Introduction
Gastroesophageal reflux disease (GERD) in children
has attracted increasingly more attention from clinicians due to
its high morbidity rate and serious influence on the quality of
life of child patients (1,2). Reflux esophagitis (RE) can be diagnosed
when the abnormalities in the esophageal mucosa are detected in
gastroscopy. Inflammatory mediators and other factors can destroy
the esophageal mucosal barrier, reduce the lower esophageal
sphincter pressure (LESP) and lower the esophageal peristalsis,
ultimately leading to RE (3,4). The role of oxidative stress in
esophageal mucosal injury is gradually receiving increased
attention. Excessive reactive oxygen species (ROS) are produced by
mucosal epithelial cells under stress due to the reflux of gastric
contents, causing mucosal damage (5). The present study detected the changes
in the levels of serum malondialdehyde (MDA), total superoxide
dismutase (T-SOD), vasoactive intestinal peptide (VIP), motilin,
interleukin-1β (IL-1β), IL-8 and tumor necrosis factor-α (TNF-α) in
child patients with different severity of RE, evaluated their
associations with the severity of disease and explored their
clinical significance.
Patients and methods
Clinical data
A total of 60 child patients (aged 7 months to 16
years) with such upper digestive tract symptoms such as vomiting,
acid reflux and heartburn and diagnosed with GERD via gastroscopy
and mucosal pathological changes in Changzhou Jintan District
People's Hospital (Changzhou, China) from March 2018 to December
2018 were selected as the objects of study. Among them, there were
39 boys and 21 girls, 5 cases were younger than 1 year of age, 20
cases were aged 1 to 3 years, 10 cases were aged 3 to 6 years, 6
cases were aged 6 to 9 years, 11 cases were aged 9 to 12 years, and
8 cases were aged 12 to 16 years. None of them took PPI and
domperidone within 2 weeks before enrollment and they all had good
compliance. The patients were divided into non-erosive reflux
disease group (NERD group, n=12) and (RE group n=48) according to
whether there was esophageal mucosal injury. In RE group, the
patients were further divided into grade I RE group (n=15), grade
II RE group (n=18) and grade III RE group (n=15) based on the
severity of mucosal injury. The sex and age were comparable in each
group (P>0.05). This study was explained to the family members
of the patients and approved by the Ethics Committee of Changzhou
Jintan District People's Hospital. Informed consents were signed by
the child parents or guardians.
Research methods
After admission, the examinations were performed,
and the body weight was controlled through low-sugar and low-fat
diet. After the serum and esophageal mucosa specimens were
collected, the patients were treated with drugs (proton pump
inhibitor, mucosal protective agent and gastrointestinal prokinetic
agent) or underwent operation according to the severity of
disease.
Detection indexes
Detection of serum VIP, motilin,
IL-1β, IL-8 and TNF-α levels
The serum IL-1β, IL-8 and TNF-α levels were measured
using the Multiskan Sky microplate reader (Thermo Fisher
Scientific, Inc.), and the VIP and motilin levels were measured
using the UniCelDxI 600 full-automatic immunoassay analyzer
(Beckman Coulter, Inc.).
Monitoring of 24 h esophageal pH
indexes
The DeMeester score, longest time of reflux, severe
acid exposure ratio and total times of reflux were determined.
LESP
LESP was measured using the XDJ-S8 upper digestive
tract dynamic monitoring system (Anhui Kaili Science &
Technology group Co., Ltd.).
Lower esophageal mucosa
The lower esophageal mucosa was collected under the
EG-600WR electronic gastroscope (Fuji), and prepared into mucosal
homogenate to determine the MDA using thiobarbituric acid (TBA)
assay and T-SOD using hydroxylamine assay.
Correlation
The correlation between each index and DeMeester
score was analyzed.
