Retinoic acid enhances ovarian steroidogenesis by regulating granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway.

INTRODUCTION: Ovarian steroidogenesis not only affects the embryonic development and pregnancy outcome, but also associates with many diseases in mammals and women. Exploring the nutrients and mechanisms influencing ovarian steroidogenesis is critical to maintaining the optimal reproductive performance, as well as guaranteeing body health. OBJECTIVES: This research aimed to explore the effect of retinol metabolism on ovarian steroidogenesis and the underlying mechanisms. METHODS: Comparative transcriptomic analysis of ovaries from normal and low reproductive performance sows were performed to identify the main causes leading to low fertility. The metabolites regulating steroid hormones synthesis were investigated in ovarian granulosa cells. Gene interference, overexpression, dual-luciferase reporter assays, chromatin immunoprecipitation and transcriptome analysis were further conducted to explore the underlying mechanisms of Aldh1a1 mediating ovarian steroidogenesis. RESULTS: Transcriptome analysis of ovaries from normal and low reproductive performance sows showed the significant differences in both retinol metabolism and steroid hormones synthesis, indicating retinol metabolism probably influenced steroid hormones synthesis. The related metabolite retinoic acid was furtherly proven a highly active and potent substance strengthening estrogen and progesterone synthesis in ovarian granulosa cells. For the first time, we revealed that retinoic acid synthesis in porcine and human ovarian granulosa cells was dominated by Aldh1a1, and required the assistance of Aldh1a2. Importantly, we demonstrated that Aldh1a1 enhanced the proliferation of ovarian granulosa cells by activating PI3K-Akt-hedgehog signaling pathways. In addition, Aldh1a1 regulated the expression of transcription factor MESP2, which targeted the transcription of Star and Cyp11a1 through binding to corresponding promoter regions. CONCLUSION: Our data identified Aldh1a1 modulates ovarian steroidogenesis through enhancing granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway. These findings provide valuable clues for improving ovarian health in mammals.

DDIT3 regulates key enzymes in the methionine cycle and flux during embryonic development.

One-carbon metabolism is a key metabolic network that integrates nutritional signals with embryonic development. However, the response of one-carbon metabolism to methionine status and the regulatory mechanisms are poorly understood. Herein, we found that methionine supplementation during pregnancy significantly increased fetal number and average fetal weight. In addition, methionine modulated one-carbon metabolism primarily through 2 metabolic enzymes, cystathionine ß-synthase (CBS) and methionine adenosyltransferase 2A (MAT2A), which were significantly increased in fetal liver tissues and porcine trophoblast (pTr) cells in response to proper methionine supplementation. CBS and MAT2A overexpression enhanced the DNA synthesis in pTr cells. More importantly, we identified a transcription factor, DNA damage-inducible transcript 3 (DDIT3), that was the primary regulator of CBS and MAT2A, which bound directly to promoters and negatively regulated the expression of CBS and MAT2A. Taken together, our findings identified that DDIT3 targeting CBS and MAT2A was a novel regulatory pathway that mediated cellular one-carbon metabolism in response to methionine signal and provided promising targets to improve pregnancy health.

The Combined Use of Medium- and Short-Chain Fatty Acids Improves the Pregnancy Outcomes of Sows by Enhancing Ovarian Steroidogenesis and Endometrial Receptivity.

