Porcine Intestinal Epithelial Cells Research Articles

Lnc001776 Affects CPB2 Toxin-Induced Excessive Injury of Porcine Intestinal Epithelial Cells via Activating JNK/NF-kB Pathway through ssc-let-7i-5p/IL-6 Axis

Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea.

Metabolomic and Proteomic Profiling of Porcine Intestinal Epithelial Cells Infected with Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) infection results in severe epidemic diarrhea and the death of suckling pigs. Although new knowledge about the pathogenesis of PEDV has been improved, alterations in metabolic processes and the functional regulators involved in PEDV infection with host cells remain largely unknow. To identify cellular metabolites and proteins related to PEDV pathogenesis, we synergistically investigated the metabolome and proteome profiles of PEDV-infected porcine intestinal epithelial cells by liquid chromatography tandem mass spectrometry and isobaric tags for relative and absolute quantification techniques. We identified 522 differential metabolites in positive and negative ion modes and 295 differentially expressed proteins after PEDV infection. Pathways of cysteine and methionine metabolism, glycine, serine and threonine metabolism, and mineral absorption were significantly enriched by differential metabolites and differentially expressed proteins. The betaine-homocysteine S-methyltransferase (BHMT) was indicated as a potential regulator involved in these metabolic processes. We then knocked down the BHMT gene and observed that down-expression of BHMT obviously decreased copy numbers of PEDV and virus titers (p < 0.01). Our findings provide new insights into the metabolic and proteomic profiles in PEDV-infected host cells and contribute to our further understanding of PEDV pathogenesis.

Transcriptomic and antiviral analyses of PoIFN-Delta5 against porcine enteric viruses in porcine intestinal epithelial cells

The interferon-delta family was first reported in domestic pigs and belongs to the type I interferon (IFN-I) family. The enteric viruses could cause diarrhea in newborn piglets with high morbidity and mortality. We researched the function of the porcine IFN-delta (PoIFN-δ) family in the porcine intestinal epithelial cells (IPEC-J2) cells infected with porcine epidemic diarrhea virus (PEDV). Our study found that all PoIFN-δs shared a typical IFN-I signature and could be divided into five branches in the phylogenic tree. Different strains of PEDV could induce typical IFN transitorily, and the virulent strain AH2012/12 had the strongest induction of porcine IFN-δ and IFN-alpha (PoIFN-α) in the early stage of infection. In addition, it was found that PoIFN-δ5/6/9/11 and PoIFN-δ1/2 were highly expressed in the intestine. PoIFN-δ5 had a better antiviral effect on PEDV compared to PoIFN-δ1 due to its higher induction of ISGs. PoIFN-δ1 and PoIFN-δ5 also activated JAK-STAT and IRS signaling. For other enteric viruses, transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (PoRV), PoIFN-δ1 and PoIFN-δ5 both showed an excellent antiviral effect. Transcriptome analyses uncovered the differences in host responses to PoIFN-α and PoIFN-δ5 and revealed thousands of differentially expressed genes were mainly enriched in the inflammatory response, antigen processing and presentation, and other immune-related pathways. PoIFN-δ5 would be a potential antiviral drug, especially against porcine enteric viruses. These studies were the first to report the antiviral function against porcine enteric viruses and broaden the new acquaintances of this type of interferon though not novelly discovered.

Fructo-oligosaccharides Ameliorate Intestinal Mechanical Barrier Injury in Piglets Induced by Soybean Antigen in vitro and in vivo.

Fructose oligosaccharides (FOS) have been shown to reduce soybean antigeninduced hypersensitivity in piglets, but their effects on intestinal epithelial barrier function have not been characterized. Therefore, this study aimed to determine the effects of FOS on intestinal barrier injury induced by soybean antigen in piglets in vitro and in vivo. We studied the protective effects of FOS against mechanical barrier dysfunction induced using β-conglycinin or glycinin in porcine intestinal epithelial cells (IPEC-J2), and measured the serum concentrations of diamine oxidase (DAO), D-lactic acid, and endotoxin, and the expression of tight junction (TJ) proteins, in piglets. We found that FOS concentration dependently increases cell activity, trans-epithelial electrical resistance, and TJ protein expression (P < 0.05) and reduces alkaline phosphatase (AP) activity (P < 0.05) in vitro. In addition, the serum DAO, D-lactic acid, and endotoxin concentrations were reduced by FOS administration in piglets (P < 0.05). Both in vitro and in vivo, the expression levels of TJ proteins (zona occludens 1 and occludin) were increased significantly by FOS (P < 0.05). Therefore, FOS protect against intestinal injury induced by soybean antigen in piglets, which may provide a basis for the prevention of allergy.

