Introduction
The main biological effect of erythropoietin (EPO)
has been manifested through the promotion of proliferation,
differentiation, and maturation of erythroid progenitor cells, as
well as an increase in the number of red blood cells in the
circulation (1). Therefore, EPO is
widely used in the treatment of neonatal, cancer and renal anemia.
Recent findings have shown that EPO and EPO receptors are widely
distributed (2). They are expressed
in a variety of non-hematopoietic tissues, and are also equipped
with a variety of non-hematopoietic biological effects, such as
angiogenic promotion, anti-inflammation, anti-apoptosis and
neuroprotection and are considered to be a systemic protective
cytokine (2). It has been found that
breast milk contains EPO, and intestines of neonatals contain EPO
receptors, which indicate that EPO may play an important
physiological role in the growth and development of the
gastrointestinal tract (3). Previous
studies suggested that the recombinant EPO in a mouse model of
neonatal necrotizing enterocolitis (NEC) can reduce the
inflammatory reaction and improve NEC injury (4).
The aim of the present study was to examine the
clinical effect of EPO on the inflammatory response and possible
mechanism of action of the TLR4/NF-κB signaling pathway, through
clinical trials, to provide a new basis for the clinical treatment
of NEC.
Patients and methods
Patients
In total, 94 cases diagnosed with NEC between
August, 2013 and August, 2015 were selected for the present study.
The diagnosis of NEC conformed to the standard of the American
Society for Parenteral and Enteral Nutrition (5). The study was approved by the Ethics
Committee of The Fourth Hospital of Shijiazhuang. Informed consent
from the guardian, according to the order of admission was
obtained. The 94 patients with NEC were randomly divided into the
control (42 cases) and observation (52 cases) groups. A comparison
of the baseline information identified that differences were
statistically significant (P<0.05). Table I shows that NEC staging was performed
in accordance with the standard of Bell's NEC staging.
 | Table I.Comparison of baseline information
between the control and observation groups. |
Table I.
Comparison of baseline information
between the control and observation groups.
| Group | Control group,
n=42 | Observation group,
n=52 | t (χ2
test) | P-value |
|---|
| Male/female | 23/19 | 28/24 | 0.008 | 0.929 |
| Age, day | 13.4±2.5 | 12.5±2.4 | 0.624 | 0.337 |
| Weight, kg | 2.2±0.6 | 2.1±0.5 | 0.421 | 0.564 |
| 1 min Apgar
score | 8.2±0.7 | 8.3±0.6 | 0.519 | 0.768 |
| NEC staging |
|
|
|
|
| I | 6 (14.3) | 6 (11.5) | 0.920 | 0.631 |
| II | 26 (61.9) | 37 (71.2) |
|
|
| III | 10 (23.8) | 9 (17.3) |
|
|
| Premature birth | 25 (59.5) | 28 (53.8) | 0.305 | 0.581 |
| Caesarean
section | 20 (47.6) | 23 (44.2) | 0.107 | 0.743 |
| Breast feeding | 29 (69.0) | 33 (63.5) | 0.373 | 0.830 |
| Milk powder
feeding | 6 (14.3) | 8 (15.4) |
|
|
| Breast + milk powder
feeding | 7 (16.7) | 11 (21.2) |
|
|
| Hemoglobin, g/l | 185.6±13.7 | 184.2±14.5 | 0.967 | 0.325 |
| Hematocrit | 62.4±1.7 | 62.5±1.6 | 0.624 | 0.857 |
Research methods
The control group were given the standard medical
treatment plan, which included fasting, no hydration,
gastrointestinal decompression, parenteral nutrition, selection of
sensitive antibiotics, early stage, full weight, appropriate amount
of rehydration, maintainance of gastrointestinal tissue perfusion,
correct anemia, electrolyte disorder, acid-base imbalance, blood
pressure, heart rate, blood glucose and respiratory support. The
abdominal X-ray was regularly performed to observe changes of the
disease. If the disease was not relieved or continued to
deteriorate with conservative treatment of internal medicine,
surgical resection was subsequently performed. The observation
group was treated with a combined application of recombinant EPO
(National Medical License no. S20000007, 2,000 IU/dose; Shandong
Kexing Bioproducts Co., Ltd., Shandong, China) for intramuscular
injection, at a dose of 200 IU/kg, twice a week for a total of 1
week. Any allergic reaction or other adverse reactions were
observed closely.