Statistical analysis
SPSS 20.0 software (IBM) was used for statistical
analysis. The measurement data were expressed as mean ± standard
deviation, one-way analysis of variance was performed for the
comparison among multiple groups, and LSD test was adopted as a
post hoc test for the comparison between two groups. The
enumeration data were expressed as rate (%), and χ2 test
was performed. Pearson's correlation analysis was used for the
correlation. P<0.05 was considered to indicate a statistically
significant difference.
Results
Changes in oxidative stress
indexes
The results showed that the content of MDA was
8.1±3.6, 12.3±4.9, 21.2±6.7 and 35.5±5.7 nmol/g protein in NERD,
grade I RE, grade II RE and grade III RE group, respectively, and
the T-SOD activity was 56±21, 41±14, 30±15 and 19±9 U/mg protein in
the four groups, respectively, displaying statistically significant
differences (P<0.05). The above findings indicate that the
levels of oxidative stress indexes are related to the degree of
mucosal injury (Table I).
 | Table I.Changes in oxidative stress indexes in
RE groups and NERD group (mean ± SD). |
Table I.
Changes in oxidative stress indexes in
RE groups and NERD group (mean ± SD).
| Index | NERD group
(n=12) | Grade I RE group
(n=15) | Grade II RE group
(n=18) | Grade III RE group
(n=15) |
|---|
| MDA content (nmol/g
prot) | 8.1±3.6 | 12.3±4.9 | 21.2±6.7a,b | 35.5±5.7a–c |
| T-SOD activity (U/mg
prot) | 56±21 | 41±14a | 30±15a,b | 19±9a–c |
Changes in levels of serum
inflammatory factors
With the aggravation of disease, the expression
levels of serum IL-1β, IL-8 and TNF-α in RE group were gradually
increased. The levels of inflammatory factors had statistically
significant differences between RE group and NERD group and among
the three RE groups (P<0.05).
The levels of serum inflammatory factors were
gradually elevated in patients with different severity of RE, which
were significantly higher than those in NERD group. Besides, the
more severe the mucosal injury was, the higher the levels of
inflammatory factors would be (P<0.05) (Table II).
| Table II.Comparison of levels of serum
inflammatory factors between RE group and NERD group (mean ± SD,
pg/ml). |
Table II.
Comparison of levels of serum
inflammatory factors between RE group and NERD group (mean ± SD,
pg/ml).
| Group | n | IL-1β | IL-8 | TNF-α |
|---|
| NERD | 12 | 26.8±3.7 | 18.6±3.3 | 30.4±4.2 |
| Grade I RE | 15 | 42.5±8.2a | 37.5±4.5a | 44.6±5.7a |
| Grade II RE | 18 | 54.8±7.2a,b | 46.7±5.5a,b | 57.4±5.6a,b |
| Grade III RE | 15 | 65.6±8.3a,b | 72.2±6.1a–c | 63.2±6.3a,b |
Changes in levels of VIP, motilin and
LESP
The results revealed that the VIP level was
15.2±3.92, 21.4±5.12, 29.2±5.32 and 37.8±7.23 pg/l in NERD, grade I
RE, grade II RE and grade III RE group, respectively, and the level
of motilin was 298.26±89.11, 245.22±80.49, 215.07±78.45 and
171.32±45.60 ng/l in the four groups, respectively. Thus, the
results show that with the increase in severity of disease, the VIP
level was gradually increased and the level of motilin was
gradually decreased in RE group. The levels of VIP, motilin and
LESP had statistically significant differences between RE group and
NERD group and the levels of VIP had statistically significant
differences among the three RE groups (P<0.05) (Table III).
| Table III.LESP, VIP and motilin levels in RE
group and NERD group (mean ± SD). |
Table III.
