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Archives of Animal Nutrition

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Effects of glutamine supplementation

on the immune status in weaning
piglets with intrauterine growth
retardation
a a a a
Xiang Zhong , Wei Li , Xuexin Huang , Yuanxiao Wang , Lili
a a a a
Zhang , Yanmin Zhou , Ahmad Hussain & Tian Wang
a
College of Animal Science and Technology, Nanjing Agricultural
University, Nanjing, Jiangsu, China

Version of record first published: 16 May 2012

To cite this article: Xiang Zhong, Wei Li, Xuexin Huang, Yuanxiao Wang, Lili Zhang, Yanmin
Zhou, Ahmad Hussain & Tian Wang (2012): Effects of glutamine supplementation on the immune
status in weaning piglets with intrauterine growth retardation, Archives of Animal Nutrition,
DOI:10.1080/1745039X.2012.683325

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 Archives of Animal Nutrition
iFirst article 2012, 110

Eects of glutamine supplementation on the immune status in weaning

piglets with intrauterine growth retardation
Xiang Zhong, Wei Li, Xuexin Huang, Yuanxiao Wang, Lili Zhang, Yanmin Zhou,
Ahmad Hussain and Tian Wang*

College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu,
China
(Received 20 January 2012; accepted 20 March 2012)
Downloaded by [Southeast University] at 23:42 28 June 2012

Neonates with intrauterine growth retardation (IUGR) often suer from

impaired cellular immunity, and weaning may further aggravate adverse eects
of IUGR on development and function of the immune system. In this study, we
investigated eects of glutamine supplementation on immune status in the
intestines of weaning pigs with IUGR, focusing on molecular mechanisms
underlying altered immune response. Piglets with IUGR were weaned at 21 days
of age and received orally 1.22 g alanine or 1 g glutamine per kg body weight
every 12 h. Weight gain and intestinal weight of weaning piglets were increased by
glutamine supplementation. Levels of serum IgG in piglets supplemented with
glutamine were increased compared with Control piglets. The production of IL-1
and IL-8 in the serum and jejunum was decreased by glutamine supplementation,
whereas the levels of IL-4 in the serum and the concentrations of IL-4 and IL-10
in the jejunum were increased. The expression of heat shock protein 70 (Hsp70) in
the jejunum was increased by glutamine supplementation, but the degradation of
inhibitor kB and the activity of nuclear factor-kB (NF-kB) were decreased. In
conclusion, glutamine supplementation enhanced immune response in weaning
piglets with IUGR. The eects of glutamine in IUGR are associated with
increased Hsp70 expression and suppression of NF-kB activation.
Keywords: cytokines; glutamine; heat shock proteins; piglets; growth retardation;
immune responses; immune globulins

1. Introduction
Intrauterine growth retardation (IUGR) is dened as retarded growth and
development of the mammalian embryo/foetus or its organs during gestation due
to intrauterine infection, genetic causes, congenital malformations, environmental
insults or severe malnutrition (Wu et al. 2006). IUGR aects 510% of human infants
and 1520% of newborn pigs and is considered a major problem in human medicine
and animal production (McMillen and Robinson 2005; Wu et al. 2006). IUGR
increases neonatal mortality and morbidity, the latter including poor physical
growth, permanent maldevelopment and increased susceptibility to infection (Wang
et al. 2008a, 2008b; DInca et al. 2010; Zhong et al. 2012). In humans, IUGR infants
suer from persistent immunological impairment throughout childhood (Raqib et al.

