Protective effects and mechanisms of ellagic acid on intestinal injury in piglets infected with porcine epidemic diarrhea virus.

The present study was conducted to decipher the protection effects of ellagic acid (EA) on piglets infected with porcine epidemic diarrhea virus (PEDV). Thirty 7-day-old piglets were randomly assigned to three treatment groups: control, PEDV, and EA + PEDV groups. After a 3-day period of adaption, piglets in the EA + PEDV group were orally administered with 20 mg/kg·BW EA during days 4-11 of the trial. On day 8, piglets were orally administered with PEDV at a dose of 106 TCID50 (50% tissue culture infectious dose) per pig. Additionally, intestinal porcine epithelial (IPEC-1) cells infected with PEDV were used to investigate the anti-PEDV effect of EA in vitro. The results showed that EA at a dose of 10-40 µmol/L increased the viability of PEDV-infected IPEC-1 cells, and EA administration mitigated intestinal edema in piglets challenged with PEDV. Further studies indicated that EA treatment significantly increased the proportion of white blood cells in blood and concentrations of IL-6, IL-1ß, and IL-10 in the serum, but decreased the TNF-α content and gene expression of IL-6, IL-1ß, TNF-α, and CXCL2 in the jejunum. Moreover, EA intervention considerably elevated the activity of total superoxide dismutase (T-SOD), but decreased the H2O2 concentration in the ileum of piglets. Importantly, EA suppressed the increased expression of antiviral-related genes and proteins (including MXI, ISG15, HSP70, and p-IRF7) induced by PEDV challenge in the jejunum. Furthermore, PEDV infection increased the protein abundance of p-JAK2 and p-STAT3, which were further enhanced by EA supplementation. In conclusion, our results revealed that EA could promote the restoration of intestinal homeostasis by regulating the interferon pathway that was interrelated with the activation of JAK2/STAT3 signaling. These findings provide theoretical basis for the use of EA as a therapy targeting PEDV infection in piglets.

Effects of Dietary Limosilactobacillus fermentum and Lacticaseibacillus paracasei Supplementation on the Intestinal Stem Cell Proliferation, Immunity, and Ileal Microbiota of Broiler Chickens Challenged by Coccidia and Clostridium perfringens.

This study was conducted to investigate effects of dietary Limosilactobacillus fermentum and Lacticaseibacillus paracasei supplementation on the intestinal stem cell proliferation, immunity, and ileal microbiota of broiler chickens challenged by coccidia and Clostridium perfringens. A total of 336 one-day-old Ross 308 chickens were randomly assigned into four groups. Chickens in the control (CTR) group were fed basal diet, and chickens in the three challenged groups were fed basal diets supplemented with nothing (CCP group), 1.0 × 109 CFU/kg L. fermentum (LF_CCP group), and 1.0 × 109 CFU/kg L. paracasei (LP_CCP group), respectively. All challenged birds were infected with coccildia on day 9 and Clostridium perfringens during days 13-18. The serum and intestinal samples were collected on days 13 and 19. The results showed that L. fermentum significantly increased jejunal gene expression of cdxB (one of the intestinal stem cell marker genes) on day 13. Additionally, L. fermentum significantly up-regulated mRNA levels of JAK3 and TYK2 and tended to increase STAT6 mRNA expression in jejunum on day 19. In the cecal tonsil, both L. fermentum and L. paracasei decreased mRNA expression of JAK2 on day 13, and L. fermentum down-regulated JAK1-2, STAT1, and STAT5-6 gene expressions on day 19. Ileal microbiological analysis showed that coccidial infection increased the Escherichia-Shigella, Lactobacillus, and Romboutsia abundance and decreased Candidatus_Arthromitus richness on day 13, which were reversed by Lactobacillus intervention. Moreover, Lactobacilli increased ileal Lactobacillus richness on day 19. In conclusion, Lactobacilli alleviated the impairment of intestinal stem cell proliferation and immunity in coccidia- and C. perfringens-challenged birds via modulating JAK/STAT signaling and reshaping intestinal microflora.

Dietary pectin attenuates Salmonella typhimurium-induced colitis by modulating the TLR2-NF-κB pathway and intestinal microbiota in mice.

