Alternariol Monomethyl-Ether Induces Toxicity via Cell Death and Oxidative Stress in Swine Intestinal Epithelial Cells.

Alternariol monomethyl-ether (AME), together with altenuene and alternariol, belongs to the Alternaria mycotoxins group, which can contaminate different substrates, including cereals. The aim of the present study was to obtain a deeper understanding concerning the effects of AME on pig intestinal health using epithelial intestinal cell lines as the data concerning the possible effects of Alternaria toxins on swine are scarce and insufficient for assessing the risk represented by Alternaria toxins for animal health. Our results have shown a dose-related effect on IPEC-1 cell viability, with an IC50 value of 10.5 µM. Exposure to the toxin induced an increase in total apoptotic cells, suggesting that AME induces programmed cell death through apoptosis based on caspase-3/7 activation in IPEC-1 cells. DNA and protein oxidative damage triggered by AME were associated with an alteration of the antioxidant response, as shown by a decrease in the enzymatic activity of catalase and superoxide dismutase. These effects on the oxidative response can be related to an inhibition of the Akt/Nrf2/HO-1 signaling pathway; however, further studies are needed in order to validate these in vitro data using in vivo trials in swine.

Using In Silico Approach for Metabolomic and Toxicity Prediction of Alternariol.

Alternariol is a metabolite produced by Alternaria fungus that can contaminate a variety of food and feed materials. The objective of the present paper was to provide a prediction of Phase I and II metabolites of alternariol and a detailed ADME/Tox profile for alternariol and its metabolites using an in silico working model based on the MetaTox, SwissADME, pKCMS, and PASS online computational programs. A number of 12 metabolites were identified as corresponding to the metabolomic profile of alternariol. ADME profile for AOH and predicted metabolites indicated a moderate or high intestinal absorption probability but a low probability to penetrate the blood-brain barrier. In addition to cytotoxic, mutagenic, carcinogenic, and endocrine disruptor effects, the computational model has predicted other toxicological endpoints for the analyzed compounds, such as vascular toxicity, haemato-toxicity, diarrhea, and nephrotoxicity. AOH and its metabolites have been predicted to act as a substrate for different isoforms of phase I and II drug-metabolizing enzymes and to interact with the response to oxidative stress. In conclusion, in silico methods can represent a viable alternative to in vitro and in vivo tests for the prediction of mycotoxins metabolism and toxicity.

Grape seed meal by-product is able to counteract oxidative stress induced by lipopolysaccharide and dextran sulphate in IPEC cells and piglets after weaning.

Oxidative stress is a pivotal factor in the pathogenesis of intestinal inflammation, leading to cellular damage and tissue injury. Natural antioxidants compounds found in agro-industrial by-products have proven their effectiveness in treatment of intestinal inflammation and oxidative stress, exhibiting many favourable effects. The aim of this study was to evaluate the capacity of a grape seed meal byproduct (GSM) to counteract the effects induced by E. coli lipopolysaccharide (LPS, 5µg/ml) in vitro on IPEC-1 cells and by dextran sulphate sodium (DSS, 1g/b.w./day) in vivo on piglets after weaning. Reactive oxygen species (ROS), pro-oxidant markers (malondialdehyde MDA, thiobarbituric acid reactive substances TBARS, protein carbonyl, DNA oxidative damage) antioxidant enzymes (catalase -CAT, superoxide dismutase -SOD, glutathione peroxidase -GPx, endothelial and inducible nitric oxide synthases -eNOS and iNOS) and several important components of Keap1/Nrf2 signalling pathway were analysed in IPEC-1 cells as well as in piglet's colon and lymph nodes. Our results demonstrated that GSM extract or 8% dietary GSM showed anti-oxidant properties counteracting the pro-oxidant response (ROS, MDA-TBARS, protein carbonyl, DNA/RNA damage) induced by LPS or DSS and restoring the levels of endogenous antioxidant enzymes, including CAT, SOD, GPx, eNOS and iNOS in colon and mesenteric lymph nodes. These beneficial effects were modulated via Nrf2 signalling pathway in both in vitro and in vivo studies.

