Open Access

Proteome changes in the small intestinal mucosa of growing pigs with dietary supplementation of non-starch polysaccharide enzymes

Proteome Science201715:3

https://doi.org/10.1186/s12953-016-0109-6

Received: 4 June 2016

Accepted: 20 December 2016

Published: 10 January 2017

Abstract

Background

Non-starch polysaccharide enzymes (NSPEs) have long been used in monogastric animal feed production to degrade non-starch polysaccharides (NSPs) to oligosaccharides in order to promote growth performance and gastrointestinal (GI) tract health. However, the precise molecular mechanism of NSPEs in the improvement of the mammalian small intestine remains unknown.

Methods

In this study, isobaric tags were applied to investigate alterations of the small intestinal mucosa proteome of growing pigs after 50 days of supplementation with 0.6% NSPEs (mixture of xylanase, β-glucanase and cellulose) in the diet. Bioinformatics analysis including gene ontology annotation was performed to determine the differentially expressed proteins. A protein fold-change of ≥ 1.2 and a P-value of < 0.05 were selected as thresholds.

Results

Dietary supplementation of NSPEs improved the growth performance of growing pigs. Most importantly, a total of 90 proteins were found to be differentially abundant in the small intestinal mucosa between a control group and the NSPE group. Up-regulated proteins were related to nutrient metabolism (energy, lipids, protein and mineral), immunity, redox homeostasis, detoxification and the cell cytoskeleton. Down-regulated proteins were primarily related to transcriptional and translational regulation. Our results indicate that the effect of NSPEs on the increase of nutrient availability in the intestinal lumen facilitates the efficiency of nutrient absorption and utilization, and the supplementation of NSPEs in growing pigs also modulates redox homeostasis and enhances immune response during simulating energy metabolism due to a higher uptake of nutrients in the small intestine.

Conclusions

These findings have important implications for understanding the mechanisms of NSPEs on the small intestine of pigs, which provides new information for the better utilization of this feed additive in the future.

Keywords

Non-starch polysaccharide enzymesSmall intestinal mucosaProteomicsGrowing pigs

Background

Many cereals such as soybean and wheat contain up to 15% non-starch polysaccharides (NSPs) in their outer or inner cell walls [1]. Monogastric animals lack enzymes to degrade the cell wall and NSP in these feeds. Thus, these anti-nutritive factors may interfere with digestion, nutrient absorption, and intestinal tract health by encapsuling starch and protein, as well as increase the viscosity of the chymus, which may elevate the proliferation of pathological bacteria in the small intestine and reduce the feed conversion ratio of monogastric livestock species [24].

The supplementation of exogenous enzymes such as xylanases and β-glucanases in pig diets may facilitate the hydrolysis of the main NSPs and increase the utilization of available raw materials [5, 6]. Adding exogenous enzymes to cereal diets improves both nutrient digestibility and growth performance in pigs [7, 8]. However, the exact molecular mechanisms of NSPEs, particularly in the gastrointestinal (GI) tract, are unknown [9]. There are several indications that exogenous enzymes may function in the GI tract of animals to aid digestion. The supplementation of NSPEs in the diets could increase the activities of certain types of digestive enzymes in vivo including protease, trypsin, and α-amylase [2, 4, 10]. These enzymes reduce the degradation of NSPs within the small intestine, thereby decreasing the viscosity of the digesta, which leads to a reduced bacterial load in the gut, especially potential pathogens [11]. Furthermore, the degradation of NSPs due to the supplementation of NSPEs promotes the higher availability of digestible nutrients such as energy substrates [12]. Additionally, the intestinal morphological structure and some physiological functions in animals benefit from the improvement of the changing intestinal environment due to the supplementation of NSPEs. Some research demonstrated that intestinal morphologies, including the villus height, the ratio of villus height to crypt depth, and the number of crypts and goblet cells, were changed due to the addition of xylanases alone or multiple enzymes [13, 14]. In addition to the effects of NSPEs observed on the GI tract, alterations of blood parameters related to the nutrient metabolism were also noted [15].

Previous studies reported that diet composition affected gene expression in animals [9, 16]. It is assumed that the improvement of the intestinal environment due to the supplementation of NSPEs in the diet may influence the gene expression and subsequent protein expression of epithelial-cell nutrient transporters in the GI tract mucosa, which has not been studied before. However, RNA editing and numerous options for posttranslational modifications should be taken into account [17, 18]. Hence, elucidation protein expression is important [19].

It is impractical to simultaneously measure all protein expression in the GI mucosa by classical method, such as western blotting. More research has yielded high throughput mass spectrometric proteomic technologies that can simultaneously detect hundreds of proteins [20, 21]. A proteomic analysis of the rat small intestinal proteome showed the presence of previously unrecognized proteins involved in various functions including the absorption and transport of nutrients and the maintenance of cell structure, as well as intestinal molecular chaperones [22]. There remains a great need to pursue proteomic technology to elucidate the beneficial effects of NSPEs in the GI tract mucosa. Therefore, we utilized a label-based iTRAQ (isobaric tags for relative and absolute quantitation) method, followed by LC-MS/MS, to quantitate proteins that are differentially induced in the small intestinal mucosa of growing pigs supplemented with NSPEs in the diet.

Methods

Enzyme preparation

The NSP enzyme mixture preparation supplemented in the diet was provided by the State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (Beijing, China); the mixture contained 7 × 105 U/g xylanase activity (EC 3.2.1.8), 1 × 105 U/g β-glucanase activity (EC 3.2.1.6), and 9000 U/g cellulase activity (EC 3.2.1.4). The activities of the enzymes used in the present study was measured according the methods mentioned in previous research [23].

Animals and treatments

Forty-eight crossbred (Duroc × Landrace × Large White) growing pigs had similar initial body weights (39.18 ± 0.98 kg); the pigs were obtained from a commercial farm in Beijing (Shunliang pig farm, Beijing). The pigs were randomly divided into two groups according to their littermates, sex and mean initial body weights with four replicates in each group and six pigs in each replicate (half females and half males). The following two groups were a control group (CTRL, basal diet) and a treatment group (NSPE, basal diet + 0.6% NSP enzymes). The amount of NSPEs supplementation in the present study was based on the previous results from our group [24]. Both diets were formulated to meet NRC (2012) recommendations (Table 1). All pigs were kept in eight adjacent pens covered in a fermentation bed facility. Feed and water were provided ad libitum during the 50 day experimental period. The individual pig weight and feed intake were recorded at the initiation and the termination of the experiment for the measurement of the average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR). All procedures involving animals were evaluated and approved by the Animal Ethics Committee of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences.
Table 1

