Porcine Enterocytes Research Articles

Lipidomics reveals the significance and mechanism of the cellular ceramide metabolism for rotavirus replication.

As one of the most important causative agents of severe gastroenteritis in children, piglets, and other young animals, species A rotaviruses have adversely impacted both human health and the global swine industry. Vaccines against rotaviruses (RVs) are insufficiently effective, and no specific treatment is available. To understand the relationships between porcine RV (PoRV) infection and enterocytes in terms of the cellular lipid metabolism, we performed an untargeted liquid chromatography mass spectrometry (LC-MS) lipidomics analysis of PoRV-infected IPEC-J2 cells. Herein, a total of 451 lipids (263 upregulated lipids and 188 downregulated lipids), spanning sphingolipid, glycerolipid, and glycerophospholipids, were significantly altered compared with the mock-infected group. Interestingly, almost all the ceramides among these lipids were upregulated during PoRV infection. LC-MS analysis was used to validated the lipidomics data and demonstrated that PoRV replication increased the levels of long-chain ceramides (C16-ceramide, C18-ceramide, and C24-ceramide) in cells. Furthermore, we found that these long-chain ceramides markedly inhibited PoRV infection and that their antiviral actions were exerted in the replication stage of PoRV infection. Moreover, downregulation of endogenous ceramides with the ceramide metabolic inhibitors enhanced PoRV propagation. Increasing the levels of ceramides by the addition of C6-ceramide strikingly suppressed the replication of diverse RV strains. We further found that the treatment with an apoptotic inhibitor could reverse the antiviral activity of ceramide against PoRV replication, demonstrating that ceramide restricted RV infection by inducing apoptosis. Altogether, this study revealed that ceramides played an antiviral role against RV infection, providing potential approaches for the development of antiviral therapies.IMPORTANCERotaviruses (RVs) are among the most important zoonosis viruses, which mainly infected enterocytes of the intestinal epithelium causing diarrhea in children and the young of many mammalian and avian species. Lipids play an essential role in viral infection. A comprehensive understanding of the interaction between RV and lipid metabolism in the enterocytes will be helpful to control RV infection. Here, we mapped changes in enterocyte lipids following porcine RV (PoRV) infection using an untargeted lipidomics approach. We found that PoRV infection altered the metabolism of various lipid species, especially ceramides (derivatives of the sphingosine). We further demonstrated that PoRV infection increased the accumulation of ceramides and that ceramides exerted antiviral effects on RV replication by inducing apoptosis. Our findings fill a gap in understanding the alterations of lipid metabolism in RV-infected enterocytes and highlight the antiviral effects of ceramides on RV infection, suggesting potential approaches to control RV infection.

Saturated fatty acids differently affect mitochondrial function and the intestinal epithelial barrier depending on their chain length in the in vitro model of IPEC-J2 enterocytes.

Introduction: Maintenance of the intestinal barrier mainly relies on the mitochondrial function of intestinal epithelial cells that provide ATP through oxidative phosphorylation (OXPHOS). Dietary fatty acid overload might induce mitochondrial dysfunction of enterocytes and may increase intestinal permeability as indicated by previous in vitro studies with palmitic acid (C16:0). Yet the impact of other dietary saturated fatty acids remains poorly described. Methods: To address this question, the in vitro model of porcine enterocytes IPEC-J2 was treated for 3days with 250µM of lauric (C12:0), myristic (C14:0), palmitic (C16:0) or stearic (C18:0) acids. Results and discussion: Measurement of the transepithelial electrical resistance, reflecting tight junction integrity, revealed that only C16:0 and C18:0 increased epithelial permeability, without modifying the expression of genes encoding tight junction proteins. Bioenergetic measurements indicated that C16:0 and C18:0 were barely β-oxidized by IPEC-J2. However, they rather induced significant OXPHOS uncoupling and reduced ATP production compared to C12:0 and C14:0. These bioenergetic alterations were associated with elevated mitochondrial reactive oxygen species production and mitochondrial fission. Although C12:0 and C14:0 treatment induced significant lipid storage and enhanced fusion of the mitochondrial network, it only mildly decreased ATP production without altering epithelial barrier. These results point out that the longer chain fatty acids C16:0 and C18:0 increased intestinal permeability, contrary to C12:0 and C14:0. In addition, C16:0 and C18:0 induced an important energy deprivation, notably via increased proton leaks, mitochondrial remodeling, and elevated ROS production in enterocytes compared to C12:0 and C14:0.

