Soybean Glycinin- and Î²-Conglycinin-Induced Intestinal Damage in

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Soybean Glycinin- and #-Conglycinin-Induced Intestinal Damage in Piglets via p38/JNK/NF-#B Signaling Pathway Chenglu Peng, Chengming Cao, Mengchu He, Yingshuang Shu, Xuebing Tang, Yuanhong Wang, Yu Zhang, Xiaodong Xia, Yu Li, and jin jie Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03641 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018

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Soybean Glycinin- and β-Conglycinin-Induced Intestinal Damage in Piglets via the p38/JNK/NF-κB Signaling Pathway Short title: Soybean-induced signaling in pig intestine

1

Chenglu Peng, Chengming Cao, Mengchu He, Yingshuang Shu, Xuebing Tang,

2

Yuanhong Wang, Yu Zhang, Xiaodong Xia, Yu Li, Jinjie Wu*

3 4

College of Animal Science and Technology, Anhui Agricultural University, 130 West

5

changjiang Road, Hefei 230036, China

6

*

7

Anhui Agricultural University, 130 West changjiang Road, Hefei 230036,

8

China

9

E-mail addresses: [email protected] (J. J. Wu)

Corresponding authors at college of Animal Science and Technology,

10 11 12 13 14 15

1


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Abstract 16

β-conglycinin (7S) and glycinin (11S) are known to induce a variety of

17

hypersensitivity reactions involving the skin, intestinal tract, and respiratory tract. The

18

present study aimed to identify the mechanism underlying the development of allergy

19

to soybean antigen proteins, using piglets as an animal model. Weaned “Duroc ×

20

Landrace × Yorkshire” piglets were fed a diet supplemented with 7S or 11S to

21

investigate the signaling pathway involved in intestinal damage in piglets. Results

22

showed that serum nitric oxide (NO), tumor necrosis factor-α (TNF-α), and caspase-3

23

levels were significantly higher in 7S- and 11S-fed piglets compared to that in

24

suckling or weaned ones. mRNA, protein, and phosphorylation levels of nuclear

25

factor-kappa B (NF-κB), p38, and Jun N-terminal kinase (JNK) were higher in 7S-

26

and 11S-fed piglets than in suckling and weaned ones. Overall, our results indicate

27

that 7S and 11S damaged the intestinal function in piglets through their impact on

28

NF-κB, JNK, and p38 expression.

29 30

Keywords: NF-κB, JNK, p38, β-conglycinin, glycinin, piglet, soybean, antigenic

31

proteins

32

2



Introduction 33

Soybean is rich in essential amino acids and is the most significant source of

34

vegetable protein in the human diet. However, soybean causes 90 % of all

35

immunoglobulin E (IgE)-mediated food allergies worldwide1. According to Huang et

36

al.2, the Wuzhishan minipig allergy models, induced by soybean glycinin and

37

β-conglycinin, may be used to determine the potential allergenicity of new proteins.

38

Increased use of various soybean products, owing to their excellent functional

39

properties and favorable amino acid profiles, has raised the incidence of soybean

40

allergies, both in humans and animals; piglets too suffer severe allergic reactions

41

against soybean3-8. Antigenic proteins identified in soybean include β-conglycinin

42

(7S), α-conglycinin, glycinin (11S), and γ-conglycinin, of which, 7S and 11S class

43

proteins are the essential molecular property. Previous studies had investigated the

44

effect of 7S or 11S sensitization on the growth performance5,9,10; levels of serum

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cytokine (IFN-c), and interleukins (IL)-4, IL-2, IL-6, and IL-10; and intestinal

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tight-junction proteins of piglets11-13. It was demonstrated that 7S directly induces

47

intestinal damage by inhibiting the growth of intestinal cells and destroying the

48

cytoskeleton, resulting in apoptosis in the piglet intestine14. A recent study evaluated

49

7S-induced changes in intestinal epithelial permeability and integrity, tight-junction

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distribution, and expression of tight-junction proteins, and found a linear decrease in

51

the expression of tight-junction proteins with an increase in the 7S level13.

