New N-Terminal Fatty-Acid-Modified Melittin Analogs with Potent Biological Activity.

Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation.

Porcine ß-Defensin 114: Creating a Dichotomous Response to Inflammation.

The immunity-related functions of defensins seem to be dependent on environmental stimuli, the cell type, and the concentration of peptides. However, the function and mechanism of porcine ß-defensin 114 (pBD114) in regulating the inflammatory response to macrophages are unclear. Therefore, the modulatory effects of porcine pBD114 on the inflammatory response were investigated by treating the mouse monocyte macrophage cell line RAW264.7 with different concentrations of pBD114 with or without lipopolysaccharide (LPS). RNA-seq analysis was performed to investigate the mechanisms underlying pBD114's regulation of inflammatory responses in macrophages. In addition, the inflammatory response-modulating effects of pBD114 were also further verified with a mouse assay. The results showed that 100 µg/mL of pBD114 significantly promoted the secretion of TNF-α and IL-10 in RAW264.7. However, the LPS-induced increase in TNFα in the RAW264.7 cell cultures was significantly decreased with 10 µg/mL of pBD114. These results suggest that pBD114 can exhibit pro-inflammatory activities under normal physiological conditions with 100 µg/mL of pBD114, and anti-inflammatory activities during an excessive inflammatory response with 10 µg/mL of pBD114. RNA-seq analysis was performed to gain further insights into the effects of pBD114 on the inflammatory response. Among the pBD114-promoting RAW264.7 pro-inflammatory responses, pBD114 significantly up-regulated 1170 genes and down-regulated 724 genes. KEGG enrichment showed that the differentially expressed genes (DEGs) were significantly enriched in the immune- and signal-transduction-related signaling pathways. Protein-Protein Interaction (PPI) and key driver analysis (KDA) analyses revealed that Bcl10 and Bcl3 were the key genes. In addition, pBD114 significantly up-regulated 12 genes and down-regulated 38 genes in the anti-inflammatory response. KEGG enrichment analysis revealed that the DEGs were mainly enriched in the "Cytokine-cytokine receptor interaction" signaling pathway, and PPI and KDA analyses showed that Stat1 and Csf2 were the key genes. The results of qRT-PCR verified those of RNA-seq. In vivo mouse tests also confirmed the pro- or anti-inflammatory activities of pBD114. Although the inflammatory response is a rapid and complex physiological reaction to noxious stimuli, this study found that pBD114 plays an essential role mainly by acting on the genes related to immunity, signal transduction, signaling molecules, and interactions. In conclusion, this study provides a certain theoretical basis for the research and application of defensins.

NF-κB-dependent induction of porcine ß-defensin 114 regulates intestinal epithelium homeostasis.

Intestinal epithelial cells (IECs) offer a primary physical barrier against commensal and pathogenic microorganisms in the gastrointestine. However, the influence of IECs on the development and regulation of mucosal immunity to infection is unknown. Here, we show that the porcine ß-defensin 114 (PBD114) is an endotoxin-responsive gene expressed in IECs. Analysis on expression profiling of PBD114 gene using an infected porcine model and IPEC-J2 cells unveiled a pattern of induction in response to stimulation of various toll-like receptors (TLRs). By means of promoter analysis, PBD114 was found to be a NF-κB-dependent gene. Importantly, PBD114 suppresses endotoxin-induced inflammation and apoptosis in IECs through downregulation of two critical inflammation-associated signaling proteins, NF-kappa-B inhibitor alpha (IkB-α) and extracellular signal-regulated kinase1/2 (ERK1/2). PBD114 also suppresses inflammation and IEC apoptosis in mice exposed to bacterial endotoxins. Thus, we propose that TLR-activated NF-kB rapidly increases the expression of PBD114 that operates a feedback control of the NF-kB-dependent inflammation. The NF-kB-dependent induction of PBD114 may be a key event through which the mammalian host maintains intestinal epithelium homeostasis in response to various infections or diseases.

The immunomodulatory function of the porcine ß-defensin 129: Alleviate inflammatory response induced by LPS in IPEC-J2 cells.