Composition of the basal diet and calculated proximate composition of the diet

Ingredients

Proportion (%)a

 Corn

70.70

 Soybean meal

19.82

 Soybean oil

2.10

 Wheat bran

5.00

 Limestone

0.51

 Calcium hydrophosphate

0.56

 L-Lysine

0.01

 Sodium chloride

0.30

 Premixb

1.00

 Total

100

Nutrient

 ME

13.65 (MJ/kg)

 Ether extract (EE)

4.82

 Crude protein (CP)

15.50

 Calcium

0.50

 Total phosphorus

0.45

 Available phosphorus

0.24

 Total lysine

0.75

 Total methionine

0.25

aAll data is expressed in g/kg dry weight except for metabolizable energy (ME) in MJ/kg. The amounts of nutrient were estimated based on the NRC 11th ed. swine feedstuff composition table

bProviding the following (g/kg fresh weight), Vitamin A, 8250 IU; Vitamin D3: 825 IU; Vitamin E: 40 IU; Vitamin K3, 4.0 mg; Vitamin B1, 1.0 mg; Vitamin B2, 5.0 mg; Vitamin B6, 2.0 mg; Vitamin B12, 25 μg; choline chloride, 600 mg; nicotinic acid, 35 mg; folic acid, 2.0 mg; biotin, 4.0 mg; Cu, 50.0 mg; Fe, 80.0 mg; Zn, 100.0 mg; Mn, 25.0 mg; Se, 0.15 mg; I, 0.5 mg

Sample collection

At the end of the experiment (Day 50), all pigs were weighted after 12 h of fasting. One pig per replicate, a total of eight pigs (n = 8), were sacrificed by CO2 asphyxiation and then exsanguinated. Blood samples were obtained from the cervical vein by syringe before sacrifice. The whole blood was centrifuged at 2000 g for 30 min at 4 °C, followed by centrifugation at 400 g for 10 min at 4 °C. Then, the resulting supernatant was collected as sera samples, which were stored at −20 °C for further analysis. A 20-cm tissue section was rapidly excised at 50% of the length of the small intestine, rinsed with cold phosphate buffer saline, and blotted dry on paper. Mucosa from this small intestine section was sequentially obtained by careful scraping of the mucosal layer using a glass microscope slide as previously described [25]. Then, the collected mucosal samples were snap-frozen in liquid nitrogen and stored at −80 °C for proteomic analysis.

Serum biochemical analyses

Important serum biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), alkaline phosphatase (ALP), glucose (GLU), and creatine kinase (CK), were analyzed using an automatic biochemical analyzer (Hitachi 7020, Tokyo, Japan). Serum levels of total superoxide dismutase (T-SOD) and immunoglobulin G (IgG) were measured using a corresponding kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Protein extraction and sample preparation

Small intestinal mucosa samples (500 μg) were ground in liquid nitrogen using a Dounce glass grinder. Grinded powder was precipitated with 10% trichloroacetic acid (TCA) (w/v) and 90% ice-cold acetone at −20 °C for 2 h. The precipitate was obtained by centrifugation at 20,000 g for 30 min at 4 °C and subsequently washed with ice-cold acetone. Then, the precipitate was lysed in lysis buffer [8 M urea, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM phenylmethanesulfonyl fluoride (PMSF), 2 mM ethylene diamine tetraacetic acid (EDTA), and 10 mM dithiothreitol (DTT)]. The crude tissue extracts were centrifuged to remove the remaining debris. The tissue lysates were reduced for 1 h at 56 °C in a water bath using 10 mM DTT and then alkylated with 55 mM iodoacetamide for 1 h in the dark. Afterwards, the lysates were precipitated by adding four volumes of pre-chilled acetone. The pellets were then washed three times with pre-chilled pure acetone and resuspended in the buffer (50% TEAB and 0.1% SDS). The centrifugation was repeated to remove the undissolved pellets. Subsequently, protein quantitation was determined using a Bio-Rad Bradford Protein Assay Kit (Hercules, CA, USA). Each sample was digested with modified sequence grade trypsin (Promega Corporation, Madison, WI) at a 1: 30 ratio (3.3 μg trypsin : 100 μg target) overnight at 37 °C. Each isobaric tag (113, 114, 115, 116, 117, 118, 119, and 121) was solubilized in 70 μL isopropanol and then added to each respective sample (4 samples per group). Incubation continued for 2 h at room temperature.

Strong cation exchange chromatography

The strong cation exchange fractionation was performed according to a previous report [26] with slight modification. Briefly, 800 μg of labeled sample was loaded onto a strong cation exchange column (Phenomenex Luna SCX 100A) installed in an Agilent 1100 (Santa Clara, CA) system and equilibrated with buffer A (25% acetonitrile and 10 mM KH2PO4, pH 3.0). The peptides were separated by a linear gradient of buffer B (25% acetonitrile, 2 M KCl and 10 mM KH2PO4, pH 3.0) according to this procedure (increasing to 5% after 41 min, 50% after 66 min and 100% after 71 min with a flow rate of 1 ml/min). Elution was monitored by setting the absorbance at 214 nm. A total of 10 fractions were obtained, then desalted with a Strata X C18 column (Phenomenex) and dried under a vacuum. The pellets were resuspended by adding 0.1% formic acid before the LC-MS/MS run.

Mass spectrometry

LC-MS/MS was conducted according to a previous report [27], and the detailed process and parameters are shown in Additional file 1.

Data processing and protein quantification

All the detailed parameters are shown in the Supporting Information (Additional file 1). MS/MS data for iTRAQ protein identification and quantitation were analyzed using Proteome Discover 1.3 (Thermo Fisher Scientific, Bremen, Germany) and in-house MASCOT software (Matrix Science, London, UK; Version 2.3.0) against the database Uniprot_pig (Apr. 11th, 2014). Median ratio normalization was performed in intra-sample channels to normalize each channel across all proteins. Protein quantitative ratios for each iTRAQ labeled sample were obtained, using a sample in the control group (sample tagged with 113) as the denominator. Quantitative ratios were then log transformed to base two and presented as the fold change relative to the denominator in the control group for final quantitative testing. Differentially expressed proteins were identified using Student’s t-test corrected for multiple testing using the Benjamini and Hochberg correction [21, 25, 28, 29]. Based on above the relative quantification, statistical analysis, and a number of previous reports regarding to iTRAQ experiments [2931], we set a 1.2-fold change or greater as the threshold for differentially expressed proteins.

Bioinformatics analysis and validation of protein expression

The databases and software for bioinformatics analysis are shown in Additional file 1. Real-time qPCR was used to verify six small intestinal mucosal proteins of differential abundance at the mRNA level. All detailed procedures are described in the Supporting Information (Additional file 1). The primer sequences used in this study are shown in Additional file 2: Table S1.

