Quantitative Proteomic Analysis of Zearalenone-Induced Intestinal Damage in Weaned Piglets

Zearalenone (ZEN), also known as the F-2 toxin, is a common contaminant in cereal crops and livestock products. This experiment aimed to reveal the changes in the proteomics of ZEN-induced intestinal damage in weaned piglets by tandem mass spectrometry tags. Sixteen weaned piglets either received a basal diet or a basal diet supplemented with 3.0 mg/kg ZEN in a 32 d study. The results showed that the serum levels of ZEN, α-zearalenol, and β-zearalenol were increased in weaned piglets exposed to ZEN (p < 0.05). Zearalenone exposure reduced apparent nutrient digestibility, increased intestinal permeability, and caused intestinal damage in weaned piglets. Meanwhile, a total of 174 differential proteins (DEPs) were identified between control and ZEN groups, with 60 up-regulated DEPs and 114 down-regulated DEPs (FC > 1.20 or <0.83, p < 0.05). Gene ontology analysis revealed that DEPs were mainly involved in substance transport and metabolism, gene expression, inflammatory, and oxidative stress. The Kyoto Encyclopedia of Genes and Genomes analysis revealed that DEPs were significantly enriched in 25 signaling pathways (p < 0.05), most of which were related to inflammation and amino acid metabolism. Our study provides valuable clues to elucidate the possible mechanism of ZEN-induced intestinal injury.


Introduction
Zearalenone (ZEN) is one of the most prevalent mycotoxins currently contaminating food and feed synthesized by the fungus Fusarium spp. [1]. Zearalenone and its metabolites are classified as xenoestrogens due to their structural similarity to endogenous estrogens [2,3]. Previous studies have shown that ZEN exposure can induce an immune response, oxidative damage, and other pathological changes in different tissues of the body, causing organ damage, decreased productive and reproductive performance, and even acute animal death in severe cases [4][5][6].
The small intestine is the most important site related to nutrient digestion and absorption, and it also serves as the first barrier to defense against pathogen invasion and mycotoxin hazards [7,8]. Previous studies have shown that the intestine is a major target organ of ZEN metabolization, and is highly vulnerable to ZEN-induced damage [9]. Weaning is one of the most stressful periods in the pig's life that can contribute to intestinal dysfunctions, leading to reduced pig health and growth retardation [10]. Therefore, weaned piglets are more susceptible to ZEN contamination [11,12]. Saenz et al. [13] found that Table 1. Effects of zearalenone (ZEN) on serum toxins of weaned piglets 1 .

Apparent Nutrient Digestibility
As shown in Table 2, compared to the control, the apparent digestibility of ether extract (EE) and crude protein (CP) was significantly lower in the ZEN piglets (p < 0.05), while no significant differences were observed in the apparent digestibility of dry matter (DM) and organic matter (OM) between the two groups (p > 0.05).

Histopathological Examination
Histopathological examination was used to observe the damage caused by ZEN in jejunum tissue. As shown in Figure 1A, compared to the control, the jejunum villi were significantly damaged and disorganized, and the lamina propria of the epithelium was detached in the ZEN. Piglets in the ZEN group had a significantly decreased jejunal villus height and the ratio of villus height to crypt depth (p < 0.05) and had significantly increased crypt depth compared to the control (p < 0.05) ( Figure 1B).

Histopathological Examination
Histopathological examination was used to observe the damage caus jejunum tissue. As shown in Figure 1A, compared to the control, the jejun significantly damaged and disorganized, and the lamina propria of the ep detached in the ZEN. Piglets in the ZEN group had a significantly decreased height and the ratio of villus height to crypt depth (p < 0.05) and had si creased crypt depth compared to the control (p < 0.05) ( Figure 1B). The red frames represent the parts of (a1, b1) enla respectively. The (a1, b1) and (a2, b1) represent the jejunal structures at different mag and ×100), respectively. IV denotes small intestinal villi; IG denotes small intestin bars were 100 μm for a-b. *** means p < 0.001.