Effects of Exposure to Low Zearalenone Concentrations Close to the EU Recommended Value on Weaned Piglets' Colon.

Pigs are the most sensitive animal to zearalenone (ZEN) contamination, especially after weaning, with acute deleterious effects on different health parameters. Although recommendations not to exceed 100 µg/kg in piglets feed exists (2006/576/EC), there are no clear regulations concerning the maximum limit in feed for piglets, which means that more investigations are necessary to establish a guidance value. Due to these reasons, the present study aims to investigate if ZEN, at a concentration lower than the EC recommendation for piglets, might affect the microbiota or induce changes in SCFA synthesis and can trigger modifications of nutritional, physiological, and immunological markers in the colon (intestinal integrity through junction protein analysis and local immunity through IgA production). Consequently, the effect of two concentrations of zearalenone were tested, one below the limit recommended by the EC (75 µg/kg) and a higher one (290 µg/kg) for comparison reasons. Although exposure to contaminated feed with 75 µg ZEN/kg feed did not significantly affect the observed parameters, the 290 µg/kg feed altered several microbiota population abundances and the secretory IgA levels. The obtained results contribute to a better understanding of the adverse effects that ZEN can have in the colon of young pigs in a dose-dependent manner.

The Use of Fruit and Vegetable by-Products as Enhancers of Health Status of Piglets after Weaning: The Role of Bioactive Compounds from Apple and Carrot Industrial Wastes.

At weaning, piglets are exposed to a large variety of stressors, from environmental/behavioral factors to nutritional stress. Weaning transition affects the gastrointestinal tract especially, resulting in specific disturbances at the level of intestinal morphology, barrier function and integrity, mucosal immunity and gut microbiota. All these alterations are associated with intestinal inflammation, oxidative stress and perturbation of intracellular signaling pathways. The nutritional management of the weaning period aims to achieve the reinforcement of intestinal integrity and functioning to positively modulate the intestinal immunity and that of the gut microbiota and to enhance the health status of piglets. That is why the current research is focused on the raw materials rich in phytochemicals which could positively modulate animal health. The composition analysis of fruit, vegetable and their by-products showed that identified phytochemicals could act as bioactive compounds, which can be used as modulators of weaning-induced disturbances in piglets. This review describes nutritional studies which investigated the effects of bioactive compounds derived from fruit (apple) and vegetables (carrot) or their by-products on the intestinal architecture and function, inflammatory processes and oxidative stress at the intestinal level. Data on the associated signaling pathways and on the microbiota modulation by bioactive compounds from these by-products are also presented.

Yeast-Fermented Rapeseed Meal Extract Is Able to Reduce Inflammation and Oxidative Stress Caused by Escherichia coli Lipopolysaccharides and to Replace ZnO in Caco-2/HTX29 Co-Culture Cells.

(1) The present study tested in vitro the capacity of a fermented rapeseed meal extract to reduce medicinal ZnO, which will be banned at the EU level from 2023 onwards because of its potential to cause environmental pollution and the development of Zn resistance in gut bacteria. Rapeseed meal could be an important ZnO substitute as it has antioxidant/radical scavenging properties due to its content of bioactive compounds (e.g., polyphenols). (2) Protein array and flow cytometry were used to detect apoptosis, oxidative stress production, and inflammatory and signaling-related molecules in Caco-2 and goblet HT29-MTX co-culture cells challenged with Escherichia coli lipopolysaccharides and treated with ZnO and FRSM. (3) LPS induced cell death (21.1% vs. 12.7% in control, p < 0.005); apoptosis (16.6%); ROS production; and overexpression of biomarkers related to inflammation (63.15% cytokines and 66.67% chemokines), oxidative stress, and signaling proteins when compared to untreated cells. ZnO was effective in counteracting the effect of LPS, and 73.68% cytokines and 91.67% of chemokines were recovered. FRSM was better at restoring normal protein expression for 78.94% of cytokines, 91.67% of chemokines, and 61.11% of signaling molecules. FRSM was able to mitigate negative effects of LPS and might be an alternative to ZnO in pig diets.

