ARTICLE IN PRESS

CLINICAL INVESTIGATION

Correlation of Intestinal
Mucosal Healing and Tight Junction
Protein Expression in Ulcerative
Colitis Patients
D1X XYue Tan, D2X XMD, D3X XYadi Guan, D4X XMD, D5X XYan Sun, D6X XMD and D7X XChangqing Zheng, D8X XMD

Department of Gastroenterology, Shengjing Hospital of China Medical University,

Shenyang Liaoning Province, China

ABSTRACT
Background: The aim of this study was to investigate the relationship between intestinal mucosal healing and tight junction
(TJ) protein expression in patients with ulcerative colitis (UC).
Materials and Methods: A total of 40 patients with UC were included as an experimental group and UC disease activity was
evaluated using the Mayo clinic score (MCS) and 8 patients with normal distal colon served as the control group. The expres-
sion of TJ proteins including occludin, ZO-1 and claudin-2 were determined by immunohistochemistry and their correlation
with clinical characteristics were also analyzed.
Results: Statistically significant differences regarding the MCS and Mayo endoscopic subscore (MES) were observed in both
groups (P < 0.01). The Geboes index was significantly increased in patients with active UC compared to patients with quies-
cent UC and normal controls (P < 0.01). Patients with active and quiescent UC had upregulated expression of claudin-2 and
reduced expression of occludin and ZO-1 compared to those of normal controls. The expression of ZO-1 was significantly
higher in patients with quiescent UC with mucosa healing (P < 0.05) compared with those without mucosal healing. The
expression of ZO-1 and occludin was negatively correlated with MCS, MES, Geboes, C-reaction protein and erythrocyte sed-
imentation rate. The expression of claudin-2 was positively correlated with MCS, MES, Geboes, C-reaction protein and eryth-
rocyte sedimentation rate.
Conclusions: These findings suggest that TJ proteins play a crucial role in mucosal healing, and may be a potential marker
of response when evaluating therapeutic interventions.
Keywords: Ulcerative colitis; Inflammatory bowel disease; Tight junction; Mucosal healing. [Am J Med Sci 2018;&(&):1
−10.]

INTRODUCTION claudins and junctional adhesion molecules, as well as

I
nflammatory bowel disease (IBD) is a disorder of the
gastrointestinal tract characterized by uncontrolled
chronic inflammation in a genetically susceptible
population exposed to environmental antigens. Ulcera-
tive colitis (UC) is one of the most common subtypes of
IBD, and the incidence of UC has been increasing all
over the world, especially in the Asia-Pacific region and
Eastern Europe.1,2 The pathogenesis of UC has not been
fully elucidated. Currently, it is considered to be the com-
bination of various factors, including genetic, environ-
mental, intestinal microorganisms and others.3-5
Tight junctions (TJ) are one of the most important
apical junctional complexes in many types of epithelial
and endothelial cells.6 As a key component of the intesti-
nal mucosal epithelial barrier, TJs play a crucial role in
maintaining intestinal permeability, tissue differentiation
and homeostasis. The TJs are mainly composed by
transmembrane proteins such as occludin, various
peripheral membrane proteins such as ZO-1. They are
linked to the cytoskeleton of the cell by F-actin and myo-
sin II.7,8 Previous studies have shown a decrease in main
TJ proteins, such as occludin and ZO-1, a corresponding
increase in intestinal permeability and a decrease in
transepithelial resistance (TER) in patients with IBD.9-11
In addition, several studies have demonstrated a higher
expression of claudin-2 in colonic samples from patients
with UC.11,12
Recently, mucosal healing has become an important
therapeutic goal for UC disease,13,14 and has been con-
sidered an important indicator for evaluating therapeutic
efficacy of drugs.15 Mucosal healing has been demon-
strated to be associated with long-term clinical remission
and a lower incidence of recurrence.16 Mucosal healing,
routinely assessed by endoscopy, generally refers to the
absence of ulcers and erosions in all visualized segments
of the colonic mucosa.17 Intestinal epithelial barrier