52

Additionally, 11S stimulated local and systemic immune responses in allergic piglets

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and had an adverse effect on piglet performance12. Till date, reports focused on the

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signal transduction mechanism of intestinal damage in soybean-allergic piglets have

55

been limited. Thus, in this study, we investigated the p38, JNK, and NF-κB signaling 3


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pathways of intestinal damage, induced by 7S or 11S, in piglets.

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Mitogen-activated protein kinases (MAPKs) is responsible for regulating a variety

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of cellular processes, including growth, metabolism, apoptosis, and innate immune

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responses15-17. JNK and p38 are subgroups of MAPK family, which have been

60

reported to play an important role in cell apoptosis and proliferation18. Moreover, the

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activation of MAPK is crucial to the production of inflammatory mediators by

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controlling NF-κB activity 19. In addition, soybean is rich in L-arginine, the precursor

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of nitric oxide (NO) 20, which is produced during the catalysis of L-arginine by nitric

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oxide synthase (NOS) and acts as a cellular inflammatory mediator21 Furthermore,

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tumor necrosis factor (TNF)-α may stimulate inducible NOS (iNOS) in the body.

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High concentrations of iNOS may bind to receptors on the cell surface and eventually

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activate NF-κB. TNF-α is also known to activate NF-κB22. The production of

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TNF-α–activated iNOS results in the generation of excessive NO, forming a positive

69

feedback loop that maintains cytokine secretion and NO level. As a consequence, the

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inflammatory response is intensified and retained for a longer period. Caspase-3, the

71

core protease that mediates apoptosis and the most effective protease in the caspase

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cascade, is usually found in the form of zymogen and induces apoptosis by activating

73

specific substrates. The transcription factor NF-κB plays a vital role in the immune

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system and regulates the expression of cytokines, growth factors, and effector

75

enzymes in response to ligation of many receptors involved in immunity23. It also

76

regulates the expression of more than 500 genes involved in inflammatory and

77

immune responses24, 25.

78

Owing to the complex phenomena of allergic responses, the present study aimed 4



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to indirectly identify the mechanism underlying the development of allergy to

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soybean antigen proteins, using piglets as an animal model. The piglet model is more

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physiologically relevant in the study of nutrition and soybean allergy, compared to the

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rodent model, since its digestive system is more anatomically and functionally similar

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to that of human infants7. Additionally, multiple sampling of piglets makes the kinetic

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observation of serum NO, TNF-α, and caspase-3 levels more feasible and convenient.

85

1. Materials and methods 1.1. Preparation of 7S and 11S 86

Both 7S and 11S were kind gifts from Professor Shuntang Guo of the College of

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Food science and nutritional engineering of China Agricultural University (patent

88

number: 200,410,029,589.4, China). We used crude soy protein as a raw material to

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purify 7S and 11S with alkaline saponification, isoelectric point precipitation, and gel

90

filtration according to a previous report26. After lyophilisation, the protein contents

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were determined, and the purity of 7S and 11S was analysed according to Hao et al.

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(2009)27. The purity of 7S and 11S was 90% and 90.6%, respectively. 1.2. Piglets and diets

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The experimental piglets were obtained from the College of Animal Science and

94

Technology, Anhui Agricultural University, Hefei, China. All experiments were

95

performed according to the Regulations for the Administration of Affairs Concerning

96

Experimental Animals (Ministry of Science and Technology, China; revised in June

97

2004) and approved by the ethics committee of Anhui Agricultural University, Anhui, 5


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China. A total of forty 20-day-old “Duroc × Landrace × Yorkshire” piglets with initial

99

body weight (BW) of 6.2 ± 0.09 kg, were used in the present study. Ten piglets were

100

kept in each pen and had free access to water and feed. Diets for the weaned piglets

101

(Table 1) were formulated to meet or exceed the nutrient requirements suggested by

102

the National Research Council (1998). The piglets were housed in an environmentally

103

controlled room with a 12 h/12 h light/dark cycle, controlled temperature ranging

104

between 26 and 28 °C, and humidity between 50 % and 60 %. The piglets were

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allowed to move freely in the environmentally controlled room and had visual contact

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with each other.

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Feed consumption and BW of individual piglets were determined every day to

108

calculate average daily gain (ADG), average daily feed intake (ADFI), and the feed to

109

gain ratio (G/F).

