1. Introduction
Rotavirus (RV) is a zoonotic pathogen that causes acute enteritis in infants and animals, including suckling piglets and weaned piglets, which is characterized by fever, diarrhea, and dehydration [
1,
2,
3]. The incomplete development of the immune and digestive systems of weaned piglets leads to higher morbidity and mortality rates, with a morbidity rate of 80% and a mortality rate of 20%, causing huge economic losses to the global pig industry [
4]. Intestinal epithelial cells (IECs) serve as the primary barrier against RV infection. RV infection can damage intestinal epithelial cells, then result in villus atrophy, apoptosis of IECs, eventually leading to diarrhea [
5]. Previous studies have shown that the susceptible site of RV is mainly located in the small intestine, where the virus directly damages the villous epithelial cells, leading to cell destruction and shedding [
5,
6,
7]. This destruction results in the accumulation of lactose in the intestinal lumen, creating high osmotic pressure in both the small and large intestine, which in turn causes diarrhea and vomiting [
8]. The damage to intestinal cells impairs the epithelial barrier, leading to a loss of digestive and absorptive functions, which can accelerate the death of piglets [
9]. A previous study confirmed that RV can infect and replicate in the colon of humans and mice [
10]. However, it is not fully understood whether RV affects the intestinal barrier of the colon in piglets. Therefore, this study aims to investigate the role of the colonic intestinal barrier in resisting RV infection.
The intestinal barrier, composed of intestinal epithelial cells, tight junctions, related secretions, and intestinal microbiota, serves as a crucial defense mechanism against the invasion of pathogenic microorganisms into the internal environment of the body [
11]. In weaned piglets, intestinal barrier dysfunction is often observed, accompanied by increased permeability, which subsequently leads to diarrhea and growth retardation [
6]. Therefore, effective and safe methods need to be developed to maintain the intestinal barrier function in piglets to resist pathogen invasion.
Previous studies have shown that amino acids in the diet can significantly impact intestinal health [
12,
13,
14]. Isoleucine (Ile) is one of the essential amino acids in animals, serves as a regulatory factor for the three major nutritional metabolism and conversion processes. It acts as an energy source for the immune system and a substrate for the synthesis of immune proteins [
15]. Studies have shown that isoleucine can improve the growth performance of weaned piglets on protein-restricted diets [
16,
17,
18]. Additional isoleucine supplementation promotes intestinal development in weaned piglets, increases immunoglobulin concentrations in the jejunal mucosa, and reduces the expression of pro-inflammatory cytokines [
19]. However, it remains unclear whether isoleucine can regulate intestinal barrier function. Therefore, this study aims to investigate the effects of isoleucine on colonic barrier function in RV-infected weaned piglets, as well as the changes in colonic fecal microbiota, to explore its mechanism on intestinal barrier function. These findings will provide a theoretical basis for the role of isoleucine in disease resistance and prevention.
2. Materials and Methods
2.1. Virus Culture
MA104 cells (ATCC CRL-2378), derived from African green monkey kidney cells, were purchased from the American type culture collection (ATCC). The MA104 cells were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) in a 37 °C incubator with 5% CO
2. The cells were seeded in cell culture plates and incubated at 37 °C with 5% CO
2. Once the cells reached 60% to 70% confluence, the cells were digested with 0.25% trypsin-EDTA (Merck, Burlington, MA, USA) and inoculated with RV virus (preserved at the National Veterinary Microbiological Culture Collection Management Center, Beijing, China). After two hours of incubation, the MEM medium without fetal bovine serum was replaced, and the cells were cultured for an additional 3 to 5 days before collecting the virus. This infection procedure was repeated with the collected virus until over 70% of the MA104 cells in the culture plate exhibited significant cytopathic effects within 3 days post inoculation [
20]. The virus titer was then determined to be 10
6 TCID
50/mL, and the virus was frozen for subsequent challenge experiments.
2.2. Experimental Design
All animal experiments were approved by the Animal Ethics Committee of Anhui Science and Technology University, under protocol number AK2023013. All experimental procedures were carried out in strict accordance with the “Guidelines for the Care and Use of Test Animals” of Anhui Province.
Thirty-two healthy Duroc×Landrace×Yorkshire weaned barrows, with similar body weights (6.88 ± 0.54 kg) at 21-days of age, were randomly divided into four groups (eight replicates in each group). The RV-CON and RV+CON groups were fed a basal diet, while the RV-Ile and RV+ Ile received a basal diet supplemented with 1% Ile. On the 15th day of feeding, all piglets were orogastrically inoculated with 5 mL of 100 mmol/L sodium bicarbonate (NaHCO
3) solution, and 30 min later, the infected group was orogastrically inoculated with 5 mL of RV (1 × 10
6 TCID
50/mL), while the uninfected group was orogastrically inoculated with 5 mL of saline. At 5 days post infection (dpi), all piglets were euthanized, and samples were collected. Approximately 2 cm of the colon was collected, and any fatty tissue on the surface of the intestine was carefully peeled off to avoid contamination during the sampling process. An additional 2 cm of fresh colon was collected and stored in liquid nitrogen, while another 2 cm was rinsed with saline and promptly fixed in 4% paraformaldehyde. The mucosa of colon was scraped into centrifuge tubes, frozen in liquid nitrogen, and subsequently stored at −80 °C. The animal experimental design and treatments are summarized in
Table 1 and
Figure 1.