Dietary Grape Seed Meal Bioactive Compounds Alleviate Epithelial Dysfunctions and Attenuates Inflammation in Colon of DSS-Treated Piglets.

Inflammatory Bowel Diseases (IBD) are chronic inflammations associated with progressive degradation of intestinal epithelium and impairment of the local innate immune response. Restoring of epithelial integrity and of the mucosal barrier function, together with modulation of inflammatory and innate immune markers, represent targets for alternative strategies in IBD. The aim of our study was to evaluate the effects of a diet including 8% grape seed meal (GSM), rich in bioactive compounds (polyphenols, polyunsaturated fatty acids (PUFAs), fiber) on the markers of colonic epithelial integrity, mucosal barrier function, pro-inflammatory, and innate immunity in DSS-treated piglets used as animal models of intestinal inflammation. Our results have demonstrated the beneficial effects of bioactive compounds from dietary GSM, exerted at three complementary levels: (a) restoration of the epithelial integrity and mucosal barrier reinforcement by modulation of claudins, Occludin (OCCL) and Zonula-1 (ZO-1) tight junction genes and proteins, myosin IXB (MYO9B) and protein tyrosine phosphatase (PTPN) tight junction regulators and mucin-2 (MUC2) gene; (b) reduction of pro-inflammatory MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) genes and activities; and (c) suppression of the innate immune TLR-2 (Toll-like receptor-2) and TLR-4 (Toll-like receptor-4) genes and attenuation of the expression of MyD88 (Myeloid Differentiation Primary Response 88)/MD-2 (Myeloid differentiation factor-2) signaling molecules. These beneficial effects of GSM could further attenuate the transition of chronic colitis to carcinogenesis, by modulating the in-depth signaling mediators belonging to the Wnt pathway.

Zearalenone and the Immune Response.

Zearalenone (ZEA) is an estrogenic fusariotoxin, being classified as a phytoestrogen, or as a mycoestrogen. ZEA and its metabolites are able to bind to estrogen receptors, 17ß-estradiol specific receptors, leading to reproductive disorders which include low fertility, abnormal fetal development, reduced litter size and modification at the level of reproductive hormones especially in female pigs. ZEA has also significant effects on immune response with immunostimulatory or immunosuppressive results. This review presents the effects of ZEA and its derivatives on all levels of the immune response such as innate immunity with its principal component inflammatory response as well as the acquired immunity with two components, humoral and cellular immune response. The mechanisms involved by ZEA in triggering its effects are addressed. The review cited more than 150 publications and discuss the results obtained from in vitro and in vivo experiments exploring the immunotoxicity produced by ZEA on different type of immune cells (phagocytes related to innate immunity and lymphocytes related to acquired immunity) as well as on immune organs. The review indicates that despite the increasing number of studies analyzing the mechanisms used by ZEA to modulate the immune response the available data are unsubstantial and needs further works.

Grape Seed Waste Counteracts Aflatoxin B1 Toxicity in Piglet Mesenteric Lymph Nodes.

Aflatoxin B1 (AFB1) is a mycotoxin that frequently contaminates cereals and cereal byproducts. This study investigates the effect of AFB1 on the mesenteric lymph nodes (MLNs) of piglets and evaluates if a diet containing grape seed meal (GSM) can counteract the negative effect of AFB1 on inflammation and oxidative stress. Twenty-four weaned piglets were fed the following diets: Control, AFB1 group (320 µg AFB1/kg feed), GSM group (8% GSM), and AFB1 + GSM group (8% GSM + 320 µg AFB1/kg feed) for 30 days. AFB1 has an important antioxidative effect by decreasing the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and total antioxidant status. As a result of the exposure to AFB1, an increase of MAP kinases, metalloproteinases, and cytokines, as effectors of an inflammatory response, were observed in the MLNs of intoxicated piglets. GSM induced a reduction of AFB1-induced oxidative stress by increasing the activity of GPx and SOD and by decreasing lipid peroxidation. GSM decreased the inflammatory markers increased by AFB1. These results represent an important and promising way to valorize this waste, which is rich in bioactive compounds, for decreasing AFB1 toxic effects in mesenteric lymph nodes.