Fatty acids play important roles in maintaining ovarian steroidogenesis and endometrial receptivity. Porcine primary ovarian granulosa cells (PGCs) and endometrial epithelial cells (PEECs) were treated with or without medium- and short-chain fatty acids (MSFAs) for 24 h. The mRNA abundance of genes was detected by fluorescence quantitative PCR. The hormone levels in the PGCs supernatant and the rate of adhesion of porcine trophoblast cells (pTrs) to PEECs were measured. Sows were fed diets with or without MSFAs supplementation during early gestation. The fecal and vaginal microbiomes were identified using 16S sequencing. Reproductive performance was recorded at parturition. MSFAs increased the mRNA abundance of genes involved in steroidogenesis, luteinization in PGCs and endometrial receptivity in PEECs (p < 0.05). The estrogen level in the PGC supernatant and the rate of adhesion increased (p < 0.05). Dietary supplementation with MSFAs increased serum estrogen levels and the total number of live piglets per litter (p < 0.01). Moreover, MSFAs reduced the fecal Trueperella abundance and vaginal Escherichia-Shigella and Clostridium_sensu_stricto_1 abundance. These data revealed that MSFAs improved pregnancy outcomes in sows by enhancing ovarian steroidogenesis and endometrial receptivity while limiting the abundance of several intestinal and vaginal pathogens at early stages of pregnancy.

A Novel Nano-Antimicrobial Polymer Engineered with Chitosan Nanoparticles and Bioactive Peptides as Promising Food Biopreservative Effective against Foodborne Pathogen E. coli O157-Caused Epithelial Barrier Dysfunction and Inflammatory Responses.

For food quality and safety issues, the emergence of foodborne pathogenic bacteria has further accelerated the spread of antibiotic residues and drug resistance genes. To alleviate the harm caused by bacterial infections, it is necessary to seek novel antimicrobial agents as biopreservatives to prevent microbial spoilage. Nanoantimicrobials have been widely used in the direct treatment of bacterial infections. CNMs, formed by chitosan nanoparticles and peptides, are promising antibiotic alternatives for use as excellent new antibacterial drugs against pathogenic bacteria. Herein, the current study evaluated the function of CNMs in the protection of foodborne pathogen Escherichia coli (E. coli) O157 infection using an intestinal epithelial cell model. Antibacterial activity assays indicated that CNMs exerted excellent bactericidal activity against E. coli O157. Assessment of the cytotoxicity risks toward cells demonstrated that 0.0125-0.02% of CNMs did not cause toxicity, but 0.4% of CNMs caused cytotoxicity. Additionally, CNMs did not induced genotoxicity either. CNMs protected against E. coli O157-induced barrier dysfunction by increasing transepithelial electrical resistance, decreasing lactate dehydrogenase and promoting the protein expression of occludin. CNMs were further found to ameliorate inflammation via modulation of tumor factor α, toll-like receptor 4 and nuclear factor κB (NF-κB) expression via inhibition of mitogen-activated protein kinase and NF-κB activation and improved antioxidant activity. Taken together, CNMs could protect the host against E. coli O157-induced intestinal barrier damage and inflammation, showing that CNMs have great advantages and potential application as novel antimicrobial polymers in the food industry as food biopreservatives, bringing new hope for the treatment of bacterial infections.

Butyrate drives the acetylation of histone H3K9 to activate steroidogenesis through PPARγ and PGC1α pathways in ovarian granulosa cells.

Maintaining ovarian steroidogenesis is of critical importance, considering that steroid hormones are required for successful establishment and maintenance of pregnancy and proper development of embryos and fetuses. Investigating the mechanism that butyrate modulates the ovarian steroidogenesis is beneficial for understanding the impact of lipid nutrition on steroidogenesis. Herein, we identified that butyrate improved estradiol and progesterone synthesis in rat primary ovarian granulosa cells and human granulosa KGN cells and discovered the related mechanism. Our data indicated that butyrate was sensed by GPR41 and GPR43 in ovarian granulosa cells. Butyrate primarily upregulated the acetylation of histone H3K9 (H3K9ac). Chromatin immune-precipitation and sequencing (ChIP-seq) data of H3K9ac revealed the influenced pathways involving in the mitochondrial function (including cellular metabolism and steroidogenesis) and cellular antioxidant capacity. Additionally, increasing H3K9ac by butyrate further stimulated the PPARγ/CD36/StAR pathways to increase ovarian steroidogenesis and activated PGC1α to enhance mitochondrial dynamics and alleviate oxidative damage. The improvement in antioxidant capacity and mitochondrial dynamics by butyrate enhanced ovarian steroidogenesis. Collectively, butyrate triggers histone H3K9ac to activate steroidogenesis through PPARγ and PGC1α pathways in ovarian granulosa cells.