110 1.3. Experimental design and sample collection 111

Forty healthy “Duroc × Landrace × Yorkshire” piglets (20-day-old) were selected

112

and randomly divided into four groups, with 10 piglets in each, as follows: groups A

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(suckling group), B (weaned group), C (weaned + 7S-sensitized group), and D

114

(weaned + 11S-sensitized group). Besides that of group A, the piglets of group B were

115

fed a basal diet at the age of 21 days, while the piglets in groups C and D were fed a

116

basal diet supplemented with 7S or 11S, respectively. The experiment lasted for seven

117

days, 7S or 11S (at 4 %) was added at 21 days of age and gradually increased to 8 %

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from 23 days of age. The study included pretreatment adaptation and control, during

119

which period, the pigs were fed a basal diet with subsequent transition to 7S or 11S 6



120

feeding within 2 days.

121

At 20 and 27 days of age, blood samples of 10 piglets from each group were obtained

122

from the anterior vena cava in the morning (7:00-9:00 A.M), the serum samples were

123

obtained by centrifugation at 2000 g for 15 min at 4 ˚C and analysed immediately. At

124

27 days of age, five piglets from each group were sacrificed by an intracardiac

125

injection of sodium pentobarbital (50 mg/kg body weight), followed by jugular

126

exsanguination. The duodenum, jejunum, and ileum were collected and divided into

127

three portions. Two portions were fixed in a 3% glutaraldehyde and 4 %

128

paraformaldehyde fixative solution to measure the expression of JNK, p38, and

129

NF-κB in the intestine by immunohistochemistry and to observe the small intestine

130

villus by electron microscopy. The other intestinal portion was transferred to liquid

131

nitrogen for preservation and detection of the levels of JNK, p38, and NF-κB proteins,

132

their phosphorylation, and mRNAs by western blotting and quantitative reverse

133

transcription polymerase chain reaction (qRT-PCR).

134 1.4. Determination of serum NO, TNF-α, and caspase-3 levels by an enzyme-linked

immunosorbent assay (ELISA) 135

Levels of NO, TNF-α, and caspase-3 were determined by ELISA using sera from

136

piglets, as per the instructions mentioned in the kit (Nanjing SenBeiJia Biological

137

Technology Co., Ltd., Nanjing, China). Their concentrations in different samples were

138

evaluated based on the standard curve.

139 7


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1.5. Determination of relative mRNA expression of JNK, p38, and NF-κB by RT-PCR 140

Total RNA from intestinal samples was extracted using RNAiso Plus (TaKaRa

141

Biotechnology [Dalian] Co., Ltd., Dalian, China), according to the manufacturer’s

142

instructions. The relative gene expression levels were analyzed using the 2−∆∆Ct

143

method. The primer sequences used for PCR amplification are shown in Table 2.

144 1.6. Observation of villus morphology under transmission electron microscope 145

Tissues were washed with phosphate buffer, fixed in 1 % osmic acid for 2 h,

146

followed by another wash in phosphate buffer, dehydration in a graded alcohol series

147

(30 %, 50 %, 70 %, and 90 %) for 20 min, and subsequently dehydration in graded

148

acetone (90 % and 100 %) for another 20 min. The tissues were washed with absolute

149

acetone, infiltrated (1 h) by low-viscosity embedding media 1:1 and 2:1, and left

150

overnight; it was then embedded in pure Spurr’s resin mixture and polymerized at

151

37 ℃ for 12 h and 60 ℃ for 72 h. Ultrathin sections were cut and stained to observe

152

changes in villi, mitochondria, and endoplasmic reticulum in the small intestinal

153

mucus layer with JEM-1230 transmission electron microscope (TEM) (JEOL,

154

Akishima, Tokyo, Japan). TEM protocols were used as per the method described by

155

Wang et al. (2018), with some modifications28.