Assessment of Food By-Products' Potential for Simultaneous Binding of Aflatoxin B1 and Zearalenone.

In this study, eight food by-products were investigated as biosorbent approaches in removing mycotoxin load towards potential dietary inclusion in animal feed. Among these food-derived by-products, grape seed (GSM) and seabuckthorn (SBM) meals showed the most promising binding capacity for Aflatoxin B1 (AFB1) and Zearalenone (ZEA), measured as percent of adsorbed mycotoxin. Furthermore, we explored the mycotoxin sequestering potential by screening the effect of time, concentration, temperature and pH. Comparative binding efficacy was addressed by carrying out adsorption experiments in vitro. The highest mycotoxin adsorption was attained using 30 mg of by-product for both GSM (85.9% AFB1 and 83.7% ZEA) and SBM (68% AFB1 and 84.5% ZEA). Optimal settings for the experimental factors were predicted employing the response surface design. GSM was estimated to adsorb AFB1 optimally at a concentration of 29 mg/mL, pH 5.95 and 33.6 °C, and ZEA using 28 mg/mL at pH 5.76 and 31.7 °C. Favorable adsorption of AFB1 was estimated at 37.5 mg of SBM (pH 8.1; 35.6 °C), and of ZEA at 30.2 mg of SBM (pH 5.6; 29.3 °C). Overall, GSM revealed a higher binding capacity compared with SBM. In addition, the two by-products showed different specificity for the binary-mycotoxin system, with SBM having higher affinity towards ZEA than AFB1 (Kf = 0.418 and 1/n = 0.213 vs. Kf = 0.217 and 1/n = 0.341) and GSM for AFB1 in comparison with ZEA (Kf = 0.367 and 1/n = 0.248 vs. Kf = 0.343 and 1/n = 0.264). In conclusion, this study suggests that GSM and SBM represent viable alternatives to commercial biosorbent products.

Diet containing grape seed meal by-product counteracts AFB1 toxicity in liver of pig after weaning.

Liver is the earliest target for AFB1 toxicity in both human and animals. In the last decade, plant derived by-products have been used in animal feed to reduce AFB1 induced toxicity. In the present study we investigated whether the presence of 8% grape seed meal by-product is able to counteract the hepatotoxic effects produced by AFB1 in liver of pig after weaning exposed to the toxin through the contaminated feed for 28 days. Twenty four weaned cross-bred TOPIGS-40 piglets with an average body weight of 9.13±0.03 were allocated to the following experimentally treatments: control diet without AFB1 (normal compound feed for weaned pigs); contaminated diet with 320 mg kg-1 AFB1; GSM diet (compound feed plus 8% grape seed meal) and AFB1+GSM diet (320 mg kg-1 AFB1 contaminated feed plus 8% grape seed meal). Pigs fed AFB1 diet had altered performance, body weight decreasing with 25.1% (b.w.: 17.17 kg for AFB1 vs 22.92 kg for control). Exposure of piglets to AFB1 contaminated diet caused liver oxidative stress as well as liver histological damage, manly characterized by inflammatory infiltrate, fibrosis and parenchyma cells vacuolation when compared to control and GSM meal group. 94.12% of the total analysed genes (34) related to inflammation and immune response was up-regulated. The addition of GSM into the AFB1 diet diminished the gene overexpression and ameliorate histological liver injuries and oxidative stress. The protective effect of GSM diet in diminishing the AFB1 harmful effect was mediated through the decreasing of gene and protein expression of MAPKs and NF-κB signalling overexpressed by AFB1 diet. The inclusion of grape seed by-products in the diet of pigs after weaning might be used as a novel nutritional intervention to reduce aflatoxin toxicity.