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Tan et al

dysfunction can broadly activate mucosal immune was approved by the institutional review board of
responses and trigger or accelerate the onset and sever- Shengjing Hospital of China Medical University, and
ity of immune-mediated colitis.18 In addition, inflamma- informed consent was obtained from each patient.
tion in UC can cause intestinal mucosal barrier
dysfunction, followed by alterations in TJ structures.
Endoscopic Examination
Thus altered TJ structures may affect mucosal healing.19
All patients in both groups received a polyethylene
However, there are few studies regarding the relationship
glycol-based electrolyte solution for bowel preparation
between the TJ protein expression and mucosal healing.
according to the instructions. Endoscopic examination
In this study, we aimed to investigate the relationship
was performed by an experienced professor of Gastro-
between mucosal healing and TJ protein expression in
enterology. The endoscopic mucosal status of UC was
patients with UC.
assessed using Mayo Clinic endoscopic subscore
(MES). Endoscopic mucosal healing was defined as a
Mayo endoscopic score of 0 or 1 in patients with UC.20
MATERIALS AND METHODS
Patients
Histological Evaluation
A total of 40 patients with UC from Shengjing Hospi-
Colonic biopsy specimens were taken from the rec-
tal of China Medical University were collected between
tum, ulcer margin of sigmoid colon and inflamed por-
September 2014 and November 2016, and served as the
tions, and fixed in 4% formaldehyde over 24 hours and
experimental group. UC disease activity was evaluated
then embedded in paraffin. Specimens were sliced into
using the Mayo clinic score (MCS) of 6-12 points
4 mm thick sections, and stained by hematoxylin and
(Table 1), which assigned a disease activity category for
eosin. The corresponding hematoxylin and eosin slides
remissive or quiescent, mild, moderate and severe activ-
at high magnification (400£) were randomly selected and
ity.17 All eligible patients had an established diagnosis of
examined by 2 independent gastrointestinal pathologists
UC according to endoscopic and histologic assess-
according to the Geboes grading system (Table 2). Histo-
ments. Patients were excluded if they met the following
logical mucosal healing was defined as a grade 0 or 1 on
criteria: (1) patients who received a diagnosis of indeter-
the Geboes index.21 In this study, more stringent criteria
minate colitis, Crohn’s disease or clinical findings sug-
for the definition of mucosal healing were used, which
gestive of Crohn’s disease (i.e., fistula or granulomas on
means the criteria for endoscopic and histological muco-
biopsy); (2) patients with tumor or epithelial neoplasia in
sal healing should be met simultaneously.22
intestinal tissue; (3) patients with serious gastrointestinal
complications, such as intestinal perforation, local steno-
sis and intestinal obstruction etc.; (4) colonoscopy intol- Immunohistochemistry Analysis
erance; (5) associated with cerebral hemorrhage, Immunohistochemical analysis was performed for
cerebral infarction and other central nervous system dis- determining the expression of occludin, ZO-1 and clau-
eases; (6) associated with rheumatoid arthritis, Graves’ din-2. After fixing in 4% formaldehyde overnight, all the
disease, psoriasis and other autoimmune diseases and blocks of colonic tissue were sectioned (2.5 mm), depar-
(7) patients with cognitive impairment and mental illness. affinized and rehydrated in graded ethanol concentra-
Eight patients with normal distal colon confirmed by sur- tions, followed by suitable antigen retrieval (boiling in
gical pathology served as the control group. The study 0.01 M citrate buffer). Endogenous peroxidase was

TABLE 1. Mayo clinic score system for assessment of ulcerative colitis activity.

Item/score 0 1 2 3
Stool frequency Normal number for patient 1-2 stools more than normal 3-4 stools more than normal ≥5 stools more than normal
Rectal bleeding No blood seen Streaks of blood with stool Obvious blood with stool most Blood alone passes
less than half the time of the time
Endoscopic Normal or inactive disease Mild disease with erythema, Moderate disease with Severe disease with sponta-
findings decreased vascular pattern, marked erythema, absent neous bleeding, ulceration
mild friability vascular pattern, friability,
erosions
Physician’s Normal Mild disease Moderate disease Severe disease
global
assessment
Scores can range from 0 to 12, with higher scores indicating more severe disease activity.
Clinical remission was defined as a total Mayo score of 2 or lower, with no individual subscore exceeding 1.
Mild activity was defined as a Mayo score 3-5.
Moderate activity was defined as a Mayo score 6-10.
Severe activity was defined as a Mayo score 11-12.