Effects of Antimicrobial Peptide Microcin C7 on Growth Performance, Immune and Intestinal Barrier Functions, and Cecal Microbiota of Broilers.

Microcin C7 is an antimicrobial peptide produced by Escherichia coli, composed of a heptapeptide with a modified adenosine monophosphate. This study was performed to evaluate the effects of Microcin C7 as a potential substrate to traditional antibiotics on growth performance, immune functions, intestinal barrier, and cecal microbiota of broilers. In the current study, 300 healthy Arbor Acres broiler chicks were randomly assigned to one of five treatments including a corn-soybean basal diet and basal diet supplemented with antibiotic or 2, 4, and 6 mg/kg Microcin C7. Results showed that Microcin C7 significantly decreased the F/G ratio of broilers; significantly increased the levels of serum cytokine IL-10, immunoglobulins IgG and IgM, and ileal sIgA secretion; significantly decreased the level of serum cytokine TNF-α. Microcin C7 significantly increased villus height and V/C ratio and significantly decreased crypt depth in small intestine of broilers. Microcin C7 significantly increased gene expression of tight junction protein Occludin and ZO-1 and significantly decreased gene expression of pro-inflammatory and chemokine TNF-α, IL-8, IFN-γ, Toll-like receptors TLR2 and TLR4, and downstream molecular MyD88 in the jejunum of broilers. Microcin C7 significantly increased the number of Lactobacillus and decreased the number of total bacteria and Escherichia coli in the cecum of broilers. Microcin C7 also significantly increased short-chain fatty acid (SCFA) and lactic acid levels in the ileum and cecum of broilers. In conclusion, diet supplemented with Microcin C7 significantly improved growth performance, strengthened immune functions, enhanced intestinal barrier, and regulated cecal microbiota of broilers. Therefore, the antimicrobial peptide Microcin C7 may have the potential to be an ideal alternative to antibiotic.

CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

OBJECTIVES: Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected. MATERIALS AND METHODS: We performed a series of experiments with primiparous rats fed diets containing different levels of methionine during early pregnancy to investigate the role of methionine in embryonic implantation and pregnancy outcomes, and used them to perform in vivo metabolic assessments and in vitro uterine explant culture. In addition, through transcriptome analysis and silencing the expression of cystathionine ß-synthase (CBS, the key enzyme in transsulfuration pathway) and cell adhesion assay, we measured signalling within Ishikawa, pTr and JAR cells. RESULTS: We determined the relevance and underlying mechanism of methionine on embryo implantation. We showed that methionine deprivation sharply decreased embryo implantation sites, expression of CBS and transsulfuration pathway end products, which were reversed by maternal methionine supplementation during early pregnancy. Moreover, we found CBS improved methionine-mediated cell proliferation and DNA synthesis by CBS inhibition or interference. In addition, transcriptome analysis also revealed that CBS influenced the signalling pathway-associated cell proliferation and DNA synthesis, as well as a correlation between CBS and methionine adenosyltransferase 2A (MAT2A), implying that MAT2A was possibly involved in cell proliferation and DNA synthesis. Further analysis revealed that MAT2A influenced S-adenosylmethionine receptor SAMTOR expression, and SAMTOR activated mTORC1 and its downstream S6K1 and CAD, ultimately enhancing DNA synthesis in the embryo and uterus. CONCLUSIONS: Taken together, these studies demonstrate that CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