Intestinal Permeability of Weaned Piglets
The effect of ZEN on the intestinal permeability of weaned piglets is sh 3. Piglets in the ZEN group had significantly higher serum endotoxin (ET) dase (DAO), and D-lactate contents than piglets in the control group (p < 0.  The red frames represent the parts of (a1,b1) enlarged to (a2,b2) respectively. The (a1,b1) and (a2,b1) represent the jejunal structures at different magnifications (×40 and ×100), respectively. IV denotes small intestinal villi; IG denotes small intestinal glands. Scale bars were 100 µm for a-b. *** means p < 0.001.

Intestinal Permeability of Weaned Piglets
The effect of ZEN on the intestinal permeability of weaned piglets is shown in Table 3. Piglets in the ZEN group had significantly higher serum endotoxin (ET), diamine oxidase (DAO), and D-lactate contents than piglets in the control group (p < 0.05).

Quantitative Mapping of Jejunum Proteome in the Control and ZEN Treatment
To identify proteomic changes of ZEN-induced damages in the jejunum of piglets, a powerful TMT-labeled quantitative proteomics analysis technique was applied in this experiment. A total of 306,099 bands, 39,827 peptide numbers, and 7145 proteins (Table S1) were identified and obtained. A total of 174 differentiable expressed proteins (DEPs, fold change (FC) > 1.20 or < 0.83, p < 0.05) were identified in the ZEN compared with the control (Table S2), including 60 up-regulated DEPs and 114 down-regulated DEPs ( Figure 2A). Notably, we identified 22 uncharacterized or unassigned proteins among the 174 DEPs, because the current porcine genome database was not fully annotated compared to the human genome database. Therefore, the functional analysis of these proteins warrants further investigation.

Quantitative Mapping of Jejunum Proteome in the Control and ZEN Treatment
To identify proteomic changes of ZEN-induced damages in the jejunum of piglets, a powerful TMT-labeled quantitative proteomics analysis technique was applied in this experiment. A total of 306,099 bands, 39,827 peptide numbers, and 7145 proteins (Table S1) were identified and obtained. A total of 174 differentiable expressed proteins (DEPs, fold change (FC) > 1.20 or < 0.83, p < 0.05) were identified in the ZEN compared with the control (Table S2), including 60 up-regulated DEPs and 114 down-regulated DEPs (Figure 2A). Notably, we identified 22 uncharacterized or unassigned proteins among the 174 DEPs, because the current porcine genome database was not fully annotated compared to the human genome database. Therefore, the functional analysis of these proteins warrants further investigation.
As shown in Figure 2C,D, six proteins were randomly selected from the DEPs to validate the results of the TMT proteomic analysis, including serum amyloid P-component (APCS), 15-oxoprostaglandin 13-reductase (PTGR1), glutamate carboxypeptidase 2 (FOLH1), bifunctional glutamate/proline-tRNA ligase (EPRS), eukaryotic elongation factor 2 kinase (EEF2K), and S100 calcium-binding protein A16 (S100A16). The results of the Western blot were in high agreement with the data of the TMT analysis, indicating the high reliability of the results of this proteomics analysis ( Figure 2B).  and FC > 1.20. Black dots indicate that there are no significant changes in protein expression between the Control and ZEN groups. (B) Relative expressions of serum amyloid P-component (APCS), 15-oxoprostaglandin 13-reductase (PTGR1), glutamate carboxypeptidase 2 (FOLH1), bifunctional glutamate/proline-tRNA ligase (EPRS), and eukaryotic elongation factor 2 kinase (EEF2K), and S100 calcium-binding protein A16 (S100A16) analyzed by TMT proteomics. The red and blue boxes represent up-regulated and down-regulated differentially expressed proteins, respectively. The same in (D). (C,D) The relative abundance of proteins between the control and ZEN groups analyzed by Western blot. a, b Means differ significantly (p < 0.05).
As shown in Figure 2C,D, six proteins were randomly selected from the DEPs to validate the results of the TMT proteomic analysis, including serum amyloid P-component (APCS), 15-oxoprostaglandin 13-reductase (PTGR1), glutamate carboxypeptidase 2 (FOLH1), bifunctional glutamate/proline-tRNA ligase (EPRS), eukaryotic elongation factor 2 kinase (EEF2K), and S100 calcium-binding protein A16 (S100A16). The results of the Western blot were in high agreement with the data of the TMT analysis, indicating the high reliability of the results of this proteomics analysis ( Figure 2B).