Anti-Salmonella Defence and Intestinal Homeostatic Maintenance In Vitro of a Consortium Containing Limosilactobacillus fermentum 3872 and Ligilactobacillus salivarius 7247 Strains in Human, Porcine, and Chicken Enterocytes.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti-Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor-intestinal alkaline phosphatase (IAP)-in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1β, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

Comprehensive proteomic analysis reveals omega-3 fatty acids to counteract endotoxin-stimulated metabolic dysregulation in porcine enterocytes

Omega-3 polyunsaturated fatty acids (n-3 PUFA), such as the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are reported to beneficially affect the intestinal immunity. The biological pathways modulated by n-3 PUFA during an infection, at the level of intestinal epithelial barrier remain elusive. To address this gap, we investigated the proteomic changes induced by n-3 PUFA in porcine enterocyte cell line (IPEC-J2), in the presence and absence of lipopolysaccharide (LPS) stress conditions using shotgun proteomics analysis integrated with RNA-sequencing technology. A total of 33, 85, and 88 differentially abundant proteins (DAPs) were identified in cells exposed to n-3 PUFA (DHA:EPA), LPS, and n-3 PUFA treatment followed by LPS stimulation, respectively. Functional annotation and pathway analysis of DAPs revealed the modulation of central carbon metabolism, including the glycolysis/gluconeogenesis, pentose phosphate pathway, and oxidative phosphorylation processes. Specifically, LPS caused metabolic dysregulation in enterocytes, which was abated upon prior treatment with n-3 PUFA. Besides, n-3 PUFA supplementation facilitated enterocyte development and lipid homeostasis. Altogether, this work for the first time comprehensively described the biological pathways regulated by n-3 PUFA in enterocytes, particularly during endotoxin-stimulated metabolic dysregulation. Additionally, this study may provide nutritional biomarkers in monitoring the intestinal health of human and animals on n-3 PUFA-based diets.

PSI-16 Intestinal Porcine Enterocyte Cell (Ipec-J2) as an In-Vitro Model for Investigating the Mechanisms of Ferroptosis

Abstract Ferroptosis is a newly identified form of regulated cell death that has been implicated in various intestinal diseases. However, there is limited research on ferroptosis in porcine models. The aim of this study was to establish an in vitro model of ferroptosis in the IPEC-J2 cell line. Ferroptosis is a form of regulated cell death that was first identified in 2012 and is characterized by unique morphological and physiological features that distinguish it from other types of cell death. This process is triggered by a complex interplay of multiple factors, including excess ferrous iron accumulation, oxidation of polyunsaturated fatty acids, and dysregulation of redox reactions. These events ultimately lead to the accumulation of lipid peroxides and subsequent membrane damage, resulting in ferroptosis. In pig production, weaning piglets often experience significant stress that can cause damage to the intestinal barrier due to changes in diet and environment, leading to diarrhea and growth retardation. Whether ferroptosis is involved in the development of intestinal diseases in pigs is still unclear, this study will establish a ferroptosis model using porcine intestinal epithelial cells. To induce ferroptosis in the IPEC-J2 cells, we applied hydrogen peroxide (H2O2), lipopolysaccharide (LPS), ferrous sulfate (FeSO4), and erastin, a ferroptosis inducer. The concentration of each treatment was determined by analyzing cell viability using the CCK-8 assay. Lipid peroxidation was evaluated using the TBARS assay, and the data indicated that compared with the control samples, IPEC-J2 cells treated with H2O2, FeSO4 and erastin had greater malondialdehyde levels (fold of control; CON = 1, H2O2 = 1.70, FeSO4 = 2.32, erastin = 1.68; P <0.05). Iron concentrations were measured using the Iron Assay kit (cat. no. ab83366; Abcam). Greater total iron concentrations were observed in IPEC-J2 cells treated with FeSO4 and erastin (fold of control; CON = 1, FeSO4 = 14.91, erastin = 1.10; P <0.05), and higher ferrous iron level on IPEC-J2 cells treated with erastin (fold of control; CON = 1, erastin = 1.17; P <0.05). In conclusion, the above results suggest that treatment with H2O2, FeSO4 and erastin is likely to lead to ferroptosis. However, further investigation is needed to confirm these findings. A ferroptosis model on intestinal porcine epithelial cells will be established in this study.