The role of dietary pectin on microbial-induced colitis, oxidative status, barrier function, and microbial composition, as well as the underlying mechanisms, is scarce. In this study, we aimed to investigate whether dietary pectin alleviates Salmonella typhimurium-induced colitis in mice. Male C57BL/6J mice fed an isocaloric and isofibrous diet with 7% pectin or cellulose were administered sterile water or Salmonella typhimurium to induce colitis, which is equal to a human food dose of 0.57% (5.68 g/kg). Dietary pectin alleviated Salmonella typhimurium-induced colitis and oxidative stress as shown by the reduced disease activity index score, decreased colon shortening and histological damage score, colonic hydrogen peroxide, malondialdehyde concentrations, and relative mRNA expressions of coenzyme Q-binding protein COQ10 homologue B (Coq10b), Ccl-2, Ccl-3, Ccl-8, Tnf-α, Il-1ß, Ifn-γ, Ifn-ß, and serum TNF-α protein level. Moreover, pectin administration ameliorated the downregulated colonic abundances of occludin, zonula occludens-1, zonula occludens-2, and the upregulated abundances of TLR2 and p-NF-κB in Salmonella-infected mice. Additionally, 16S rRNA analysis demonstrated that pectin altered the microbial beta-diversity and reduced Salmonella levels. Collectively, pectin ameliorated Salmonella typhimurium-induced colitis, oxidative stress, and tight junction, which may be related to the inactivation of TLR2-NF-κB signalling and reduced abundance of Salmonella.

Amuc attenuates high-fat diet-induced metabolic disorders linked to the regulation of fatty acid metabolism, bile acid metabolism, and the gut microbiota in mice.

Amuc_1100 (hereafter called Amuc) is a highly abundant pili-like protein on the outer membrane of Akkermansia muciniphila and has been found to be effective for in anti-obesity, which is probably through the activation of TLR2. However, the precise mechanisms underlying the contributions of TLR2 to obesity resistance remain unknown. Here, TLR2 knockout mice were used to decipher the anti-obesity mechanism of Amuc. Mice exposed to a high-fat diet (HFD) were treated with Amuc (60 µg) every other day for 8 weeks. The results showed that Amuc supplementation decreased mouse body weight and lipid deposition by regulating fatty acid metabolism and reducing bile acid synthesis by activating TGR5 and FXR and strengthening the intestinal barrier function. The ablation of TLR2 partially reversed the positive effect of Amuc on obesity. Furthermore, we revealed that Amuc altered the gut microbiota composition by increasing the relative abundance of Peptostreptococcaceae, Faecalibaculum, Butyricicoccus, and Mucispirillum_schaedleri_ASF457, and decreasing Desulfovibrionaceae, which may serve as a contributor for Amuc to reinforce the intestinal barrier in HFD-induced mice. Therefore, the anti-obesity effect of Amuc was accompanied by the mitigation of gut microbes. These findings provide support for the use of Amuc as a therapy targeting obesity-associated metabolic syndrome.

Amuc Prevents Liver Inflammation and Oxidative Stress in Mice Challenged with Salmonella Typhimurium.

BACKGROUND: Salmonella typhimurium is a pathogen that causes gastroenteritis in humans and animals. Amuc_1100 (hereafter called Amuc), the outer membrane protein of Akkermansia muciniphila, alleviates metabolic disorders and maintains immune homeostasis. OBJECTIVE: This study was conducted to determine whether there is a protective effect of Amuc administration. METHODS: Male 6-wk-old C57BL6J mice were randomly allocated into 4 groups: CON (control), Amuc (gavaged with Amuc, 100 µg/d for 14 d), ST (oral administration of 1.0 × 106 CFU S. typhimurium on day 7), and ST + Amuc (Amuc supplementation for 14 d, S. typhimurium administration on day 7). Serum and tissue samples were collected 14 d after treatment. Histological damage, inflammatory cell infiltration, apoptosis, and protein levels of genes associated with inflammation and antioxidant stress were analyzed. Data were analyzed by 2-way ANOVA and Duncan's multiple comparisons using SPSS software. RESULTS: The ST group mice had 17.1% lower body weight, 1.3-3.6-fold greater organ index (organ weight/body weight for organs including the liver and spleen), 10-fold greater liver damage score, and 3.4-10.1-fold enhanced aspartate transaminase, alanine transaminase, and myeloperoxidase activities, and malondialdehyde and hydrogen peroxide concentrations compared with controls (P < 0.05). The S. typhimurium-induced abnormalities were prevented by Amuc supplementation. Furthermore, the ST + Amuc group mice had 1.44-1.89-fold lower mRNA levels of proinflammatory cytokines (interleukin [Il]6, Il1b, and tumor necrosis factor-α) and chemokines (chemokine ligand [Ccl]2, Ccl3, and Ccl8) and 27.1%-68.5% lower levels of inflammation-related proteins in the liver than ST group mice (P < 0.05). CONCLUSIONS: Amuc treatment prevents S. typhimurium-induced liver damage partly through the toll-like receptor (TLR)2/TLR4/myeloid differentiation factor 88 and nuclear factor-κB signaling as well as nuclear factor erythroid-2 related factor signaling pathways. Thus, Amuc supplementation may be effective in treating liver injury in S. typhimurium-challenged mice.