Top Ten Up-Regulated or Down-Regulated DEPs
The top ten up-regulated proteins in the ZEN group are listed in Table 4. Among the top ten up-regulated proteins, the SERPIN domain-containing protein and uncharacterized protein exert enzyme inhibitory activity; the glutathione S-transferase kappa 1 isoform a (fragment), 15-oxoprostaglandin 13-reductase, and the threonyl-tRNA synthetase exert enzymatic activity; the beta-parvin isoform X3 binds to actin; and the apolipoprotein C-II exerts enzyme activator activity. The main functions of the Ig-like domain-containing protein, uncharacterized protein, and nuclear autoantigenic sperm protein have not been reported. The top ten down-regulated proteins in the ZEN group are listed in Table 5. Of the top ten down-regulated proteins, the glutathione S-transferase kappa 1 isoform a (fragment), enoyl-CoA hydratase, glutamyl-tRNA synthetase, and threonyl-tRNA synthetase exert enzymatic activity; the EF-hand domain-containing protein binds to calcium ions; the lysozyme C-2 plays an immune-enhancing role; the translation initiation factor eIF-2B subunit delta isoform 2 exerts translation initiation factor activity; the eukaryotic elongation factor 2 kinase binds to calmodulin; the HIT domain-containing protein plays a catalytic activity. In addition, there is one down-regulated protein whose function has not been described.

Gene Ontology (GO) Functional Annotation and Enrichment Analysis of DEPs
To characterize the DEPs, we performed GO functional annotation statistics on the DEPs, which were divided into three major categories at the functional level: biological process (BP), cellular component (CC), and molecular function (MF) [21].
Within the BP category, the DEPs were predicted to be linked with 16 biological processes, such as cellular process, biological regulation, and metabolic process ( Figure 3, Table S3). Among them, proteins associated with the regulation of the modulation of the molecular function in other organisms, the modulation of the molecular function in other organisms involved in symbiotic interaction, modulation by a host of symbiont molecular function, response to thyroid hormone, and cellular response to thyroid hormone stimulus were the top five significantly enriched in the BP category ( Figure 4, Table S4).    Within the CC category, the DEPs were predicted to be primarily distributed w two different cellular components, including the cellular anatomical entity and pr containing complex ( Figure 3, Table S3). Especially, proteins associated with the r tion of nuclear pericentric heterochromatin, chromocenter, pericentric heterochro Within the CC category, the DEPs were predicted to be primarily distributed within two different cellular components, including the cellular anatomical entity and proteincontaining complex ( Figure 3, Table S3). Especially, proteins associated with the regulation of nuclear pericentric heterochromatin, chromocenter, pericentric heterochromatin, histone deacetylase complex, and nuclear speck were the top five significantly enriched in the CC category ( Figure 4, Table S4).
Additionally, within the MF category, the DEPs were predicted to be linked with ten molecular functions, for instance, binding, catalytic activity, and the molecular function regulator ( Figure 3, Table S3). Particularly, proteins associated with the regulation of proteoglycan binding, supercoiled DNA binding, threonine-tRNA ligase activity, glutathione peroxidase activity, and SH3 domain binding were the top five significantly enriched in the MF category ( Figure 4, Table S4).

Kyoto Encyclopedia of Gene and Genomes (KEGG) Enrichment Analysis of DEPs
KEGG enrichment analysis was performed on 174 DEPs, and a total of 25 significantly different pathways were enriched for these proteins. Those pathways related to inflammation and amino acid metabolism were more enriched. The major pathways related to inflammation included the Rap1 signaling pathway, B cell receptor signaling pathway, and Fc gamma R-mediated phagocytosis. These three pathways contain two identical DEPs, including the Ig-like domain-containing protein (LOC100523213) and protein kinase domain-containing protein (PBK). The pathways related to amino acid metabolism mainly included lysine degradation, beta-alanine metabolism, and tryptophan metabolism ( Figure 5, Table S5). The aldehyde dehydrogenase (NAD + ) (ALDH-NAD + ) of DEPs was involved in the three pathways.
identical DEPs, including the Ig-like domain-containing protein (LOC100523213) and protein kinase domain-containing protein (PBK). The pathways related to amino acid metabolism mainly included lysine degradation, beta-alanine metabolism, and tryptophan metabolism ( Figure 5, Table S5). The aldehyde dehydrogenase (NAD + ) (ALDH-NAD + ) of DEPs was involved in the three pathways.

