Soybean is nutrient-rich, not only because of its high protein content but
because it also contains more balanced amino acids (Liener,1994;
Naismith, 1955). Thus, soybeans and soybean meals are
high-quality plant protein sources for livestock and poultry. However, soy protein
that contains antigens can cause allergic reactions and reduce the absorption
of nutrients by young animals. Sissons and Smith (1976)
reported that the main allergy-causing antigenic components of soy protein for
weanling pigs are glycinin and β-conglycinin. Glycinin and β-conglycinin,
which exhibit antigen activity in soybean, primarily belong to cotyledon proteins.
Glycinin, also known as 11S protein, is composed of six subunits; each subunit
contains an acidic chain and a basic chain linked with each other through a
disulfide bond (Golubovic et al., 2005). β-conglycinin,
also known as 7S protein, is a class of glycosylated protein (Hou
and Chang, 2004) that contains three subunits associated with each other
by hydrophobic interactions; it has a relative molecular mass of 180 Kda, containing
4-5% of carbohydrates (Ogawa et al., 1995; Yamauchi
et al., 1975).
Peptide transporter 1 (Pept1), is a member of the family of solute carriers
that rely on the production of a peptide (POT) and plays a key role in the absorption
of a peptideDivalent metal transporter 1 (DMTl), also known as soluble carrier
family 11 number 2 (SLC11A2), is the ion transporter in the protons of mammals.
Zheng et al. (2011) DMTl can transport Fe2+,
Zn2+, Mn2+, Co2+, Cd2+, Cu2+,
and so on. No studies on the effect of glycinin and β-conglycinin on the
expression of PEPT1 and DMTl in young animals have been reported. In this study,
therefore, purified glycinin, β-conglycinin and in vitro cultured
mouse intestinal epithelial cells were used as experimental materials. The effects
of glycinin and β-conglycinin on the absorbing capacity of mouse intestinal
epithelial cells were investigated.
MATERIALS AND METHODS
Test animals: Healthy 10-day-old Kunming mice were obtained from the Center
of Animal Science, Si Chuan Agricultural University.
Reagents: Glycinin and β-conglycinin were extracted and isolated
in the laboratory.
DMEM high glucose medium (GIBCO U.S. companies), trypsin XI, medium protease
I, insulin, epidermal growth factor, fetal calf serum, sorbitol, L-glutamyl
ammonium, HEPES, pyruvate, penicillin, streptomycin, etc. were purchased from
Sigma (USA). The Minim Na-K ATP enzyme test kit was bought from Nanjing Jiancheng
Bioengineering Institute, while the SV Total RNA Isolation system, SYBR Premix
Ex Taq II, DNase (RNase-free), gel extraction kit, Taq DNA Polymerase, SDS,
proteinase K and dNTP were purchased from Takara Bio Co., Ltd. The reverse transcription
kit was purchased from Promega Corporation.
Instruments and equipment: The instruments and equipment used were as
follows: CO2 incubator (Heraeus, Germany), enzyme-linked immunoassay
analyzer (Thermo Corporation USA), UV spectrophotometer (Beckman Inc.), Clean
Benches (Harbin East Union Electronic Technology Development Co., Ltd.), inverted
microscope (Zeiss, Germany company), ordinary centrifuge, micro pipette, real-time
PCR instrument (Bio-Rad company), protein nucleic acid detector (BeckmanCoulter)
and gel imaging system (Bio-Rad).
The isolation of intestinal epithelial cells was conducted following the methods
of Evans et al. (1992) with modifications. First,
the duodenum and jejunum were cut using eye scissors and then shredded into
small pieces of approximately 1 mm3. The shredded pieces were then
digested with collagenase and neutral protease. The residual enzyme solution
was discarded and the cells were washed and resuspended. The end result was
a culture of intestinal epithelial cells in fluid suspension with a concentration
of 105 cells mL-1. Cell suspension of 500 μL volume
was inoculated into each well of a 24 hole cell culture plate. The original
culture medium was replenished with a new culture medium every 48 h. Cells were
observed every day until the culture plate bottom showed a large colony and
then the cell culture plates were covered with the end of the forthcoming. Processing
was then implemented.