Allelic functional variation of FimH among Salmonella enterica subspecies

Salmonella enterica has a wide diversity, with numerous serovars belonging to six different subspecies with dynamic animal-host tropism. The FimH protein is the adhesin mediating binding to various cells, and slight amino acid discrepancy significantly affects the adherence capacities. To date, the general function of FimH variability across different subspecies of Salmonella enterica has not been addressed. To investigate the biological functions of FimH among the six Salmonella enterica subspecies, the present study performed several assays to determine biofilm formation, Caenorhabditis elegans killing, and intestinal porcine enterocyte cell IPEC-J2 adhesion by using various FimH allele mutants. In general, allelic mutations in both the lectin and pilin domains of FimH could cause changes in binding affinity, such as the N79S mutation. We also observed that the N79S variation in Salmonella Dublin increased the adhesive ability of IPEC-J2 cells. Moreover, a new amino acid substitution, T260M, within the pilin domain in one subspecies IIIb strain beneficial to binding to cells was highlighted in this study, even though the biofilm-forming and Caenorhabditis elegans-killing abilities exhibited no significant differences in variants. Combined with point mutations being a natural tendency due to positive selection in harsh environments, we speculate that allelic variation T260M probably contributes to pathoadaptive evolution in Salmonella enterica subspecies IIIb.

MicroRNA-223 Inhibits Soybean Glycinin- and β-Conglycinin-Induced Apoptosis of IPEC-J2 Cells by Targeting NLRP-3 in the IEL/IPEC-J2 Co-culture System.

The apoptosis of intestinal porcine epithelial cells induced by soybean antigen protein allergy is one of the most important mechanisms responsible for enteritis. MicroRNAs (miRNAs) affect the cellular and physiological functions of all multicellular organisms. We hypothesize that microRNA-223 inhibits soybean glycinin- and β-conglycinin-induced apoptosis of intestinal porcine enterocytes (IPEC-J2) by targeting the NLR family pyrin domain containing 3 (NLRP-3). Using the intestinal interepithelial lymphocyte (IEL)/IPEC-J2 co-culture system as an in vitro model, we investigate the role of microRNA-223 in the regulation of soybean glycinin- and β-conglycinin-induced apoptosis. In co-cultured IEL/IPEC-J2 cells incubated with glycinin or β-conglycinin, microRNA-223 decreased NLRP-3, ASC, caspase-1, caspase-3, FAS, BCL-2, and APAF-1 expressions in IPEC-J2 cells; decreased cytokine and cyclooxygenase-2 levels; significantly increased cell activity; and inhibited apoptosis. These data supported a novel antiallergic mechanism to mitigate the sensitization of soybean antigenic protein, which involves the upregulation of microRNA-223-targeting NLRP-3.

3-Acetyldeoxynivalenol induces apoptosis, barrier dysfunction and endoplasmic reticulum stress by inhibiting mTORC1-dependent autophagy in porcine enterocytes

3-Acetyldeoxynivalenol (3-Ac-DON), an acetylated form of deoxynivalenol, is widely present in mycotoxin-contaminated food, feed as well as in other natural sources. Ingestion of 3-Ac-DON may result in intestinal dysfunction, leading to gut diseases in humans and animals. Nevertheless, the molecular mechanism of 3-Ac-DON in intestinal epithelial cytotoxicity remains unclear. In this study, intestinal porcine epithelial cell line 1 (IPEC-1) cells were treated with different concentrations of 3-Ac-DON for 12 h or 24 h, respectively. The results showed that 3-Ac-DON caused decreased cell viability, cell cycle arrest in G1 phase and depolarization of mitochondrial membrane potential. Western blotting analysis showed that 3-Ac-DON significantly decreased the expression of tight junction proteins, inhibited autophagy and activated endoplasmic reticulum (ER) stress in IPEC-1 cells (P < 0.05). Further investigation demonstrated that 3-Ac-DON caused apoptosis, ER stress and barrier dysfunction were reversed after co-treatment with the autophagy activator rapamycin (100 nM), indicating that autophagy plays a key role in the process of 3-Ac-DON-induced cell damage. In addition, we demonstrated that 3-Ac-DON inhibits the occurrence of autophagy mediated by mTORC1 protein. In conclusion, our research indicated that the mTORC1 protein and autophagy played a key role in the 3-Ac-DON-induced cytotoxic in IPEC-1 cells, which would provide new therapeutic targets and ideas for 3-Ac-DON-mediated intestinal injury.