ß-defensin family plays a critical role in host defense against infections. In this study, we found that pBD129 are widely expressed in porcine tissues such as the intestine, liver, and spleen. Interestingly, the expression level of pBD129 in most tissues was higher in Tibetan pigs than in DLY (Duroc × Landrace × Yorkshire) pigs (P < 0.05), and was significantly upregulated upon E. coli K88 infection (P < 0.05). The pBD129 protein was successfully expressed in E. coli and the molecule weight was estimated by SDS-PAGE to be 37.2 kDa. Mass spectrometry verified the protein as a pBD129. The protein showed antibacterial activities against Streptococcus and E. coli DH5α with a minimal inhibitory concentration (MIC) of 32 µg/mL. Hemolytic and cytotoxicity assays indicated that pBD129 had no detrimental effect on cell viability. Importantly, pBD129 significantly reduced the apoptosis of porcine intestinal epithelial cells exposure to bacterial endotoxins, which was associated with down-regulation of inflammatory cytokines such as the IL-1ß, IL-6 and TNFα (P < 0.05), and down-regulation of apoptosis-related genes such as the caspase-3, caspase-8, and caspase-9 (P < 0.05). These results suggested that pBD129 is a novel modulator of innate immunity involved in mammalian inflammatory responses.

Effects of essential oil on growth performance, digestibility, immunity, and intestinal health in broilers.

Essential oils (EO) are concentrated hydrophobic liquids containing volatile aromatic compounds obtained from plants, which have properties as withdrawn antibiotic growth promoters. The objective of this study was to explore the effects of EO on growth performance, digestibility, immunity and intestinal health in broilers. A total of 500 1-day-old Arbor Acre broilers were randomly put into five groups with 10 replicate cages containing 10 birds each. Birds in the 5 groups were fed a basal diet (CON), and basal diet with 50, 100, 200 or 400 mg/kg EO (EO0.5, EO1, EO2 and EO4) for 42 d respectively. Birds were euthanized at 21d and 42 d, blood and tissue samples were collected. In the study, the digestibility of DM, GE and EE in groups with EO supplementation were significantly increased compared with CON group (P < 0.05). However, only EO2 and EO4 significantly increased the digestibility of CP compared with CON group (P < 0.05). In contrast to CON group, EO0.5 and EO1 in jejunum at 21 d, and EO1 in jejunum at 42 d markedly increased the activity of sucrase (P < 0.05). In addition, the level of SOD of EO2 and EO4 in serum at 21 d was significantly increased compared with CON group (P < 0.05). What's more, the concentration of intestinal mucosa SIgA in jejunum and ileum at 21 d of groups with EO supplementation was significantly increased compared with CON group (P < 0.05). Moreover, V/C in jejunum at 21 d of groups with EO supplementation, CD in jejunum at 42 d was also significantly increased to compare with CON group (P < 0.05). Furthermore, the expression levels of critical genes associated with nutrient transportation (i.e., GLUT2, SGLT1, SLC38A, SLC79A and SLC27A4) and barrier function (TJP1) were quadratically and linearly up-regulated in jejunum and ileum with EO supplementation (P < 0.05). These results suggest that EO has a positive impact on growth, immunity and intestinal health in broilers, and 200 mg/kg of EO was recommended in broiler diet.

Protective effect of Bombyx mori gloverin on intestinal epithelial cells exposure to enterotoxigenic E. coli.

Bombyx mori gloverin A2 (BMGlvA2) is an induced antimicrobial insect protein isolated from Bombyx mori. This study was conducted to explore the effect and potential mechanisms of BMGlvA2 on inflammatory responses and cellular functions in intestinal epithelial cells (IPEC-J2) exposure to enterotoxigenic E. coli (ETEC). IPEC-J2 cells pretreated with or without BMGlvA2 (12.5 µg/mL) were challenged by ETEC K88 (1×106 CFU/well) or culture medium. We show that BMGlvA2 pretreatment increased the cell viability and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells exposure to ETEC (P < 0.05). Interestingly, BMGlvA2 not only decreased the expression levels of inflammatory cytokines such as the tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), but also decreased the expression level of Caspase3 and the apoptosis rate in the ETEC-challenged cells (P < 0.05). Importantly, BMGlvA2 decreased the protein abundances of two critical inflammation-associated signaling proteins, phosphorylated nuclear factor-kappa-B inhibitor alpha (p-IκBα) and phosphorylated nuclear factor-kappa B (p-NF-κB), in the ETEC-challenged cells. These results indicate that BMGlvA2 attenuates ETEC-induced inflammation in the IPEC-J2 cells by regulating the NF-κB signaling pathway, resulting in decreased secretion of inflammatory cytokine and reduced cell apoptosis.

Dietary supplementation of plant essential oil improves growth performance, intestinal morphology and health in weaned pigs.