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Mucosal Healing and Tight Junction Proteins in Ulcerative Colitis

TABLE 2. Geboes index for evaluation of disease severity in ulcerative randomly selected, and image analysis was performed
colitis. with Image Plus (Media Cybernetics, INC., Maryland).
Grade Histological change
0 Structural Statistical Analysis
Subgrades Statistical analysis was performed using SPSS (ver-
0.0 No abnormality sion 20.0, Inc., Chicago, IL). The quantitative data are
0.1 Mild abnormality presented as mean § standard deviation and compared
0.2 Mild or moderate diffuse or multifocal abnormalities using 1-way analysis of variance followed by least signifi-
0.3 Severe diffuse or multifocal abnormalities cant difference test. Correlation between the parameters
1 Chronic inflammatory infiltrate was performed by Pearson correlation analysis. Qualita-
Subgrades tive data were expressed as numbers and analyzed by
1.0 No increase chi-square test. P values < 0.05 were considered statisti-
1.1 Mild but unequivocal increase cally significant.
1.2 Moderate increase
1.3 Marked increase
2 Lamina propria neutrophils and eosinophils RESULTS
2A Eosinophils
2A.0 No increase
Clinical Characteristics of the Patients
2A.1 Mild but unequivocal increase A total of 40 patients with UC were included between
2A.2 Moderate increase September 2014 and November 2016. Among these, 10
2A.3 Marked increase (25%) were in remission, while the remaining 30 (75%)
2B Neutrophils had mild and moderate/severe disease activity according
2B.0 None to the MCS. The clinical characteristics of all patients are
2B.1 Mild but unequivocal increase shown in Table 3. There were no significant differences
2B.2 Moderate increase with regard to age and sex between the experimental
2B.3 Marked increase and control groups (P > 0.05). There was also no signifi-
3 Neutrophils in epithelium cant difference in disease duration among various dis-
3.0 None ease activity groups (P > 0.05).
3.1 <5% crypts involved
3.2 <50% crypts involved
3.3 >50% crypts involved Endoscopic Examination
4 Crypt destruction The representative endoscopic images from patients
4.0 None are showed in Figure 1. In the control group, colonic
4.1 Probable-local excess of neutrophils in part of crypt mucosa are characterized as smooth surfaces without
4.2 Probable-marked attenuation the evidence of erosion, ulceration and bleeding. Similar
4.3 Unequivocal crypt destruction endoscopic profiles can be shown in patients with UC in
5 Erosion or ulceration remission, however, inflammatory polyp and mucosal
5.0 No erosion, ulceration or granulation tissue scarring can be observed in some patients. In patients
5.1 Recovering epithelium + adjacent inflammation with UC with mild disease activity, colonoscopy shows
5.2 Probable erosion-focally stripped intestinal mucosal hyperemia, edema, superficial erosion
5.3 Unequivocal erosion and ulceration. In patients with UC with moderate dis-
5.4 Ulcer or granulation tissue ease activity, similar profiles can be seen as in those with
mild disease activity, except that the ulcerated surface is
coated with fibrinous exudate. In patients with UC with
blocked by 3% H2O2 (Shengjing Hospital of China Medi- severe disease activity, similar profiles can be seen as in
cal University, China) for 15-20 minutes. Nonspecific those with moderate disease activity, except that
antibody binding sites were blocked with 5% goat serum mucosa is friable, rough and unsmooth.
albumin (SPN-9002,ZSGB-BIO CO, Beijing, China) for 30
minutes. Then, the sections were incubated with anti-
bodies (Santa Cruz Biotechnology, Santa Cruz, CA) Histological Features
against rabbit polyclonal ZO-1 (1:75), rabbit polyclonal Typical features can be observed in biopsies of
claudin-2 (1:100) and mouse monoclonal occludin patients with clinically proven UC. In the normal controls,
(1:100) and incubated overnight. Thereafter, the slides the structure of the intestinal epithelium was intact and
were incubated with horseradish peroxidase-labeled uni- the glands were arranged in a regular fashion. A small
versal secondary antibody (SPN-9002, ZSGB-BIO CO, number of lymphocytes were identified in the lamina
Beijing, China). Immunodetection was performed with propria. No neutrophilic or eosinophilic infiltration was
3,30 -diaminobenzidine (DAB) as chromogen and H2O2 as observed. In patients with UC in the remission stage, the
substrate, followed by counter-staining with hematoxy- structure of the intestinal epithelium was intact and the
lin. More than 3 images in magnification (40£) were glands were arranged in a regular fashion. The number of

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Tan et al

TABLE 3. Clinical characteristics of the patients.