Molecular mechanism of valine and its metabolite in improving triglyceride synthesis of porcine intestinal epithelial cells

An insufficient energy supply to intestinal epithelial cells decreases production performance in weaned piglets. Triglycerides are the main energy source for intestinal epithelial cells in piglets. The present study aimed to investigate the effects and mechanisms of valine supplementation on triglyceride synthesis in porcine intestinal epithelial (IPEC-J2) cells. Valine supplementation in the medium significantly increased the content of triglycerides, fat droplets, and long-chain fatty acids (C17:0, C18:0, C20:0, C18:1, C20:1, and C22:1) (P < 0.05). Valine metabolite (3-hydroxyisobutyrate [3-HIB]) concentration increased significantly in the valine-supplemented group (P < 0.05). Silencing of the 3-HIB synthase enzyme 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) in IPEC-J2 cells significantly reduced the triglyceride concentration and lipid droplet synthesis. Further studies found that 3-HIB supplementation in the medium significantly increased the concentration of triglycerides, lipid droplets, and unsaturated fatty acids (C16:1, C18:1, C18:2, C18:3, C20:3, C20:4, and C20:5) (P < 0.05) by upregulating the expression of proteins involved in fatty acid transport (CD36) and fatty acid binding protein 3 (FABP3) or triglyceride synthesis (DGAT1) (P < 0.05), indicating that 3-HIB mediates valine-enhanced triglyceride synthesis in IPEC-J2 cells. In conclusion, our results demonstrated that valine enhanced triglyceride synthesis in IPEC-J2 cells via increasing the 3-HIB concentration, which may promote fatty acid transport via upregulation of proteins related to fatty acid transporter. These findings provide new insights into the mechanisms through which valine participates in lipid metabolism.

Air–liquid interface cultures trigger a metabolic shift in intestinal epithelial cells (IPEC-1)

An improved oxygen availability in air–liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-d-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.

Silybin Showed Higher Cytotoxic, Antiproliferative, and Anti-Inflammatory Activities in the CaCo Cancer Cell Line while Retaining Viability and Proliferation in Normal Intestinal IPEC-1 Cells.

The anticancer potential of silymarin is well known, including its anti-inflammatory as well as antiproliferative effect mediated by influencing the cell cycle, suppression of apoptosis, and inhibition of cell-survival kinases. However, less is known about silybin, the main component of the silymarin complex, where studies indicate its dual effect on the proliferation and immune response of various cell types in a dose-dependent manner. Moreover, there is a lack of studies comparing the effect of silybin on the same type of healthy and tumor cells, especially intestinal ones. Therefore, our study aimed to investigate the concentration-dependent effect of silybin on the normal intestinal porcine epithelial cell line-1 (IPEC-1) and the human epithelial colorectal adenocarcinoma cell line (CaCo-2). The metabolic viability, cell cycle, mitochondrial membrane potential, apoptosis, and the relative gene expression for pro- and anti-inflammatory cytokines were monitored in cells treated with silybin. Silybin stimulates metabolic viability as well as proliferation in IPEC-1 cells, protects the mitochondrial membrane, and thus exerts a cytoprotective effect, and has only a minimal effect on the gene expression of pro-inflammatory cytokines but significantly increases the expression of anti-inflammatory TGF-β. In contrast, it inhibits metabolic viability in tumor intestinal CaCo-2 cells, has an antiproliferative effect accompanied by increased apoptosis, and significantly reduces the expression of genes for pro-inflammatory interleukins as well as TGF-β. The antiproliferative and anti-inflammatory effect of silybin on tumor intestinal cells without a negative effect on healthy cells is a prerequisite for its potential use in the adjuvant therapy of colon cancer; however, further studies are necessary.