LESP, VIP and motilin levels in RE
group and NERD group (mean ± SD).
| Group | n | LESP (mmHg) | VIP (pg/l) | Motilin (ng/l) |
|---|
| NERD | 12 | 23.55±4.53 | 15.2±3.92 | 298.26±89.11 |
| Grade I RE | 15 |
15.32±5.01a |
21.4±5.12a |
245.22±80.49a |
| Grade II RE | 18 |
11.21±4.56a |
29.2±5.32a,b |
215.07±78.45a |
| Grade III RE | 15 |
5.43±5.24a |
37.8±7.23a–c |
171.32±45.60a |
Changes in 24 h esophageal pH
indexes
The results manifested that the DeMeester score was
42.3±9.9, 44.9±9.1, 58.6±10.2 and 70.7±12.5 points in NERD, grade I
RE, grade II RE and grade III RE group, respectively, suggesting
that the more severe injury corresponds to the higher DeMeester
score. The DeMeester score had statistically significant
differences in grade II RE group and grade III RE group compared
with NERD group, and between grade II RE group and grade III RE
group (P<0.05). Besides, the longest time of reflux, severe acid
exposure ratio and total times of reflux in RE group were greater
than those in NERD group (P<0.05) (Table IV).
| Table IV.Changes in 24 h esophageal pH indexes
in RE group and NERD group. |
Table IV.
Changes in 24 h esophageal pH indexes
in RE group and NERD group.
| Group | n | DeMeester score
(points) | Longest time of
reflux (min) | Severe acid exposure
ratio [n (%)] | Total times of
reflux |
|---|
| NERD | 12 | 42.3±9.9 | 15.9±4.9 | 2 (16.6) | 2.38±1.17 |
| Grade I RE | 15 | 44.9±9.1 | 20.2±6.1 | 3 (20.0) | 3.22±1.49 |
| Grade II RE | 18 |
58.6±10.2a |
24.6±5.5a | 5 (27.7) |
4.38±1.55a |
| Grade III RE | 15 |
70.7±12.5a,b |
33.5±7.2a,b | 8
(53.3)a,b |
6.12±1.67a,b |
Correlation of DeMeester score with
inflammatory factors and motilin
According to the correlation analysis, the serum
T-SOD, IL-8, TNF-α and VIP levels in RE patients were positively
correlated with the DeMeester score (r=0.6376, 0.5241, 0.5958 and
0.6118, P<0.01), confirming that the abnormal expression levels
of inflammatory factors in vivo are important factors
leading to progression of RE. The more severe the disease was, the
stronger the inflammatory response state in vivo would be.
Moreover, the motilin level in RE patients was negatively
correlated with the DeMeester score (r=−0.2283, P<0.05),
indicating that the abnormal expression of gastrointestinal
hormones is also an important factor causing upper gastrointestinal
motility disorders in RE (Figs.
1–5).
Discussion
RE is an upper gastrointestinal motility disorder,
whose common clinical symptom is gastrointestinal dysfunction. RE
is mainly diagnosed using gastroscopy and esophageal pH value. In
recent years, a large number of studies have focused on the
etiology and pathogenesis of RE, and scholars have found that the
occurrence of esophageal mucosal injury and extraoesophageal
complications in RE patients are closely associated with ILs and
oxidative stress factors (6–8).
In the present study, the detection results of 24 h
esophageal pH indexes showed that the DeMeester score was 42.3±9.9,
44.9±9.1, 58.6±10.2 and 70.7±12.5 points in NERD, grade I RE, grade
II RE and grade III RE group, respectively, suggesting that the
more severe injury corresponds to the higher DeMeester score. The
DeMeester score had statistically significant differences in grade
II RE group and grade III RE group compared with NERD group, and
between grade II RE group and grade III RE group (P<0.05).
In the pathogenesis of GERD, ROS plays a crucial
role, which can alter the normal function of enzymes and proteins
(9). The results of this study
revealed that the content of MDA was 8.1±3.6, 12.3±4.9, 21.2±6.7
and 35.5±5.7 nmol/g protein in NERD, grade I RE, grade II RE and
grade III RE group, respectively, and the T-SOD activity was 56±21,
41±14, 30±15 and 19±9 U/mg protein, respectively, in the four
groups, displaying statistically significant differences
(P<0.05). It can be seen that the content of MDA in the
esophageal mucosa was higher in RE group than that in NERD group,
and the T-SOD activity declined with the increased severity of
injury (P<0.05), indicating that the levels of oxidative stress
indexes are related to the degree of mucosal injury.