*Corresponding author. Email: tianwangnjau@163.com

ISSN 1745-039X print/ISSN 1477-2817 online

2012 Taylor & Francis
http://dx.doi.org/10.1080/1745039X.2012.683325
http://www.tandfonline.com
 2 X. Zhong et al.

2007). IUGR infants exhibit low IgG levels and low numbers of B and T cells in cord
blood or thymus (Contreras et al. 2011). Furthermore, IUGR leads to altered
cytokine proles in placenta and in the foetus (Hahn-Zoric et al. 2002; Amu et al.
2006).
Glutamine is the most abundant free amino acid in the body and is involved in
several important functions in the intestine including energy metabolism, synthesis of
nucleotides, hexosamines and other amino acids, and cellcell signalling (Wu et al.
1995). Glutamine acts as a main nutritional product for enterocytes and as an
activation marker in lymphocytes in the immune system (Horio et al. 2008). A
previous study showed that glutamine reduced pro-inammatory cytokine release
and decreased multiple organ system dysfunction and mortality in a rat model of
endotoxemia (Wischmeyer et al. 2001). In humans, glutamine treatment signicantly
decreased production of pro-inammatory cytokines (IL-6 and IL-8) in the intestinal
mucosa (Coeer et al. 2002). In addition, glutamine preserved expression of anti-
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inammatory cytokines in lipopolysaccharide (LPS)-stimulated intestinal lamina

propria (Fukatsu et al. 2001). Therefore, glutamine is thought to enhance immune
response in humans and other animals or cell lines; however, little is known about
the eects of glutamine supplementation on IUGR neonates in postnatal life,
especially during weaning periods.
The intestine is not merely a digestive absorptive organ; it is also the largest
immune organ of the body. Moreover, the intestinal barrier is involved in the rst
steps of postnatal immune system maturation and lymphocytes rst appear in the
intestinal subepithelium early in development (Macpherson and Harris 2004).
Notably, IUGR impairs morphological and functional maturation of the intestine
(DInca et al. 2010; Zhong et al. 2010), leading to subsequent impairment of the
intestinal immune system due to the strong connection between the digestive and
immune systems. Therefore, appropriate postnatal nutrition is likely to be essential
for enhancing immune response in IUGR neonates.
In this study, we used the newborn piglet, a widely used animal model for
studying physiology and nutrition in the human infant, to investigate the eects of
glutamine supplementation on immune response and to probe the molecular
mechanisms underlying these eects in the intestine of weaning pigs with IUGR.

2. Material and methods

2.1. Animals and experimental design
All experiments were conducted in accordance with the Guide for the Care and Use
of Laboratory Animals prepared by the Institutional Animal Care and Use
Committee, Nanjing Agricultural University, China. Pregnant sows (Large
White 6 Landrace) were fed a gestating diet (2 kg/d) during the entire period of
pregnancy and had free access to drinking water. After farrowing, newborn pigs with
IUGR were marked and remained with the sow. Piglets with birth weight two
standard deviations below the mean birth weight of the total population were dened
as IUGR (Wang et al. 2005). At 21 days of age, 10 piglets with IUGR
(3.75 + 0.50 kg body weight [BW]) taken from 10 litters were weaned and assigned
randomly into one of the two treatment groups (Control or oral glutamine
supplementation) with ve piglets per group. Piglets were housed in pens with slotted
steel oors in an environmentally controlled room (27.0 + 0.58C) and had access to
feed and water ad libitum. The diet (Table 1) met the NRC (1998) requirements for
 Archives of Animal Nutrition 3

Table 1. Composition and nutrient levels of the basal diet (as-fed basis).

Ingredients Content [%]

Corn 58.0
Soybean meal 17.0
Extruded full-fat soybean 7.0
Fish meal 4.5
Dried whey 4.5
Wheat middling 5.0
CaHPO4 1.5
Salt 0.25
Limestone 1.1
Lys-HCl 0.15
Premix* 1.0
Nutrients Content
Analysed
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Crude protein [%] 20.1

Lysine [%] 1.12
Methionine cystine [%] 0.65
Calcium [%] 0.90
Phosphorus [%] 0.75
Calculated
Digestible energy [MJ/kg] 13.9

Note: *Provided per kg of diet: vitamin A (trans-retinyl acetate), 2200 IU; vitamin D3 (cholecalciferol),
220 IU; vitamin E (all-rac-tocopherol-acetate), 16 IU; vitamin K3 (bisulfate menadione complex), 0.5 mg;
biotin, 0.05 mg; folic acid, 0.3 mg; pantothenic acid (D-Ca pantothenate), 10 mg; niacin, 15 mg; vitamine
B2 (riboavin), 3.6 mg; vitamine B1 (thiamine-mononitrate), 1.0 mg; vitamine B6 (pyridoxine
hydrochloride), 1.5 mg; Cu (CuSO4  5H2O), 6 mg; Fe (FeSO4  7H2O), 100 mg; Zn (ZnSO4  7H2O),
110 mg; Mn (MnSO4  H2O), 4 mg; Se (Na2SeO3), 0.3 mg; I (KI), 0.14 mg.