Experimental group (n = 40)

Characteristics Control group (n = 8) Clinical Mild Moderate Severe P
remission activity activity activity
(n = 10) (n = 10) (n = 9) (n = 11)
Age (years) 44.4 § 12.7 41.5 § 14.3 43.5 § 13.1 41.9 § 15.2 44.4 § 12.8 0.984
Course of disease (years) — 3.4 § 2.5 3.5 § 2.7 3.8 § 2.3 3.5 § 2.6 0.957
Gender (male/female) 4/4 4/6 4/6 5/4 4/7 0.951
Montreal classification —
E1 — — 5 0 0 —
E2 — — 4 3 1 —
E3 — — 1 6 10 —
MCS score 0.6 § 0.5 4.1 § 0.78*,^ 8.4 § 1.1*,# 11.7 § 0.5*,#,^
MES score 0.5 § 0.5 1.7 § 0.4*,^ 2.3 § 0.5*,# 3.0 § 0.0*,#,^
CRP(mg/L) 3.4 § 0.5 3.9 § 1.3 9.9 § 1.8 64.8 § 12.8*,#,^
ESR(mm/h) 3.7 § 2.5 12.3 § 9.8 25.9 § 17.0* 39.0 § 22.2*,#
Abbreviations: CRP, C-reaction protein; ESR, erythrocyte sedimentation rate; MCS, Mayo clinic score; MES, Mayo endoscopic subscore.
P < 0.05 was considered statistically significant.
* P < 0.01, vs. clinical remission.
# P < 0.01, vs. mild activity.
^ P < 0.01, vs. moderate activity.

goblet cells was increased, the inflammatory cells in the
lamina propria were significantly decreased or had disap-
peared and the surface was infiltrated by scattered
eosinophil granulocytes. No neutrophilic infiltration was
observed. For patients with UC with mild disease activity,
the epithelium structure of the colonic mucosa was
destroyed, and the number of goblet cells was reduced.
Infiltration of neutrophils, eosinophils and lymphocytes
could be observed in the lamina propria. In patients with
UC with moderate disease activity, colonic epithelial cells

FIGURE 1. Endoscopic images of patients in the control group and experimental group. A, Control group, the colonic mucosa are characterized
as smooth surfaces without the evidence of erosion, ulceration and bleeding; (BI) and (BII) Clinical remission, (BI) similar endoscopic profiles can
be seen similar to control group, (BII) intestinal mucosa scarring (yellow arrow), inflammatory polyp (red arrow); C, mild activity, the colonic
mucosa appeared congested and edematous, with superficial erosions, superficial ulcers and bleeding spots; D, moderate activity, the ulcerated
surface is coated with fibrinous exudate; E, Severe activity, mucosa was friable, rough and unsmooth, accumulation of blood in the intestinal
cavity can be observed. UC, ulcerative colitis

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Mucosal Healing and Tight Junction Proteins in Ulcerative Colitis

FIGURE 2. Histological changes of colon (£400). (A) Control group, the glands were arranged regularly, and there are abundant goblet cells. No
neutrophilic or eosinophilic infiltration was observed; (B) clinical remission, the inflammatory cells in the lamina propria was decreased or disap-
peared significantly, and the surface was infiltrated by scattered eosinophil granulocyte. No neutrophilic infiltration was observed; (C) mild activ-
ity, the epithelium structure of colonic mucosa was destroyed, and the number of goblet cells was reduced; (D) moderate activity, diffuse
infiltration of inflammatory cells can be observed in the lamina propria; (E) severe activity, mucosal glands were destroyed by the inflammatory
cell infiltration and crypt-abscesses were found. Mucosal erosions, superficial ulcers and granulation tissue hyperplasia occurred. Scale
bar = 50 mm.