Benzo[a]pyrene induces epithelial tight junction disruption and apoptosis via inhibiting the initiation of autophagy in intestinal porcine epithelial cells

Ingestion of food contaminated with benzo[a]pyrene (B[a]P) poses health risks to animals and humans. However, the toxicity of B[a]P exposure on the intestinal barrier function and underlying mechanisms remain obscure. In the present study, intestinal porcine epithelial cells (IPEC-1) were challenged with different doses of B[a]P and its deleterious effects were determined. We found that B[a]P exposure led to impaired intestinal tight junction function as evidenced by reduced transepithelial electric resistance, increased permeability, and downregulated intestinal tight junction protein levels. Further study demonstrated that B[a]P treatment induced cell cycle arrest, and resulted in oxidative damage-related apoptosis in IPEC-1 cells. Intriguingly, we observed an inhibition of autophagy and an activation of unfolded protein response (UPR) in B[a]P-challenged cells, when compared with controls. To investigate the role of autophagy on B[a]P-induced epithelial tight junction disruption and apoptosis, cells were cotreated with B[a]P and rapamycin, and rapamycin dramatically improved intestinal tight junction and reduced apoptosis, indicating a protective effect of autophagy for the cells in response to B[a]P treatment. We also explored the role of UPR in B[a]P-induced cellular damage by using 4-phenylbutyric acid, an antagonist of UPR. Interestingly, B[a]P-induced apoptosis and dysfunction of the intestinal tight junction were exacerbated by 4-phenylbutyric acid, and the 4-phenylbutyric acid didn't ameliorate the inhibitory effects of B[a]P on microtubule-associated protein 1 light chain 3 (LC3-II) and lysosomal-associated membrane protein 2 (LAMP2) in IPEC-1 cells. These novel findings provided herein indicated that B[a]P induces intestinal epithelial tight junction disruption and apoptotic cell death via inhibiting autophagy in IPEC-1 cells.

Protocatechuic acid and quercetin attenuate ETEC-caused IPEC-1 cell inflammation and injury associated with inhibition of necroptosis and pyroptosis signaling pathways

BackgroundNecroptosis and pyroptosis are newly identified forms of programmed cell death, which play a vital role in development of many gastrointestinal disorders. Although plant polyphenols have been reported to protect intestinal health, it is still unclear whether there is a beneficial role of plant polyphenols in modulating necroptosis and pyroptosis in intestinal porcine epithelial cell line (IPEC-1) infected with enterotoxigenic Escherichia coli (ETEC) K88. This research was conducted to explore whether plant polyphenols including protocatechuic acid (PCA) and quercetin (Que), attenuated inflammation and injury of IPEC-1 caused by ETEC K88 through regulating necroptosis and pyroptosis signaling pathways.MethodsIPEC-1 cells were treated with PCA (40 μmol/L) or Que (10 μmol/L) in the presence or absence of ETEC K88.ResultsPCA and Que decreased ETEC K88 adhesion and endotoxin level (P < 0.05) in cell supernatant. PCA and Que increased cell number (P < 0.001) and decreased lactate dehydrogenases (LDH) activity (P < 0.05) in cell supernatant after ETEC infection. PCA and Que improved transepithelial electrical resistance (TEER) (P < 0.001) and reduced fluorescein isothiocyanate-labeled dextran (FD4) flux (P < 0.001), and enhanced membrane protein abundance of occludin, claudin-1 and ZO-1 (P < 0.05), and rescued distribution of these tight junction proteins (P < 0.05) after ETEC infection. PCA and Que also declined cell necrosis ratio (P < 0.05). PCA and Que reduced mRNA abundance and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8 (P < 0.001), and down-regulated gene expression of toll-like receptors 4 (TLR4) and its downstream signals (P < 0.001) after ETEC infection. PCA and Que down-regulated protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated-RIP1 (p-RIP1), p-RIP1/t-RIP1, t-RIP3, p-RIP3, mixed lineage kinase domain-like protein (MLKL), p-MLKL, dynamin- related protein 1 (DRP1), phosphoglycerate mutase 5 (PGAM5) and high mobility group box 1 (HMGB1) (P < 0.05) after ETEC infection. Moreover, PCA and Que reduced protein abundance of nod-like receptor protein 3 (NLRP3), nod-like receptors family CARD domain-containing protein 4 (NLRC4), apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D (GSDMD) and caspase-1 (P < 0.05) after ETEC infection.ConclusionsIn general, our data suggest that PCA and Que are capable of attenuating ETEC-caused intestinal inflammation and damage via inhibiting necroptosis and pyroptosis signaling pathways.