nutrient content. Piglets received 1.22 g alanine or 1 g glutamine per kg BW every

12 h. Glutamine was dissolved in distilled water at room temperature and
administered orally using a 10-ml syringe. Control piglets received alanine as an
isonitrogenous control supplement as described by Kim et al. (2004).

2.2. Sample collection

One hour after feeding on day 14 postweaning, ve piglets per treatment were
sacriced and blood samples were collected as previously described (Zhong et al.
2010). Serum was obtained after centrifugation at 3000 g for 15 min at 48C and
stored at 7808C. In neonatal pig, the small intestine was dened as the portion of
the digestive tract between the pylorus and the ileocaecal valve, with the rst 10-cm
segment being duodenum, and was divided into duodenum, jejunum and ileum. The
duodenum, jejunum and ileum were rinsed with saline and weighed after removal of
luminal contents. The middle portion of the duodenum, jejunum and ileum was
immediately placed in liquid nitrogen for protein analysis.

2.3. Determination of IgG and IgM

Serum concentrations of IgG and IgM were measured using immunoassay kits from
Triple J Farms (Bellingham, WA, USA) according to the manufacturers
instructions.
 4 X. Zhong et al.

2.4. Cytokine analysis

Frozen intestine samples were homogenised as previously described (Zhong et al.
2010). The homogenate was centrifuged at 1200 g for 15 min at 48C. The
supernatant was collected and total protein was determined by the BCA method.
Concentrations of cytokines (IFN-g, IL-4, IL-10, IL-1 and IL-8) in the serum and
intestinal lysates were determined using a commercially available pig ELISA kit
according to the manufacturers instructions (RapidBio Lab, Calabasas, CA, USA).
The sensitivities of the IFN-g, IL-4, IL-10, IL-1 and IL-8 assays were 30 pg/ml, 5 pg/
ml, 10 pg/ml, 10 pg/ml and 10 pg/ml, respectively. Samples were assayed in triplicate.
Assays were performed using a microplate ELISA reader at 450 nm and
concentrations were expressed as picograms per millilitre in the serum and picograms
per microgram protein in the intestine.
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2.5. Protein immunoblot analysis

Protein was isolated from 200 mg of frozen tissue using a total protein extraction
kit and cytoplasmic and nuclear protein extraction kit (Beyotime Biotechnology,
Haimen, China). Protein concentrations were determined using a BCA protein
assay kit according to manufacturers protocol (Beyotime Biotechnology, Haimen,
China). Proteins (40 mg per lane) were resolved by 10% SDS-PAGE and
transferred to nitrocellulose membranes. Membranes were blocked with 5% skim
milkPBS/0.1% Tween 20 for 1 h before overnight incubation at 48C with mouse
monoclonal anti-Hsp70 (SPA-810, Stressgen, San Diego, CA, USA), rabbit
polyclonal anti-p65 (Bioworld, Dublin, OH, USA), mouse polyclonal anti-IkBa
(Cell Signaling Technology, Beverly, MA, USA) or rabbit polyclonal anti-b-actin
antibody (Cell Signaling Technology, Beverly, MA, USA), diluted 1:1000 in 5%
skim milk-PBS/0.1% Tween 20. Membranes were washed thrice in PBS/0.1%
Tween 20 and incubated with horseradish peroxidase-conjugated secondary
antibody diluted 1:3000 in 5% skim milkPBS/0.1% Tween 20 for 1 h. Following
successive washes, the membranes were developed using an ECL kit (Pierce,
Rockford, IL, USA).

2.6. Statistical analysis

All data are expressed as mean + standard error of the mean. The results were
statistically analysed by the t test, using the Statistical Package for Social Sciences
16.0 software. Dierences between the Control group and the Glutamine group were
considered signicant at p 5 0.05.