Integrated lncRNA transcriptomics, proteomics, and metabolomics to identify early cellular response variation in deoxynivalenol-treated IPEC-J2 cells.

Mycotoxins, especially deoxynivalenol (DON), are common contaminants of food and feed, which also has serious threaten to human health and livestock production. Moreover, DON severely impair intestinal epithelial barrier function. Therefore, it is necessary to investigate the mechanism of intestinal epithelial cell injury induced by DON. Here, intestinal porcine enterocyte cell (IPEC-J2) was incubated with 200ng/ml or 2000ng/ml DON for 6h, then lncRNA sequencing, metabolomics and proteomics were applied. Combined with long coding transcriptomics, and proteomics, 200ng/ml DON treatment (LDON group) significantly upregulated ribosome biogenesis in eukaryotes, spliceosome, and ubiquitin mediated proteolysis, RNA transport, and downregulated metabolic pathways in IPEC-J2, 2000ng/ml of DON treatment (HDON group) significantly upregulated ribosome biogenesis in eukaryotes, and spliceosome, and downregulated base excision repair, cell cycle, DNA replication, homologous recombination, and mismatch repair in IPEC-J2. Combined with long coding transcriptomics, and proteomics, as compared with LDON group, HDON group significantly upregulated adherens junction, hippo signaling pathway, and pathways in cancer, and downregulated DNA replication pathways in IPEC-J2. In metabolomics, LDON group and HDON group was mainly downregulated biosynthesis of unsaturated fatty acids, and fatty acid metabolism. These results provide a new insight to prevent and treat DON induced intestinal epithelial cell injury.

Angelica sinensis aboveground part polysaccharide and its metabolite 5-MT ameliorate colitis via modulating gut microbiota and TLR4/MyD88/NF-κB pathway

The roots of Angelica sinensis have been used in Traditional Chinese Medicine for thousands of years. However, tons of aerial parts of this herb (aboveground part) are commonly discarded during the process of root preparations. A polysaccharide (ASP-Ag-AP) in the aboveground parts of A. sinensis was isolated and preliminarily characterized as typical plant pectin. ASP-Ag-AP exhibited noticeable protective effects against dextran sodium sulfate (DSS)-induced colitis, including reduction of colonic inflammation, modulation of barrier function, and alteration of gut microbiota and serum metabolite profile. Anti-inflammatory effects of ASP-Ag-AP were observed by inhibiting TLR4/MyD88/NF-κB signaling pathway in vitro and in vivo. Additionally, the level of serum metabolite 5-methyl-dl-tryptophan (5-MT) was reduced by DSS and restored by ASP-Ag-AP, which also negatively correlated with Bacteroides, Alistipes, Staphylococcus and pro-inflammatory factors. The protection from inflammatory stress on intestinal porcine enterocytes cells (IPEC-J2) of 5-MT was observed through the inhibition of TLR4/MyD88/NF-κB pathway. Besides, 5-MT also exhibited robust anti-inflammatory effect in colitis mice with improving colitis symptoms, barrier function and gut microbiota, which was the same as presented by ASP-Ag-AP. Therefore, ASP-Ag-AP could be a promising agent for colitis prevention and 5-MT could be the signal metabolite of ASP-Ag-AP on defending against intestinal inflammatory stress.

Impact of Quorum Sensing Signal N-3-Oxododecanoyl Homoserine Lactone on Intestinal Porcine Enterocyte Cell

