy showed epithelia
impairment, intestinal gland loss and inflammatory
cellular infiltration. These findings suggested that
the basic structure of the epithelial mucosa was
damaged and that mucosal inflammation occurred.
Furthermore, analysis of the tissue ultrastructure by
electron microscopy showed intercellular connection
loss and broadened intercellular spaces. These
results suggested that TJs were damaged and failed
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3000
2000
1000
0
2500
2000
1500
1000
500
0
2500
2000
1500
1000
500
0
Claudin-1 mRNA levels (%)
P
= 0.388
P
= 0.017
P
= 0.041
HMA MESA
Occludin mRNA levels (%)
P
= 0.748
P
= 0.017
P
= 0.042
HMA MESA
ZO-1 mRNA levels (%)
P
= 0.023
P
= 0.000
P
= 0.000
HMA MESA
Before After Before After Before After
D
C
BA
Claudin-1 Occludin ZO-1
Figure 4 Expression of zonula occludens-1 mRNA, occludin mRNA and claudin-1 mRNA in two groups before and after the treatment session. A: HMA
group before treatment; B: HMA group after treatment; C: MESA group before treatment; D: MESA group after treatment; E: Bar graphs of the expression of zonula
occludens-1 mRNA, occludin mRNA and claudin-1 mRNA in HMA group and MESA group before and after the treatment session. HMA: Herb-partitioned moxibustion
combined with acupuncture group; MESA: Mesalazine group; ZO-1: Zonula occludens-1.
E
Shang HX
et al
. Moxibustion increases tight junction protein expression
to strengthen the intercellular connections and seal
the intercellular spaces. These defects may lead to a
continuous loss of solute and compromise resistance
against pathogens in the gut lumen. After the
treatments, the morphological and ultrastructural
observations of the MESA group showed partially
recovered but still disorganized intestinal glands.
There was also limited inammatory cellular inltration
and comparatively strengthened intercellular
connections with partially narrowed intercellular
spaces. These results suggest that mesalazine
ameliorate inammation in the intestinal mucosa and
can restore the TJ barrier function. In the HMA group,
the morphological and ultrastructural observations
revealed regularly arranged intestinal glands, mild
and localized inflammatory cellular infiltration,
tight intercellular connections and few broadened
intercellular spaces. These ndings suggest that HMA
induces mucosa inflammation remission and repairs
the TJ barrier structure. Our comparisons between the
MESA group and the HMA group indicate that HMA
achieves inammatory remission similar to mesalazine
and surpasses mesalazine in repairing the TJ barrier
structure in intestinal epithelial mucosa.
We compared the expression of the TJ proteins
and their mRNAs before and after treatment. The data
show that the expression of occludin, claudin-1, and
ZO-1 and their mRNAs are significantly increased in
both the MESA and HMA group after treatment. This
result in combination with morphological evidence
suggests the mechanism of mesalazine and HMA is to
repair the TJ barrier by upregulating the expression of
TJ proteins such as occludin, claudin-1, and ZO-1 and
their mRNAs. The expression of occludin and claudin-1
and their mRNAs showed no significant differences
between the MESA gr ou p and the HMA group.
However, the HMA group showed a signicant increase
in ZO-1 (P = 0.047) and ZO-1 mRNA (P = 0.023)
expression compared to the MESA group.
ZO-1 mediates the assembly of TJs by organizing
components of TJs and linking them to the cortical
actin cytoskeleton[46]. We found that there was a
signicant difference between groups in the expression
ZO-1 and its mRNA, which may suggest a different
mechanism of HMA and mesalazine in CD patients.
However, the specific functional mechanism of HMA
still requires further investigation.
This study is only an initial attempt to investigate
the effectiveness of HMA treatment and its underlying
mechanism. We should examine several questions
in future studies. What is the indication for HMA in
CD treatment - mild or moderate CD? Is HMA alone
effective in controlling CD symptoms, or does HMA only
function as a supplement to conventional management?
Is there any short-term or long-term adverse reaction
to HMA, and what is the corresponding measurement?
In conclusion, HMA improves intestinal epithelial
barrier repair and reduces inammation in CD patients
by upregulating the expression of the TJ proteins
occludin, claudin-1, and ZO-1 and their mRNAs.
COMMENTS
Background
Crohn’s disease (CD) is a chronic, recurrent inflammatory bowel disease.
Although its etiopathogenesis remains obscure, compromised permeability
of the intestinal epithelial barrier is recognized to play a pivotal role in CD
pathology.
Research frontiers
Tight junctions (TJs) within intestinal epithelial cells form the structural basis
of the intestinal epithelial barrier and are the major determinant of epithelial
permeability. Recent studies have conrmed that the decreased expression of
TJ proteins is positively correlated with CD severity.
Innovations and breakthroughs
Previous studies by Shang et al have demonstrated the efficiency of
acupuncture and herb-partitioned moxibustion in improving CD patients’ clinical
symptoms and pathological changes. In the current study, they found that
herb-partitioned moxibustion combined with acupuncture (HMA) can improve
epithelial barrier repair and reduce inammation by upregulating the expression
of TJ proteins in CD patients.
Applications
This study provides initial evidence of the therapeutic effect of HMA on CD by
upregulating the expression of intestinal epithelial TJ proteins and their mRNAs.
Therefore, HMA might be considered as an alternative option to treat mild to
moderate CD.
Terminology
The intestinal epithelial barrier is a monolayer lining the entire digestive
tract. Tight junctions are fibril-like structures connecting adjacent cells,
sealing the intercellular spaces to control paracellular transportation of ions,
macromolecules and immune cells.
Peer-review
This study evaluated whether herb-partitioned moxibustion combined with
acupuncture changes the permeability of the intestinal epithelial barrier by
affecting the expression of colonic epithelial TJ-related proteins in mild to
moderate CD patients. The question posed by the authors is well defined
and the methods are described appropriately. The data and figures in the
manuscript appear to be genuine.
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P- Reviewer: Jang SH, Shi Y S- Editor: Yu J L- Editor: Logan S
E- Editor: Zhang DN
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Shang HX
et al
. Moxibustion increases tight junction protein expression
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