Enhancement of Macrophage Function by the Antimicrobial Peptide Sublancin Protects Mice from Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is the major pathogen responsible for community and hospital bacterial infections. Sublancin, a glucosylated antimicrobial peptide isolated from Bacillus subtilis 168, possesses antibacterial infective effects. In this study, we investigated the role and anti-infection mechanism of sublancin in a mouse model of MRSA-induced sublethal infection. Sublancin could modulate innate immunity by inducing the production of IL-1ß, IL-6, TNF-α, and nitric oxide, enhancing phagocytosis and MRSA-killing activity in both RAW264.7 cells and mouse peritoneal macrophages. The enhanced macrophage function by the peptide in vitro correlated with stronger protective activity in vivo in the MRSA-invasive sublethal infection model. Macrophage activation by sublancin was found to be partly dependent on TLR4 and the NF-κB and MAPK signaling pathways. Moreover, oral administration of sublancin increased the frequencies of CD4+ and CD8+ T cells in mesenteric lymph nodes. The protective activity of sublancin was associated with in vivo augmenting phagocytic activity of peritoneal macrophages and partly improving T cell-mediated immunity. Macrophages thus represent a potentially pivotal and novel target for future development of innate defense regulator therapeutics against S. aureus infection.

Isoleucine attenuates infection induced by E. coli challenge through the modulation of intestinal endogenous antimicrobial peptide expression and the inhibition of the increase in plasma endotoxin and IL-6 in weaned pigs.

Enteric infection is a major cause of morbidity and mortality in both humans and animals worldwide. Immunotherapy against intestinal infection is a well-known alternative to the antibiotic strategy. Herein, we demonstrated that isoleucine significantly suppressed the multiplication of E. coli in the presence of IPEC-J2 cells. Isoleucine supplementation enhanced the concentrations of total plasma protein and IgA in pigs compared to the alanine control diet, while inhibiting the increase in plasma endotoxin and IL-6 contents induced by E. coli challenge. A significant interaction between the E. coli challenge and the diet treatment was found in the red blood cell volume. Isoleucine improved the expression of porcine ß-defensin-1 (pBD-1), pBD-2, pBD-3, pBD-114 and pBD-129 in the jejunum and ileum of pigs with or without E. coli challenge. Conclusively, isoleucine attenuated the infection caused by the E. coli challenge possibly through increasing the intestinal ß-defensin expression and inhibiting the increase in plasma endotoxin and IL-6 in weaned pigs.

Maternal short and medium chain fatty acids supply during early pregnancy improves embryo survival through enhancing progesterone synthesis in rats.

Exploring strategies to prevent miscarriage in women or early pregnancy loss in mammals is of great importance. Manipulating maternal lipid metabolism to maintain sufficient progesterone level is an effective way. To investigated the embryo loss and progesterone synthesis impacts of short and medium chain fatty acids on the lipid metabolism, pregnancy outcome and embryo implantation were investigated in rats fed the pregnancy diets supplemented without or with 0.1% sodium butyrate (SB), 0.1% sodium hexanoate (SH), or 0.1% sodium caprylate (SC) during the entire pregnancy and early pregnancy, respectively, followed with evaluation of potential mechanisms. Maternal SB, SH, or SC supply significantly improved live litter size and embryo implantation in rats. Serum progesterone, arachidonic acid, and phospholipid metabolites levels were significantly increased in response to maternal SB, SH, and SC supply. The expression of key genes involved in ovarian steroidogenesis and granulosa cell luteinization were elevated in ovaries and primary cultured granulosa cells, including cluster of differentiation 36 (CD36), steroidogenic acute regulatory protein (StAR), and cholesterol side-chain cleavage enzyme (CYP11A1). Additionally, the expression of lysophosphatidic acid receptor 3 (LPA3) and cyclooxygenase-2 (COX2) related with phospholipid metabolism were enhanced in uterus in vivo and in in vitro cultured uterine tissue. In conclusion, maternal SB, SH and SC supply reduced early pregnancy loss through modulating maternal phospholipid metabolism and ovarian progesterone synthesis in rats. Our results have important implications that short or medium chain fatty acids have the potential to prevent miscarriage in women or early pregnancy loss in mammals.

Functions of Macrophages in the Maintenance of Intestinal Homeostasis.