Discussion
Zearalenone can be converted to α-ZOL and β-ZOL after being absorbed by the intestine. Once the rate of ZEN deposition exceeded that of metabolism, ZEN and its metabolites might be accumulated in the body [22][23][24]. However, a previous study showed that the positive detection rate of ZEN could reach 69.15%, and the highest value of ZEN in compound feed samples was 4.33 mg/kg [6]. In our study, significantly increased serum ZEN, α-ZOL, and β-ZOL levels of weaned piglets were observed in the piglets fed the diet

Discussion
Zearalenone can be converted to α-ZOL and β-ZOL after being absorbed by the intestine. Once the rate of ZEN deposition exceeded that of metabolism, ZEN and its metabolites might be accumulated in the body [22][23][24]. However, a previous study showed that the positive detection rate of ZEN could reach 69.15%, and the highest value of ZEN in compound feed samples was 4.33 mg/kg [6]. In our study, significantly increased serum ZEN, α-ZOL, and β-ZOL levels of weaned piglets were observed in the piglets fed the diet containing 3.0 mg/kg ZEN, which was in line with our previous study [25]. Zhang et al. [24] found that 970 µg/kg ZEN could increase the levels of ZEN, α-ZOL, and β-ZOL of serum in gilts. Moreover, it was also reported that oral 1.0 mg/kg BW ZEN increased serum levels of ZEN, α-ZOL, and β-ZOL in juvenile female pigs [26]. Long-term ZEN deposition can damage intestinal development and function, negatively influencing the digestive system of animals [27]. In the present study, the apparent digestibility of CP and EE in ZEN piglets was significantly reduced. Consistently, our previous study in piglets showed that the apparent digestibility of CP, gross energy (GE), the metabolic rate of GE, and net protein utilization were decreased by ZEN (more than 1.0 mg/kg) treatment [28]. Wang et al. [29] also reported that the apparent digestibility of DM and nitrogen was decreased with increasing concentrations of ZEN (0.2 to 0.8 mg/kg) in the weaned piglets.
The small intestine is the principal organ in charge of nutrient absorption, and intestinal morphology and structural integrity were fundamental to maintaining normal function [9]. In this study, ZEN exposure disrupted the jejunal villus and glands and decreased the villus height and villus height/crypt depth ratio, showing intestinal barrier damage and nutrient absorption area reduction. The villi height, crypt depth, and the villi height/crypt depth ratio were considered important indicators to evaluate the ability of the animal to digest and absorb nutrients [30]. The shortening or loss of the intestinal villus usually led to a reduction in nutrient absorption area, resulting in malnutrition, diarrhea, and decreased disease resistance [31]. Liu et al. [32] also found that ZEN (0.3 to 146 mg/kg) Toxins 2022, 14, 702 9 of 17 damaged the jejunal villus of pregnant dams in a dose-dependent manner, leading to a reduction in functional mucosal surface area. Moreover, a previous study in rats showed that 1.0 and 5.0 mg/kg of ZEN caused intestinal villus and gland injury with the separation of the submucosa and lamina propria [9]. In addition, in the present study, we found that ZEN exposure increased the serum concentrations of ET, DAO, and D-lactate in weaned piglets. A study in rats also proved that ZEN (1.0 and 5.0 mg/kg) exposure significantly elevated serum DAO and D-lactate concentrations [9]. Endotoxin was a component of the cell wall of Gram-negative bacteria [33], and D-lactate was a specific metabolite of intestinal bacteria [34]. The DAO was an intracellular enzyme present in Mammalian intestinal mucosal cells [35]. The ET, DAO, and D-lactate would be released into the circulation system when the intestinal barrier was compromised [36]. Therefore, Blood ET, DAO, and D-lactate levels were considered key markers to assess intestinal permeability [36]. Above all, our results suggested that ZEN induced the decrease in the apparent digestibility of nutrients in weaned piglets partly through damaging the small intestinal morphology and barrier integrity.
To characterize the underlying mechanisms of ZEN-induced intestinal damage in weaned piglets, the TMT proteomics approach was employed in this study. We further confirmed the results of TMT proteomics using Western blot and found the related proteins significantly altered with up-regulated APCS, PTGR1, and FOLH1 expressions and down-regulated EPRS, EEF2K, and S100A16 expressions in ZEN piglets. Gene ontology and KEGG enrichment analysis provide important references and clues for understanding the functions of DEPs [37]. In the present study, GO terms and signaling pathways related to inflammation and substance metabolism were more enriched with ZEN treatment. Consistently, Gajęcka et al. [38] indicated that ZEN stimulated energy and protein metabolic processes in pre-pubertal gilts. Besides, a previous study in mice showed that ZEN increased intestinal inflammatory factors [39]. Therefore, inflammatory response and metabolic disorders induced might be contributed to the intestinal injury induced by ZEN in this study.