The present study was conducted to explore the effect of plant essential oil (PEO) on growth performance, intestinal morphology and health in weaned pigs. Twenty-four weaned pigs were assigned into four groups fed with a basal diet (CON) or basal diet containing PEO at a concentration of 50 (PEO50), 100 (PEO100) or 200 ppm (PEO200). After 21 days, pigs were slaughtered and blood and tissue samples were collected. Result showed that PEO200 group significantly increased the average daily gain (ADG) compared with CON group (p < .05). Moreover, PEO supplementation significantly improved the digestibility of DM (p < .05). However, it significantly decreased the serum triglyceride and cholesterol concentration (p < .05). Interestingly, PEO supplementation significantly increased the activity of sucrase in the duodenal and the activity of lactase in the jejunal mucosa (p < .05). Moreover, PEO supplementation improved the growth of intestinal mucosa. As compared to the CON group, the jejunum and ileum villus height were significantly elevated in the PEO200 group (p < .05). Importantly, the expression levels of critical genes associated with nutrient transportation (i.e., GLUT2 and SGLT1) and barrier function (occludin) were significantly elevated in the PEO200 group (p < .05). Moreover, the PEO100 and PEO200 group had higher propionic acid concentration and higher total bacterial gene copies in colon digesta than the CON group (p < .05) respectively. These results not only suggest that PEO has a positive role in the regulation of growth and intestinal health in weaned pigs, but also offer a potential candidate substituting the conventionally used antibiotics in the livestock industry.

Bombyx mori gloverin A2 alleviates enterotoxigenic Escherichia coli-induced inflammation and intestinal mucosa disruption.

Background: Enterotoxigenic Escherichia coli (ETEC) is one of the leading bacterial causes of intestinal inflammation and diarrhea. However, the ETEC is frequently resistant to common antibiotics. In this study, we explored the role of a novel antibacterial peptide Bombyx mori gloverin A2 (BMGlvA2) in alleviating ETEC-induced inflammation and intestinal epithelium disruption in mice. Methods: An ETEC-challenged mice model was used, and the ETEC-challenged mice and non-challenged mice were treated by the BMGlvA2 at different doses. Results: ETEC challenge not only elevated the concentrations of serum inflammatory cytokines such as the IL-6 and TNF-α (P < 0.01), but also elevated the concentrations of serum creatinine and urea (P < 0.05). However, BMGlvA2 attenuated the inflammatory responses by decreasing the serum inflammatory cytokines and improving the metabolisms in ETEC-challenged mice, and alleviated the ETEC-induced tissue damage in spleen. Moreover, BMGlvA2 treatment significantly elevated the duodenum villus height and decreased the crypt depth in the duodenum and ileum in ETEC-challenged mice (P < 0.05). Interestingly, BMGlvA2 improved the distribution and abundance of tight-junction protein ZO1 in duodenum and ileum epithelium after ETEC-challenge. Moreover, BMGlvA2 significantly down-regulated the expression levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and the apoptosis-related genes (Caspase 8 and Caspase 9) in jejunal mucosa (P < 0.05) in the TETC-challenged mice. Importantly, BMGlvA2 significantly elevated the expression levels of critical genes related to mucosal barrier functions such as the mucins (MUC1 and MUC2) and glucose transporter (GLUT2) in the intestinal mucosa (P < 0.05). Conclusion: Our results suggested a novel function of the conventional antibacterial peptides, and the anti-bacterial and anti-inflammatory properties of BMGlvA2 may allow it a potential substitute for conventionally used antibiotics or drugs.

ß-Defensin 129 Attenuates Bacterial Endotoxin-Induced Inflammation and Intestinal Epithelial Cell Apoptosis.

Defensins have attracted considerable research interest worldwide because of their potential to serve as a substitute for antibiotics. In this study, we characterized a novel porcine ß-defensin (pBD129) and explored its role in alleviating bacterial endotoxin-induced inflammation and intestinal epithelium atrophy. The pBD129 gene was cloned and expressed in Escherichia coli. A recombinant pBD129 protein was also purified. To explore its role in alleviating the endotoxin-induced inflammation, mice, with or without lipopolysaccharide (LPS) challenge were treated by pBD129 at different doses. The recombinant pBD129 showed significant antimicrobial activities against the E. coli and Streptococcus with a minimal inhibitory concentration (MICs) of 32 µg/mL. Hemolytic assays showed that the pBD129 had no detrimental impact on cell viabilities. Interestingly, we found that pBD129 attenuated LPS-induced inflammatory responses by decreasing serum concentrations of inflammatory cytokines, such as the IL-1ß, IL-6, and TNF-α (P < 0.05). Moreover, pBD129 elevated the intestinal villus height (P < 0.05) and enhanced the expression and localization of the major tight junction-associated protein ZO-1 in LPS-challenged mice. Additionally, pDB129 at a high dose significantly decreased serum diamine oxidase (DAO) concentration (P < 0.05) and reduced intestinal epithelium cell apoptosis (P < 0.05) in LPS-challenged mice. Importantly, pBD129 elevated the expression level of Bcl-2-associated death promoter (Bcl-2), but down-regulated the expression levels of apoptosis-related genes such as the B-cell lymphoma-2-associated X protein (Bax), BH3-interacting domain death agonist (Bid), cysteinyl aspartate-specific proteinase-3 (Caspase-3), and caspase-9 in the intestinal mucosa (P < 0.05). These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of pBD129 may allow it a potential substitute for conventionally used antibiotics or drugs.