Intestinal macrophages constitute the largest pool of macrophages in the body and have emerged as crucial sentinels for pathogen recognition and elimination. The source and development of intestinal macrophages, as well as their distinct properties have been well documented. Intestinal macrophages exert their functions in the maintenance of intestinal homeostasis by shaping host-microbiota symbiosis, managing gut inflammation, crosstalking with T cells, and facilitating wound repair. Recently, nutritional regulation of intestinal macrophages has attracted substantial attention and is becoming a promising approach to disease prevention and control. Understanding the mechanisms employed by intestinal macrophages in mediating intestinal immune homeostasis and inflammation, as well as the mode of action of dietary nutrients in the modulating functions of intestinal macrophages, represents an opportunity to prevent and control inflammatory bowel diseases.

Metabolic disorder of amino acids, fatty acids and purines reflects the decreases in oocyte quality and potential in sows.

Oocyte quality is closely related to female fertility. Nevertheless, core nutritional metabolites influencing oocyte quality are unclear. Herein, comprehensive metabolomics analysis of follicular fluid, serum, and urine from low reproductive performance (LRP) and normal reproductive performance (NRP) sows was conducted. Twenty-seven, fourteen and sixteen metabolites (involved in metabolism of amino acids, fatty acids, purine and pyrimidine) were altered in follicular fluid, serum and urine, respectively, in LRP compared with NRP sows, and could decrease oocyte quality and developmental potential, ultimately leading to low fertility. Deoxyinosine, guanidine acetate, thymidine, 5,6-epoxy-eicosatrienoic acid, carnosine, docosahexaenoic acid and carbamoyl phosphate in follicular fluid, cysteine, carnitine, serotonin, hypoxanthine, valine and arginine in serum, as well as carnitine, phenyl glycine, N-acetyl glutamine, propionyl carnitine and choline in urine could be selected as diagnostic markers to indicate oocyte quality. Consistent with metabolomics data, we confirmed changes in concentrations of fatty acids and amino acids in follicular fluid. Targeting purine metabolism, elevating levels of deoxyinosine in in-vitro maturation medium of porcine oocyte significantly promoted the blastocyst rate. Collectively, this study provided new information of potential targets for predicting oocyte quality and developmental potential, and may help with strategies for early diagnosis or therapeutic/dietary intervention in improving reproductive outcomes.

Protective Ability of Biogenic Antimicrobial Peptide Microcin J25 Against Enterotoxigenic Escherichia Coli-Induced Intestinal Epithelial Dysfunction and Inflammatory Responses IPEC-J2 Cells.

Poison of intestinal induce severe health problems in human infants and young animals due to contaminating foods and feedstuffs. With the emergence of public health concerns and high-speed diffuse of drug-opposition of bacteria, the adoption of antimicrobial peptides as potential candidates in treating pathogen infections raised up. Nature Microcin J25 (MccJ25), a class of lasso peptides separated from a fecal strain of E. coli, has been replied to display powerful antimicrobial behavior. Herein, the study was to assess the usefulness of biogenic MccJ25 in the prophylaxis of ETEC K88 infection in IPEC-J2 cells. In vitro antimicrobial activity against ETEC K88 and cytotoxicity of biogenic MccJ25 were determined first. To further understand how biogenic MccJ25 mediates its impact, ETEC K88 adhesion in cells, membrane permeability [as indicated by reduced release of lactate dehydrogenase (LDH)], transepithelial electrical resistance (TEER), barrier function, and proinflammatory cytokines levels were determined in IPEC-J2 cells after treatment with biogenic MccJ25 and challenge with ETEC K88. Biogenic MccJ25 had a minimum inhibitory concentration of 0.25 µg/mL against ETEC K88, decreased ETEC K88 adhesion in cells and did not cause cytotoxicity toward cells. Furthermore, biogenic MccJ25 protects against ETEC-induced barrier dysfunction by increasing the TEER, decreasing the LDH and promoting tight junction proteins (TJPs) by promoting the assembly of occludin and claudin-1 in the tight junction complex. Biogenic MccJ25 was further found to relieve inflammation responses through modulation of interleukine-6, IL-8 and tumor necrosis factor-α levels via inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor κB activation. In summary, biogenic MccJ25 can protects against ETEC K88-induced intestinal damage and inflammatory response, recommend the hidden adoption of biogenic MccJ25 as a novel prophylactic agent to reduce pathogen infection in animals, food or humans.