Statistical analysis

The data for growth parameters, serum parameters, and gene expression were analyzed by one-way ANOVA using block as a covariate (SAS Version 9.2, SAS institute Inc., Cary, NC) according the previous studies [21, 31], and a t-test was used for independent samples in MS data analysis. A group difference was assumed statistically significant when P < 0.05.

Results

Growth performance of growing pigs

During the entire experimental period (50 days), NSPE pigs had 15.5% greater ADG (P < 0.05) compared with the control group; however, the ADFI between the two groups was not significantly different (P > 0.05). It is notable that pig fed NSPEs had an 8.7% greater FCR compared with the control group (P < 0.05; Table 2).
Table 2

Effects of NSP enzymes on growth performance of growing pigs

 

Groups

Control

Treatment

P value

Initial weight (kg)

38.80 ± 0.99

39.55 ± 0.63

0.1245

Final weight (kg)

74.04 ± 1.77b

78.42 ± 1.06a

0.0318

ADG (kg/d)c

0.71 ± 0.05b

0.82 ± 0.05a

0.0437

ADFI (kg/d)d

1.97 ± 0.09

2.07 ± 0.06

0.0423

FCR (kg feed/kg weight gain)e

2.77 ± 0.02a

2.53 ± 0.03b

0.0352

a, b Values within a column having different superscript letters indicate a significant difference at P < 0.05. Numbers are mean ± S.D. (n = 24 for ADG; n = 4 for ADFI and FCR)

c ADG = average daily gain

d ADFI = average daily feed intake

e FCR = feed conversion ratio

Serum parameters of growing pigs

In NSPE pigs, serum concentration of CK was significantly lower (P < 0.05) than the control group (Table 3). Furthermore, the serum concentrations of T-SOD, IgG, and glucose were significantly elevated compared with the control group (P < 0.05) (Table 3). Serum levels of TP, ALT and AST were similar between the two groups (Table 3).
Table 3

Effect of NSPEs on serum biochemical parameters of growing pigs

 

Groups

Control

Treatment

P value

ALT (IU/L)c

49.01 ± 7.96

49.00 ± 9.30

0.4768

AST (IU/L)d

79.60 ± 10.70

63.80 ± 16.05

0.2240

TP (mmol/L)e

67.31 ± 5.44

69.50 ± 2.44

0.5331

ALP (U/L)f

131.83 ± 36.14

126.40 ± 22.06

0.2565

GLU (mmol/L)g

6.37 ± 2.24b

9.73 ± 2.34a

0.0479

T-SOD (U/mL)h

61.55 ± 2.67b

67.44 ± 3.64a

0.0002

CK (U/L)i

3117 ± 274a

2188 ± 218b

0.0089

IgG (g/L)j

3.19 ± 0.16b

3.43 ± 0.20a

0.0392

a, bValues within a column not sharing a common superscript letter indicate significant difference at P < 0.05. Numbers are means ± S.D. (n = 4)

cALT = alanine aminotransferase

dAST = aspartate aminotransferase

eTP = total protein

fALP = alkaline phosphatase

gGLU = glucose

hT-SOD = total superoxide dismutase

iCK = creatine kinase

jIgG = immunoglobulin G

Identification and comparison of proteins of differential abundance

Using iTRAQ analysis, a total of 2634 proteins were identified within the FDR (false discovery rate) of 1% (Additional file 3: Table S2). Following statistical analysis, 104 proteins were found to be differentially expressed in the small intestinal mucosa between CTRL and NSPE pigs, with 43 up-regulated and 61 down-regulated (Additional file 4: Table S3).

A total of 90 proteins of differential abundance were grouped into eight classes based on putative functions: transcriptional and translational regulation (44.4%), miscellaneous (16.7%), redox homeostasis and detoxification (10.0%), immune response and inflammation (8.9%), energy metabolism (7.8%), protein metabolism and modification (5.6%), lipid metabolism (3.3%), and cell cytoskeleton (3.3%) (Fig. 1). Those related to transcriptional and translational regulation, redox homeostasis, and immune response were predominant, accounting for approximately 63% of the differentially expressed proteins. A comparison of proteins of differential abundance with functional groupings between the two groups indicated that a smaller number of protein species were up-regulated in NSPE pigs (36 versus 54) (Table 4).
Fig. 1

Functional classification of the proteins of differential abundance identified from the small intestinal mucosa of growing pigs supplemented with NSPE

Table 4

List of differentially expressed proteins in small intestinal mucosal samples from treatment group and control group

Accessiona

Descriptionb

Gene symbol

Scorec

Pep. Nod

Log2 fold change

P-valuee

Biological process GO term

Transcriptional and translational regulation

 F1S419

Uncharacterized protein OS = Sus scrofa GN = SF3B3 PE = 4 SV = 2 - [F1S419_PIG]

None

85.61

3

−0.37

0.0007

RNA binding

 K9J4V0

U5 small nuclear ribonucleoprotein 200 kDa helicase OS = Sus scrofa GN = SNRNP200 PE = 2 SV = 1 - [K9J4V0_PIG]

SNRNP200

248.18

9

−0.32

0.0012

Nucleic acid binding

 F2Z5Q6

40S ribosomal protein S6 (Fragment) OS = Sus scrofa GN = RPS6 PE = 3 SV = 2 - [F2Z5Q6_PIG]

RPS6

140.42

4

−0.81

0.0013

Structural constituent of ribosome

 F1SD96

Uncharacterized protein (Fragment) OS = Sus scrofa GN = RAD23A PE = 4 SV = 1 - [F1SD96_PIG]

RAD23A

85.25

3

1.06

0.0026

Nucleotide excision repair

 F1S8K5

Uncharacterized protein OS = Sus scrofa GN = SUPT16H PE = 4 SV = 1 - [F1S8K5_PIG]

SUPT16H

35.43

2

−0.40

0.0028

RNA binding

 F1RZH4

Uncharacterized protein OS = Sus scrofa PE = 4 SV = 1 - [F1RZH4_PIG]

ADAM10

32.67

1

−0.82

0.0048

Structural constituent of ribosome

 F1SD98

Uncharacterized protein OS = Sus scrofa GN = TRMT1 PE = 4 SV = 2 - [F1SD98_PIG]

TRMT1

27.72

1

−0.30

0.0065

Poly(A) RNA binding

 I3LHZ6

Uncharacterized protein OS = Sus scrofa GN = DHX9 PE = 4 SV = 1 - [I3LHZ6_PIG]

DHX9

994.71

27

−0.30

0.0075

ATP-dependent RNA helicase activity

 F1SDV7

Uncharacterized protein (Fragment) OS = Sus scrofa GN = TOP1 PE = 4 SV = 1 - [F1SDV7_PIG]