Evaluation of the role of clathrin and bacterial viability in the endocytosis of Lawsonia intracellularis.

Lawsonia intracellularis is an obligate intracellular bacterium and causative agent of proliferative enteropathy. The pathogenesis of L. intracellularis is not completely understood, including the endocytic mechanisms to access the host cell cytoplasm. In this study, we evaluated the mechanisms involved in endocytosis of L. intracellularis in vitro using intestinal porcine epithelial cells (IPEC-J2). Confocal microscopy was used to co-localize L. intracellularis and clathrin. Clathrin gene knockdown was then applied to verify whether L. intracellularis endocytosis is clathrin-dependent. Finally, internalization of viable and non-viable (bacteria were inactivated by heat) L. intracellularis organisms were assessed to study the role of the host cell during bacterial endocytosis. L. intracellularis organisms were observed co-localized with clathrin by confocal microscopy but the amount of L. intracellularis internalized in cells, with and without clathrin knockdown, did not differ statistically. The internalization of non-viable L. intracellularis showed a decrease in the internalization in cells with less clathrin synthesis (P<0.05). The present study is the first to elucidate the involvement of clathrin in the endocytosis of L. intracellularis. Clathrin-mediated endocytosis was shown to be an important, but not required, process for L. intracellularis internalization in porcine intestinal epithelial cells. Independence of bacterial viability for host cell internalization was also confirmed.

Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED). In particular, the virus causes severe diarrhea, dehydration, and death in neonatal piglets. Maternal immunity effectively protects neonatal piglets from PEDV infection; however, maternal antibodies can only prevent PEDV attachment and entry into target cells, but have no effects on intracellular viruses. Intracellular antibodies targeting virus-encoded proteins are effective in preventing viral infection. We previously identified four single chain variable fragments (scFvs), ZW1-16, ZW3-21, ZW1-41, and ZW4-16, which specifically targeted the PEDV N protein and significantly inhibited PEDV replication and up-regulated interferon-λ1 (IFN-λ1) expression in host cells. In our current study, the four scFvs were subcloned into replication-defective adenovirus vectors to generate recombinant adenoviruses rAdV-ZW1-16, rAdV-ZW3-21, rAdV-ZW1-41, and rAdV-ZW4-16. ScFvs were successfully expressed in Human Embryonic Kidney 293 (HEK293) cells and intestinal porcine epithelial cell line J2 (IPEC-J2) and were biosafe for piglets as indicated by body temperature and weight, scFv excretion in feces, IFN-γ and interleukin-4 (IL-4) expression in jejunum, and pathological changes in porcine tissue after oral administration. Western blotting, immunofluorescence, and immunohistochemical analyses showed that scFvs were expressed in porcine jejunum. The prophylactic effects of rAdV-ZW, a cocktail of the four rAdV-scFvs, on piglet diarrhea caused by PEDV was investigated. Clinical symptoms in piglets orally challenged with PEDV, following a two-time treatment with rAdV-ZW, were significantly reduced when compared with PEDV-infected piglets treated with phosphate buffered saline (PBS) or rAdV-wild-type. Also, no death and jejunal lesions were observed. ScFv co-localization with the PEDV N protein in vivo was also observed. Next, the expression of pro-inflammatory serum cytokines such as tumor necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, and IFN-λ was assessed by enzyme-linked immunosorbent assay (ELISA), which showed that scFvs significantly suppressed PEDV-induced pro-inflammatory cytokine expression and restored PEDV-inhibited IFN-λ expression. Therefore, our study supported a promising role for intracellular scFvs targeting the PEDV N protein to prevent and treat diarrhea in PEDV-infected piglets.