3. Results
3.1. Growth and intestinal weight
Table 2 summarises the eect of oral glutamine supplementation on growth in
weaned piglets with IUGR. The daily weight gain of piglets supplemented with
glutamine was 27% greater (p 5 0.05) than that of the Control piglets. There was no
dierence in daily feed intake between the groups. The feed conversion ratio was
lower in piglets administered glutamine (p 5 0.05). Average jejunum weight, relative
to body weight, was greater in piglets administered glutamine (p 5 0.05) (Table 2).
 Archives of Animal Nutrition 5

Table 2. Eect of oral glutamine supplementation on growth performance and intestinal

weight in weaned piglets with intrauterine growth retardation on day 14 postweaning (n 5
per treatment).

Experimental groups
Control Glutamine
BW at birth [kg] 0.98 + 0.025 0.96 + 0.031
BW at weaning [kg] 3.62 + 0.24 3.89 + 0.11
BW gain after weaning [g/d] 91.4 + 4.9a 116.2 + 8.1b
Feed intake after weaning [g/d] 179 + 10 194 + 11
Feed conversion ratio [kg feed/kg gain] 1.97 + 0.12a 1.67 + 0.03b
Intestinal weight [g/kg BW]
Duodenum 1.12 + 0.18 1.13 + 0.02
Jejunum 15.4 + 0.40a 17.2 + 0.55b
Ileum 15.4 + 0.65 16.8 + 0.74
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Note: Means with dierent superscripts dier signicantly at p 5 0.05.

There was no dierence in the duodenum and ileum weight relative to body weight
between the treatment groups.

3.2. Concentrations of IgG, IgM and cytokines

Compared with the Control group, the levels of serum IgG and IgM in the
Glutamine group were increased (8.36 + 1.06 vs. 11.73 + 1.02 [p 5 0.05] and
1.21 + 0.10 vs. 1.56 + 0.11 [p 0.065], respectively). In the serum, there was no
dierence in the concentrations of IFN-g and IL-10 between the groups. Serum
levels of IL-1 and IL-8 were lower in the Glutamine group than in Controls
(p 5 0.05) (Table 3). Conversely, serum levels of IL-4 were increased in the
Glutamine group compared with the Control group (p 5 0.05). In the jejunum, the
concentrations of IL-1 and IL-8 were decreased in the Glutamine group (p 5 0.05);
however, the levels of IL-4 and IL-10 were increased compared with the Control
group (p 5 0.05) (Table 3). There was no dierence between the groups in the level
of any cytokine in the ileum.

3.3. Expression of Hsp70, NF-kB and I-kB

The expression of Hsp70 in the jejunum of weaning piglets supplemented with
glutamine was increased compared with control piglets (p 5 0.05) (Table 4). There
were no dierences in the levels of Hsp70 protein in the ileum between the groups.
The degradation of I-kB in the jejunum and ileum of the Glutamine group was
decreased compared to Control piglets (p 5 0.05) (Table 4). The activity of NF-kB
in the jejunum and ileum was lower in the Glutamine group (p 5 0.05) (Table 4).

4. Discussion
Intrauterine growth retardation has been linked to decits in several aspects of
adaptive immunity, including involution of lymphoid tissues such as the thymus,
suppression of antibody responses to vaccinations and the development of atopy and
autoimmune disease during postnatal life (Cromi et al. 2009; Contreras et al. 2011).
 6 X. Zhong et al.

Table 3. Concentrations of cytokines in blood serum, jejunum and ileum of weaned piglets
with intrauterine growth retardation on day 14 post weaning (n 5 per treatment).