Prevention of Cyclophosphamide-Induced Immunosuppression in Mice with the Antimicrobial Peptide Sublancin.

Sublancin is a glycosylated antimicrobial peptide produced by Bacillus subtilis 168 with combined antibacterial and immunomodulatory activities. The purpose of this study was to evaluate the protective effects of sublancin on immunosuppression in cyclophosphamide-treated mice. In normal mice, the phagocytic activity of mouse peritoneal macrophages was significantly enhanced by oral administration of sublancin (1.0 mg/kg body weight) to BALB/c mice for 7 days (P < 0.01). In addition, the mRNA expression of IL-1ß, IL-6, and TNF-α in peritoneal macrophages from sublancin- (1.0 mg/kg body weight) administered mice was significantly increased (P < 0.05). In cyclophosphamide-treated mice, oral sublancin administration accelerated the recovery of peripheral white blood cells, red blood cells, hemoglobins, and platelets and enhanced the macrophage phagocytic activity. Furthermore, sublancin restored the mRNA levels of IL-2, IL-4, and IL-6 in the spleen. Finally, the intestinal absorption of sublancin was poor as detected in the Caco-2 transwell system. Taken together, these findings suggest that sublancin plays a crucial role in the protection against immunosuppression in cyclophosphamide-treated mice and could be a potential candidate for use in immune therapy regimens.

Lactobacillus reuteri I5007 Modulates Intestinal Host Defense Peptide Expression in the Model of IPEC-J2 Cells and Neonatal Piglets.

Modulation of the synthesis of endogenous host defense peptides (HDPs) by probiotics represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections in human and animals. However, the extent of HDP modulation by probiotics is species dependent and strain specific. In the present study, The porcine small intestinal epithelial cell line (IPEC-J2) cells and neonatal piglets were used as in-vitro and in-vivo models to test whether Lactobacillus reuteri I5007 could modulate intestinal HDP expression. Gene expressions of HDPs, toll-like receptors, and fatty acid receptors were determined, as well as colonic short chain fatty acid concentrations and microbiota. Exposure to 108 colony forming units (CFU)/mL of L. reuteri I5007 for 6 h significantly increased the expression of porcine ß-Defensin2 (PBD2), pBD3, pBD114, pBD129, and protegrins (PG) 1-5 in IPEC-J2 cells. Similarly, L. reuteri I5007 administration significantly increased the expression of jejunal pBD2 as well as colonic pBD2, pBD3, pBD114, and pBD129 in neonatal piglets (p < 0.05). This was probably associated with the increase in colonic butyric acid concentration and up-regulating expression of Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and G Protein-Coupled Receptor 41 (GPR41) (p < 0.05), but not with stimulation of Pattern-Recognition Receptors. Additionally, supplementation with L. reuteri I5007 in the piglets did not affect the colonic microbiota structure. Our findings suggested that L. reuteri I5007 could modulate intestinal HDP expression and improve the gut health of neonatal piglets, probably through the increase in colonic butyric acid concentration and the up-regulation of the downstream molecules of butyric acid, PPAR-γ and GPR41, but not through modifying gut microbiota structure.

Maternal N-Carbamylglutamate Supplementation during Early Pregnancy Enhances Embryonic Survival and Development through Modulation of the Endometrial Proteome in Gilts.