2.3. Diet Composition
The basal diet was a corn–soybean meal formulation, designed according to the NRC (2012) guidelines (
Table 2). The isoleucine nutritional level in the basal diet was set at 0.72%. For the different dietary groups, either 1% L-isoleucine (provided by Evonik Industries AG, Essen, Germany) or 1% L-alanine (Evonik Industries AG) was added, with alanine serving as the isonitrogenous control for isoleucine.
2.4. Tissue Staining
Fixed colonic tissues were subjected to a series of procedures, including gradient alcohol dehydration, clearing, wax impregnation, embedding, slicing, slide spreading, and baking, followed by hematoxylin and eosin (HE) staining according to the manufacturer’s protocol to assess tissue morphology. Subsequently, five visual fields were uniformly selected from each slice and photographed under the same magnification. The muscular layer thickness and crypt depth of the colon were measured using Image-Pro Plus (Media Cybernetics, Version 6.0, USA) microscopic image analysis software.
2.5. The Cytokines and sIgA Analysis
The levels of interleukin (IL)-2, IL-4, IL-10, IL-12, IL-18, IL-22, TNF-α, and sIgA in the colonic mucosa were determined using ELISA kits following the manufacturer’s instructions. All the ELISA kits were purchased from Shanghai Xinle Biotechnology Co., Ltd. (Shanghai, China) (
Table 3).
2.6. Western Blot
The total protein of the colon was extracted using RIPA lysate supplemented with 1% PMSF (Beyotime, Shanghai, China) at 4 °C. The protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). A total of 20 μg of protein from each sample was loaded onto a 10% SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. To prevent non-specific binding, the PVDF membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h. Next, the membranes were incubated overnight at 4 °C with the appropriate primary antibody. Following this incubation, the membranes were washed three times with Tris-buffered saline containing Tween 20 (TBST), and then incubated with HRP-linked goat anti-rabbit antibodies at room temperature for 2 h. Visualization was performed using an ECL detection kit (Solarbio, Beijing, China). The antibodies used for Western blot are listed in
Table 4.
2.7. Colonic Microbiota Analysis
Middle-section colonic digesta samples were collected and sent to Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). DNA was extracted using a DNA extraction kit, and the V3-V4 region of 16S rRNA was targeted for PCR amplification. The primers were synthesized by Beijing Novogene Bioinformatics Technology Co., Ltd. Sequences from different samples were split using the OIIME2 demux plugin, and the split sequences underwent quality control, trimming, denoising, splicing, and chimera removal using the OIIME2 dada2 plugin to obtain the final feature sequences.
2.8. Colonic Contents Metabolomics Analysis
Samples were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) for GC-MS untargeted metabolomics analysis. The raw data were processed using the metabolomics processing software Progenesis QI 2.4 (Waters, Milford, MA, USA) to generate a data matrix. The mass spectrometry information was matched with the metabolic public database HMDB to obtain metabolite information. Differentially abundant metabolites were selected based on the criterion of p < 0.05 and VIP > 1, and volcano plots were generated using R language. The KEGG database was used for metabolic pathway annotation to identify pathways associated with differentially abundant metabolites, and enrichment analysis was performed.
2.9. Statistical Analysis
For the general data, the obtained results are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using multiple t-tests in GraphPad Prism 6.0 (GraphPad Software Inc., USA). A p value of <0.05 was considered statistically significant (*), while p values of <0.01 (**) were considered highly significant. For microbiota and metabolomics data, the analysis was conducted using the free online platform provided by the sequencing company.