Effects of Dietary Grape Seed Meal Bioactive Compounds on the Colonic Microbiota of Weaned Piglets With Dextran Sodium Sulfate-Induced Colitis Used as an Inflammatory Model.

Microbiota affects host health and plays an important role in dysbiosis. The study examined the effect of diet including grape seed meal (GSM) with its mixture of bioactive compounds on the large intestine microbiota and short-chain fatty acid synthesis in weaned piglets treated with dextran sodium sulfate (DSS) as a model for inflammatory bowel diseases. Twenty-two piglets were included in four experimental groups based on their diet: control, DSS (1 g/kg/b.w.+control diet), GSM (8% grape seed meal inclusion in control diet), and DSS+GSM (1 g/kg/b.w., 8% grape seed meal in control diet). After 30 days, the colon content was isolated and used for microbiota sequencing on an Illumina MiSeq platform. QIIME 1.9.1 pipeline was used to process the raw sequences. Both GSM and DSS alone and in combination affected the diversity indices and Firmicutes:Bacteroidetes ratio, with significantly higher values in the DSS-afflicted piglets for Proteobacteria phylum, Roseburia, Megasphera and CF231 genus, and lower values for Lactobacillus. GSM with high-fiber, polyphenol and polyunsaturated fatty acid (PUFA) content increased the production of butyrate and isobutyrate, stimulated the growth of beneficial genera like Prevotella and Megasphaera, while countering the relative abundance of Roseburia, reducing it to half of the DSS value and contributing to the management of the DSS effects.

Phytogenic actives supplemented in hyperprolific sows: effects on maternal transfer of phytogenic compounds, colostrum and milk features, performance and antioxidant status of sows and their offspring, and piglet intestinal gene expression.

Phytogenic actives (PA) are plant-derived natural bioactive compounds that may promote livestock health and well-being, as well as improve growth performance and production efficiency. The current study aims to evaluate their effects on sows and their offspring. Eighty-one hyperprolific sows (up to parity 7) were assigned to 3 experimental treatments. Control sows were offered a nonsupplemented diet during gestation and lactation, and treated sows were fed the control diet supplemented with 1 g/kg of a blend of PA (BPA) in lactation (L) or during gestation and lactation (GL). An evaluation was made of placental and milk maternal transfer of these BPA and colostrum-milk features, sows and piglets antioxidant status, reproductive performance (litter size), body weight (BW) changes, weaning-estrus interval, and litter performance. Finally, piglet´s jejunum gene expression was measured. The BPA supplementation during gestation (GL) increased the number of piglets born alive (P = 0.020) and reduced (P < 0.05) the newborn piglets BW, while there were no differences among treatments on the suckling (day 20) and weaned (day 7) piglets BW (P > 0.05). Dietary phytogenic volatile compounds reached GL placental fluid, and milk of L and GL sows (P < 0.05). Moreover, colostrum protein in GL and milk fat content in L and GL were increased (P < 0.05). Milk of GL showed inhibitory activity against Bacillus subtilis and Staphylococcus aureus (P < 0.05). Antioxidant status of GL sows showed an enhanced (P < 0.05) of catalase (CAT) and total antioxidant capacity levels at early gestation (day 35), whereas higher levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes at late gestation (day 110). Likewise, GL newborn piglets showed higher CAT levels, whereas both CAT and SOD levels in suckling piglets, as well as CAT, SOD, and GSH-Px in weaned piglets, were increased in L and GL (P < 0.05). Jejunum messenger ribonucleic acid abundance of suckling piglets in L and GL groups showed overexpression of barrier function MUC2, digestive enzyme IDO, and immune response PPARGC-α, TNF-α, TGF-ß1, and IL-10 genes (P < 0.05). In conclusion, dietary BPA supplementation in hyperprolific sows increased the litter size (born alive) and improved the composition and bioactivity of colostrum and milk, besides, modified the antioxidant status of sows and their offspring, as well as the suckling piglets gut health gene expression. Several BPA volatile compounds were prenatal and postnatal maternally transferred (placental fluid and milk).