BACKGROUND: Early pregnancy loss is a major concern in humans and animals. N-carbamylglutamate (NCG) has been found to enhance embryonic survival during early pregnancy in rats. However, little is known about the key factors in the endometrium involved in the improvement of embryonic implantation and development induced by maternal NCG supplementation. OBJECTIVES: Our objectives were to investigate whether NCG supplementation during early gestation enhanced embryonic survival and development in gilts and to uncover the related factors using the approach of endometrium proteome analysis with isobaric tags for relative and absolute quantification (iTRAQ). METHODS: Uteruses and embryos/fetuses were obtained on days 14 and 28 of gestation from gilts fed a basal diet that was or was not supplemented with 0.05% NCG. The iTRAQ-based quantitative proteomics approach was performed to explore the endometrium proteome altered by NCG supplementation. RESULTS: Maternal NCG supplementation significantly increased the number of total fetuses and live fetuses on day 28 of gestation by 1.32 and 1.29, respectively (P < 0.05), with a significant decrease in embryonic mortality (P < 0.05). iTRAQ results indicated that a total of 59 proteins showed at least 2-fold differences (P < 0.05), including 52 proteins that were present at higher abundance and 7 proteins present at lower abundance in NCG-supplemented gilts. The differentially expressed proteins primarily are involved in cell adhesion, energy metabolism, lipid metabolism, protein metabolism, antioxidative stress, and immune response. On day 14 of gestation, several proteins closely related to embryonic implantation and development, such as integrin-αv, integrin-ß3, talin, and endothelial nitric oxide synthase, were upregulated (3.7-, 4.1-, 2.4-, and 5.4-fold increases, respectively) by NCG supplementation. CONCLUSION: To our knowledge, our results provide the first evidence that altered abundance of the endometrial proteome induced by NCG supplementation is highly associated with the improvement of embryonic survival and development in gilts.

Lactobacillus reuteri I5007 modulates tight junction protein expression in IPEC-J2 cells with LPS stimulation and in newborn piglets under normal conditions.

BACKGROUND: Tight junctions (TJs) maintain the intestinal mucosal barrier, dysfunction of which plays a vital role in the pathophysiology of a variety of gastrointestinal disorders. Previously, we have shown that L. reuteri I5007 maintained the gut epithelial barrier in newborn piglets. Here we aimed to decipher the influence of L. reuteri I5007 on tight junction (TJ) protein expression both in vivo and in vitro. RESULTS: We found that L. reuteri I5007 significantly increased the protein abundance of intestinal epithelial claudin-1, occludin and zonula occluden-1 (ZO-1) in newborn piglets (orally administrated with 6 × 10(9) CFU of L. reuteri I5007 daily for 14 days). In vitro, treatment with L. reuteri I5007 alone maintained the transepithelial electrical resistance (TEER) of IPEC-J2 cells with time. In addition, IPEC-J2 cells were stimulated with 1 µg/mL lipopolysaccharide (LPS) for 1, 4, 8, 12 or 24 h, following pre-treatment with L. reuteri I5007 or its culture supernatant for 2 h. The results showed that LPS time-dependently induced (significantly after 4 or 8 h) the expression of TNF-α and IL-6, and decreased TJ proteins, which was reversed by pre-treatment of L. reuteri I5007 or its culture supernatant. CONCLUSIONS: L. reuteri I5007 had beneficial effects on the expression of TJ proteins in newborn piglets and the in-vitro results showed this strain had a positive effect on TEER of cells and inhibited the reduction of TJ proteins expression induced by LPS. These findings indicated L. reuteri I5007 may have potential roles in protection TJ proteins in TJ-deficient conditions.

Intestinal microbiota succession and immunomodulatory consequences after introduction of Lactobacillus reuteri I5007 in neonatal piglets.