Quorum sensing (QS) is an autoinducer (AI) molecules mediated signaling system in bacteria. It is generally believed that bacteria can regulate their own characteristics or virulence through OS. This study investigated the direct effects of a quorum sensing signal molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL, synonyms: OdDHL) on host cells, to further reveal the function of QS system in the interaction between bacteria and host cells. Intestinal porcine enterocyte cell line (IPEC-J2) were used as model cells, and the AHL signal molecule OdDHL was used to stimulate the cells to observe the effect of quorum sensing signals on host cells. The results showed that OdDHL had an obvious effect on the morphology of IPEC-J2 cells, and a lower concentration and shorter time of stimulation could induce cell shrinkage and rounding. MTT assay revealed that OdDHL had no effect on IPEC-J2 cell viability, suggesting that the cell morphological changes were not from signaling molecule toxicity. Using different concentrations and different exposure times of OdDHL to stimulate IPEC-J2 cells, we demonstrated that changes in cell morphology were positively correlated with the concentration of AHL and exposure time. Bacterial adhesion assay showed that OdDHL could enhance the adhesion of enteropathogenic E. coli to host cell IPEC-J2, while reducing its invasion into host cell. Fluorescent quantitative PCR and Western blot revealed that the expression of tight junction protein ZO-1 was significantly increased. The expression levels of multiple genes that related to cytoskeletons and cell junction were all regulated by OdDHL. In conclusion, OdDHL could directly affect the biological characteristics of IPEC-J2 cells such as morphology and tight junction, reduce the intestinal defences of hosts, thereby promoting the adhesion and injury of pathogenic bacteria to the intestine of piglets. This study provides a basic reference for further understanding of the pathogenic role of regulating pathogenic bacterial populations and their prevention against diseases.

Salvia miltiorrhiza polysaccharide and its related metabolite 5-methoxyindole-3-carboxaldehyde ameliorate experimental colitis by regulating Nrf2/Keap1 signaling pathway

The roots of Salvia miltiorrhiza have been used in Traditional Chinese Medicine for thousands of years. However, tons of aerial parts of this plant are usually discarded in the production of roots preparation. To make better use of these plant resources, the polysaccharide isolated from the aerial part of S. miltiorrhiza was investigated for its potential protection against intestinal diseases. A pectic polysaccharide (SMAP-1) was isolated and characterized being composed of homogalacturonan as the main chain and rhamnogalacturonan type I as ramified region, with side chains including arabinans and possible arabinogalactan type I and II. SMAP-1 exhibited robust protective effects against dextran sodium sulfate (DSS)-induced colitis and restored colitis symptoms, colonic inflammation, and barrier functions. Anti-oxidative effects were also observed by up-regulating Nrf2/Keap1 signaling pathway. Additionally, the level of serum 5-methoxyindole-3-carboxaldehyde (5-MC) was restored by SMAP-1 identified in metabolomic analysis, being correlated with the aforementioned effects. Protection against oxidative stress on intestinal porcine enterocyte cells (IPEC-J2) by 5-MC was observed through the activation of Nrf2/Keap1 system, as also shown by SMAP-1. In conclusion, SMAP-1 could be a promising candidate for colitis prevention, and 5-MC could be the signal metabolite of SMAP-1 in protecting against oxidative stress in the intestine.

Impact of heat-inactivated Lactobacillus on inflammatory response in endotoxin- and chemotherapeutic-treated porcine enterocytes

Several factors such as pathogen bacteria, or oral chemotherapy disturb the intestinal integrity, leading to several undesirable effects. Inactivated probiotics may be beneficial in safely redress the physiological functions of the intestinal epithelium.Our aim is to determine the effect of tyndallized Lactobacillus on LPS- and 5-fluorouracil-treated porcine jejunal cells.IPEC-J2 cells derived from porcine jejunal epithelium were used as the in vitro model. The enterocyte cell cultures were treated with 109Lactobacillus reuteri cells/ml or 10 μg/ml lipopolysaccharides (LPS) or 100 μM 5-fluorouracil separately and simultaneously. We determined the alterations in mRNA levels of inflammatory mediators IL6, CXCL8/IL8, TNF. Furthermore, the protein level of IL-6 and IL-8 were measured.The fluorouracil treatment upregulated the IL6 gene expression, the endotoxin treatment upregulated the IL8 and TNF level. The heat-inactivated Lactobacillus increased the IL-8 level both at the gene expression and protein level. The co-administration of the non-viable probiotic with the 5-fluorouracil and the LPS resulted in decrease of IL6, IL8, and TNF level.The immune-modulator effect of tyndallized probiotic product is demonstrated in porcine jejunal cells. The inactivated Lactobacillus was able to prevent the accumulation of the selected inflammatory mediators in LPS- or 5-fluorouracil-exposed enterocytes.