Differential expression, molecular cloning, and characterization of porcine beta defensin 114.

BACKGROUND: ß-defensins have attracted considerable research interest because of their roles in protecting hosts from various pathogens. This study was conducted to investigate the expression profiles of the porcine ß-defensin 114 (PBD114) in different breeds and in response to infections. Moreover, the function of PBD114 protein was partially investigated. METHODS: Six Tibetan pigs (TP) and six DLY (Duroc×Landrace×Yorkshire) pigs were slaughtered to explore the expression profiles of PBD114 in different breeds and tissues. For infection models, sixteen DLY pigs were divided into two groups and challenged either with sterile saline or E. coli K88. The recombinant protein PBD114 (rPBD114) was obtained by using a heterologous expression system in E. coli. RESULTS: PBD114 gene was highly expressed in tissues such as the intestine, liver, spleen, and thymus. Interestingly, the expression level of PBD114 gene was higher in the TP pigs than in the DLY pigs (P < 0.05), and was significantly elevated upon E. coli K88 challenge (P < 0.05). The nucleotide sequences of PBD114 from Tibetan and DLY pigs was identical, and both showed a 210-bp open reading frame encoding a 69-amino acid mature peptide. To explaore the function of PBD114 protein, PBD114 gene was successfully expressed in E. coli Origami B (DE3) and the molecular weight of the rPBD114 was estimated by SDS-PAGE to be 25 kDa. The rPBD114 was purified and mass spectrometry verified the protein as PBD114. Importantly, rPBD114 showed antimicrobial activities against E. coli DH5α and E. coli K88, and the minimal inhibitory concentrations (MICs) were 64 and 128 µg/mL, respectively. Hemolytic and cytotoxicity assays showed that rPBD114 did not affect cell viability under physiological concentrations. CONCLUSIONS: PBD114 is an infection response gene that is differentially-expressed between different porcine breeds and tissues. The antimicrobial activity of PBD114 protein, against pathogens such as the E. coli K88, suggested that it may serve as a candidate for the substitution of conventionally used antibiotics.

Effects of plant essential oil supplementation on growth performance, immune function and antioxidant activities in weaned pigs.

BACKGROUND: The aim of this study was to determine the effects of plant essential oil supplementation on growth performance, immune function and antioxidant activities in weaned pigs. METHODS: In the study, 24 weaned pigs were used to explore the effects of plant essential oil (PEO) on growth performance, immune properties and antioxidant activities. Pigs were fed with a basal diet (CON) or basal diet containing different concentrations of PEO (PEO50: 50 ppm; PEO100: 100 ppm; PEO200: 200 ppm). After 3 weeks, all pigs were slaughtered and blood and tissue samples were collected for biochemical analysis. RESULTS: The results showed that PEO supplementation quadratically increased body weight gain (BWG) (P = 0.031), linearly (P <  0.05) and quadratically (P <  0.05) decreased F:G. In addition, IgG increased linearly (P <  0.05) and IgM increased linearly (P <  0.05) and quadratically (P < 0.05) as PEO supplementation. Similarly, MDA in serum, jejunal mucosa and pancreas were linearly decreased (P < 0.05) and GSH in serum (linear and quadratic, P < 0.05), duodenal mucosa (linear and quadratic, P < 0.05) and in ileal mucosa (linear and quadratic, P < 0.05) were notably increased. Futhermore, antioxidant-related genes expression levels of GST in spleen (linear and quadratic, P < 0.05), GPX1 (quadratic, P < 0.05) and SOD1 (linear, P < 0.05) in spleen and GST in liver (quadratic, P < 0.05) were markedly upregulated by PEO supplementation increasing. CONCLUSIONS: These results suggest that PEO improves growth performance, immune function, and antioxidant activities in weaned pigs, and it may also relieve weaning stress if used as a feed additive in the livestock industry. And that supplementation 200 ppm PEO in diet would seem to be economically feasible.