Linking serotonin homeostasis to gut function: Nutrition, gut microbiota and beyond.

Serotonin (5-HT) produced by enterochromaffin (EC) cells in the digestive tract is crucial for maintaining gut function and homeostasis. Nutritional and non-nutritional stimuli in the gut lumen can modulate the ability of EC cells to produce 5-HT in a temporal- and spatial-specific manner that toning gut physiology and immune response. Of particular interest, the interactions between dietary factors and the gut microbiota exert distinct impacts on gut 5-HT homeostasis and signaling in metabolism and the gut immune response. However, the underlying mechanisms need to be unraveled. This review aims to summarize and discuss the importance of gut 5-HT homeostasis and its regulation in maintaining gut metabolism and immune function in health and disease with special emphasis on different types of nutrients, dietary supplements, processing, and gut microbiota. Cutting-edge discoveries in this area will provide the basis for the development of new nutritional and pharmaceutical strategies for the prevention and treatment of serotonin homeostasis-related gut and systematic disorders and diseases.

l-Proline improves the cytoplasmic maturation of mouse oocyte by regulating glutathione-related redox homeostasis.

Recent studies have shown that l-proline (proline) is an antioxidant to protect cells from oxidative stress in vivo and in vitro. Glutathione (GSH) is a major cellular redox regulator involved in controlling redox balance and is regarded as one of the key indices to predict the cytoplasmic maturation of oocytes. The objectives of this study are to investigate the effect of proline on the developmental potential of mouse oocytes and to determine the role of gap junctional communication (GJC) on intraoocyte GSH concentration during in vitro maturation (IVM). Compared with control (0 mmol/L), 0.5 mmol/L proline supplementation enhanced rates of activated oocytes, 2-cell and 4-cell embryos, and blastocysts. Furthermore, 0.25 and 0.5 mmol/L proline supplementation markedly upregulated mRNA expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) in oocytes and cumulus cells, enhanced GSH concentration in oocytes, and reduced reactive oxygen species (ROS) level in oocytes. Interestingly, carbenoxolone disodium salt (CBX) treatment reduced GSH concentration in oocytes and the rate of early embryo development without proline incubation. Notably, CBX-triggered reduction in the rates of the number of 2-cell and 4-cell embryos and blastocysts were rescued by 0.5 mmol/L proline supplementation. Collectively, these results indicate a novel functional role of proline in oocyte cytoplasmic maturation and regulation of glutathione-related redox homeostasis.

Protective effect of glucosamine on zearalenone-induced reproductive toxicity and placental dysfunction in mice.

This study was conducted to determine the effects of glucosamine (GlcN) on zearalenone (ZEA)-induced reproductive toxicity and placental dysfunction in mice. The pregnant mice were randomly divided into one of the four groups, such as the control group, the ZEA group, the GlcN group, and the GlcN plus ZEA group. Reproductive toxicity was induced by consecutive gavages of ZEA at 5 mg/kg body weight during gestational days (GDs 0-14) and in the presence or absence of oral administration of GlcN (0.5 mM). The results showed that GlcN significantly alleviated the decrease of growth performance induced by ZEA exposure of pregnant mice. Meanwhile, ZEA ingestion significantly reduced the number and weight of fetuses, and reduction of placenta weight. Moreover, results of blood biochemical markers indicated that ZEA exposure led to increased oxidative stress levels in pregnant mice. Further analyses demonstrated that ZEA inhibited placental development, resulted in placental inflammation, increased the expression of pro-apoptotic proteins, and decreased the expression of placental tight junction proteins, which were reversed by the administration of GlcN. Results of western blot revealed that GlcN reversed ZEA-mediated phenotype by activating PI3K, while inhibiting MAPK signaling pathway. All these findings showed that GlcN was effective in the protection against ZEA-induced placental dysfunction and reproductive toxicity in pregnant mice. Supplementation of GlcN might be potential nutritional intervention with an ability to alleviate ZEA-induced toxicity in pregnant mice.

Knockout of ICAT in Adipose Tissue Alleviates Fibro-inflammation in Obese Mice.