MicroRNA profiling in kidney in pigs fed ochratoxin A contaminated diet.

OTA is a toxic metabolite produced by fungus belonging to Aspergillus and Penicillium genera. Kidney is the main target of this toxin; OTA is considered as one of the etiological factors at the origin of the human Balkan endemic nephropathy. microRNA are short non-coding transcrips (18-22 nucleotides in length) regulating key cellular processes. Various miRNAs have been established to play important roles in development of renal carcinoma and urothelial cancer. The objective of this study is to analyse the miRNA profiling in the kidney of piglets experimentally intoxicated with feed contaminated with OTA. Fifteen piglets (five pigs/group) were randomly distributed into 3 groups, fed normal diet (Group 1: control), or diets contaminated with OTA in two concentrations: 50 µg OTA/kg feed (Group 2: 50 µg OTA/kg feed) or 200 µg OTA/kg feed (Group 3: 200 µg OTA/kg feed) for 28 days. At the end of the experiment blood samples were taken for serological analyses. Animals from control group and 200 µg OTA/kg feed were sacrificed and kidney samples were taken for histological and molecular analyses. As resulted from molecular profiling study there are 8 miRNA differentially expressed in OTA kidney vs control kidney, in which five miRNA were overexpressed in the kidney of OTA intoxicated animals: miR-497 (FC = 6.34), miR-133a-3p (FC = 5.75), miR-423-3p (FC = 5.48), miR-34a (FC = 1.68), miR-542-3p (1.65) while three miRNA were downregulated: miR-421-3p (FC = -3.96); miR-490 (FC = -3.87); miR-9840-3p (FC = -2.13). The altered miRNAs as effect of OTA are strongly connected to the engine of cancer, disturbing nodal points in different pathways, as TP53 signalling. This proof-of-concept study proves the actual utility of miRNAs as biomarkers of mycotoxin exposure, including OTA.

Red Grape Pomace Rich in Polyphenols Diet Increases the Antioxidant Status in Key Organs-Kidneys, Liver, and Spleen of Piglets.

The aim of this work was to evaluate the influence of a diet containing 5% dried GP on the antioxidant status (total antioxidant status (TAS), antioxidant enzyme activity (catalase-CAT, superoxide dismutase-SOD, and gluthatione peroxidase-GPx), and lipid peroxidation) on the key organs of the liver, kidneys, and spleen in relation to health status as indicated by blood biochemical parameters and total polyphenol content in the blood, organs (liver, spleen, kidney, mesenteric lymph nodes, heart, and brain) and Longissimus dorsi muscle in piglets. The GP diet results in a significant increase of TAS in the liver, spleen, and kidneys, with increased CAT activity in the spleen and kidneys, increased SOD activity in the liver, kidneys, and spleen, and increased GPx activity in the kidneys, as well as a decrease in lipid peroxidation in the liver and kidneys. The GP included in the piglets' feed contained polyphenols that showed antioxidant activity and were absorbed in the plasma, contributing to maintaining the good health of the animals. The inclusion of 5% GP inclusion in the diets of piglets is beneficial for overall normal blood constituent metabolism and helps to maintain piglet health by increasing the polyphenol content in blood plasma and antioxidant activity in the liver, spleen, and kidneys.

Effect of Dietary Hemp Seed on Oxidative Status in Sows during Late Gestation and Lactation and Their Offspring.