Seventy-two, suckling piglets, obtained from 9 litters standardized to 8 piglets, were assigned to 1 of 3 treatments (n = 24) to compare short-term, early administration with intermittent, longer-term administration of Lactobacillus reuteri I5007. The treatments were a control (given a placebo of 0.1% peptone water from day 1 to 5) or treatments in which 1.7 × 1010 CFU L. reuteri was administrated either daily for 4 days starting on day 1 or every 4th day from day 1 to 17. Five piglets per treatment were killed at 3 time points (day 7, 14 and 21). Denaturing Gradient Electrophoresis of ileal digesta revealed an increase in the presence of L. reuteri I5007 and Clostridium lentocellum (on day 14 and 21) in the every 4th-day treatment and Actinobacillus porcinus (on day 7 and 14) in both L. reuteri treatments, while reducing the abundance of E. coli on day 21 in the every 4th-day treatment. Real-time qPCR of ileal digesta showed an increase in Bifidobacterium spp. on day 14 for both L. reuteri I5007 treatments. An increase in the concentration of lactic acid and a lower pH was observed in the first 4-day treatment on day 7 and the every 4th day treatment on day 14. The relative abundance of mRNA for TGF-ß was increased while that for IFN-γ was decreased in the mesenteric lymph nodes of piglets treated with L. reuteri every 4th day. In conclusion, early intervention with L. reuteri increases the presence of beneficial bacteria and decreases the presence of undesirable microbes in the lower gastrointestinal tract. The changes appear to be mediated by altering the intestinal pH through lactic acid production resulting in favorable bacterial species colonization. A prolonged duration of treatment (i.e. every 4th day) would appear to be superior to treatment only during the first 4 days.

Leucine stimulates ASCT2 amino acid transporter expression in porcine jejunal epithelial cell line (IPEC-J2) through PI3K/Akt/mTOR and ERK signaling pathways.

Leucine has been shown to influence intestinal protein metabolism, cell proliferation and migration. Furthermore, our previous study demonstrated that branched-chain amino acids could modulate the intestinal amino acid and peptide transporters in vivo. As the possible mechanisms are still largely unknown, in the present work, we studied the transcriptional and translational regulation of leucine on amino acid transporter production in IPEC-J2 cells and the signaling pathways involved. Treatment of IPEC-J2 cells with 7.5 mM leucine enhanced the mRNA expression of the Na(+)-neutral AA exchanger 2 (ASCT2) and 4F2 heavy chain (4F2hc) and caused an increase in ASCT2 protein expression. Leucine also activated phosphorylation of 4E-BP1 and eIF4E through the phosphorylation of mTOR, Akt and ERK signaling pathways in IPEC-J2 cells. Pre-treatment of IPEC-J2 cells with inhibitors of mTOR and Akt (rapamycin and wortmannin) or an inhibitor of ERK (PD098059) for 30 min before leucine treatment attenuated the positive effect of leucine in enhancing the protein abundance of ASCT2. These results demonstrate that leucine could up-regulate the expression of the amino acid transporters (ASCT2) through transcriptional and translational regulation by ERK and PI3K/Akt/mTOR activation.

The bacteriocin sublancin attenuates intestinal injury in young mice infected with Staphylococcus aureus.

Sublancin, a bacteriocin, has bactericidal activity against a broad spectrum of gram-positive bacteria. However, studies have not been conducted to determine its in vivo efficacy against potential pathogens. The objective of this study was to investigate the effects of sublancin in a Staphylococcus aureus (S. aureus) infected mouse model which induced intestinal injury. A total of 160, 4-week-old mice were randomly assigned to one of eight treatments. Mice in the control group were injected intraperitoneally with 0.5 mL of 0.9% saline. Mice in the other seven groups were given an intraperitoneal injection of 0.5 mL saline containing 1.0 × 10(10) colony-forming units (CFU)/mL S. aureus. Six hours after inoculation, mice in the control group were again injected with 0.5 mL of 0.9% saline. Mice in the other seven groups were injected intraperitoneally with 0.5 mL of 0.9% saline containing 0, 0.5, 1.0, 2.0, or 4.0 mg/kg body weight (BW) sublancin or 1.0 or 2.0 mg/kg BW ampicillin. The results showed that 4.0 mg/kg sublancin and 2.0 mg/kg ampicillin significantly reduced mice mortality from 55 to 10%. The height and the number of proliferated cells from the intestinal villi in the sublancin and ampicillin treated mice were higher than in the control. We conclude that sublancin has potent antibacterial activity against S. aureus. Therefore, sublancin could find use as an alternative antimicrobial agent for the treatment of gram-positive bacterial infections.