The E2 promoter binding factor 1 (E2F1) and the Wnt/ß-catenin signaling are crucial in regulating metabolic homeostasis including obesity. The ß-catenin interacting protein 1 (CTNNBIP1), also known as the inhibitor of ß-catenin and TCF4 (ICAT), is required for E2F1 to inhibit the activity of ß-catenin. However, the role of ICAT in E2F1 regulating obesity-related metabolic disorders remains unknown. In the present study, male adipose tissue-specific ICAT knockout (ICATadi-/-) C57BL/6 J mice and control littermates aged 6-8 weeks were fed with high-fat diet (HFD) for 12 weeks to explore the effect of ICAT on lipid metabolism and obesity-related disorders. Results showed that the adipose tissue-specific ICAT knockout had negligible effect on lipid metabolism, reflected by no difference in body weight, fat mass, and the expression of proteins involved in lipid metabolism in white adipose tissue (WAT) and the liver between the ICATadi-/- mice and the control littermate (ICATfl/fl) mice. However, the knockout of ICAT reduced inflammatory response in WAT and the liver. Additionally, Sirius red staining results showed that deletion of ICAT attenuated fibrosis and reduced mRNA levels of transforming growth factor ß1(TGF-ß1), matrix metallopeptidase 2 (Mmp2), Mmp3, and collagen, type V, alpha 1 (Col5a1) in WAT and the liver. These results suggested that knockout of ICAT improved the metabolic abnormalities of obese mice through attenuating adipose tissue and the liver inflammation as well as fibrosis. Our findings may provide a new insight to understand the role of ICAT in inflammation and fibrosis.

α-Ketoglutarate Restores Intestinal Barrier Function through Promoting Intestinal Stem Cells-Mediated Epithelial Regeneration in Colitis.

This study investigated the preventive effects of α-ketoglutarate (α-KG, in the form of sodium salt) on a Citrobacter rodentium (CR)-induced colitis and explored potential mechanisms. The results demonstrated that CR caused body weight loss and colon length shortening, which were abrogated by the α-KG administration. The colon length of mice in the α-KG plus CR group was significantly higher than that of mice in the CR group (6.9 ± 0.59 (mean ± SD) vs 6.1 ± 0.55; P < 0.05). This beneficial effect was associated with regulating endoplasmic reticulum (ER) stress signaling. In addition, small intestinal organoids generated from intestinal crypts of mice were exposed to α-KG in the presence of TNF-α or IWR-1 to assess stem cell activity in vitro. The results demonstrated that TNF-α exposure decreased the viability of organoids and impaired barrier function by suppressing Wnt signaling, which was abolished by α-KG. Interestingly, the protective effect of α-KG on intestinal barrier function was abrogated by the inhibitor of Wnt signaling in the intestinal organoids. Taken together, α-KG restored barrier function by regulating ER stress and activating Wnt/ß-catenin-medicated intestinal stem cell proliferation and differentiation.

Preventive Effects of l-Glutamine on High-Fat Diet-Induced Metabolic Disorders Linking with Regulation of Intestinal Barrier Integrity, Hepatic Lipid Metabolism, and Gut Microbiota in Rats.

The present study was conducted to investigate the effects of l-glutamine (Gln) on a high-fat diet (HFD)-induced lipid metabolic abnormality and explore its possible mechanisms. The results demonstrated that Gln administration reduced body weight, improved serum lipids, and decreased glucose tolerance in HFD-fed rats. Meanwhile, Gln administration alleviated liver injury, reduced the hepatic inflammatory response by inhibiting NLRP3 inflammasome activation, and decreased hepatic lipid accumulation by promoting VLDL secretion and fatty acid ß-oxidation, as well as reduced bile acid synthesis by activating hepatic and ileal FXR in HFD-fed rats. Moreover, Gln administration restored HFD-induced intestinal barrier dysfunction, promoted intestinal fat absorption, suppressed intestinal inflammation, and also reshaped the gut microbiota composition in HFD-fed rats by downregulating the abundance of potential pathogens Escherichia-Shigella and upregulating the abundance of beneficial bacteria such as Akkermansia. To conclude, the present results showed that Gln may be a potential option for preventing HFD-induced metabolic disorders via the gut-liver axis.

Resveratrol alleviates enterotoxigenic Escherichia coli K88-induced damage by regulating SIRT-1 signaling in intestinal porcine epithelial cells.