In our study, almost half of the signaling pathways were related to inflammation. The inflammatory pathways of the Rap1 signaling pathway, B cell receptor signaling pathway, and Fc gamma R-mediated phagocytosis were activated by ZEN. The Rap1 signaling pathway was activated to increase cell adhesion, polarization, and chemotaxis, and could regulate the inflammatory process by activating monocytes [40]. Cai et al. [41] demonstrated that the Rap1 could induce cytokine production in pro-inflammatory macrophages through NF-κB signaling in human atherosclerotic lesions. The function and maturation of B cells receptor (BCR) were closely connected with the proliferation and differentiation of B cells [42]. BCR-mediated antigen recognition was thought to regulate B cell differentiation, and the activation and amplification of BCR signaling benefited to promote B cell survival and growth [43,44]. Fcγ receptors (FcγRs), receptors for IgG, classically regulated the course of the immune response [45] and B cell activation signals delivered by the BCR [46]. Interestingly, two identical DEPs, LOC100523213 and PBK, were both involved in the three pathways. In this study, ZEN exposure up-regulated the LOC100523213 expression and down-regulated the PBK expression in the intestine. These results further demonstrated that ZEN could induce intestinal damage by activating the inflammatory response in the intestine of weaned piglets. The LOC100523213 belongs to the immunoglobulin superfamily [47]. Inflammation stimulation or infection could result in the activation of B cells and increased immunoglobulins, which improves the ability of the body to remove pathogens that induce humoral immune responses [48,49]. A recent study reported that the LOC100523213 expression was elevated in the serum of meningitis piglets [50]. The PBK is a novel serine/threonine kinase that is highly expressed in proliferating cells and tissues [51]. A recent study demonstrated a negative correlation between PBK expression and immune suppressor cells, including regulatory T cells and M2 macrophages [52]. Moreover, Zhu et al. [53] found that PBK expression was suppressed after the development of inflammation in thin endometrium. Therefore, ZEN-induced inflammatory response might be one reason for intestinal injury in weaned piglets.
Other highly enriched signaling pathways were associated with amino acid metabolism. In this study, the amino acid metabolic pathways of lysine, beta-alanine, and tryptophan were activated under ZEN treatment. The intestine is the main site of amino acid absorption and metabolism [54]. The activation of amino acid metabolic pathways further demonstrated that ZEN could regulate amino acid absorption and metabolism in weaned piglets. Lysine and tryptophan are not only essential amino acids but also serve as the first and second limiting amino acids for pigs [55,56]. Lysine and tryptophan deficiencies in piglet diets could cause reduced feed intake and feed utilization and suppress immune function, leading to increased morbidity and mortality [57][58][59][60]. A previous study also indicated that the ileal apparent digestibility of tryptophan was significantly reduced in piglets feeding the basal diet supplemented with 10 mg/kg of ZEN compared with those fed the basal diet [61]. Beta-alanine was the precursor for the synthesis of functional compounds such as coenzyme A and pantothenic acid [62]. As a potentially functional amino acid, betaalanine plays an important role in maintaining the normal growth of organisms [63,64]. In addition, the ALDH-NAD + from the three pathways mentioned above was suppressed in the ZEN-treated piglets. The ALDH-NAD + was the one of ALDH [65], and its deficiency enhanced the ethanol-induced disruption of intestinal epithelial tight junctions and barrier dysfunction [66]. Therefore, we hypothesized that ZEN could cause damage to the intestine by regulating amino acid metabolic processes via inhibiting ALDH expression in weaned piglets. However, precise functions of those pathways and DEPs in ZEN-induced intestinal damage remain to be elucidated by cellular experiments.