Molecular characterization of gut microbiome in weaning pigs supplemented with multi-strain probiotics using metagenomic, culturomic, and metabolomic approaches

BackgroundProbiotics have been reported to exhibit positive effects on host health, including improved intestinal barrier function, preventing pathogenic infection, and promoting nutrient digestion efficiency. These internal changes are reflected to the fecal microbiota composition and, bacterial metabolites production. In accordance, the application of probiotics has been broadened to industrial animals, including swine, which makes people to pursue better knowledge of the correlation between changes in the fecal microbiota and metabolites. Therefore, this study evaluated the effect of multi-strain probiotics (MSP) supplementation to piglets utilizing multiomics analytical approaches including metagenomics, culturomics, and metabolomics.ResultsSix-week-old piglets were supplemented with MSP composed of Lactobacillus isolated from the feces of healthy piglets. To examine the effect of MSP supplement, piglets of the same age were selected and divided into two groups; one with MSP supplement (MSP group) and the other one without MSP supplement (Control group). MSP feeding altered the composition of the fecal microbiota, as demonstrated by metagenomics analysis. The abundance of commensal Lactobacillus was increased by 2.39%, while Clostridium was decreased, which revealed the similar pattern to the culturomic approach. Next, we investigated the microbial metabolite profiles, specifically SCFAs using HPLC–MS/MS and others using GC–MS, respectively. MSP supplement elevated the abundance of amino acids, including valine, isoleucine and proline as well as the concentration of acetic acid. According to the correlation analyses, these alterations were found out to be crucial in energy synthesizing metabolism, such as branched-chain amino acid (BCAA) metabolism and coenzyme A biosynthesis. Furthermore, we isolated commensal Lactobacillus strains enriched by MSP supplement, and analyzed the metabolites and evaluated the functional improvement, related to tight junction from intestinal porcine enterocyte cell line (IPEC-J2).ConclusionsIn conclusion, MSP administration to piglets altered their fecal microbiota, by enriching commensal Lactobacillus strains. This change contributed amino acid, acetic acid, and BCAA concentrations to be increased, and energy metabolism pathway was also increased at in vivo and in vitro. These changes produced by MSP supplement suggests the correlation between the various physiological energy metabolism functions induced by health-promoting Lactobacillus and the growth performance of piglets.

The synergy of thanatin and cathelicidin-BF-15a3 combats Escherichia coli O157:H7

Escherichia coli O157:H7 is a pathogen that commonly causes foodborne illness and represents a health hazard to consumers. The combined use of synergistic antimicrobial peptides (AMPs) is a promising way to improve the microbiological safety of foods. In this study, we detected the synergistic interactions between thanatin and BF-15a3 to reduce their usage and obtain more efficient antibacterial activity. The minimal inhibitory concentrations (MICs) of thanatin and BF-15a3 against 49 E. coli O157:H7 strains were ranged from 2 to 8 μg/mL and 4–32 μg/mL, showed a general inhibitory effect on E. coli O157:H7 strains, respectively, even multidrug-resistant strains. Their fractional inhibitory concentration index (FICI) was 0.375, which suggested that their combination presented synergistic antibacterial effect against E. coli O157:H7. The killing kinetic curves indicated that the 0.25 × MIC combination had equivalent bactericidal effects to 1 × MIC thanatin or BF-15a3. When AMP combinations were used to treat eukaryotic cells to evaluate the hemolytic characteristics against rabbit erythrocytes and cytotoxicity against human embryonic kidney 293T (HEK-293T) cells and intestinal porcine enterocyte J2 (IPEC-J2) cells, no magnified adverse effects were observed, exhibiting higher specificity to bacteria and lower toxicity to eukaryotic cells. Compared with bacteriostasis of thanatin or BF-15a3 alone, the proportion of membrane-damaged bacteria treated with the synergetic combination did not appear a significant rise, interestingly the Zeta potential of them greatly decreased and their cell membrane permeability significantly increased. Besides, more release of ions and cytoplasm were detected, confirming a more severe loss of membrane integrity. These results suggested that the synergistic action mode of thanatin and BF-15a3 is likely attributed to damage aggravation to E. coli membrane. When applying in fresh-cut lettuce and cucumber, their combination allowed for 2.5 log CFU/piece reductions of E. coli O157:H7 in 24 h. In conclusion, the combination of thanatin and BF-15a3 showed excellent synthetic efficacy to kill E. coli O157:H7 in vitro under lower MICs than single use of them.

