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Indian Pediatr 2017;54:
825-829 |
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Relationship Between Immune Parameters and
Non-alcoholic Fatty Liver Disease in Obese Children
|
Jun-Qi Shi, Wen-Xia Shen, Xiang-Zhi Wang, Ke Huang
and Chao-Chun Zou
From Department of Endocrinology, Children’s
Hospital, Zhejiang University School of Medicine; Zhejiang Province;
China.
Correspondence to: Dr Chao-Chun Zou, Children’s
Hospital, Zhejiang University School of Medicine, Binsheng Road,
Hangzhou 310051, Zhejiang Province, China.
Email: [email protected]
Received: May 27, 2016;
Initial Review: November 03, 2016;
Accepted: June 30, 2017.
Published online: July 11, 2017.
PII:S097475591600073
|
Objective: To investigate the
relationship between immune parameters and non-alcoholic fatty liver
disease (NAFLD) in obese children.
Design: Cross-sectional study.
Setting: Hospital-based study in
Zhejiang Province, China between July to September 2015.
Participants: A total of 117
obese children and 209 healthy non-obese children were studied as the
obese and control groups. Depending on the severity of NAFLD, the obese
group was divided into subgroups 1 (without NAFLD), 2 (with simple fatty
liver) and 3 (with steatohepatitis).
Outcome Measures: Glucose
metabolism, lipid metabolism and immune parameters.
Results: In the obese group,
body mass index (BMI), waist-and hip-circumferences, fasting insulin,
Homeostasis model of assessment for insulin resistance (HOMA-IR),
triglyceride, total cholesterol, low density lipoprotein cholesterol
(LDL-C), apolipoprotein (Apo)B/ApoA1, alanine aminotransferase, uric
acid, white blood cells, neutrophils percentage, platelet and
interleukin (IL)-6 were significantly higher than those in the controls
(P<0.05), while lower high density lipoprotein cholesterol and
lymphocyte percentage were noted (P<0.05). IL-10 in the subgroup
3 was higher than those in the control group, subgroup 1 and 2 (P<0.05).
Logistic regression analysis showed that BMI, LDL-C, HOMA-IR and IL-10
were independent factors of NAFLD (P<0.05).
Conclusion: These results
support a low-grade chronic inflammation in obese children. Moreover,
obesity, dyslipidaemia and IR are risk factors while IL-10 may be a
protective factor for NAFLD.
Keywords: Cytokine, Interleukin-6,
Interleukin-10, Metabolic syndrome, Overweight
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N on-alcoholic fatty liver
disease (NAFLD) is a severe complication of obesity. It is a kind of
hepatic steatosis, inflammation and fibrosis, which is characterized by
fatty infiltration in over 5% hepatocytes without a history of alcohol
intake, viral infections, autoimmune hepatitis or drug-induced liver
disease. According to pathologic changes and liver function, NAFLD is
divided into simple fatty liver (SFL), non-alcoholic steatohepatitis
(NASH) and cirrhosis [1]. With the increase of childhood obesity, the
incidence of NAFLD has increased recently. Studies on NAFLD have
suggested that free fatty acid, triacylglycerol and insulin resistance
(IR) were involved in pathogenesis of NAFLD [2,3]. However, the
pathogenesis of NAFLD is not fully understood till now.
Recently, multitude evidence has revealed that
obesity was a low-grade chronic inflammation process. Interleukin
(IL)-6, tumor necrosis factor-alpha (TNF- a)
may contribute to inflammation [4-6]. TNF-a
was reported to be closely related to IR. In addition, some cytokines
may play a protective role in the progression of NAFLD, such as IL-4 and
IL-10 [7]. However, studies on the cytokines and NAFLD in children were
rare. We studied the relationship between immune parameters and NAFLD to
explore the risk and protective factors for NAFLD.
Methods
A total of 117 obese children (81 males), were
studied as the obese group aged between 5.2 to 14.8 years. They were
hospitalized in the Endocrinology Department of Children’s Hospital,
Zhejiang University School of Medicine during July to September 2015 to
definite etiology and investigate the complications of obesity, such as
disorder of glucose metabolism or lipid metabolism disorders. Their body
mass index (BMI) ranged from 22.2 to 42.2 kg/m 2.
Children with viral hepatitis, drug-induced hepatitis, autoimmune liver
disease, infection, severe heart and kidney disease, or alcohol
consumption were excluded. Another 209 non-obese healthy children aged
6-14 year from Children Health Care Department in our hospital were
enrolled as the control group. Their BMI ranged from 11.6 to 20.3 kg/m2.