Butyrate limits the replication of porcine epidemic diarrhea virus in intestine epithelial cells by enhancing GPR43-mediated IFN-III production.

Porcine epidemic diarrhea virus (PEDV) is a threat to the health of newborn piglets and has a significant impact on the swine industry. Short-chain fatty acids (SCFAs) are gut microbial metabolites that regulate intestinal function through different mechanisms to enhance the intestinal barrier and immune function. In this study, we aimed to determine whether butyrate displayed a better effect than other SCFAs on limiting PEDV replication in porcine intestinal epithelial cells. Mechanistically, butyrate treatment activated the interferon (IFN) response and interferon-stimulated gene (ISG) expression. Further experiments showed that inhibition of GPR43 (free fatty acid receptor 2) in intestinal epithelial cells increased virus infection and reduced antiviral effects through IFN λ response. Our findings revealed that butyrate exerts its antiviral effects by inducing GPR43-mediated IFN production in intestinal epithelial cells.

Beneficial Effects of Phytogenic Feed Additives on Epithelial Barrier Integrity in an In Vitro Co-Culture Model of the Piglet Gut.

Industrial farming of livestock is increasingly focused on high productivity and performance. As a result, concerns are growing regarding the safety of food and feed, and the sustainability involved in their production. Therefore, research in areas such as animal health, welfare, and the effects of feed additives on animals is of significant importance. In this study, an in vitro co-culture model of the piglet gut was used to investigate the effects of two phytogenic feed additives (PFA) with similar compositions. Intestinal porcine epithelial cells (IPEC-J2) were co-cultivated with peripheral blood mononuclear cells (PBMC) to model the complex porcine gut environment in vitro. The effects of treatments on epithelial barrier integrity were assessed by means of transepithelial electrical resistance (TEER) in the presence of an inflammatory challenge. Protective effects of PFA administration were observed, depending on treatment duration and the model compartment. After 48 h, TEER values were significantly increased by 12-13% when extracts of the PFA were applied to the basolateral compartment (p < 0.05; n = 4), while no significant effects on cell viability were observed. No significant differences in the activity of a PFA based mainly on pure chemical compounds versus a PFA based mainly on complex, natural essential oils, and extracts were found. Overall, the co-culture model was used successfully to investigate and demonstrate beneficial effects of PFAs on intestinal epithelial barrier function during an inflammatory challenge in vitro. In addition, it demonstrates that the two PFAs are equivalent in effect. This study provides useful insights for further research on porcine gut health status even without invasive in vivo trials.

LncRNA EN-90756 promotes CPB2-induced proliferation and inhibits apoptosis in IPEC-J2 cells by affecting the JAK-STAT signaling pathway activation.

Long non-coding RNAs (lncRNAs), as key regulators, are closely associated with the development of a variety of disease. However, the mechanisms by which lncRNAs regulate Clostridium perfringens type C induced piglet diarrhea are unclear. In the present study, we explored the expression and characterization of lncRNAs in a C. perfringens beta2 (CPB2) toxin-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) using RNA-sequencing (RNA-seq). A total of 6,558 lncRNAs were identified, of which 49 lncRNAs were significantly differentially expressed between the control and CPB2 groups. Functional enrichment analysis showed that the target genes of differentially expressed lncRNA EN-90756 were mainly associated with defense response to virus, and negative regulation of apoptotic process. LncRNA EN-90756 was significantly up-regulated in IPEC-J2 cells at different time points after CPB2 treatment. Functionally, knockdown of lncRNA EN-90756 might regulate the proliferation and apoptosis of IPEC-J2 cells by affecting the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. LncRNA EN-90756 may be involved in CPB2 toxin-induced piglet diarrhea by regulating the expression of its target gene MX1 (encoding MX dynamin like GTPase 1). Long non-coding RNA EN-90756 affected the antiviral ability of IPEC-J2 cells by regulating the expression of MX1. Meanwhile, lncRNA EN-90756 might regulate cell proliferation and apoptosis by affecting JAK-STAT signaling pathway activation. These findings provide novel perspectives and directions for further exploration of the regulatory mechanisms of lncRNAs on CPB2 toxin-induced diarrhea in piglets.