Conclusions
In summary, our study showed that 3.0 mg/kg ZEN resulted in decreased nutrient digestibility and destroyed intestinal integrity. Meanwhile, ZEN could damage the intestine of weaned piglets through changing the process of substance metabolism and triggering an inflammatory response. Additionally, the DEPs, acting as intermediates or key enzymes in a variety of potential signaling pathways, were mainly involved in pathways related to inflammation and amino acid metabolism in this study. Although further studies will be required to elucidate the functions of the DEPs, our study provides valuable clues to elucidate the possible mechanism of ZEN-induced intestine intestinal injury and lays the groundwork for future research on ZEN detoxification in animals.

Animals, Treatments, and Feeding Management
A total of 16 healthy Duroc × Landrace × Yorkshire weaned piglets (35 d of age) with an average body weight (BW) of 12.45 ± 0.19 kg were randomly allotted to two treatments with eight replicates per treatment and one piglet per replicate. Weaned piglets either received a basal diet (control group) or a basal diet supplemented with 3.0 mg/kg ZEN (ZEN group). The dose of ZEN was selected according to the results of our previous study that 3.0 mg/kg of ZEN in piglet diets induced the obstruction of intestinal selfrepair [16]. The basal diet was prepared according to the National Research Council (NRC, 2012, Washington, DC, USA), and the ration formulation and its contents of various major nutrients were shown in Table S6. The weaned piglets used in this trial were housed in cages with an area of 0.48 m 2 for a 32 d experiment period after a 7 d adjustment. The room temperature was controlled at 30 • C in the first week of the experiment and then maintained between 26 • C and 28 • C until the end of the experiment. The relative humidity was kept at about 65%.
The mycotoxins of the diets were carried out by Qingdao Entry-Exit Inspection and Quarantine Bureau (Qingdao, China) as previously described [67]. The levels of ZEN and aflatoxin were quantified by liquid chromatography in conjunction with fluorescence detection, affinity column chromatography, and external standard method, and the contents of fumonisin and deoxynivalenol were quantified by high-performance liquid chromatography-tandem mass with fluorescence detection, affinity column chromatography, and external standard method [14,16]. The minimum detection limits of ZEN, fumonisins, deoxynivalenol, and aflatoxin were 0.01 mg/kg, 0.25 mg/kg, 0.1 mg/kg, and 1.0 µg/kg, respectively. The analyzed ZEN levels in the basal diet and ZEN diet were <0.01 and 3.12 ± 0.13 mg/kg, respectively, and the other toxins were not detected.

Sample Collection
Piglets were fasted for 12 h before sampling on the last day of the trial. Approximately 5 mL of blood was collected from the jugular veins into vacuum procoagulation tubes and placed at room temperature for 20 min. The serum was obtained after being centrifuged at 3000× g for 15 min in a low-temperature centrifuge and stored at -20 • C immediately. The serum was used to determine the toxin, ET, DAO, and D-lactate content. After blood samples collection, the piglets were injected intramuscularly with 0.1 mg/kg BW Zoletile 50 Vet (Virbac, Brittany, Carros, France) and immediately slaughtered for jejunum sampling. One part of the jejunum samples was fixed in 4% paraformaldehyde solution for 24 h to observe the morphological changes of the intestine, while the other part of the jejunum samples was stored in a −80 • C refrigerator for antioxidant capacity, protein expression, and proteomic analysis.