4. Discussion
The intestine is responsible for the digestion and absorption of nutrients, while its barrier function helps resist the invasion of pathogenic microorganisms, including viruses, bacteria and others [
22,
23]. The intestinal immune barrier comprises lymphocytes contained in the intestinal lamina propria, intraepithelial lymphocytes (IELs), and secretory immunoglobulin A (sIgA) secreted by some lymph nodes and plasma cells [
24]. The stability of the intestinal barrier function relies on various cytokines present within it, which, together with immunoglobulins, constitute the humoral immunity of the intestine [
22]. IL-2 is the first cytokine to be molecularly cloned and has been proven to be essential for T cell proliferation and the generation of effector and memory cells [
25]. IL-4 inhibits the inflammatory response through suppressing the differentiation of T helper 1 (Th1) cells and reducing the production of pro-inflammatory factors such as IFN-γ [
26]. IL-10 is an anti-inflammatory cytokine that plays a crucial role in limiting the immune response to pathogens, thereby preventing damage to the host [
27]. IL-12 is a heterodimeric proinflammatory cytokine that induces the production of interferon-gamma (IFN-γ) by promoting the differentiation of Th1 cells [
28,
29]. IL-18 is also a pro-inflammatory cytokine that stimulates Th1 cells to produce abundant IFN-γ [
30]. IL-22 primarily targets non-hematopoietic epithelial and stromal cells, promoting proliferation and playing a role in tissue regeneration. Additionally, IL-22 regulates host defense at barrier surfaces and is implicated in diseases involving inflammatory tissue pathology [
31]. TNF-α (Tumor Necrosis Factor-alpha) is involved in maintaining the immune system’s homeostasis, regulating inflammation, and participating in pathological processes such as chronic inflammation [
32,
33]. sIgA, as an important component of the immune barrier, plays a crucial role in humoral immunity and is vital for the overall immune competence of the body [
34,
35]. In this study, we found that Ile did not affect the concentration of pro-inflammatory cytokines including IL-2, IL-12, IL-18, IL-22, and TNF-α in the colon of RV-infected piglets. However, when compared to the RV-CON group, the addition of Ile significantly raised the levels of IL-4, IL-10, and sIgA in the colons of RV-infected piglets. These findings suggest that a diet supplemented with 1% Ile can reduce intestinal inflammation caused by RV infection in piglets. Furthermore, this supplementation also appears to enhance the immune response in RV-infected piglets.
Tight junctions are formed by various transmembrane proteins that connect epithelial cells. Intestinal tight junction proteins, including Zonula Occludens-1 (ZO-1), Occludin, and Claudin-3, are crucial for establishing and maintaining the integrity and functionality of the intestinal mechanical barrier [
36,
37]. Transmembrane glycoprotein mucin-1 (MUC-1), a member of the mucin family, is secreted by goblet cells and serves as a lubricant, moisturizer, and chemical barrier in normal cells [
38]. Lin found that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and ZO-1, leading to the intercellular structure damage of fish gills [
39]. In this study, we found that inclusion of 1% isoleucine (Ile) significantly elevated the levels of Claudin-3, Occludin, ZO-1, and MUC-1 in the colons of RV-infected piglets compared with the RV+CON group. These findings suggested that isoleucine can promote the colonic intestinal barrier function of RV-infected piglets through promoting the expression of Claudin-3, Occludin, ZO-1, and MUC-1. However, the detailed mechanism requires further investigation.
Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria are the dominant phyla in the intestines of healthy piglets [
40,
41]. Previous study had shown that the abundance of Proteobacteria increases in the intestinal tract of enteritis piglets [
42]. Additionally, in diarrheal piglets, the relative abundance of Proteobacteria decreased, while the relative abundance of Sutterella and Campylobacter increased significantly [
43]. These results are consistent with our study. We found that Firmicutes, Bacteroidetes, and Proteobacteria were the dominant phyla in piglets, with a notable increase in the relative abundance of Proteobacteria and a decrease in Firmicutes in RV-infected piglets. When 1% Ile was added to the diet, the abundance of Proteobacteria in the colon of RV-infected piglets significantly reduced, while the abundance of Firmicutes increased. In piglets with diarrhea, there was a decrease in the relative abundance of members of the Firmicutes phylum, including Lactobacillus, Enterococcus, Streptococcus, and Clostridium, which are stable members of the normal intestinal microbiota in piglets [
44]. Several members of Firmicutes phylum are known to produce short-chain fatty acids and regulate systemic immune responses [
45]. Therefore, our study suggests that Ile may promote the recovery of gut microbiota in RV-infected piglets.
Metabolomics analysis revealed that the significantly altered metabolites between the RV+Ile and RV+CON groups were primarily associated with fatty acid biosynthesis, the biosynthesis of unsaturated fatty acids, and purine metabolism. Fatty acids play a crucial role in immune responses and inflammatory processes and influence various cellular signaling and metabolic pathways [
46,
47]. Unsaturated fatty acids are essential for protecting cells and maintaining their normal physiological functions [
48,
49]. Purine metabolites provide the energy and cofactors necessary for cell survival and proliferation [
50]. RV infection can cause intestinal mucosal damage, and the mechanism by which isoleucine mitigates this damage may involve promoting the synthesis of fatty acids and purine metabolites, thereby protecting normal cells. The decline in intestinal barrier function caused by RV infection, along with the partial alleviation observed with isoleucine supplementation, may be related to the significant upregulation of metabolites involved in fatty acid biosynthesis, the biosynthesis of unsaturated fatty acids, and purine metabolism.