Glutamine alleviated heat stress-induced damage of porcine intestinal epithelium associated with the mitochondrial apoptosis pathway mediated by heat shock protein 70.

The present study aimed to investigate the effect of glutamine (Gln) addition on the damage of porcine intestinal epithelial cells (IPEC-J2) induced by heat stress (HS). IPEC-J2 cultured in logarithmic growth period in vitro were firstly exposed to 42 °C for 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h for cell viability and cultured with 1, 2, 4, 6, 8, or 10 mmol Gln per L of culture media for heat shock protein 70 (HSP70) expression to determine the optimal disposal strategy (HS, 42 °C for 12 h and HSP70 expression, 6 mmol/L Gln treatment for 24 h). Then IPEC-J2 cells were divided into three groups: control group (Con, cultured at 37 °C), HS group (HS, cultured at 42 °C for 12 h), and glutamine group (Gln+HS, cultured at 42 °C for 12 h combined with 6 mmol/L Gln treatment for 24 h). The results showed that HS treatment for 12 h significantly decreased the cell viability of IPEC-J2 (P < 0.05) and 6 mmol/L Gln treatment for 12 h increased HSP70 expression (P < 0.05). HS treatment increased the permeability of IPEC-J2, evidenced by the increased fluorescent yellow flux rates (P < 0.05) and the decreased transepithelial electrical resistance (P < 0.05). Moreover, the downregulated protein expression of occludin, claudin-1, and zonula occludens-1 was observed in HS group (P < 0.05), but Gln addition alleviated the negative effects on permeability and the integrity of intestinal mucosal barrier induced by HS (P < 0.05). In addition, HS resulted in the elevations in HSP70 expression, cell apoptosis, cytoplasmic cytochrome c potential expression, and the protein expressions of apoptosis-related factors (apoptotic protease-activating factor-1, cysteinyl aspartate-specific proteinase-3, and cysteinyl aspartate-specific proteinase-9) (P < 0.05); however, the reductions in mitochondrial membrane potential expression and B-cell lymphoma-2 expression were induced by HS (P < 0.05). But Gln treatment attenuated HS-induced adverse effects mentioned above (P < 0.05). Taken together, Gln treatment exhibited protective effects in protecting IPEC-J2 from cell apoptosis and the damaged integrity of epithelial mucosal barrier induced by HS, which may be associated with the mitochondrial apoptosis pathway mediated by HSP70.

PPARγ activation inhibits endocytosis of claudin-4 and protects against deoxynivalenol-induced intestinal barrier dysfunction in IPEC-J2 cells and weaned piglets

The role of peroxisome proliferator activated receptor gamma (PPARγ) in the regulation of adipocyte differentiation has been well characterized. Besides adipose tissue, PPARγ is also highly expressed in the intestine. However, the functional role of PPARγ in the regulation of intestinal function still remains poorly understood. In the present study, we sought to understand the role of PPARγ activation on regulation of intestinal barrier function in intestinal porcine epithelial cells (IPEC-J2) and weaned piglets exposed to the mycotoxin, deoxynivalenol (DON). PPARγ activation by rosiglitazone and troglitazone, two pharmacological PPARγ ligands, increased the protein expression of tight junction proteins (TJP), claudin-3 and 4. PPARγ inhibition increased endocytosis of claudin-4 which was reversed by its activation with troglitazone. DON exposure decreased the protein expression of TJP, and also significantly suppressed PPARγ transcriptional activity. Interestingly, PPARγ activation reversed the reduction of claudin-3 and 4 caused by DON in vitro and in vivo. PPARγ activation also partially restored the transepithelial electrical resistance (TEER) and reduced the permeability of fluorescein isothiocyanate-dextran (FITC-dextran) that have been negatively impacted by DON. These effects were lost in the presence of a specific PPARγ antagonist or in PPARγ knockout cells, confirming the importance of PPARγ in the regulation of intestinal barrier function and integrity. Likewise, in weaned pigs exposed to DON, the PPARγ agonist pioglitazone mitigated the impaired villus-crypt morphology caused by DON. Therefore, pharmacological and natural bioactive compounds with PPARγ stimulatory activities could be effective in preventing DON-induced gut barrier dysfunction.