139 Calcium-Sensing Receptor and the Regulation of Nutrient Absorption and gut Health in Pigs

Abstract The gut has a network of G protein-coupled receptors (GPCR) that can recognize nutrients, other chemicals, and microbes and then initiate a cascade of signals to regulate nutrition, gut function, and metabolism. Calcium sensing receptors (CaSR) belong to the GPCR family and are expressed in various animal tissues, and CaSR plays important role in nutrient sensing and the physiology, growth, and development of animals. Our results demonstrated that pCaSR is widely expressed in different tissues including intestinal segments in weaned piglets and the ileum has a higher expression level of pCaSR. However, pCaSR is not expressed in either isolated porcine ileal enterocytes or intestinal porcine enterocyte cell lines (IPEC-J2). We also found that the activation of pCaSR showed biased agonism through multiple signaling with a dual-luciferase reporter assay system. Besides, α-casein (90-95) and L-Tryptophan (L-Trp) are type II agonists for pCaSR. We investigated the roles of pig calcium-sensing receptor (pCaSR) ligands in gut hormone secretion and mechanism and the effects of pCaSR ligands on inflammation response, nutrient absorption and barrier function in the intestinal epithelial cells co-culture system challenged with peptidoglycan (PGN). Our results demonstrated that the pCaSR ligands (L-Trp and α-casein (90-95)) to STC-1 cells could induce the secretion of gut hormones through the regulation of CaSR in STC-cells. Plus, the GLP-1 secretion triggered by L-Trp and α-casein (90-95) in STC-cells attenuated the inflammation response, improved the nutrient absorption and barrier function in the IPEC-J2 cells. Further research is still needed to elucidate the roles of L-Trp and α-casein (90-95) on nutrient absorption and barrier function in pigs under physiological challenges. Collectively, the pCaSR is widely expressed in the porcine tissues, and the activation of pCaSR has the potential to improve gut health and growth performance in swine production.

Milk lactose protects against porcine group A rotavirus infection.

Rotavirus A (RVA) is an important pathogen causing acute gastroenteritis in animals and humans. Attachment to the host receptor is a crucial step for virus replication. The VP8* domain is the distal terminal region of the RVA spike protein VP4 (expressed by the P gene) and is important for rotavirus binding and infectivity. Recent studies have indicated a role for non-sialylated glycans, including mucin core 2 and histo-blood group antigens (HBGAs), in the infectivity of human and animal group A rotaviruses. In the present study, we determined if porcine rotavirus-derived recombinant VP8* of the endemic strains 14R103 G5P[6], 13R054 G5P[7], 12R010 G4P[13], 12R046 G9P[23], and 12R022 G2P[27] interact with hitherto uncharacterized glycans. We successfully produced five recombinant GST-VP8* proteins of genotype P[6], P[7], P[13], P[23], and P[27]. The hemagglutination assay showed genotypes P[7] and P[23] hemagglutinate porcine and human red blood cells. In an array screen of > 300 glycans, recombinant VP8* of rotavirus genotype P[6], P[7], and P[13] showed specific binding to glycans with a Gal-β-1,4-Glc (β-lactose) motif, which forms the core structure of HBGAs. The specificity of glycan-binding was confirmed through an ELISA-based oligosaccharide binding assay. Further, 13R054 G5P[7] and 12R046 G9P[23] infectivity was significantly reduced by β-lactose in MA104 cells and primary porcine enterocytes. These data suggest that lactose, the main natural sugar in milk, plays an important role in protecting piglets from enteric viral replication and diarrhea.

Effects of Spermidine on Cell Proliferation, Migration, and Inflammatory Response in Porcine Enterocytes.