TOP1

99.93

4

−0.44

0.0075

DNA binding

 P62802

Histone H4 OS = Sus scrofa PE = 1 SV = 2 - [H4_PIG]

None

358.11

7

−0.67

0.0103

DNA binding

 F1S1V1

Uncharacterized protein OS = Sus scrofa GN = SSB PE = 4 SV = 2 - [F1S1V1_PIG]

SSB

196.5

6

−0.73

0.0111

Nucleotide binding

 F1RS45

DNA topoisomerase 2 OS = Sus scrofa PE = 3 SV = 2 - [F1RS45_PIG]

TOP2B

116.62

6

−0.27

0.0117

DNA binding

 F1S1X3

Uncharacterized protein OS = Sus scrofa GN = NARS PE = 3 SV = 2 - [F1S1X3_PIG]

NARS

262.65

7

−0.30

0.0119

Nucleotide binding

 F2Z576

Histone H3 OS = Sus scrofa GN = LOC100525821 PE = 2 SV = 1 - [F2Z576_PIG]

HIST1H3E

159.44

6

−0.77

0.0120

DNA binding

 Q29194

Ribosomal protein S2 (Fragment) OS = Sus scrofa PE = 2 SV = 1 - [Q29194_PIG]

None

46.59

1

−0.45

0.0138

Structural constituent of ribosome

 I3LFV4

Uncharacterized protein OS = Sus scrofa GN = YBX1 PE = 4 SV = 1 - [I3LFV4_PIG]

YBX1

157.89

4

0.41

0.0148

DNA repair

 I3LIN8

Histone H2A OS = Sus scrofa GN = H2AFY PE = 3 SV = 1 - [I3LIN8_PIG]

H2AFY

224.89

6

−0.52

0.0149

Chromatin DNA binding

 B0FWK5

Ribosomal protein L5 OS = Sus scrofa GN = RPL5 PE = 2 SV = 1 - [B0FWK5_PIG]

RPL5

178.57

8

−0.34

0.0165

Structural constituent of ribosome

 I3LCI4

Uncharacterized protein OS = Sus scrofa GN = ZFR PE = 4 SV = 1 - [I3LCI4_PIG]

ZFR

41.93

2

−0.28

0.0167

Poly(A) RNA binding

 F1S8A5

Uncharacterized protein OS = Sus scrofa GN = MRPS26 PE = 4 SV = 1 - [F1S8A5_PIG]

MRPS26

38.63

1

−0.36

0.0181

Poly(A) RNA binding

 A5GFY4

Negative elongation factor D OS = Sus scrofa GN = NELFCD PE = 3 SV = 1 - [NELFD_PIG]

NELFCD

43.67

1

−0.32

0.0189

Negative regulation of transcription

 F1S5A8

Uncharacterized protein OS = Sus scrofa GN = DHX15 PE = 4 SV = 1 - [F1S5A8_PIG]

DHX15

259.43

8

−0.26

0.0198

ATP-dependent RNA helicase activity

 F1RRG9

Uncharacterized protein OS = Sus scrofa GN = SMARCA5 PE = 4 SV = 1 - [F1RRG9_PIG]

SMARCA5

99.44

3

−0.39

0.0201

DNA binding

 F1RGP1

Uncharacterized protein OS = Sus scrofa GN = MYBBP1A PE = 4 SV = 1 - [F1RGP1_PIG]

MYBBP1A

445.89

12

−0.50

0.0208

Poly(A) RNA binding

 F2Z5Q8

Uncharacterized protein OS = Sus scrofa GN = LOC100519675 PE = 4 SV = 1 - [F2Z5Q8_PIG]

RPL35A

57.33

2

−0.45

0.0209

Structural constituent of ribosome

 I3L7T6

Histone H2A OS = Sus scrofa GN = H2AFX PE = 3 SV = 1 - [I3L7T6_PIG]

H2AFX

357.7

7

−0.56

0.0231

DNA binding

 F1SMZ9

Uncharacterized protein (Fragment) OS = Sus scrofa GN = SF3B1 PE = 4 SV = 2 - [F1SMZ9_PIG]

SF3B1

267.33

9

−0.26

0.0245

mRNA binding

 F2Z5K9

Histone H3 OS = Sus scrofa GN = LOC100622412 PE = 3 SV = 1 - [F2Z5K9_PIG]

LOC100622412

178.75

6

−0.76

0.0270

DNA binding

 P53027

60S ribosomal protein L10a (Fragment) OS = Sus scrofa GN = RPL10A PE = 2 SV = 3 - [RL10A_PIG]

RPL10A

154.25

5

−0.34

0.0272

RNA binding

 K9IVG8

DEAD (Asp-Glu-Ala-Asp) box helicase 21 OS = Sus scrofa GN = DDX21 PE = 2 SV = 1 - [K9IVG8_PIG]

DDX21

44.62

1

−0.38

0.0292

RNA binding

 F2Z554

Uncharacterized protein OS = Sus scrofa GN = RPL30 PE = 3 SV = 1 - [F2Z554_PIG]

RPL30

105.87

4

−0.26

0.0323

RNA binding

 Q29195

60S ribosomal protein L10 OS = Sus scrofa GN = RPL10 PE = 2 SV = 3 - [RL10_PIG]

RPL10

105.8

4

−0.39

0.0350

Structural constituent of ribosome

 P67985

60S ribosomal protein L22 OS = Sus scrofa GN = RPL22 PE = 2 SV = 2 - [RL22_PIG]

RPL22

113.83

3

−0.48

0.0355

Structural constituent of ribosome

 I7GF95

Guanine nucleotide binding protein-like 1 OS = Sus scrofa GN = GNL1 PE = 4 SV = 1 - [I7GF95_PIG]

GNL1

58.56

1

−0.35

0.0371

Ribosome biogenesis

 F1S8L9

Uncharacterized protein OS = Sus scrofa GN = HNRNPU PE = 4 SV = 2 - [F1S8L9_PIG]

HNRNPU

883.61

23

−0.34

0.0377

Poly(A) RNA binding

 Q53DY5

Histone H1.3-like protein OS = Sus scrofa GN = LOC595122 PE = 2 SV = 1 - [Q53DY5_PIG]

HIST1H1D

251.92

7

1.29

0.0384

Chromatin DNA binding

 F1S2G3

Uncharacterized protein (Fragment) OS = Sus scrofa GN = TBCA PE = 4 SV = 1 - [F1S2G3_PIG]

TBCA

78.18

2

0.31

0.0389

Poly(A) RNA binding

 F2Z5P1

Histone H2A (Fragment) OS = Sus scrofa GN = H2AFV PE = 3 SV = 1 - [F2Z5P1_PIG]