This study shows the antioxidant effect of a dietary hemp seed diet rich in ω-6 polyunsaturated fatty acid (PUFA) on oxidative status in sows during late gestation and lactation and their offspring. Ten pregnant sows were divided into two groups and fed either a control diet (CD) or a hemp diet (HD) containing 2% hemp seed meal for a period of 10 days before farrowing and 5% throughout the lactation period (21 d). After farrowing, 16 of their resulting piglets were divided into two groups: control group CD (eight piglets derived from control sows) and HD group (eight piglets derived from HD sows), respectively. Blood collected from sows and piglets at day 1, 7 and 21 was used for the measurement of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), glutathione (GPx)), nitric oxide production (NO), lipid peroxidation (thiobarbituric acid reactive substances-TBARS), reactive oxygen species (ROS) generation and total antioxidant capacity (TAC) in plasma. The results showed a significant improvement in the oxidative status of sows fed HD throughout lactation compared with CD. Similarly, in piglets, HD positively influenced the activities of antioxidant enzymes, TAC and NO levels and significantly decreased lipid peroxidation in plasma until weaning, in comparison with the CD group. This study suggests the potential of hemp seed diet to improve the overall antioxidant status of the lactating sows and their progeny.

Assessment of the efficacy of a grape seed waste in counteracting the changes induced by aflatoxin B1 contaminated diet on performance, plasma, liver and intestinal tissues of pigs after weaning.

The aim of this study was to investigate the potential of a grape seed byproduct to mitigate the harmful damage produced by aflatoxin B1 (AFB1) at systemic level in plasma and liver as well as at local level in the gastrointestinal tract in weaned piglets. Twenty four crossbred pigs (TOPIG) were randomly assigned to one of four experimental diets: 1)- control diet (normal compound feed for starter piglets without mycotoxin), 2)- AFB1 diet (compound feed contaminated with 320 ppb pure AFB1), 3)- GS diet (compound feed including 8% of grape seed meal), 4)- AFB1+GS diet (compound feed containing 8% of grape seed meal contaminated with 320 ppb AFB1) for 30 days. The results showed that pigs fed AFB1 diet had altered performance (-25.1%), increased the thiobarbituric substances (TBARS) concentration wile reduced total antioxidant capacity and activity of antioxidant enzymes (CAT, SOD and GPx) in plasma and organs. AFB1 produced a dual effect on inflammatory response by increasing the level of pro-inflammatory cytokines in liver and colon and decreasing these cytokines in duodenum. The inclusion of grape seed in the diet of AFB1 intoxicated pigs enhanced the antioxidant enzymes activity, decreased the pro-inflammatory cytokines and TBARS level and ameliorated the growth performance of AFB1-treated animals. These findings suggest that grape waste is a promising feed source in counteracting the harmful effect of aflatoxin B1.

The Impact of Dietary Grape Seed Meal on Healthy and Aflatoxin B1 Afflicted Microbiota of Pigs after Weaning.

The study investigated the effect of grape seed (GS) meal, aflatoxin (AFB1), or their combination on the large intestine microbiota of weanling piglets. Twenty-four piglets were allocated into four groups based on diet composition: (1) Control group; (2) AFB1 (320 g/kg feed) group; (3) GS group (8% inclusion in the diet); (4) AFB1 + GS group. After 30 days of experiment, the colon content was used for microbiota analyses; after isolation of total bacterial genomic DNA, V3/V4 regions of the 16S rRNA amplicons were sequenced using the Illumina MiSeq platform. The raw sequences were analyzed using the v.1.9.1 QIIME pipeline software. 157 numbers of OTUs were identified among all four dietary groups with 26 of them being prevalent above 0.05% in the total relative abundance. GS and AFB1 increase the relative abundance of phylum Bacteroidetes and Proteobacteria, while decreasing the Firmicutes abundance in a synergic manner as compared with the individual treatments. An additive or synergistic action of the two treatments was identified for Lactobacillus, Prevotella and Campylobacter, while rather an antagonistic effect was observed on Lachnospira. The action mechanisms of aflatoxin B1 and grape seed meal that drive the large intestine microbiota to these changes are not known and need further investigations.

Synbiotic combination of prebiotic grape pomace extract and probiotic Lactobacillus sp. reduced important intestinal inflammatory markers and in-depth signalling mediators in lipopolysaccharide-treated Caco-2 cells.