This study found that resveratrol pretreatment attenuated porcine intestinal epithelial cell damage caused by enterotoxigenic Escherichia coli (ETEC) K88 in vitro and the protective effects of resveratrol were associated with SIRT-1 signaling. ETEC K88 is a main intestinal pathogen for post-weaning diarrhea (PWD) in piglets. With the strict ban on antibiotics in animal feed, people are seeking effective antibiotic substitutes to protect the intestinal system against harmful pathogenic bacteria. This study was conducted to evaluate the effects of resveratrol, a natural plant polyphenol, on ETEC K88-induced cellular damage in porcine enterocytes and underlying mechanisms. Intestinal porcine epithelial cell line 1 (IPEC-1) cells, pretreated with or without resveratrol (30 µM, 4 h), were challenged with ETEC K88 (MOI = 1 : 10) for 3 h. The results showed that ETEC K88 infection induced severe damage and dysfunction in IPEC-1 cells, as evidenced by a reduced cell viability, decreased tight junctions, mitochondrial dysfunction, and autophagy. It is noteworthy that IPEC-1 cells pre-treated with resveratrol improved their capacity for resistance to most of these abnormal phenotypes caused by ETEC K88 infection. Furthermore, we found that the activation of SIRT-1 signaling was associated with the benefits of resveratrol, as demonstrated by EX-527, an inhibitor of SIRT-1, which reversed most of the protective effects of resveratrol. In conclusion, these results indicated that resveratrol could protect intestinal epithelial cells against ETEC K88 infection by activating SIRT-1 signaling. These findings provide new insights into the role of resveratrol in maintaining intestinal physiological functions.

Regulation of protein synthesis in porcine mammary epithelial cells by L-valine.

This study was conducted to determine the catabolism of L-valine in porcine mammary epithelial cells (PMECs) and its role in stimulating protein synthesis in these cells. PMECs were incubated with 0.05-, 0.10-, 0.25-, 0.5-, and 1.0-mM L-valine at 37 oC for 2 h. Cell viability and expressions of α-lactalbumin and ß-casein were measured after culture with L-valine for 3 days. L-[1-14C]valine was used to study valine catabolism, whereas [3H]phenylalanine was employed as a tracer to determine protein synthesis and degradation in PMECs. The abundances of proteins involved in the mTOR signaling pathway and the mRNA levels for the related key genes were determined using the western blot and RT-PCR techniques, respectively. Cell numbers and the synthesis of proteins (including α-lactalbumin and ß-casein) were greater (P < 0.05) in the presence of 0.5-mM L-valine, compared with 0.05- or 0.1-mM L-valine. L-Valine at 0.5 mM also enhanced (P < 0.05) the production of α-lactalbumin by PMECs, in comparison with 0.25 mM L-valine. Increasing the extracellular concentration of L-valine from 0.05 to 0.5 mM stimulated protein synthesis in a concentration-dependent manner without affecting proteolysis. Although L-valine was actively transaminated in PMECs, its α-ketoacid product (α-ketoisovalerate) at 0.05-0.2 mM did not affect protein synthesis or degradation in the cells. Thus, the effect of L-valine on protein synthesis was independent of its metabolism to yield α-ketoisovalerate. At the molecular level, 0.5-mM L-valine increased (P < 0.05) the mRNA levels for Ras, ERK1/2, and p70S6K, and the abundances of mTOR, p-4EBP1, total 4EBP1, p-ERK1/2, and total ERK1/2 proteins. These findings establish the critical role of L-valine in enhancing PMEC growth and milk protein synthesis possibly by regulating the mTOR and Ras/ERK signaling pathways. Further studies are warranted to understand how L-valine regulates gene expression and mTOR activation in PMECs.

A triphenylamine-based colorimetric and fluorescent probe with donor­bridge­acceptor structure for detection of G-quadruplex DNA.

In this Letter, three triphenylamine-based dyes (TPA-1, TPA-2a and TPA-2b) with donor­bridge­acceptor (D­p­A) structure were designed and synthesized for the purpose of G-quadruplexes recognition. In aqueous conditions, the interactions of the dyes with G-quadruplexes were studied with the aim to establish the influence of the geometry of the dyes on their binding and probing properties. Results indicate that TPA-2b displays significant selective colorimetric and fluorescent changes upon binding of G-quadruplex DNA. More importantly, its distinct color change enables visual detection and differentiation of G-quadruplexes from single and duplex DNA structures. CD titration date reveals that TPA-2b could induce and stabilize the formation of G-quadruplex structure. All these remarkable properties of TPA-2b suggest that it should have promising application in the field of G-quadruplexes research.