Lactiplantibacillus plantarum Strains Modulate Intestinal Innate Immune Response and Increase Resistance to Enterotoxigenic Escherichia coli Infection.

Currently, probiotic bacteria with not transferable antibiotic resistance represent a sustainable strategy for the treatment and prevention of enterotoxigenic Escherichia coli (ETEC) in farm animals. Lactiplantibacillus plantarum is among the most versatile species used in the food industry, either as starter cultures or probiotics. In the present work, the immunobiotic potential of L. plantarum CRL681 and CRL1506 was studied to evaluate their capability to improve the resistance to ETEC infection. In vitro studies using porcine intestinal epithelial (PIE) cells and in vivo experiments in mice were undertaken. Expression analysis indicated that both strains were able to trigger IL-6 and IL-8 expression in PIE cells in steady-state conditions. Furthermore, mice orally treated with these strains had significantly improved levels of IFN-γ and TNF-α in the intestine as well as enhanced activity of peritoneal macrophages. The ability of CRL681 and CRL1506 to beneficially modulate intestinal immunity was further evidenced in ETEC-challenge experiments. In vitro, the CRL1506 and CRL681 strains modulated the expression of inflammatory cytokines (IL-6) and chemokines (IL-8, CCL2, CXCL5 and CXCL9) in ETEC-stimulated PIE cells. In vivo experiments demonstrated the ability of both strains to beneficially regulate the immune response against this pathogen. Moreover, the oral treatment of mice with lactic acid bacteria (LAB) strains significantly reduced ETEC counts in jejunum and ileum and prevented the spread of the pathogen to the spleen and liver. Additionally, LAB treated-mice had improved levels of intestinal IL-10 both at steady state and after the challenge with ETEC. The protective effect against ETEC infection was not observed for the non-immunomodulatory TL2677 strain. Furthermore, the study showed that L. plantarum CRL1506 was more efficient than the CRL681 strain to modulate mucosal immunity highlighting the strain specific character of this probiotic activity. Our results suggest that the improved intestinal epithelial defenses and innate immunity induced by L. plantarum CRL1506 and CRL681 would increase the clearance of ETEC and at the same time, protect the host against detrimental inflammation. These constitute valuable features for future probiotic products able to improve the resistance to ETEC infection.

Deoxynivalenol hijacks the pathway of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT-3) to drive caspase-3-mediated apoptosis in intestinal porcine epithelial cells

Deoxynivalenol (DON) can easily injure the intestinal tract, which represents the first barrier against food contaminants. The intestinal toxicity induced by DON was mainly focused on mitogen-activated protein kinase (MAPK) activation, however, the underlying mechanisms by which DON triggers apoptosis by other pathways remain poorly understood. In this study, the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT-3) pathway was proposed to regulate the intrinsic apoptosis induced by DON and thoroughly investigated in intestinal porcine epithelial cells (IPEC-J2). First, DON was found to be able to efficiently inhibit cell viability and increase the release of lactate dehydrogenase. It could also enhance the activity of the cleaved caspase-3 in a time-dependent manner, accompanied by a loss of mitochondrial membrane potential and an up-regulation of the apoptosis rate. Then, the expression of genes associated with inflammation and apoptosis were investigated. DON increased the expression of IL-6, IL-1β, TNF-α, SOCS3 and Bax, but decreased the expression of Bcl-2 and Bcl-xL. Moreover, we discovered that DON robustly inhibited STAT-3 activity together with the down-regulation of JAK2, Bcl-2 and Bcl-xL, paralleling the increase in p38 phosphorylation. Furthermore, a pharmacological activation of JAK2/STAT-3 alleviated DON induced-apoptosis. Concurrent with the apoptotic pathway, during the initial exposure to DON (first 4 h), a survival pathway involving phosphorylated Erk1/2, Akt, and FoxO1 was also observed. Thus, apoptosis induced by DON was Janus faced: although the survival pathway was activated, the DON-induced apoptotic JAK2/STAT-3/caspase-3 pathway dominated, leading to an imbalance in cell homeostasis. This study provides a novel avenue to comprehensively reveal the pathological mechanisms of DON-induced intestinal disorders, which is promising for future applications to other contaminants in food and feed.