LOC100512448

256.74

5

−0.43

0.0427

DNA binding

 F2Z553

Uncharacterized protein OS = Sus scrofa GN = EIF1 PE = 4 SV = 1 - [F2Z553_PIG]

EIF1

103.66

2

0.82

0.0437

Translation initiation factor activity

 F2Z5L5

Histone H2A OS = Sus scrofa GN = HIST2H2AC PE = 3 SV = 1 - [F2Z5L5_PIG]

HIST2H2AC

322.1

5

−0.62

0.0448

DNA binding

Redox homeostasis and detoxification

 F1SKJ2

Uncharacterized protein OS = Sus scrofa GN = TXN2 PE = 4 SV = 1 - [F1SKJ2_PIG]

TXN2

29.86

1

0.39

0.0043

Cell redox homeostasis

 F1SGS9

Catalase OS = Sus scrofa GN = CAT PE = 3 SV = 1 - [F1SGS9_PIG]

CAT

923.56

23

0.58

0.0151

Protect cells from the toxic effects of hydrogen peroxide

 I3LDJ8

Uncharacterized protein OS = Sus scrofa PE = 3 SV = 1 - [I3LDJ8_PIG]

None

303.51

10

0.77

0.0202

Oxidoreductase activity

 P12309

Glutaredoxin-1 OS = Sus scrofa GN = GLRX PE = 1 SV = 2 - [GLRX1_PIG]

GLRX

277.83

6

0.64

0.0208

Cell redox homeostasis

 F1SCF9

Uncharacterized protein (Fragment) OS = Sus scrofa GN = TECR PE = 4 SV = 2 - [F1SCF9_PIG]

TECR

38.34

1

−0.37

0.0242

Oxidoreductase activity

 A5J2A8

Thioredoxin (Fragment) OS = Sus scrofa GN = TRX PE = 4 SV = 1 - [A5J2A8_PIG]

TRX

128.36

3

0.34

0.0303

Cell redox homeostasis

 F1SMY1

Uncharacterized protein OS = Sus scrofa GN = TMX3 PE = 4 SV = 2 - [F1SMY1_PIG]

TMX3

39.1

2

0.30

0.0345

Cell redox homeostasis

 P16549

Dimethylaniline monooxygenase [N-oxide-forming] 1 OS = Sus scrofa GN = FMO1 PE = 1 SV = 3 - [FMO1_PIG]

FMO1

39.55

2

1.64

0.0084

Oxidative metabolism of a variety of xenobiotics

 P04178

Superoxide dismutase [Cu-Zn] OS = Sus scrofa GN = SOD1 PE = 1 SV = 2 - [SODC_PIG]

SOD1

459.04

9

0.35

0.0424

Superoxide dismutase activity

Immune response and inflammation

 A3FJ41

MHC class I antigen (Fragment) OS = Sus scrofa GN = SLA-1 PE = 4 SV = 1 - [A3FJ41_PIG]

SLA-1

120.03

5

0.35

0.0050

Immune response

 F1RGC8

Uncharacterized protein OS = Sus scrofa GN = NLRP6 PE = 4 SV = 3 - [F1RGC8_PIG]

NLRP6

119.58

4

−0.32

0.0061

Activation of NF-κB

 F1RFM7

Uncharacterized protein OS = Sus scrofa GN = AIMP2 PE = 4 SV = 1 - [F1RFM7_PIG]

AIMP2

232.75

6

−0.29

0.0076

Metabolism of xenobiotics

 A2SZV5

Tax1 binding protein 3 (Fragment) OS = Sus scrofa PE = 4 SV = 1 - [A2SZV5_PIG]

None

55.14

1

0.29

0.0133

Negative regulation of NF-κB

 B8XX91

DNA-dependent activator of IFN-regulatory factor OS = Sus scrofa GN = DAI PE = 2 SV = 1 - [B8XX91_PIG]

DAI

100.5

4

0.70

0.0137

Innate immune responses

 Q8WNQ7

N-acetylgalactosamine-6-sulfatase OS = Sus scrofa GN = GALNS PE = 2 SV = 1 - [GALNS_PIG]

GALNS

52.52

1

0.60

0.0311

Degradation of the glycosaminoglycans keratan sulfate

 B8XTR8

Granzyme H OS = Sus scrofa GN = gzmH PE = 2 SV = 1 - [B8XTR8_PIG]

gzmH

168.84

6

−0.67

0.0272

Serine-type endopeptidase activity

 A5GFQ5

Protein canopy homolog 3 OS = Sus scrofa GN = CNPY3 PE = 3 SV = 1 - [CNPY3_PIG]

CNPY3

40.13

2

−0.63

0.0376

Receptor binding for proper TLR folding

Energy metabolism

 Q1ACV5

Transporter associated with antigen processing 1 OS = Sus scrofa PE = 2 SV = 1 - [Q1ACV5_PIG]

None

298.67

7

−0.32

0.0030

Triggers ATP hydrolysis

 F1RIG0

Uncharacterized protein (Fragment) OS = Sus scrofa PE = 4 SV = 1 - [F1RIG0_PIG]

None

47.28

2

−0.27

0.0169

ATP binding

 Q7SIB7

Phosphoglycerate kinase 1 OS = Sus scrofa GN = PGK1 PE = 1 SV = 3 - [PGK1_PIG]

PGK1

850.39

23

0.30

0.0160

Conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate

 H9BYW2

Acyl-coenzyme A oxidase OS = Sus scrofa GN = ACOX1 PE = 2 SV = 1 - [H9BYW2_PIG]

ACOX1

370.35

10

0.91

0.0200

Fatty acid beta-oxidation

 I3LEN7

Uncharacterized protein OS = Sus scrofa GN = ALDH1L1 PE = 3 SV = 1 - [I3LEN7_PIG]

ALDH1L1

49.04

2

0.40

0.0245

Formate oxidation

 F1S0Y8

Uncharacterized protein OS = Sus scrofa GN = ADH4 PE = 3 SV = 2 - [F1S0Y8_PIG]

ADH4

40.7

2

0.67

0.0309

Oxidation of long-chain aliphatic alcohols

 A7UIU7

ATP citrate lyase OS = Sus scrofa GN = ACL PE = 2 SV = 1 - [A7UIU7_PIG]

ACL

468.98

14

−0.38

0.0374

ATP binding

Protein metabolism and modification

 F1RIF3

Uncharacterized protein OS = Sus scrofa GN = FAH PE = 4 SV = 1 - [F1RIF3_PIG]