and the grand cells were arranged in a disorderly manner.
The number of goblet cells was obviously decreased, the
gland cells were decreased and the crypt was arranged
in a disorderly manner. Diffuse infiltration of inflammatory
cells including neutrophils, eosinophils, plasmacyte and
lymphocytes can be observed in the lamina propria. In
patients with UC with severe disease activity, mucosal
glands were destroyed by the inflammatory cell infiltra-
tion and crypt-abscesses were found. Mucosal erosions,
superficial ulcers and granulation tissue hyperplasia
were observed in individual cases (Figure 2 A-E). The his-
tological scores are reported in Figure 3.
protein. In patients with active and quiescent UC, ele-
vated claudin-2 expression could be seen in the cyto-
plasm and cell membrane of the intestinal epithelial cells
and the glandular epithelial cells (Figure 6). Positive
expression was demonstrated by a yellowish-brown
staining for ZO-1, occludin and claudin-2.
Cumulative optical density of TJ proteins including
ZO-1, occludin and occludin-2 are showed in Figure 7.
Occludin and ZO-1 expressions in patients with active
and quiescent UC were significantly decreased com-
pared with the normal controls (P < 0.01). ZO-1 expres-
sion in quiescent UC patients with nonmucosal healing
was significantly decreased compared with those of the

Expression of TJ Proteins ZO-1, Occludin and

Claudin-2 Analyzed by Immunohistochemistry
In normal controls and quiescent UC patients, posi-
tive expression of occludin protein can been seen in the
cell membrane of the intestinal epithelial cells and glan-
dular epithelial cells, as well as in the cytoplasm adjacent
to the cell membrane. In patients with active UC, the pos-
itive expression of occludin protein in the intestinal epi-
thelial cells and glandular epithelial cells was decreased
when compared with those of the normal controls and
quiescent UC patients (Figure 4). In normal controls, the
positive expression of ZO-1 protein could be seen in the
cytoplasm and cell membrane of the intestinal epithelial
cells and glandular epithelial cells. Compared with the
normal controls, the positive expression of ZO-1 was
FIGURE 3. Geboes scores. *P < 0.01, compared with the control
decreased in patients with mild to severely active UC group; **P < 0.05 compared with the control group; #P < 0.01, com-
and quiescent UC (Figure 5). In normal controls, there pared with patients with UC in clinical remission; ^P < 0.01, com-
was no expression or very low expression of claudin-2 pared with patients with UC of severe activity. UC, ulcerative colitis.

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Tan et al

FIGURE 4. Occludin expression in colonic biopsies (£400). In patients with active ulcerative colitis (UC), the positive expression (yellowish-
brown staining) of occludin protein in the intestinal epithelial cells and glandular epithelial cells was decreased compared with those of the nor-
mal controls and quiescent UC patients; (A) control group; (B) clinical remission; (C) mild activity, (D) moderate activity and (E) severe activity.
Scale bar = 50 mm.

mucosal healing (P < 0.01). Claudin-2 expression was
significantly higher in patients with active and quiescent
UC than that in the normal controls (P < 0.01).
Correlation Analysis Between TJ Proteins and
Clinical characteristics
Correlation analysis between TJ proteins and clinical
characteristics revealed that the expressions of occludin,
ZO-1 and claudin-2 were not relevant to age and sex
(P > 0.05). The expressions of ZO-1 and occludin were
negatively correlated with MCS, MES, C-reaction protein
(CRP), erythrocyte sedimentation rate (ESR) and Geboes
index. However, the expression of claudin-2 was posi-
tively correlated with MCS, MES, Geboes index, CRP
and ESR (Table 4).
DISCUSSION
In this study, the relationship between intestinal muco-
sal healing and TJ protein expression in patients with UC
was investigated. We observed upregulated expression of

FIGURE 5. ZO-1 expression in colonic biopsies (£400). (A) control group; (B) clinical remission, the glands were intact, and a large number of
brownish-yellow staining was found in the epithelial cell membrane and cytoplasm of the gland; (C) mild activity, (D) moderate activity and (E)
severe activity, brownish-yellow staining gradually decreased. Scale bar = 50 mm.