Polyamines have been demonstrated to be beneficial to porcine intestinal development. Our previous study showed that putrescine mitigates intestinal atrophy in weanling piglets and suppresses inflammatory response in porcine intestinal epithelial cells, it is still unknown the role of spermidine in mediating putrescine function. The current study aimed to investigate the effect of spermidine on the proliferation, migration, and inflammatory response in porcine intestinal epithelial cells (IPEC-J2 cell line). The effects of spermidine on proliferation and migration of IPEC-J2 cells were measured. Difluoromethyl ornithine (DFMO) and diethylglyoxal bis (guanylhydrazone) (DEGBG) were used to block the production of putrescine and spermidine, respectively. A cell inflammation model was established with lipopolysaccharides (LPS) stimulation. Gene expression and protein abundance were determined by real-time quantitative PCR and western blotting, respectively. Spermidine significantly enhanced cell proliferation in DFMO (or/and) DEGBG treated IPEC-J2 cells (p < 0.05). Pretreatment with putrescine restored cell growth inhibited by DFMO but did not prevent the decrease in cell proliferation caused by DEGBG (p > 0.05). Similarly, spermidine but not putrescine significantly elevated the rate of migration in DEGBG treated IPEC-J2 cells (p < 0.05). Spermidine deprivation by DEGBG dramatically enhanced mRNA abundance of pro-inflammatory cytokines IL-8, IL-6, and TNF-α (p < 0.05), and the addition of spermidine attenuated excessive expression of those inflammatory pro-inflammatory cytokines, moreover, spermidine but not putrescine suppressed the phosphorylation of NF-κB induced by DEGBG. Spermidine supplementation also significantly suppressed LPS-induced the expression of TNF-α. The present study highlights a novel insight that putrescine may be converted into spermidine to modulate cell proliferation, migration, and inflammatory response on porcine enterocytes.

Polysaccharides from Aconitum carmichaelii leaves: Structure, immunomodulatory and anti-inflammatory activities

Roots of Aconitum carmichaelii are used in Asian countries due to its content of bioactive alkaloids. In the production of root preparations, tons of leaves are usually discarded, leading to a huge waste of herbal material. The aim of this study is to investigate the polysaccharides in these unutilized leaves. A neutral polysaccharide (AL-N) appeared to be a mixture of heteromannans, and two purified acidic polysaccharides (AL-I-I and AL-I-II) were shown to be pectins containing a homogalacturonan backbone substituted with terminal β-Xylp-units. AL-I-I consisted of a type-I rhamnogalacturonan core, with arabinan and type-II arabinogalactan domains while AL-I-II was less branched. AL-N and AL-I-I were able to modulate the complement system, while AL-I-II was inactive. Interestingly, AL-N, AL-I-I and AL-I-II were shown to exert anti-inflammatory effects on porcine enterocyte IPEC-J2 cells. AL-I-I and AL-I-II were able to down-regulate the expression of toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1).

Potential of Oral Nanoparticles Containing Cytokines as Intestinal Mucosal Immunostimulants in Pigs: A Pilot Study.

Simple SummaryAntibiotics are essential compounds to cope with bacterial infections. However, their inadequate and excessive use has triggered the rapid arising of antimicrobial-resistant bacteria. In this scenario, immunostimulants, which are molecules that boost the immune system, open up a new approach to face this problem, enhancing treatment efficacy and preventing infections by immune system response. Cytokines are central effector molecules of the immune system, and their recombinant production and administration in animals could be an interesting immune modulation strategy. The aim of this study was the development of a highly stable nanoparticle of porcine cytokines to achieve the immunostimulation of intestinal mucosa in piglets. The outcomes of the present study prove this approach is able to stimulate swine intestinal cells and macrophages in vitro and tends to modulate inflammatory responses in vivo, although further studies are required to definitively evaluate their potential in animals.Antimicrobial resistance is a global threat that is worryingly rising in the livestock sector. Among the proposed strategies, immunostimulant development appears an interesting approach to increase animal resilience at critical production points. The use of nanoparticles based on cytokine aggregates, called inclusion bodies (IBs), has been demonstrated as a new source of immunostimulants in aquaculture. Aiming to go a step further, the objective of this study was to produce cytokine nanoparticles using a food-grade microorganism and to test their applicability to stimulate intestinal mucosa in swine. Four cytokines (IL-1β, IL-6, IL-8, and TNF-α) involved in inflammatory response were produced recombinantly in Lactococcus lactis in the form of protein nanoparticles (IBs). They were able to stimulate inflammatory responses in a porcine enterocyte cell line (IPEC-J2) and alveolar macrophages, maintaining high stability at low pH and high temperature. In addition, an in vivo assay was conducted involving 20 piglets housed individually as a preliminary exploration of the potential effects of IL-1β nanoparticles in piglet intestinal mucosa after a 7 d oral administration. The treated animals tended to have greater levels of TNF-α in the blood, indicating that the tested dose of nanoparticles tended to generate an inflammatory response in the animals. Whether this response is sufficient to increase animal resilience needs further evaluation.