FAH

38.37

2

0.39

0.0010

Catabolism of the amino acid phenylalanine

 Q9GK25

Peptidyl-prolyl cis-trans isomerase (Fragment) OS = Sus scrofa PE = 2 SV = 1 - [Q9GK25_PIG]

None

266.1

7

1.43

0.0025

Accelerate the folding of proteins

 I3L739

Uncharacterized protein OS = Sus scrofa GN = JMJD6 PE = 4 SV = 1 - [I3L739_PIG]

JMJD6

39.99

1

−0.29

0.0193

Protein hydroxylases

 I3LK37

Uncharacterized protein (Fragment) OS = Sus scrofa PE = 3 SV = 1 - [I3LK37_PIG]

GALNT7

33.39

2

−0.30

0.0248

Protein glycosylation

 F1RNR6

4-hydroxyphenylpyruvate dioxygenase OS = Sus scrofa GN = HPD PE = 3 SV = 2 - [F1RNR6_PIG]

HPD

31

1

0.35

0.0391

Aromatic amino acid family metabolic process

Lipid metabolism

 I3LM15

Uncharacterized protein OS = Sus scrofa GN = AGPS PE = 4 SV = 1 - [I3LM15_PIG]

AGPS

48.77

1

−0.36

0.0019

Lipid biosynthetic process

 Q9GJX2

Diazepam binding inhibitor (Fragment) OS = Sus scrofa GN = DBI PE = 2 SV = 1 - [Q9GJX2_PIG]

DBI

80.13

3

0.91

0.0057

Long-chain fatty acyl-CoA binding, triglyceride metabolic process

 P27917

Apolipoprotein C-III OS = Sus scrofa GN = APOC3 PE = 1 SV = 2 - [APOC3_PIG]

APOC3

226.39

7

0.78

0.0241

High-density lipoprotein particle receptor binding

Cell cytoskeleton

 P10668

Cofilin-1 OS = Sus scrofa GN = CFL1 PE = 1 SV = 3 - [COF1_PIG]

CFL1

704.02

15

0.31

0.0059

Cytoskeleton organization

 Q5G6W0

Cofilin-2 (Fragment) OS = Sus scrofa PE = 2 SV = 1 - [Q5G6W0_PIG]

CFL1

48.67

2

0.43

0.0073

Cytoskeleton organization

 B5APV0

Actin-related protein 2/3 complex subunit 5 OS = Sus scrofa GN = ARPC5 PE = 2 SV = 1 - [B5APV0_PIG]

ARPC5

170.99

6

0.30

0.0167

Structural constituent of cytoskeleton

Miscellaneous

 Q9TSA7

Calmodulin (Fragments) OS = Sus scrofa PE = 4 SV = 1 - [Q9TSA7_PIG]

None

108.72

4

1.11

0.0008

Calcium ion binding

 K7GKQ1

Uncharacterized protein OS = Sus scrofa GN = RAB9A PE = 3 SV = 1 - [K7GKQ1_PIG]

RAB9A

26.6

1

−0.40

0.0071

Cytoskeletal signaling

 F1RKI3

Uncharacterized protein OS = Sus scrofa GN = HINT1 PE = 4 SV = 1 - [F1RKI3_PIG]

HINT1

80.55

3

0.32

0.0073

Tumor suppressing

 I3LSY0

Uncharacterized protein OS = Sus scrofa GN = ACSM4 PE = 4 SV = 1 - [I3LSY0_PIG]

ACSM4

21.13

1

0.86

0.0179

Catalytic activity

 D0G6R8

Phosphatidate cytidylyltransferase OS = Sus scrofa GN = CDS2 PE = 2 SV = 1 - [D0G6R8_PIG]

CDS2

33.01

1

−0.39

0.0192

Synthesis of phosphatidylglycerol

 Q95332

Betaine--homocysteine S-methyltransferase 1 (Fragment) OS = Sus scrofa GN = BHMT PE = 1 SV = 3 - [BHMT1_PIG]

BHMT

110.41

4

1.10

0.0193

Regulation of homocysteine metabolism

 F1RS34

Uncharacterized protein OS = Sus scrofa GN = GAPVD1 PE = 4 SV = 2 - [F1RS34_PIG]

GAPVD1

22.69

1

−0.40

0.0207

Signal transduction

 F1ST01

Uncharacterized protein OS = Sus scrofa GN = SELENBP1 PE = 4 SV = 1 - [F1ST01_PIG]

SELENBP1

936.42

22

0.33

0.0209

Selenium binding

 Q9TV62

Myosin-4 OS = Sus scrofa GN = MYH4 PE = 2 SV = 1 - [MYH4_PIG]

MYH4

192.94

7

−0.83

0.0336

Motor activity

 F1RN91

Uncharacterized protein (Fragment) OS = Sus scrofa PE = 4 SV = 2 - [F1RN91_PIG]

MYO18A

35.04

2

0.28

0.0355

Cell migration

 F1RPC8

Uncharacterized protein OS = Sus scrofa GN = CRYM PE = 4 SV = 2 - [F1RPC8_PIG]

CRYM

59.33

2

0.49

0.0392

Thyroid hormone binding

 F2Z5W6

Uncharacterized protein OS = Sus scrofa GN = LAMTOR1 PE = 4 SV = 1 - [F2Z5W6_PIG]

LAMTOR1

26.54

1

−0.37

0.0410

Guanyl-nucleotide exchange factor activity

 Q29069

Myosin light chain OS = Sus scrofa PE = 2 SV = 2 - [Q29069_PIG]

None

58.61

3

−0.38

0.0458

Calcium ion binding

 O19175

Casein kinase I isoform alpha (Fragment) OS = Sus scrofa GN = CSNK1A1 PE = 2 SV = 1 - [KC1A_PIG]

CSNK1A1

51.13

1

−0.44

0.0473

Protein kinase activity

 N0E654

Casein kinase II b subunit splicing isoform 476 (Fragment) OS = Sus scrofa GN = Csnk2b PE = 2 SV = 1 - [N0E654_PIG]

Csnk2b

63.97

2

−0.27

0.0039

Cell proliferation and cell differentiation

aUniprot_ Sus scrofa_9823 database accession number

bThe name of the protein exclusive of the identifier that appears in the database

cThe sum of the scores of the individual peptides

dThe number of distinct peptide sequences in the protein group

eDifferential protein expression in the treatment group was presented as a log2 fold change relative to the control group

GO annotations of proteins of differential abundance

In the cellular component group, the differentially expressed proteins were concentrated in the intracellular part and membrane-bounded organelles (Fig. 2). In the molecular functional group, the differentially expressed proteins that are binding proteins (protein, nucleotide, or nucleic acid binding) and metabolic enzymes (hydrolase, oxidoreductase, or transferase activity) were ranked at the top of the category (Fig. 2). In the biological process category, the proteins that participate in cellular process (organelle organization process), metabolic process (nitrogen compound metabolic and biosynthetic process), and biological regulation (transcriptional and translational regulation, redox homeostasis, and immune response) had the highest ratios among the differentially expressed proteins.
Fig. 2