Observation index and detection
technology
The differences of the complication and death rates
were compared between the two groups, including levels of tumor
necrosis factor-α (TNF-α), interleukin (IL)-6 and the mRNA
expression levels of Τoll-like receptor 4 (TLR4) and nuclear
factor-κB (NF-κB). Venous blood (5 ml) was collected on an empty
stomach and centrifuged at 2,800 g at high speed and the
supernatant was removed after 5 min. TNF-α and IL-6 kits were
purchased from Shanghai J&M Co. Ltd. (Shanghai, China). The
ELISA method was applied and the experiment was performed according
to the manufacturer's instructions. The expression of mRNA for TLR4
and NF-κB was detected by quantitative polymerase chain reaction
(TaqMan probe method) and the SYBR-Green I staining method
(Shinegene, Shanghai, China), respectively. Subsequently, the
standard curves of the target genes (TLR4 and NF-κB)
and housekeeping genes (GAPDH) were created. By means of the
standard curves, the expression of the target and housekeeping
genes in the samples was quantified. The relative expression amount
of TLR4 and NF-κB in each group was determined after correction of
the housekeeping genes.
Statistical analysis
SPSS 20.0 statistical software (IBM SPSS, Armonk,
NY, USA) was applied to determine statistical analysis. Measurement
data were presented as mean ± standard deviation. The t-test was
applied for comparisons between groups and the Kruskal-Wallis H was
applied to make pathological scoring of intestinal tissues.
Enumeration data were presented as a percentage (%). The
χ2 test was applied for comparisons between groups.
P<0.05 was considered to indicate a statistically significant
difference.
Results
Comparison of complication and death
rates
The complication and death rates in the observation
group were significantly lower than those in the control group and
differences were statistically significant (P<0.05) (Tables I and II).
| Table II.Comparison of complication and death
rates. |
Table II.
Comparison of complication and death
rates.
| Group | Cases | Septicemia | Acute intestinal
perforation | Infectious
peritonitis | Others | Complication
rate | Death rate |
|---|
| Control group | 42 | 7 | 6 | 6 | 2 | 21 (50.0) | 11 (26.2) |
| Observation
group | 52 | 5 | 5 | 4 | 2 | 16 (30.8) | 5 (9.6) |
| χ2
test |
|
|
|
|
| 3.868 | 4.519 |
| P-value |
|
|
|
|
| 0.049 | 0.034 |
Comparison of the expression levels of
TNF-α and IL-6
Prior to intervention, differences between the two
groups with regard to the levels of TNF-α and IL-6 were not
statistically significant (P>0.05). After intervention, the
levels of TNF-α and IL-6 of the control group were increased while
the levels of the observation group were reduced. The levels of the
observation group were significantly lower than those of the
control group and differences were statistically significant
(P<0.05) (Table III).
| Table III.Comparison of the levels of TNF-α and
IL-6 between the observation and control groups. |
Table III.
Comparison of the levels of TNF-α and
IL-6 between the observation and control groups.
|
| TNF-α, ng/l | IL-6, ng/l |
|---|
|
|
|
|
|---|
| Group | Pre-intervention |
Post-intervention | Pre-intervention |
Post-intervention |
|---|
| Control | 56.7±6.4 | 78.4±10.2 | 241.3±36.9 | 287.5±48.7 |
| Observation | 61.2±6.8 | 32.5±4.8 | 259.6±42.3 | 143.2±32.5 |
| t-test | 0.529 | 6.498 | 0.629 | 6.213 |
| P-value | 0.423 | 0.012 | 0.415 | 0.018 |
Comparison of mRNA expression levels
of TLR4 and NF-κB
Prior to intervention, differences between the two
groups with regard to the mRNA expression levels of TLR4 and NF-κB
were not statistically significant (P>0.05). After intervention,
the mRNA expression levels of TLR4 and NF-κB in the two groups were
significantly decreased and those in the observation were lower
than those in the control group. Differences were statistically
significant (P<0.05) (Table
IV).
| Table IV.Comparison of mRNA expression levels
of TLR4 and NF-κB. |
Table IV.