Design of trials: A single-factor experimental design with six treatment
groups was used. Each treatment group consisted of 4 replicates (the cell survival
test for 10 repetitions) for each well. The six treatment groups were designated
as 1-1, 1-2, 1-3, 2-1, 2-2 and 2-3; soybean globulin and β-conglycinin
extract were added at concentrations of 0, 1, 5 and 10 mg mL-1. The
design of the trials is shown in Table 1.
Na-K ATP enzyme determination: Soy protein antigen-treated cells
were cultured for 36 h, then collected. Cell samples were prepared and stored
in a refrigerator at -20°C.
||Experimental design of the trials
Determination of medium Na-K ATP enzyme was conducted according to the operating
instructions kit of the DU-800 UV spectrophotometer.
Determination of PEPT1 and DMT1 expression
Primer synthesis: The primers were designed using the Premier 5.0
software. According to the PEPT1 gene sequence, a pair of primers for the amplification
length of 136 bp is the upstream Primer F1: 5'-CAGTATCTCCAAATGCCCAGGAA-3'; Primer
R1: 5'-TTTGGCCCTAAACCAACATATCAAC-3'. According to the DMT1 gene sequence, a
pair of primers for the amplification length of 90 bp is the upstream primer
F2: 5'-CTGTCCGGCCTCAACGATCTA-3'; Primer R2: 5'-TGGCATGCTGGTGAAAGTCAA-3'. According
to the GenBank sequence of mouse β-actin mRNA internal reference sequence
primers designed to amplify a length of 171 bp, the upstream primer F3 is: 5'-CATCCGTAAAGACCTCTATGCCAAC-3';
downstream primer R3 is: 5'-ATGGAGCCACCGATCCACA-3', with an amplification length
of 171 bp. The design of the primers was determined by NCBI BLAST web tool search.
The initial validation of specificity was also searched. The primers were synthesized
by TaKaRa (Dalian) Company. Total RNA extraction, reverse transcription and
real-time quantitative PCR were performed according to the kits instructions.
Data processing and statistical analysis: Single-factor analysis was
performed on the data obtained for Na-K-ATP enzyme determination using SPSS11.0
Analysis of variance with Duncans multiple comparison method was used
to test the differences between the results. Data were presented as means±standard
deviation and significant differences were further analyzed by regression analysis
of indicators. Gene expression in the sample with β-actin as an internal
reference was analyzed using the ΔΔ Ct method: the average relative
concentration = 2-average ΔΔ Ct, which was calculated using
the time at which each genome copy number template was initiated for the average
relative content of PEPT1 and DMT1.
Effect of soybean antigen protein on cell survival rate: Soybean β-conglycinin
and glycinin extracts had significant effects (p<0.01) on the MTT OD of mouse
IEC in primary culture (Table 2). The effect on the control
group MTT OD was significantly higher than that on the 1-2, 1-3, 2-2 and 2-3
groups (p<0.01); the effect on the 1-1 group MTT OD was significantly higher
than that on the 1-2 and 1-3 groups (p<0.01); the effect on the 2-1 group
MTT OD was significantly higher than that on the 2-2 and 2-3 groups (p<0.01).
||Soybean β-conglycinin and glycinin in cultured mouse
intestinal cells Na-K-ATP enzyme activities
|Different superscript capital letters indicate level of significance
= 0.01. Different lowercase letters indicate level of significance = 0.05,
whereas same letters indicate no significant difference (p>0.05)
||PEPT1 mRNA expression dynamics
||DMT1 mRNA expression dynamics
Soy protein antigen PEPT1, DMT1 mRNA expression of genes: Glycinin and
β-conglycinin inhibited the expression of PEPT1 and DMT1 genes in mouse
intestinal epithelial cells (Table 3 and 4).
As antigen protein concentration increased, the inhibition of PEPT1 and DMT1
gene expression gradually increased. A protein antigen concentration of 5 mg
mL-1 decreased PEPT1 gene expression by 7%; at an antigen protein
concentration higher than 5 mg mL-1, the inhibition by β-conglycinin
was lower than 11% and that by glycinin was significantly reduced. The effect
of the antigen on DMT1 gene expression suggests that at low doses (5 mg mL-1)
of the antigen, DMT1 gene expression could be reduced by 50%, whereas at high
doses (5 mg mL-1 above), the inhibition effect was even more pronounced.