GO distribution analysis of differentially expressed proteins in small intestinal mucosal samples from the NSPE group and control group. The right coordinate axis indicates the number of proteins for each GO annotation, and the left one represents the proportion of proteins for every GO annotation

Validation of proteins of differential abundance

Six differentially expressed proteins superoxide dismutase (SOD1) involved in redox homeostasis; calmodulin (CALM1) involved in calcium ion binding; MHC class I antigen (SLA-1) involved in immune response; acyl-coenzyme A oxidase (ACOX1) involved in energy metabolism; 40S ribosomal protein S6 (RPS6) involved in transcriptional and translational regulation; and apolipoprotein C-III (APOC3) involved in lipid absorption, were selected for the validation of proteomic data at the mRNA level using qPCR (Fig. 3). Most protein levels were consistent with their mRNA expression levels, except for RPS6.
Fig. 3

qPCR validation of six proteins of differential abundance from the intestinal mucosa of growing pigs at the mRNA level (a, b, c, d, e and f). Samples were normalized with the reference gene β-actin. Vertical lines represent means ± S.D, and different letters denote significant difference at P < 0.05 (n = 4)

Discussion

The benefit of NSPEs supplementation is well recognized in monogastric animal production; NSPEs supplementation promotes growth performance and GI tract health, including the efficiency of nutrient utilization [2, 3, 8]. A number of studies have proven that the addtion of NSPEs to the diet reduces digesta viscosity by the partial or complete hydrolysis of soluble NSPs, which triggers the changes in microbial composition, especially the reduction of the amount of pathological bacteria within the small intestine [11, 32]. Moreover, the supplementation of NSPEs could increase the nutrient availability in the intestinal lumen (for example, energy substrates and proteins) [12, 33]. All above effects of NSPE supplementation are due to the improvements of the intestinal environment. However, it is still largely unknown how the small intestinal mucosa of the hosts responds to alterations in the luminal environment triggered by the addition of NSPEs. The present study marks the first time that the well-established quantitative iTRAQ label-based technology was applied for the proteomic analysis of the small intestinal mucosa of growing pigs with dietary supplementation of NSPEs. Various functional groupings of differentially expressed mucosal proteins related to nutrient metabolism, transcriptional and translational regulation, immune, and redox homeostasis were identified in response to NSPEs.

In former research, the utilization of β-glucanase and xylanase in the diet demonstrated that enzymes tended to increase the absorptive area and reduce cell proliferation and intraepithelial lymphocytes in the gut of pigs [34]. Both cereal grains and enzymes would affect components of gut health, including intestine morphology, bacteria populations, and microbial metabolites in the gut content [35]. It has been demonstrated that enhanced cell proliferation in the intestinal mucosa is associated with bowel diseases, cellular repair, and apoptosis [36, 37]. As shown in the present study, 89% of proteins related to transcriptional and translational regulation were down-regulated in NSPE pigs. We speculate that supplementation with NSPEs in the diet of growing pigs can reduce the possibility of intestinal infection. This is consistent with the former research result that NSPEs reduce the amount of pathological bacteria within the small intestine by lowering the viscosity of intestinal digesta [11].

The abundance of proteins CFL1 (cofilin-1), CFL2 (cofilin-2) and ARPC5 (actin-related protein 2/3 complex subunit 5), which are classified as cell cytoskeleton proteins relevant to cell structure and mobility, was increased. CFL1 and CFL2 are widely distributed intracellular actin-modulating proteins [38]. These two proteins can cause actin cytoskeleton rearrangement and membrane remodeling to the formation of phagosomes, which are recognized by Fc gamma receptors and beneficial for the host-defense in animals [39]. ARPC5 has a similar function as cofilin in the actin cytoskeleton, which is required for phagocytosis in mammals [40]. The up-regulation of these proteins might reflect the improved integrity of the intestinal mucosa.

As an important immune organ, the small intestine participates in the inflammatory response and the prevention of bacterial infection. SLA-1 (MHC class I antigen), GALNS (N-acetylgalactosamine-6-sulfatase), and DAI (DNA-dependent activator of IFN-regulatory factor) are considered to be involved in the immune response. SLA-1 alerts the immune system to virus-infected cells by presenting peptide fragments derived from intracellular proteins [41]. GALNS is located in lysosomes that digest different types of molecules and engulf viruses or bacteria within cells [42, 43]. DAI selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity [44, 45]. The abundance of these proteins was up-regulated in NSPE pigs, suggesting that the supplementation of NSPEs may improve potential immunity and reduce the chance of bacterial infection in the small intestine. This is consistent with the elevated serum level of IgG in the NSPE group. However, challenges with exogenous pathogens are still required to verify the effect of NSPEs supplementation on immunity. In contrast, proteins involved in an inflammatory response, including NLRP6 (NLR family, pyrin domain containing 6) and CNPY3 (protein canopy homolog 3), are down-regulated, which indicates that inflammation is attenuated in the small intestinal mucosa due to the supplementation of NSPEs [46]. It has been suggested that one of the performance improvement attributes of NSPEs is due to the reduced local inflammation by controlling pathogens within the small intestine [32].

In addition to affecting the immune response, the up-regulated proteins catalase (CAT), glutaredoxin (GRXS), thioredoxin (TRX), superoxide dismutase (SOD), dimethylaniline monooxygenase [N-oxide-forming] 1 (FMO1) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) are classified as redox homeostasis and detoxification proteins based on their primary functions. The up-regulation of CAT, GRXS, TRX and SOD may suggest that NSPE pigs had more potential to keep redox homeostasis in vivo [4752]. This is consistent with the increased serum level of T-SOD in the NSPE group of this study. The reason for the up-regulation of these oxidoreductases and immune factors in the present study may be the increased abundance of reactive oxygen species (ROS) and inflammatory factors during stimulating energy metabolism due to a higher uptake of nutrients with NSPEs supplementation. However, further study is required to prove the effect of NSPEs on redox homeostasis. As one of the detoxification enzymes, FMO1 is regulated by xenobiotics, as the enzyme activity markedly increases in response to the invading harmful chemicals [53]. The up-regulation of this protein suggests that the supplementation of NSPEs is helpful to eliminate xenobiotics in the small intestine, which also could be related to the improvement of the intestinal lumen due to NSPEs.