Serum ZEN, α-ZOL, and β-ZOL
Serum ZEN, α-ZOL, and β-ZOL were determined by the Institute of Quality Standards and Detection Technology of the Chinese Academy of Agricultural Sciences (Beijing, China). The specific operation method was referenced by Wan et al. [25].

Apparent Nutrient Digestibility
Feces excreted by the piglets were collected daily for four consecutive days starting on day 20 of the experiment. The feces were weighed and mixed, and representative samples were stored in a −20 • C refrigerator after the nitrogen was fixed with 10% sulfuric acid to determine CP (Kjeldahl method). The rest of the feces were baked to constant weight in a 65 • C thermostat, followed by being crushed and stored in sealed containers for the determination of DM (105 • C drying method), crude ash (CA, 550 • C scorching method), OM (OM = DM − CA), and EE (Soxhlet extraction method). The apparent nutrient digestibility of DM, OM, CP, and EE was calculated in a previous study [68].

Histopathological Examination of Jejunum
After being fixed in 4% paraformaldehyde solution for 24 h, the jejunum samples were trimmed and placed in an embedding box. Then, they were rinsed in running water for 48 h, followed by alcohol gradient dehydration, xylene transparency, and paraffin embedding. Finally, serial sections of 5 µm thickness were created and dewaxed in a gradient solution of xylene and ethanol after being dried at 37 • C, followed by staining with hematoxylin and eosin. The jejunum structures were observed using an Olympus BX41 microscope equipped with a DP25 digital camera (Olympus, Tokyo, Japan). The intestinal villus height and crypt depth of weaned piglets were carried out using Motic images 2000 software (version 1.3, Motic Incorporation, Ltd., Hong Kong, China). Eight sections were chosen from each sample, and ten well-extended, straight, and intact villi were selected from each section for measurement. The villi height and the crypt depth around them were measured and averaged to calculate the ratio of villi height to crypt depth. The detail determination methods of the villi height and the crypt depth were referred to in a previous study [35]. In short, the villi height was measured from the villi tip to the villi base, and the crypt depth was measured from the intervillous valley to the basement membrane.

Intestinal Permeability of Weaned Piglets
The levels of ET and DAO in serum were determined using commercial ELISA assay kits purchased from the Jiangsu Meimian Industrial Co., Ltd., (Yancheng, China). The ELISA experiment procedures were derived from Chen et al. [69]. Additionally, the Dlactate content of the serum was measured by a corresponding ELISA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The ELISA experiment procedures were referenced by Gao et al. [70].

Sample Preparation and TMT Labeling
To determine the expression of the entire proteome, eight jejunum samples were selected for TMT-based quantitative proteomic analysis in Majorbio Bio-pharm Technology Co., Ltd., (Shanghai, China). The screenshot of the workflow of Proteome Discoverer was shown in Figure S1. Four jejunum samples were randomly selected from each group. The samples were removed from the refrigerator at -80 • C and transferred to centrifuge tubes, followed by being shaken and mixed with protein lysis solution (8 M urea + 1% SDS with protease inhibitor). The tubes were placed in a high-throughput tissue grinder (Tissuelyser-24, Shanghai, China) and lysed on ice for 30 min. Afterward, the tubes were centrifuged (4 • C, 16,000× g, 30 min) in a low-temperature centrifuge to separate the supernatants, and the desired protein samples were obtained. The extracted proteins were quantified using the Thermo Scientific Pierce BCA kit (Waltham, MA, USA), and the protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
The 100 µg of protein sample was placed in a centrifuge tube and the volume was replenished with lysate to 90 µL. Tris (2-carboxyethyl) phosphine (TCEP) at a final concentration of 10 mM was added to each tube and incubated for 1 h at 37 • C. Iodoacetamide (IAM) at a final concentration of 40 mM was added to each tube, and the reaction was carried out for 40 min at ambient temperature and protected from light. Then, pre-chilled acetone (acetone: sample volume ratio = 6:1) was added to each tube and precipitated at -20 • C for 4 h. After centrifugation at 10,000× g for 20 min at 4 • C, the supernatant was removed, and 100 µL of 100 mM ammonium bicarbonate (TEAB) was added to the pellet. Trypsin (2 µg/µL) was added to each tube at a ratio of trypsin: protein = 1:50 and digested overnight at 37 • C, and then terminated by the addition of 1% trifluoroacetic acid. Acetonitrile was added to TMT reagent (art. NO. 90111, Thermo Fisher, Waltham, MA, USA), which was added to the previously prepared peptide after centrifugation and reacted at room temperature for 2 h. Hydroxylamine was added to each tube and left at room temperature for 30 min. The control group samples were labeled as TMT10-127N, TMT10-127C, TMT10-128N, and TMT10-128C, and the ZEN group samples were labeled as TMT10-129N, TMT10-129C, TMT10-130N, and TMT10-130C, respectively.