Li et al. (1990) and Qiao
et al. (2003) have previously reported that high dietary concentrations
of soybean antigen protein can be harmful to piglet intestinal integrity and
immune function, thereby inhibiting the growth of piglets. Burrells
et al. (1999) reported that high concentrations of soy protein undermine
the integrity of salmon gut, thus inhibiting growth. Li
et al. (1990) reported that soybean protein extract fed to 7-day-old
piglets resulted in a daily weight loss of 6 g over 5 days. At 21 days of age
after weaning, piglets fed with soy protein added to the corresponding weaning
diets showed a relatively high titer of anti-glycinin and β-conglycinin
antibody in their serum.
With an increase in the added soy protein antigen, PEPT1 expression decreased.
Correlation analysis shows that PEPT1 expression, as well as soybean β-conglycinin
and glycinin dosage showed a significant negative correlation (R = -0.94), indicating
that soy protein reduces the antigen expression of PEPT1, thereby reducing the
absorption of small peptides in the intestinal epithelial cells. Studying the
human colon cancer cell line Caco-2. Baron-Delage et
al. (1996) showed that when transfected with the proto-oncogene, the
intestinal cell-specific hexose transporter (SGLT1), specific brush border enzymes
and association with hair growth gene expression significantly decreased. The
relationship between soy protein antigen and PEPT1 gene expression may be due
to several reasons.
First, any expression or function of the carriers protein expression
regulator has its own substrate. PEPT1 substrates for transport are mainly dipeptides
and tripeptides and β-conglycinin and glycinin may have corresponding binding
sites. However, the two proteins on the intestinal epithelial cells are antigens,
which triggered cellular immune responses when combined with epithelial cells.
Consequently, the expression of PEPT1 was reduced, as well as the antigenic
protein and cells, thereby reducing cell damage. Second, as the β-conglycinin
and glycinin dosages increased, cell damage also gradually increased, which
caused cells to mobilize stored energy and proteins for damage repair and immune
functions, such as cytokine secretion and Na-K-ATP enzyme accelerated synthesis.
These cost substantial energy and amino acid cells, resulting in reduced nutrient
absorption and cell gene expression. Third, the intestinal epithelial cell function
may have been altered after reaction with the antigen, including nutrient absorption.
In addition, some severely damaged cells die, to a certain extent, affected
the absolute expression of PEPT1.
DMT1 gene expression may be regulated by many factors, such as inflammatory
mediators, TNF-α, IFN-γ, protein kinase C and developmental stage,
among others. Ludwiczek et al. (2003) confirmed
that proinflammatory cytokines and lipopolysaccharides affect DMT1 expression
in macrophages. In the present paper, increasing the amount of soy protein antigen
resulted in decreased DMTl gene expression. DMTl gene expression and the quantity
of β-conglycinin added exhibited a negative correlation (R = -0.78). Similarly,
DMT1 and the quantity of soybean globulin were negatively correlated (R = -0.75).
After the addition of soy antigens, the integrity of cell damage (part of the
antigen protein in the cell) directly interfered with DMTl of gene transcription.
Meanwhile, epithelial cells exhibiting allergic reactions increased in inflammatory
cytokines; cell metabolic disorders and the self-regulation of DMTl expression
may have affected the expression of DMTl. Reduced DMTl gene expression will
lead to a decrease in the absorption of divalent metal ions, such as Zn2+,
which are necessary for RNA transcription enzymes; these, in turn, reduces the
expression of DMTl genes. The Na-K-ATP enzyme functions as Na-K pump and enzyme,
indicating that it is involved in energy metabolism, material transport, oxidative
phosphorylation and other important biochemical processes, as well as ion balance
regulation. Thus, Na-K-ATP activity changes can affect many important cellular
In this study, the addition of soy protein antigen in mouse intestinal cells
increased Na-K-ATP enzyme activity. The self-protection mechanism of the cells
may result to the increased consumption of a large amount of energy. Thus, the
cell membrane Na and K-ATP enzyme are in a highly active metabolic state, leading
to decreased levels of ATP within the cell membrane. These decreased levels
affect other important intracellular biochemical reactions and ultimately influence
the nutrient absorption of normal cells (Lytton et al.,