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Mucosal Healing and Tight Junction Proteins in Ulcerative Colitis
FIGURE 6. Claudin-2 expression in colonic biopsies (£400). (A) control group, rare brown staining can be seen in the whole gland epithelial cell
membrane; (B) clinical remission, a few of brownish-yellow staining was found; (C) mild activity, (D) moderate activity and (E) severe activity,
brownish-yellow staining gradually increased. Scale bar = 50 mm.
claudin-2 and reduced expression of occludin and ZO-1 in
patients with active and quiescent UC compared with the
normal controls. Additionally, the expressions of ZO-1 and
occludin were significantly correlated with the clinical
mucosal healing characteristics. Similar findings regarding
TJ protein expression in patients with UC have been
described in the literature.23,24
The pathogenesis of UC has not yet been fully eluci-
dated, and several studies have shown that intestinal
barrier dysfunction is an important pathogenic factor in
UC.25,26 The intestinal barrier is composed of the intesti-
nal epithelial cells, intestinal mucosal layer and intercellu-
lar junctions.27 TJs are one of the most important apical
junctional complexes in intestinal epithelial cells. They
play a crucial role in maintaining intestinal permeability,
tissue differentiation and homeostasis.28 As studied pre-
viously, claudin-2 is responsible for forming pores in the
TJs and transcellular ion transport. In this study, we
found a significant increase in claudin-2 expression lev-
els in both groups of subjects. In addition, we found pos-
itive correlations between the claudin-2 expression
levels and MCS, MES and Geboes. Previous studies
have shown that claudin-2 was highly expressed in
patients with IBD, and its expression is positively corre-
lated with inflammatory activity.12,29 Therefore, increased
claudin-2 expression levels may be an important cause
of intestinal epithelial barrier dysfunction and intestinal
mucosal damage. Occludins are considered to maintain
the enterocyte polarity and indirectly maintain the pores
by forming hetero-polymers with different claudin pro-
teins.30,31 The transmembrane hetero-polymer claudin
and occludin proteins connect with the cytoplasmic actin
−myosin filaments by the scaffold protein ZO-1, and it
can be observed in the cytoplasmic region of enterocyte
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and glandular epithelium. It is crucial for cell penetration,
gene transcription and regulation of cell proliferation. The
study of dextran sulfate sodium-induced colitis in mice
reported by Poritz et al9 reveals that the reduced expres-
sion of ZO-1 and increased permeability preceded the
development of significant intestinal inflammation. Thus,
TJ dysfunction may be an initiating factor in the patho-
genesis of IBD. In the present study, the expressions of
ZO-1 and occludin were significantly reduced in patients
with UC compared with controls, and negatively corre-
lated with MCS, MES, CRP, ESR as well as Geboes
index. The aforementioned findings were similar to those
of Heller et al.10,32
Mucosal healing has been regarded as an important
therapeutic goal in UC. A previous study by Lau et al33
reported that mucosal healing can improve long-term
prognosis of IBD and may help to lengthen corticoste-
roid-free clinical remission. However, there has been no
definitive criterion for mucosal healing in UC at present.34
Although mucosal healing was defined as a MES of 0 or 1
in a number of previous studies,35,36 there has been no
long-term study examining whether an MES of 0 or 1
actually contributes to the maintenance of clinical remis-
sion in patients with UC. In addition, previous study has
demonstrated that patients with a MES of 0 were much
less likely to undergo colon resection and maintained
longer remission compared with patients with a MES of
1.37 In this study, more stringent criteria for the definition
of mucosal healing was used, which means endoscopic
(MES of 0 or 1) and histological (Geboes score of 0 or 1)
mucosal healing should be met simultaneously. We
found that 4 of 10 (40%) patients in remission stage
achieved mucosal healing, which was consistent with
the findings of a previous study by Arijs et al.22
7
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FIGURE 7. Mean optical density of occludin, ZO-1 and claudin-2. Mucosal healing was defined as absolute endoscopy subscore of 0 or 1, and
Geboes score of 0 or 1. Nonmucosal healing was defined as absolute subscore for endoscopy > 1, and (or) Geboes score >1. *P < 0.01, com-
pared with the control group; #P < 0.01, compared with patients with mucosal healing; DP < 0.05, compared with patients with nonmucosal
healing.

TABLE 4. Correlation analysis between tight junction proteins and clin- Increased intestinal mucosal permeability usually
ical characteristics. occurs in the early stages of disease in IBD or before
its recurrence.38 Remarkably, disease recurrence
Clinical Claudin-2 Occludin ZO-1 becomes more likely in nonactive IBD patients if they
characteristics had increased epithelial permeability.9 The TJ is the
MCS 0.8802** ¡0.9255** ¡0.7463** rate-limiting step in transepithelial transport and the
MES 0.8197** ¡0.8815** ¡0.7045** principal determinant of mucosal permeability.7 The
Geboes 0.8393** ¡0.876** ¡0.769** dysregulation of TJs may contribute to increased epi-
CRP 0.3558* ¡0.4078* ¡0.342* thelial permeability which eventually leads to mucosal
ESR 0.559* ¡0.6114** ¡0.5372* inflammation.39-41 Thus, epithelial barrier dysfunction
Age 0.001195 ¡0.0007065 0.02616
caused by the abnormal expression of TJ proteins
Sex 0.07155 ¡0.0945 ¡0.03583
may be the initiating factor in the pathogenesis of
Abbreviations: CRP, C-reaction protein; ESR, erythrocyte sedimentation IBD. Mucosal healing has been described as closely
rate; MCS, Mayo clinic score; MES, Mayo endoscopic subscore. related to the intestinal epithelial barrier function.42 In
* P < 0.05.
** P < 0.0001.
addition, several previous studies demonstrated that
quiescent IBD with irritable bowel syndrome-like