Sequence Database Searching and Protein Identification
The RAW files from the mass spectrometry downstream were searched using the Proteome Discoverer TM Software 2.4 (Thermo, Waltham, MA, USA). The RAW files were submitted to the Proteome Discoverer server; the selected species was Sus scrofa, the database was uniprot-taxonomy-9823.unique.fasta, and then the database search was performed. The false discovery rate (FDR) of peptide identification during the library search was set to FDR ≤ 0.01, and the protein contained at least one specific peptide. The relevant parameters were shown in Table 6.

Bioinformatics Analysis
GO (https://www.biobam.com/blast2go/ (accessed on 28 July 2021); http://gene ontology.org/ (accessed on 28 July 2021)) was selected for functional clustering analysis of all differential proteins. The metabolic pathways involved in differential proteins were analyzed using the KEGG (http://www.genome.jp/kegg/ (accessed on 28 July 2021)) pathway database, which was used to analyze the metabolic pathways involved in the DEPs. Fisher's exact test was used to identify the GO terms or KEGG pathways that were significantly enriched in significantly differential proteins compared to the proteomic background. p < 0.05 can be used as the threshold for significantly elevated GO or KEGG.

Statistical Analysis
To detect the difference between the control and the ZEN group, the data were statistically analyzed using an independent samples t-test with SAS 9.4 statistical software (SAS Institute Inc., Cary, NC, USA). The data are presented as means ± standard error. The difference was considered significant when p < 0.05, and the protein features were considered to be significantly changed between different jejunum samples using a statistical p < 0.05 and FC > 1.20 or <0.83.

Supplementary Materials:
The following supporting information can be downloaded at: https://ww w.mdpi.com/article/10.3390/toxins14100702/s1, Table S1: A total of 7145 proteins were identified in the jejunum tissue of weaned piglets from the control and ZEN treatment using TMT-labeled LC−MS/MS analysis; Table S2: A total of 174 differentially expressed proteins were identified in the jejunum tissue of weaned piglets from the control and ZEN treatment using TMT-labeled LC−MS/MS analysis; Table S3: GO functional annotation of 174 significantly differentially expressed proteins in the control and ZEN treatment; Table S4: GO enrichment analysis of 174 significantly differentially expressed proteins in the control and ZEN treatments; Table S5: KEGG enrichment analysis of 174 significantly differentially expressed proteins in the control and ZEN treatment; Table S6: Ingredients and nutrient contents of the basal diet (air-dry basis); Figure S1: Screenshot of the workflow of Proteome Discoverer.

Informed Consent Statement: Not applicable.
Data Availability Statement: In this study, the proteomics data have been deposited to the Pro-teomeXchange Consortium (http://proteomecentral.proteomexchange.org (accessed on 13 September 2022)) via the iProX partner repository with the dataset identifier PXD036686.

Acknowledgments:
The authors would like to express their gratitude to the pig farm in Fanzhuang Village, Dongping County, Tai'an City, Shandong Province, China. The authors are particularly grateful to Xiao Li and Huajie Zhou for their help in data collection. Additionally, particular thanks to other members of the team for their involvement and efforts in the whole experiment organization and information collection.

Conflicts of Interest:
The authors declare no conflict of interest.