The age difference between the two groups was significantly different (P=0.03)
(Table I).
TABLE I Baseline Parameters in the Control and Obese Groups
|
Control group (n=209) |
Obese group (n=117) |
P value |
Female/male |
149/60 |
81/36 |
0.706 |
Age (y) |
10.3 (1.8) |
10.8 (2.2) |
0.028 |
Body mass index (kg/m2) |
15.6 (1.9) |
28.5 (3.8) |
<0.001 |
Waist circumference (cm) |
55.7 (5.6) |
91.1 (10.7) |
<0.001 |
Hip circumference (cm) |
65.4 (6.2) |
95.8 (9.3) |
<0.001 |
*Fasting glucose (mmol/L) |
5.3 (5.1-5.5) |
5.3 (5.0-5.7) |
0.632 |
*Fasting insulin (pmol/L) |
6.9 (4.6-9.6) |
17.8 (12.7-26.3) |
<0.001 |
*HOMA-IR |
1.6 (1.1-2.2) |
4.1 (3.1-6.5) |
<0.001 |
*Triglyceride (mmol/L) |
0.8 (0.6-1.1) |
1.3 (1.0-1.9) |
<0.001 |
Cholesterol (mmol/L) |
4.4 (0.8) |
4.7 (0.8) |
0.009 |
*Lipoprotein A (mmol/L) |
89.0 (44.0-201.0) |
79.0 (47.5-158.0) |
0.369 |
HDL-C (mmol/L) |
1.6 (0.3) |
1.3 (0.3) |
<0.001 |
LDL-C (mmol/L) |
2.7 (0.5) |
2.9 (0.5) |
<0.001 |
ApoB/ApoA1 |
0.3 (0.1) |
0.5 (0.2) |
<0.001 |
*Alanine aminotransferase (U/L) |
15 (13-20) |
34 (23-69) |
<0.001 |
*Aspartate aminotransferase (U/L) |
27 (24-30) |
28 (22-44) |
0.120 |
Uric acid (µmol/L) |
271.2 (65.4) |
405.9 (94.2) |
<0.001 |
HOMA-IR, homeostasis model of assessment for insulin resistance
index; HDL-C, high density lipoprotein-cholesterol; LDL-C, low
density lipoprotein-cholesterol; Apo, apolipoprotein; *Median
(IQR). |
Depending on the severity of NAFLD, the obese group
was divided into three subgroups: Subgroup 1 (Obesity without
NAFLD, N=23), Subgroup 2 (Obesity with SFL, N=43)
and Subgroup 3 (Obesity with NASH, N=51). Subgroup 1 (mean
age, 9.8 (2.5)y, 14 males) had BMI from 22.4 to 32.0 kg/m2
(mean BMI, 26.1 (2.5) kg/m2).
Subgroup 2 (mean age, 10.8 (2.2)y, 30 males) had BMI from 22.2 to 34.9
kg/m2 (mean BMI, 28.2 (3.1)
kg/m2). Subgroup 3 (mean
age, 11.3 (1.9) y, 37 males) had BMI from 22.5 to 42.2 kg/m2
a (mean BMI, 29.7 (4.2) kg/m2).
The difference of age between the groups was statistically significant (P=0.003).
This study was approved by the Ethics Committee of
the Children’s Hospital of Zhejiang University School of Medicine.
Informed consent was obtained from the parents of these children.
Obesity was diagnosed according to the criteria that
BMI was above 95th percentile compared with the same age and sex
children (Height and weight standardized growth charts for Chinese
children and adolescents aged 0 to 18 years) [8]. NAFLD was diagnosed
according to the revised definition and treatment guidelines for NAFLD
by the Chinese Hepatology Association in 2010 [9]. NAFLD was diagnosed
as a diffusely echogenic change on liver B-mode ultrasonography (fatty
infiltration in liver), with or without elevated liver enzyme (alanine
aminotransferase and aspartate aminotransferase). SFL was defined as a
diffusely echogenic change with normal liver enzyme while NASH was
defined as a diffusely echogenic change with elevated liver enzyme.
Anthropometric indicators were measured by
professionals with unified calibrated measurement tools. Body height was
measured to the nearest 0.1 cm, after subjects took off their shoes,
standing on the measuring instrument. Body weight was measured to the
nearest 0.1 kg on a scale. Waist circumference was measured to the
nearest 0.1 cm by measuring at the midpoint between the lower rib margin
and the iliac crest using a conventional tape in centimeters. Hip
circumference was measured to the nearest 0.1 cm by measuring maximum
hip circumference. BMI was calculated as weight (kg) divided by height
(m) squared.