Comparison of mRNA expression levels
of TLR4 and NF-κB.
|
| TLR4 | NF-κB |
|---|
|
|
|
|
|---|
| Group | Pre-intervention |
Post-intervention | Pre-intervention |
Post-intervention |
|---|
| Control | 3.3±0.5 | 2.6±0.3 | 2.4±0.6 | 1.8±0.2 |
| Observation | 3.6±0.7 | 1.2±0.4 | 2.7±0.5 | 0.7±0.1 |
| t-test | 0.924 | 7.436 | 0.825 | 8.125 |
| P-value | 0.721 | <0.001 | 0.364 | <0.001 |
Discussion
EPO is a glycoprotein with a molecular weight of
30.4 kDa. In the fetus period, it is mainly synthesized by liver
and post birth, and the kidney. Ledbetter and others (6) found from a retrospective study that the
incidence of NEC on premature infants with EPO injection was
significantly lower than that of infants not treated with EPO
(7). To study the protective
mechanism of EPO on NEC, Kumral and others (8) used EOP to preprocess hypoxia
reperfusion NEC mice (EPO 750 U/kg/week, intraperitoneal injection
in 3 shots for 2 weeks). On day 15, the animals were exposed to
100% CO2 for anoxia for 5 min, followed by reoxygenation
with 100% O2 for 10 min, and then executed after 4 h.
Compared to the result of the experimental group, the level of
nitric oxide in the EPO pretreatment group was significantly
decreased, and the injury of the intestinal tissue was reduced,
indicating that EPO retarted the occurrence of NEC in mice by
inhibiting the excessive production of nitric oxide (9). Akisu and others (10) used the same method to study the role
of EPO in the production of intestinal malondialdehyde (MDA) and
platelet activating factor (PAF). Their study results showed that
although EPO cannot inhibit the production of PAF in the necrotic
intestine tissues resulting from hypoxia reperfusion, it can
significantly decrease the MDA content in the intestinal tissues of
mice, indicating that EPO may play a role in the prevention and
treatment of NEC by inhibiting the lipid peroxidation mediated by
oxygen-free radicals (11). Shiou
and others (12) have shown that
supplementing EPO via the intestinal tract upregulated the
expression of ZO-1, a tight junction protein in the enterocytes of
NEC mice, which protected the intestinal mucosal barrier function
(13). Halpern and Denning (7) used 3-day SD mice and constructed a NEC
model after artificial feeding and hypoxia cold stimulation, and
intervened by supplementing EPO of a similar physiological
concentration as breast milk through the intestinal tract. Their
study results showed that EPO downregulated the expression of TLR4
in intestinal tissues of mice and inhibited the inflammatory
cascade reaction, reduced the intestinal tissue damage, retarted
the malignant progression, and finally reduced the occurrence of
bacterial translocation (3,12).
The role of TLR4 in the pathogenesis of NEC has
aroused increasing attention (14).
The expression of TLR4 in normal intestinal epithelial cells is
low, whereas that in NEC children and animal models is high. The
high expression of TLR4 appeared even before NEC tissue injury.
High-risk factors of NEC, including premature birth, artificial
feeding, hypoxia ischemia, and bacterial factors can lead to an
increase of TLR4 expression (4). The
role of TLR4 in the pathogenesis of NEC was mainly associated with
inflammation and repair (15).
Activated TLR4 can activate NF-κB, and induce the upregulated
expression of downstream inflammatory cytokines. NF-κB in
intestinal tissues removed from animal models and NEC children
showed a sustained high expression, while the expression of the
inflammatory cytokines in the lower reaches, such as TNF-α, IL-6
and IL-8, also increased. A large number of inflammatory mediators
leads to damage in the intestinal epithelial cells and mucosal
defense mechanism, which would ultimately develop into NEC.
Inhibiting of the TLR4 pathway can significantly reduce the
expression of inflammatory mediators, alleviate macrophage
infiltration, and reduce the occurrence of bacterial translocation
(16,17).
Results of the present study show that the
complication and death rates in the observation group were lower
than those in the control group. The levels of TNF-α and IL-6 in
the observation group were significantly lower than those in the
control group. The mRNA expression levels of TLR4 and NF-κB in the
observation group were significantly lower than those in the
control group, and differences were statistically significant. In
conclusion, EPO can reduce the levels of inflammatory response of
TNF-α and IL-6 through the TLR4/NF-κB signaling pathway and improve
NEC, thereby providing a basis for the clinical treatment of
NEC.
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