Experimental groups
Control Glutamine
Serum [pg/ml]
IFN-g 117 + 5.4 122 + 4.8
IL-1 56.1 + 2.54a 45.1 + 1.68b
IL-8 116 + 4.2a 94 + 4.0b
IL-4 796 + 49a 975 + 40b
IL-10 66.7 + 4.7 52.0 + 3.8
Jejunum [pg/mg protein]
IFN-g 9.86 + 0.90 10.36 + 1.56
IL-1 7.72 + 0.57a 5.36 + 0.57b
IL-8 3.29 + 0.40a 1.35 + 0.18b
IL-4 42.1 + 2.40a 53.2 + 3.3b
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IL-10 4.40 + 0.26a 5.96 + 0.53b

Ileum [pg/mg protein]
IFN-g 7.31 + 0.67 8.96 + 1.51
IL-1 6.54 + 0.33 6.36 + 0.82
IL-8 2.10 + 0.13 2.41 + 0.44
IL-4 39.6 + 3.20 41.0 + 5.00
IL-10 4.65 + 0.42 5.04 + 0.70

Note: Means with dierent superscripts dier signicantly at p 5 0.05.

Table 4. The relative abundance of Hsp70, cytoplasmic I-kB and nuclear NF-kB p65
subunits in the jejunum and ileum of weaned piglets with intrauterine growth retardation
(means + SEM, n 5 per treatment)*.

Experimental groups
Control Glutamine
Jejunum
Hsp70/b -actin 0.518 + 0.015a 0.578 + 0.015b
I-kB/b -actin 0.315 + 0.017a 0.377 + 0.010b
p65/b-actin 0.488 + 0.015a 0.418 + 0.015b
Ileum
Hsp70/b-actin 0.406 + 0.018a 0.445 + 0.032a
I-kB/b-actin 0.348 + 0.012a 0.408 + 0.015b
p65/b-actin 0.466 + 0.018a 0.375 + 0.032b

Notes. *The results were normalised by the level of b-actin expression in each individual sample. Means
with dierent superscripts dier signicantly at p 5 0.05.

Weaning, a critical stage of postnatal growth and gut development in mammals, may
further aggravate adverse eects of IUGR on the development and functions of the
immune system in the intestine, since the weaning period is associated with
impairment of cellular immunity and an increased prevalence of gastrointestinal
infection in many species (Sun et al. 2008; Ewaschuk et al. 2011). Therefore,
appropriate postnatal nutrition appears to be essential for enhancement of immune
function in IUGR neonates. In the present study, it was shown that oral glutamine
supplementation improved growth, increased levels of serum IgG and altered
 Archives of Animal Nutrition 7

production of pro-inammatory cytokines (IL-1 and IL-8) and anti-inammatory

cytokines (IL-4 and IL-10) in weaning piglets with IUGR. Furthermore, glutamine
supplementation induced expression of Hsp70 protein, inhibited the degradation of
I-kB and decreased the activity of NF-kB in the intestine of weaning piglets with
IUGR.
Glutamine acts not only as the main nutrient for enterocytes but also as a
modulator of immune cell function and cytokine production. There are several
potential reasons for its central role. Glutamine is an important energy source for
immune cells. In addition, glutamine is one of the major precursors from which
purines and pyrimidines are synthesised, of particular importance given the high
proliferation rate in immune cells. Moreover, glutamine regulates protein
synthesis in immune cells (Newsholme 2001). Therefore, under stressful conditions
or in pathological situations, the immune system requires more glutamine (Pai
et al. 2008). During the weaning period, supplementing the diet with glutamine
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signicantly modied immune cells in the mesenteric lymph node by increasing