Gastric acid and pepsin in RE patients can stimulate
immune cells to release a variety of inflammatory mediators,
directly damaging the esophageal mucosal epithelial cells (10). Previously, it was reported that the
expression levels of IL-1β and IL-8 in the esophageal mucosa in RE
patients are increased, and the degree of mucosal injury is
positively correlated with the IL-8 level (11,12).
After effective drug or operative treatment, the IL-8 expression
level in the esophageal mucosa declines, and it is found in the
subsequent monitoring that the IL-8 level continuously increases in
recurrence patients (13). TNF-α
produced by macrophages and immune cells stimulates neutrophils to
produce large amounts of ROS, leading to peroxidation of the
esophageal mucosa (14). Moreover,
studies have found that the increased levels of inflammatory
factors are important factors leading to esophageal peristalsis
disorder (15), and TNF-α can also
promote the development of RE into Barrett's esophagitis to some
extent (16). In the present study,
the levels of serum IL-1β, IL-8 and TNF-α were the highest in grade
III RE group, followed by grade II RE group, grade I RE group and
NERD group (P<0.05). According to the Pearson's correlation
analysis, the serum T-SOD, IL-8 and TNF-α were positively
correlated with the DeMeester score (r=0.6376, 0.5241 and 0.5958,
P<0.01), confirming that the abnormal expression levels of
inflammatory factors in vivo are important leading to the
progression of RE. The severer the disease is, the stronger the
inflammatory response state in vivo will be.
VIP is a polypeptide with 28 amino acids in the
gastrointestinal tract, which can regulate the esophageal
peristalsis and esophageal sphincter relaxation under normal
conditions (17). Upon the stimuli
of pathological factors, VIP in vivo has an elevated level
and binds to a large number of gastrointestinal epithelial cell
receptors, reducing LESP and leading to GRED (18). The main role of motilin, another type
of gastrointestinal hormone secreted by the duodenal and jejunal
mucosa, is to affect the interdigestive gastrointestinal motility.
According to previous clinical studies, the serum motilin level in
patients with gastric motility disorder is lower than that in the
normal people (19). In this
experiment, the VIP level was 15.2±3.92, 21.4±5.12, 29.2±5.32 and
37.8±7.23 pg/l in NERD, grade I RE, grade II RE and grade III RE
group, respectively, and the level of motilin was 298.26±89.11,
245.22±80.49, 215.07±78.45 and 171.32±45.60 ng/l, respectively, in
the four groups. It can be seen that in the three RE groups, the
plasma VIP level was obviously higher than that in NERD group,
while the motilin level was evidently lower than that in NERD group
(P<0.05). The positive correlation between motilin level and
LESP has been previously confirmed (20). In addition, the Pearson's correlation
analysis showed that the motilin level in RE patients was
negatively correlated with the DeMeester score (r=−0.2283,
P<0.05), indicating that the abnormal expression of
gastrointestinal hormones is also an important factor causing upper
gastrointestinal motility disorders in RE.
In conclusion, RE patients have strong oxidative
stress and inflammatory response, an increased level of serum
regulator of gastrointestinal motility (VIP) and a decreased level
of motilin, and the expression levels of these indexes are
associated with the severity of RE. Controlling the changes in the
above factors using effective treatment means can improve the
development of GERD.
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current
study are available from the corresponding author on reasonable
request.
Authors' contributions
YD wrote the manuscript. YD and LP collected and
analyzed general data of patients. WQ helped with indexes detection
and analysis. All the authors read and approved the final
manuscript.
Ethics approval and consent to
participate
The study was approved by the Ethics Committee of
Changzhou Jintan District People's Hospital (Changzhou, China) and
informed consents were signed by the child parents or
guardians.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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