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Mucosal Healing and Tight Junction Proteins in Ulcerative Colitis
symptoms had more inflammatory cell infiltration, sig-
nificantly higher paracellular permeability as well as
significantly lower expression of ZO-1 and occudin
compared to quiescent IBD without irritable bowel
syndrome-like symptoms.40,41 Thus, we speculated
that the integrity of TJ proteins is crucial to maintain
mucosal healing and expression of TJ proteins may
differ in patients with UC during the remission stage
depending on wound healing. In the present study,
we found that a significant increase in the expression
of ZO-1 in patients with quiescent UC with mucosal
healing compared with those without mucosal healing
(P < 0.05). In addition, we found a trending increase
in occludin expression levels in patients with mucosal
healing compared to those without mucosal healing,
although this trend was not statistically significant.
However, there was no obvious difference regarding
claudin-2 expression between patients with and with-
out mucosal healing. These findings support the idea
that the abnormal expression of TJ proteins in the
intestinal mucosa correlates with patients with UC
who achieved clinical remission but failed to achieve
mucosal healing. These findings suggest that the
expression of TJ proteins could be an important indi-
cator for the evaluation of mucosal healing. Further-
more, therapy targeting TJ may help to achieve and
maintain mucosal healing in IBD patients.
Further research regarding the correlation between
TJ proteins and mucosal healing needs to be carried out
and validated by western blot and reverse transcription
polymerase chain reaction analysis. In addition, larger
sample size and study of patients with Crohn’s disease
will be also needed.
CONCLUSIONS
In summary, the expression of claudin-2 in active and
quiescent patients with UC was upregulated, while the
expression of occludin and ZO-1 was decreased in com-
parison to normal controls. In addition, we found that the
expression of ZO-1 significantly correlated with the muco-
sal healing index (MCS and MES). Furthermore, we found
a significant increase in the expression of ZO-1 in patients
with quiescent UC with mucosal healing compared to
those without mucosal healing. These finding suggest that
TJ proteins play a crucial role in mucosal healing, and may
be a potential marker of response when evaluating thera-
peutic interventions. The detection of TJ proteins can be
used to assess the integrity of the intestinal mucosal bar-
rier and perhaps predict the tendency for recurrence in qui-
escent UC patients. Thus, for patients with UC with an
elevated risk of recurrence, anti-inflammatory therapy
could be administered prophylactically to reduce the risk
of recurrence. For patients with active UC undergoing ther-
apy to induce remission, detection of TJ proteins may help
to evaluate the condition of mucosal healing to judge the
therapeutic effect of drugs and to determine when to con-
sider stopping the intervention.
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AUTHOR CONTRIBUTIONS
All the authors contributed toward the study by mak-
ing substantial contributions to conception, design,
acquisition of data, or analysis and interpretation of data.
Y.T. and Y-D.G. made substantial contributions to con-
ception and design, were in charge of data collection
and wrote the manuscript. Y.S. and C.Z. were in charge
of statistical analysis.
SUPPLEMENTARY MATERIALS
Supplementary material associated with this article
can be found, in the online version, at https://doi.org/
10.1016/j.amjms.2018.11.011.
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Submitted March 14, 2018; accepted November 21, 2018.
Disclosures: The authors declare that they have nothing to disclose.
Funding: This work was supported by the Science and Technology
Program of Liaoning Province (no. 2014021083).
The author has no financial or other conflicts of interest to disclose.
Correspondence: Changqing Zheng, MD, Department of Gastroenterol-
ogy, Shengjing Hospital of China Medical University, 39 Huaxiang Road,
Tiexi District, Shenyang, Liaoning Province 110022, China. (E-mail:
zhengchangqing2007@163.com).
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