the proliferative response to pokeweed mitogen (Johnson et al. 2006). In infected
early weaned pigs, glutamine supplementation can normalise lymphocyte function
(Yoo et al. 1997). In the present study, we observed that the levels of serum IgG
in the Glutamine group were increased compared to the Control group. Yu et al.
(2002) also reported that supplementation with glutamine combined with
nucleotides enhanced the concentration of IgG and IgA in weaning piglets.
Glutamine has been shown to reduce proinammatory cytokine release and
enhance the production of anti-inammatory cytokines in intestinal mucosa
(Coeer et al. 2003). In human gut mucosa, glutamine decreased the in vitro
production of the pro-inammatory cytokines, IL-6 and IL-8, and increased the
production of the anti-inammatory cytokine, IL-10, during experimental stimula-
tion of the inammatory response (Coeer et al. 2003). In the present study, we
found that the production of IL-1 and IL-8 in the serum of weaning piglets was
decreased by supplementation with glutamine, but the concentrations of IL-4 and
IL-10 in the serum were increased. In addition, we observed similar results in the
jejunum; however, there was no dierence in cytokine production in the ileum of
weaning piglets. This discrepancy may be associated with glutamine ux in the small
intestine, since most glutamine is taken up by the proximal part of the small intestine
(James et al. 1998). Moreover, the present data showed that glutamine
supplementation did not aect the concentrations of IFN-g in the serum and
intestine. However, Johnson et al (2006) found that the production of IFN-g was
decreased by treatment with glutamine in peripheral blood mononuclear cells from
weaning piglets. This discrepancy could be due to dierent experimental procedures
such as the dose and route of glutamine supplementation.
Glutamine aects expression of several transcription factors involved in
inammation, cell proliferation, apoptosis and cell survival (Joon Suh et al. 2003;
Wang et al. 2008b). NF-kB is an essential transcription factor that regulates
transcription of genes involved in the early inammatory response such as
cytokines, chemokines and adhesion molecules and plays a central role in the
pathobiology of inammation (Dokladny et al. 2010). Treatment with glutamine
decreases NF-kB protein expression in the intestine of irradiated rats (Erbil et al.
2005). In an experimental model of colitis in the rat, glutamine prevented the
decrease in I-kB and the subsequent increase in nuclear p65 and inhibited
overexpression of pro-inammatory genes (Fillmann et al. 2007). Interestingly,
 8 X. Zhong et al.

weaning induced degradation of I-kB and activated NF-kB translocation to the

nucleus, where it binds to specic target genes, such as pro-inammatory genes
(Clarkson et al. 2000; Zaragoza et al. 2005). In the present study, the degradation
of I-kB and the concentrations of NF-kB in the jejunum and ileum of weaning
piglets supplemented with glutamine were decreased, suggesting that the inhibition
of NF-kB activation is one of the mechanisms whereby glutamine exerts its
protective eect.
The mechanism by which glutamine modulates cytokine production and inhibits
NF-kB activation may be associated with overexpression of Hsp70. Hsp70 is one of
the most abundant and best-characterised proteins in the heat shock proteins family
and is a molecular chaperone involved in the folding of nascent and misfolded
proteins under normal conditions. A number of studies have showed that exogenous
glutamine induces the expression of Hsp70 mRNA and protein under normal
conditions or in stressful situations (Zhang et al. 2009). The present data indicated
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that the expression of Hsp70 in the jejunum of weaning piglets with IUGR was
increased by supplementation with glutamine, whereas there was no dierence in the
ileum. The varying expression of Hsp70 among intestinal segments may be
associated with glutamine ux in the small intestine. It has been shown that
Hsp70, as an anti-inammatory molecule, suppresses cytokine gene transcription
and decreases the release of inammatory mediators (Hall 1994). LPS-stimulated
increases in TNF-a and IL-6 were prevented by Hsp70 overexpression in cultured
macrophages (Hagiwara et al. 2007). Overexpression of Hsp70 in animals is sucient
to inhibit LPS-induced increases in cytokine expression (Dokladny et al. 2010).
Notably, upregulation of Hsp70 can impair NF-kB signalling. Dokladny et al (2010)
found that Hsp70 inhibited LPS-induced NF-kB p65 nuclear translocation and
IkBa degradation in vivo. Zheng et al. (2007) also revealed that Hsp70 bound to the
NF-kB:IkB complex, preventing I kB phosphorylation and decreasing expression of
several NF-kB-regulated genes. Taken together, these results indicated that
glutamine modulates cytokine production by inducing overexpression of Hsp70
and inhibiting activation of NF-kB.
In conclusion, glutamine supplementation enhanced immune response in
weaning piglets with IUGR. The mechanisms of glutamine action may be associated
with an increase in Hsp70 expression and suppression of NF-kB activation. The
results suggest a potential strategy for enhancing neonatal immune response using
nutritional modication.

Acknowledgments
This project was supported by a grant from National Natural Science Foundation of China
(no. 30972116), Foundation of Nanjing Agricultural University (no. KJ2011010), the
Specialized Research Fund for the Doctoral Program of Higher Education of China (no.
20110097120033) and a project funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions.

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