Inflammatory bowel diseases (IBD) are a major problem for public health, with an increased incidence and impact on life quality. The effect of pre- and probiotic combination has been less studied in IBD. Using genomic and proteomic array technologies, this study examined the efficacy of a new combination of natural alternatives: prebiotics (grape pomace extract, GP) and probiotics (lactobacilli mixture, Lb mix) on inflammation and intracellular signalling routes in a cellular model of inflammation. Caco-2 cells challenged with lipopolysaccharide (LPS) for 4 h were treated with GP extract (50 µg/ml gallic acid equivalent) and Lb combination (3 × 108 colony-forming units/ml total Lb) for 24 h. The profile expressions of forty key inflammatory markers and twenty-six signalling kinases were analysed. Other markers involved in inflammation were also investigated (NF-κB/RELA, Nrf2, aryl hydrocarbon receptor, Cyp1A1, Cyp1B1); 57·5 and 60 % of investigated genes and proteins, respectively, were down-regulated by the synbiotic combination. Relevant cytokines and chemokines involved in response to microbial infection and inflammation were reduced under the level induced by LPS treatment and toward the unchallenged control. As expected, the reduction effect seems to imply mitogen-activated protein kinase and NF-κB pathway. Most of the signalling molecules activated by LPS were decreased by GP extract and Lb mix. Our study indicates that the synbiotic combination of GP extract and Lactobacillus sp. mixture exerted anti-inflammatory properties, which are able to decrease the majority of inflammatory genes, their proteins and associated signalling markers. Due to protective role of GP compounds on lactobacilli probiotic, this synbiotic combination might serve as a promising adjunctive therapy in intestinal inflammations.

In Vitro Transcriptome Response to a Mixture of Lactobacilli Strains in Intestinal Porcine Epithelial Cell Line.

BACKGROUND: Food and feed supplements containing microorganisms with probiotic potential are of increasing interest due to their healthy promoting effect on human and animals. Their mechanism of action is still unknown. Using a microarray approach, the aim of this study was to investigate the differences in genome-wide gene expression induced by a mixture of three Lactobacillus strains (L. rhamnosus, L. plantarum, and L. paracasei) in intestinal porcine epithelial cells (IPEC-1) and to identify the genes and pathways involved in intestinal barrier functions. METHODS: Undifferentiated IPEC-1 cells seeded at a density of 2.0 × 105/mL in 24-wells culture plates were cultivated at 37 °C and 5% CO2 until they reached confluence (2⁻3 days). Confluent cells monolayer were then cultivated with 1 mL of fresh lactobacilli (LB) mixture suspension prepared for a concentration of approximately 3.3 × 107 CFU/mL for each strain (1 × 108 CFU/mL in total) for 3 h and analyzed by microarray using Gene Spring GX v.11.5. RESULTS: The functional analysis showed that 1811 of the genes modulated by LB treatment are involved in signaling (95% up-regulation, 121 genes with a fold change higher than 10). The most enhanced expression was registered for AXIN2 (axis inhibition protein 2-AXIN2) gene (13.93 Fc, p = 0.043), a negative regulator of ß-catenin with a key role in human cancer. LB affected the cellular proliferation by increasing 10 times (Fc) the NF1 gene encoding for the neurofibromin protein, a tumor suppressor that prevent cells from uncontrolled proliferation. The induction of genes like serpin peptidase inhibitor, clade A member 3 (SERPINA 3), interleukin-20 (IL-20), oncostatin M(OSM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the suppression of chemokine (C-X-C motif) ligand 2/macrophage inflammatory protein 2-alpha (CXCL-2/MIP-2), regulator of G-protein signaling 2 (RGS2), and of pro-inflammatory interleukin-18 (IL-18) genes highlights the protective role of lactobacilli in epithelial barrier function against inflammation and in the activation of immune response. CONCLUSION: Gene overexpression was the predominant effect produced by lactobacilli treatment in IPEC-1 cells, genes related to signaling pathways being the most affected. The protective role of lactobacilli in epithelial barrier function against inflammation and in the activation of immune response was also noticed.