156 1.7. Determination of relative protein expression of JNK, p38, and NF-κB by

immunohistochemistry 157

Paraformaldehyde-fixed

tissues

(duodenum, jejunum, and 8


ileum)

were


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paraffin-embedded, sectioned, deparaffinized, dehydrated, and rinsed thrice with PBS,

159

Hydrogen peroxide (3 %) was added dropwise for 25 min to block endogenous

160

peroxidase activity. After three washes with PBS, the antigen was microwave-repaired

161

with

162

and washed thrice with PBS. The sections were blocked with 5% BSA at 26 ℃ for 25

163

min in the dark, then incubated overnight with diluted NF-κB (SC-33020) antibodies

164

(1:500 dilution, Santa Cruz Biotech, Dallas, TX, USA), and JNK1/2/3 (YT2441) and

165

p38 (YT3513) antibodies (1:500 dilution, Immunoway Biotechnology Company,

166

Plano,

167

used as the negative control. The incubated sections were placed in a 37℃ and

168

incubator for 45 min, washed with PBS thrice, followed by drop-wise addition

169

of the secondary antibody (1:500 dilution, Goat anti-rabbit IgG). After incubation at

170

37℃

171

to coloration with DAB (Zsgb Biotechnology, Beijing, China) for 3 min, washed with

172

distilled water, stained with hematoxylin, dehydrated and mounted with neutral gums.

173

Finally, the immune-labeled sections were observed under a light microscope and

174

photos were taken.

10

mM

TX,

for

30

sodium

citrate

US) at 4℃;

min,

buffer

solution

(pH

PBS, instead of the

6.0),

cooled to 26°C,

primary antibody,

the sections were rinsed thrice with

PBS,

was

subjected

1.8. Determination of JNK, p38, and NF-κB protein and phosphorylation levels by

western blot analysis 175

Intestinal proteins were separated by electrophoresis (Bio-Rad, Hercules,

176

CA,USA) on a 12 % acrylamide resolving gel with 5 % stacking gel and then

177

electroblotted (Bio-Rad) onto a polyvinylidene fluoride membrane (Millipore,

178

Temecula, CA, USA). The membrane was blocked with 5 % bovine serum albumin 9


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for 4 h at 26℃ and incubated with the appropriately diluted NF-κB (YT-3107), p-p38

180

(sc-166182) (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), JNK1/2/3

181

(YT2441), and p38 (YT3513), p-NF-κB (YP-0188), p-JNK1/2/3 (YP0157) (1:1000

182

dilution, Immunoway Biotechnology Company, Plano, TX, USA) antibodies for 16 h.

183

Following three washes with Tris-buffered saline (TBS) containing 0.05 % Tween-20

184

(TBST), the membrane was incubated with rabbit anti-pig IgG secondary antibody

185

(1:5,000 dilution; Beijing Biosynthesis Biotechnology, Beijing, China). The

186

membrane was washed in TBST thrice, and results were analyzed using Quantity One

187

1-D analysis software (Bio-Rad).

188 1.9. Statistical analysis 189

Statistical analysis was performed using analysis of variance (ANOVA) to

190

evaluate differences within each group, over three time points, or among the four

191

groups, according to the general linear procedure of SAS (SAS 2002; Version 9.1.3,

192

SAS Institute, Inc., Cary, NC). Data were expressed as means ± standard deviation

193

(SD), with a two-sided 5 % significance level. Statistical software GraphPad Prism

194

version 5.01 (GraphPad Software, San Diego, CA) was used to make histograms.

195

2. Results 2.1. Effect of 7S and 11S on performance and gastrointestinal symptoms in piglets 196

As shown in Table 3, 7S and 11S showed adverse effects on growth performance.

197

Piglets in groups C and D had lower average daily weight-gain (P < 0.01); the average 10



198

daily feed intake of groups B, C, and D was not significantly different. At 24 days of

199

age, five piglets in group C and three piglets in group D exhibited diarrhea, which

200

lasted for 3–5 days. Groups A and B did not show any such symptom.

201 2.2. Effect of 7S and 11S on serum NO, TNF-α, and caspase-3 levels in piglets 202

As shown in Table 4, no significant difference was observed in serum NO, TNF-α,

203

and caspase-3 levels among the four groups at the age of 20 days, the levels were

204

significantly higher in groups C and D than in group B (P < 0.01). In particular, the

205

serum level of caspase-3 in group C was significantly higher than that in group D (P

Soybean Glycinin- and Î²-Conglycinin-Induced Intestinal Damage in

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