Furthermore, the up-regulated abundance of proteins was observed in the NSPE group, including multiple nutrient metabolism processes such as energy, lipid, amino acid and mineral. These proteins included phosphoglycerate kinase 1 (PGK1), diazepam binding inhibitor (DBI), and acyl-coenzyme A oxidase (ACOX1). PGK1 plays a vital role in glycolysis or gluconeogenesis [54]. The up-regulation of ACOX1 indicates the elevation of glucose synthesis in the small intestine, which is consistent with the increased serum glucose level in the NSPE group. Likewise, higher abundance of DBI and ACOX1 was observed in this study, suggesting the stimulation of lipids β-oxidation for nutrient absorption to meet the energy requirement in the small intestine of NSPE pigs [55, 56]. Apolipoprotein C-III (APOC3) is an important modulator that is secreted from the intestine on the chylomicron upon lipid absorption [57]. The up-regulation of APOC3 implies the enhanced absorption of dietary lipids in the NSPE group.

Two differentially expressed proteins related to the permeability of the tight junction (TJ), including casein kinase II beta subunit splicing isoform 476 (Csnk2b) and myosin-4 (MYH4), were identified in the present study. The tight junctions (TJs) in the small intestine are not only a physical and biological barrier but also a passive diffusion system that depends on the permeability of the TJs [58]. Paracellular transport is one of the passive diffusion systems providing an absorption way for small molecular compounds [59], which are regulated by the permeability of the TJs and are thought to be important for mineral absorption [60]. Additionally, the transepithelial transport of oligosaccharides, but not polysaccharides, also occurs via the paracellular pathway [61]. Previous research has demonstrated that NSPEs are capable of hydrolyzing polysaccharides from the food to oligosaccharides in the gut [62]. Thus, the down-regulation of these two proteins in this study, in addition to former studies, indicates an increased permeability of the TJs in the NSPE group, which is beneficial to small molecular compounds absorption in the small intestine.

Calmodulin regulates cellular calcium concentration as a primary calcium-binding protein [63]. Calcium absorption is reduced if the bioavailability of dietary calcium is lowered by calcium-binding agents like cellulose because nearly all dietary calcium intake occurs in the upper intestine [64]. The up-regulation of this protein observed in this study suggests that calcium absorption in the small intestine is facilitated in the NSPE group by the degradation of calcium-binding agents in the diet, which could be conductive to bone health.

It has been demonstrated that one of the important roles of NSPEs within the small intestine is the elimination of the nutrient-encapsulating effect of cell wall polysaccharides, which increases the availability of starches, amino acids, and minerals. These results are consistent with our results from the present study that the levels of proteins related to nutrient absorption and utilization (energy, lipid, amino acid and mineral) are up-regulated. A fully understanding of the mechanisms of NSPEs supplementation will require the determination of protein modifications and protein regulation such as phosphorylation or glycosylation [65]. However, this part was not involved in the present study due to the technical limitation. Thus, further study is required to prove the effect of NSPEs on regulatory proteins using specific method, for example, the phosphoproteome.

Conclusions

The results of this study provide the first evidence that the small intestinal mucosa proteome is altered in growing pigs supplemented with NSPEs. Growing pigs most likely responded to the increased reactive oxygen species (ROS) and inflammatory factors during stimulating energy metabolism due to NSPEs supplementation by changing the abundance of certain mucosal proteins that modulate redox homeostasis and enhance immune response. Most important of all, the effect of NSPEs on the increase of nutrient availability in the intestinal lumen provided additional benefits to facilitate protein expressions related to the efficiency of nutrient absorption and utilization, such as energy metabolism, amino acid metabolism, mineral metabolism, lipid absorption, and cell structure and mobility. These novel findings show the mechanisms whereby dietary supplementation with NSPEs promotes growth performance and improves the GI health of growing pigs, which also has important implications for the better utilization of this feed additive.

Abbreviations

ACOX1: 

Acyl-coenzyme A oxidase

ADFI: 

Average daily feed intake

ADG: 

Average daily gain

ALP: 

Alkaline phosphatase

ALT: 

Alanine aminotransferase

APOC3: 

Apolipoprotein C-III

ARPC5: 

Actin-related protein 2/3 complex subunit 5

AST: 

Aspartate aminotransferase

CALM1: 

Calmodulin

CAT: 

Catalase

CFL: 

Cofilin

CK: 

Creatine kinase

CNPY3: 

Protein canopy homolog 3

Csnk2b: 

Casein kinase II beta subunit splicing isoform 476 MYH4: myosin-4

DAI: 

DNA-dependent activator of IFN-regulatory factor

DTT: 

Dithiothreitol

FCR: 

Feed conversion ratio

FMO1: 

Dimethylaniline monooxygenase [N-oxide-forming] 1

GALNS: 

N-acetylgalactosamine-6-sulfatase

GI: 

Gastrointestinal

GRXS: 

Glutaredoxin

HEPES: 

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPPD: 

4-hydroxyphenylpyruvate dioxygenase

IgG: 

Immunoglobulin G

iTRAQ: 

Isobaric tags for relative and absolute quantitation

NSPEs: 

Non-starch polysaccharide enzymes

PGK1: 

Phosphoglycerate kinase 1

PMSF: 

Phenylmethanesulfonyl fluoride

RPS6: 

40S ribosomal protein S6

SLA-1: 

MHC class I antigen

SOD1: 

Superoxide dismutase

TP: 

Total protein

TRX: 

Thioredoxin

T-SOD: 

Total superoxide dismutase

Declarations

Acknowledgements

This research was supported by the Chinese National Science and Technology Pillar Program (No: 2012BAD39B0), the Special Fund for Innovation Team of the Chinese Academy of Agricultural Sciences (No: ASTTP-IAS07), and the Chinese National Key Basic Research and Development Program (No: 2014CB138804).

Funding

This research was supported by the Chinese National Science and Technology Pillar Program (No: 2012BAD39B0), the Special Fund for Innovation Team of the Chinese Academy of Agricultural Sciences (No: ASTTP-IAS07), and the Chinese National Key Basic Research and Development Program (No: 2014CB138804). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

All relevant data are within the paper and its Additional files.

Authors’ contributions

JZ and HZ designed the study. JZ and YG performed the experiments and analyzed the data. JZ, QL and RS contributed reagents/materials/analysis tools. JZ prepared the manuscript and all of the authors contributed to, read and approved the final manuscript.

Competing interest

The authors declare that there is no competing interest.

Consent for publication

Not applicable.

Ethics approval

This study was conducted in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Council of the People’s Republic of China. The protocol was approved by the Committee on Experimental Animal Management of the Chinese Academy of Agricultural Sciences.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Grassland Research, Chinese Academy of Agricultural Sciences
(2)
State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
(3)
College of Animal Science and Technology, Jilin Agricultural University

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