Blood samples were taken after overnight fasting and
placed into tubes at room temperature. Then serum samples were isolated
and stored at -70°C until use. Fasting blood glucose, insulin,
triglyceride, total cholesterol, high density lipoprotein cholesterol
(HDL-C), low density lipoprotein cholesterol (LDL-C), alanine
aminotransferase (ALT), aspartate aminotransferase (AST), lipoprotein A,
uric acid, apolipoprotein (Apo) A1 and ApoB were measured in our
clinical laboratory center. IR was individually estimated by the
homeostasis model of assessment for IR (HOMA-IR) by considering as
(fasting insulin×fasting glucose)/22.5.
Serum Th1/Th2 cytokines were measured by flow
cytometry. IL-2, IL-4, IL-6, IL-10, TNF- a
and interferon (IFN)–a
were quantitatively determined by the CBA kit-BDTM
CBA Human Th1/Th2 Cytokine Kit II (BD Biosciences, San Jose, CA).
Peripheral blood cell count, including white blood cells, percentage of
neutrophils, percentage of lymphocytes and platelet, were measured in
our clinical laboratory center.
Statistical analysis: Statistical analyses were
conducted by using SPSS software (SPSS Inc. Chicago, USA). The Pearson
Chi-square test was used to analyze the difference of categorical data
between different groups. Quantitative data with normal distribution
were analyzed by the independent t test or One-way ANOVA followed by LSD
test when appropriate. Fasting insulin, HOMA-IR, triglyceride,
lipoprotein A, ApoB/ApoA1, IL-2, IL-4, IL-6 and TNF- a
were analyzed after logarithmic transformation. Skewed data were
compared with Mann-Whitney U test. Logistic regression analysis was
performed to analyze the independent factors of NAFLD (assignment:
control group = 1, subgroup 1 =2, subgroup 2=3, subgroup 3=4). Factors
with P<0.3 in the comparison between obese and control groups
were enrolled in the logistic regression analysis. The differences were
considered significant at P<0.05.
Results
In this study, 125 obese children were screened and 8
children were excluded becuuse of hepatitis B or Prader-Willi syndrome.
Also, 221 non-obese children were screened and 12 children were excluded
because of hepatitis B, atrial septal defect or hypothyroidism. A total
of 117 obese children and 209 non-obese children were enrolled in the
obese and control groups in the following analysis. We diagnosed NAFLD
in 94 (80.3%) obese children.
Compared to the control group, higher BMI, waist
circumference, hip circumference, fasting insulin, HOMA-IR,
triglyceride, cholesterol, LDL-C, ApoB/ApoA1, ALT and uric acid, and
lower HDL-C were found in the obese group (P<0.05, respectively).
However, fasting glucose, lipoprotein A and AST were not significantly
different between the obese and control groups (Table I).
Among the 117 obese children, NAFLD was found in 94 (80.3%), including
43 (36.7%) in subgroup 2 and 51 (43.6%) in subgroup 3. Comparing the
control and obese subgroups, BMI, waist circumference, hip
circumference, fasting insulin, HOMA-IR, triglyceride, total
cholesterol, LDL-C, HDL-C, ApoB/ApoA1, uric acid and ALT were
significantly different (P<0.05, respectively), although fasting
blood glucose and lipoprotein A were not different among these groups (P>0.05,
respectively). BMI, waist circumference, hip circumference, fasting
insulin, HOMA-IR, triglyceride, LDL-C, ALT and uric acid have increased
tendency while HDL-C have decreased tendency among the control group and
subgroups of obese group.
Higher white blood cells, percentage of neutrophils,
platelet and IL-6, and lower percentage of lymphocytes in the obese
group were noted (P<0.05). The difference of IL-10 was marginal
between the control and obese groups (P=0.07). IL-2, IL-4, TNF- a
and INF-a were
not significantly different between the obese and control groups (Table
II). Moreover, white blood cells and platelet in the 3 subgroups
were higher than those in the control group (P<0.05).