Gut-Derived Metabolites from Dietary Tryptophan Supplementation Quench Intestinal Inflammation through the AMPK-SIRT1-Autophagy Pathway

Tryptophan has drawn wide attention due to its involvement in improving intestinal immune defense directly and indirectly by regulating metabolic pathways. The study aims to elucidate the potential modulating roles of tryptophan to protect against intestinal inflammation and elucidate the underlying molecular mechanisms. The protective effects of tryptophan against intestinal inflammation are examined in the lipopolysaccharide (LPS)-induced inflammatory model. We first found that tryptophan markedly (p < 0.01) inhibited proinflammatory cytokines production and nuclear factor κB (NF-κB) pathway activation upon LPS challenge. Next, we demonstrated that tryptophan (p < 0.05) attenuated LPS-caused intestinal mucosal barrier damage by increasing the number of goblet cells, mucins, and antimicrobial peptides (AMPs) in the ileum of mice. In addition, tryptophan (p < 0.05) inhibited LPS-induced autophagic flux through the AMP-activated protein kinase (AMPK)-sirtuin 1 (SIRT1) pathway in the intestinal systems to maintain autophagy homeostasis. Meanwhile, tryptophan also reshaped the gut microbiota composition in LPS-challenge mice by increasing the abundance of short-chain fatty acid (SCFA)-producing bacteria such as Acetivibrio (0.053 ± 0.017 to 0.21 ± 0.0041%). Notably, dietary tryptophan resulted in the activation of metabolic pathways during the inflammatory response. Furthermore, exogenous treatment of tryptophan metabolites kynurenine (Kyn) and 5-HT in porcine intestinal epithelial cells (IPEC-J2 cells) reproduced similar protective effects as tryptophan to attenuate LPS-induced intestinal inflammation through regulating the AMPK-SIRT1-autophagy. Taken together, the present study indicates that tryptophan exhibits intestinal protective and immunoregulatory effects resulting from the activation of metabolic pathways, maintenance of gut mucosal barrier integrity, microbiota composition, and AMPK-SIRT1-autophagy level.

METTL3 Regulates the Inflammatory Response in CPB2 Toxin-Exposed IPEC-J2 Cells through the TLR2/NF-κB Signaling Pathway.

Clostridium perfringens beta2 (CPB2) toxin is one of the main pathogenic toxins produced by Clostridium perfringens, which causes intestinal diseases in animals and humans. The N6-methyladenosine (m6A) modification is the most common reversible modification in eukaryotic disease processes. Methyltransferase-like 3 (METTL3) regulates immunity and inflammatory responses induced by the bacterial infections in animals. However, METTL3's involvement in CPB2-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) remains unclear. In the current study, we used methylated RNA immunoprecipitation-quantitative polymerase chain reaction, Western blotting and immunofluorescence assay to determine the role of METTL3 in CPB2-exposed IPEC-J2 cells. The findings revealed that m6A and METTL3 levels were increased in CPB2 treated IPEC-J2 cells. Functionally, METTL3 overexpression promoted the release of inflammatory factors, increased cytotoxicity, decreased cell viability and disrupted tight junctions between cells, while the knockdown of METTL3 reversed these results. Furthermore, METTL3 was involved in the inflammatory response of IPEC-J2 cells by activating the TLR2/NF-κB signaling pathway through regulating TLR2 m6A levels. In conclusion, METTL3 overexpression triggered the TLR2/NF-κB signaling pathway and promoted CPB2-induced inflammatory responses in IPEC-J2 cells. These findings may provide a new strategy for the prevention and treatment of diarrhea caused by Clostridium perfringens.