TABLE II Immune Parameters Between the Control Group and Obese Group
|
Control group |
Obese group |
|
N=209 |
N=117 |
*White blood cell (×109/L) |
7.5 (1.6) |
9.0 (2.1) |
*Neutrophils (%) |
54.2 (9.7) |
58.5 (7.5) |
*Lymphocyte (%) |
37.5 (9.0) |
33.2 (6.8) |
*Platelet (×109/L) |
287.8 (63.3) |
316.5 (67.4) |
#IL-2 (pg/mL) |
3.5 (3.1-4.0) |
3.4 (3.0-4.0) |
#IL-4 (pg/mL) |
3.0 (2.6-3.6) |
2.9 (2.5-3.3) |
*#IL-6 (pg/mL) |
2.7 (2.2-3.5) |
3.8 (2.9-5.3) |
#IL-10 (pg/mL) |
2.8 (2.4-3.2) |
2.9 (2.4-3.5) |
#TNF-a (pg/mL) |
2.8 (2.4-3.2) |
2.8 (2.4-3.2) |
#IFN-a (pg/mL) |
6.4 (5.1-8.0) |
6.1 (4.6-8.6) |
IL: interleukin; TNF: tumor necrosis factor; IFN: interferon;
*P<0.001; All values in mean (SD) except # in Median (IQR). |
Logistic regression analysis showed that BMI, LDL-C,
HOMA-IR and IL-10 were independent determinants for NAFLD (P<0.001,
P=0.024, P=0.009 and P=0.024) (Web
Table I).
Discussion
We found NAFLD in 94 (80.3%) of the 117 obese
children. Logistic analysis revealed that BMI was the most significant
independent determinant of NAFLD in these children, which confirmed that
obesity was a main risk factor for NAFLD in children [10,11].
There were several limitations in our study. Firstly,
the study was not a prospective study, but a cross-sectional analysis.
Secondly, NAFLD, SFL or NASH was diagnosed only by ultrasonic and serum
liver enzymes without pathological biopsy of the liver. Thirdly, the
sample size in our study was small. Also, we did not investigate the
accurate role of IL-10 on the NAFLD.
In our study, we noted higher white blood cells and
neutrophils percentages in obese children, which were consistent with
studies from adults and mice [12-14]. Moreover, higher levels of IL-6
were noted in obese children, which suggested that IL-6 might play a
role in obesity. Although the white blood cells, neutrophils, platelet
and IL-6 were not independent factor by the logistic analysis, these
data supported systemic low-grade inflammation in obese children and
white blood cells may be a cause of increased production of cytokines
[15].
Several studies in adults or animal model have found
that imbalance between pro- and anti-inflammatory cytokines was involved
in the second hit of NAFLD [16]. In this study, we noted that IL-10 and
IL-6 levels increased with the severity of NAFLD in obese children.
Several studies have reported that IL-10 played a role as a protective
factor for liver injury induced by alcoholic, hepatitis C and liver
steatosis [17]. Also, animal model studies suggested protective effect
of IL-10 against liver steatosis, fibrosis and other metabolic disorders
[18,19]. Our results seem to be contrary against the above studies, but
consistent with a human study, which found that the IL-10 mRNA was
higher in obese patients with NAFLD, and especially in obese patients
with NASH [20]. Also, higher IL-10 levels were noted in mice after
long-term high fat diet fed, and IL-10-/-
mice displayed greater liver inflammatory response after high fat diet
feeding [21]. These implied that IL-10 is a protective factor, which may
increase to compensate inflammation and NAFLD in obesity.
The effect of IL-6 on the NAFLD reported from
different studies was seen to be contradictory. It was reported to play
a protective effect in early stage of hepatic steatosis by inhibiting
oxidative stress and preventing mitochondrial dysfunction [16]. However,
it was reported to mediate inflammatory of hepatocyte, induce apoptosis
of hepatocytes and IR in the subsequent progress of fatty liver [22,23].
Animal model found that hepatocytes exposed to exogenous IL-6
accelerated self-repairing in short-term, but aggravated injury in
long-term [24].
In summary, our data support low-grade chronic
inflammation in obese children. Moreover, obesity, dyslipidemia and IR
are risk factors of NAFLD in children. IL-10 may be a protective factor
for liver, with increased compensatory levels in children with NAFLD.
Acknowledgments: Guan-Ping Dong, Wei Wu, and Li
Zhang for patient-care and organization.
Contributors : CCZ: concept and design; JQS, WXS:
data collection and analysis; JQS: drafting the manuscript; XZW, KH:
help in patient organization and data collection. Jun-Qi Shi and Wen-Xia
Shen contributed equally to the article.
Funding : This work is supported, in part, by
National Natural Science Foundation of China (81170787 & 81670786),
Zhejiang Health and Family Planning Commission (2016KYA128) and Zhejiang
Provincial Program for the Cultivation of High-Level Innovative Health
Talents (2015).
Competing interest: None stated.
What is Already Known?
•
The roles of cytokines in NAFLD
in obese children is not well-elucidated.
What This Study Adds?
•
Obesity, dyslipidemia and insulin-resistance are risk
factors of non-alcoholic fatty liver disease in children.
|
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