Effects of oil quality and antioxidant supplementation on sow performance, milk composition and oxidative status in serum and placenta.

BACKGROUND: The aim of this study was to determine the effects of oil quality and antioxidant (AOX) supplementation on sow performance, milk composition and oxidative status. METHODS: A total of 80 PIC (PIC breeding, 3 ~ 5 parities) sows with similar body condition were allocated to four groups (n = 20), receiving diets including fresh corn oil, oxidized corn oil, fresh corn oil plus AOX and oxidized corn oil plus AOX, respectively, from d 85 of gestation to d 21 of lactation. AOX was provided at 200 mg/kg diet and mixed with corn oil prior to dietary formulation. RESULTS: The results showed that sows fed oxidized corn oil had significantly lower feed intake (P < 0.05) during lactation period. Feeding oxidized corn oil markedly decreased (P < 0.05) the contents of protein and fat in colostrums and milk, but the addition of AOX in oxidized corn oil prevented the decrease on protein content of colostrums. Moreover, sows fed oxidized corn oil had significantly lower serum activities of total SOD and Mn-SOD across lactation (P < 0.05). In contrast, addition of AOX to oxidized corn oil tended to inhibit the production of MDA (P = 0.08) in sows across lactation relative to fresh oil. Intriguingly, the placental oxidative status was affected by oil quality and AOX supplementation, as indicated by the markedly increased placental gene expression of GPX and SOD (P < 0.05) in sows fed oxidized corn oil but normalized by supplementation of AOX. CONCLUSION: In conclusion, feeding oxidized corn oil did not markedly affect reproductive performance in addition to decreasing feed intake during lactation. Milk composition and systemic oxidative status were deteriorated in sows fed oxidized corn oil and partially improved by AOX supplementation. Moreover, placental antioxidant system of sows may have an adaptive response to oxidative stress, but normalized by AOX.

Dietary supplementation with ß-hydroxy-ß-methylbutyrate calcium during the early postnatal period accelerates skeletal muscle fibre growth and maturity in intra-uterine growth-retarded and normal-birth-weight piglets.

Intra-uterine growth restriction (IUGR) impairs postnatal growth and skeletal muscle development in neonatal infants. This study evaluated whether dietary ß-hydroxy-ß-methylbutyrate Ca (HMB-Ca) supplementation during the early postnatal period could improve muscle growth in IUGR neonates using piglets as a model. A total of twelve pairs of IUGR and normal-birth-weight (NBW) male piglets with average initial weights (1·85 (sem 0·36) and 2·51 (sem 0·39) kg, respectively) were randomly allotted to groups that received milk-based diets (CON) or milk-based diets supplemented with 800 mg/kg HMB-Ca (HMB) during days 7-28 after birth. Blood and longissimus dorsi (LD) samples were collected and analysed for plasma amino acid content, fibre morphology and the expression of genes related to muscle development. The results indicate that, regardless of diet, IUGR piglets had a significantly decreased average daily weight gain (ADG) compared with that of NBW piglets (P<0·05). However, IUGR piglets fed HMB-Ca had a net weight and ADG similar to that of NBW piglets fed the CON diet. Irrespective of body weight (BW), HMB-Ca supplementation markedly increased the type II fibre cross-sectional area and the mRNA expression of mammalian target of rapamycin (mTOR), insulin-like growth factor-1 and myosin heavy-chain isoform IIb in the LD of piglets (P<0·05). Moreover, there was a significant interaction between the effects of BW and HMB on mTOR expression in the LD (P<0·05). In conclusion, HMB-Ca supplementation during the early postnatal period could improve skeletal muscle growth and maturity by accelerating fast-twitch glycolytic fibre development in piglets.

Effects of maternal over- and undernutrition on intestinal morphology, enzyme activity, and gene expression of nutrient transporters in newborn and weaned pigs.

OBJECTIVES: It has been suggested that maternal nutrition during gestation is involved in the offspring's intestinal development. The aim of this study was to evaluate the effects of maternal nutrition on digestive and absorptive function of offspring at both birth and weaning with pig as model. METHODS: Eighteen primiparous Landrace × Yorkshire gilts (ages 253 ± 9 d; body weight 148.6 ± 6.9 kg) were allocated to a normal nutrition (control) group as recommended by the National Research Council (NRC; 1998), an undernutrition (UN) group (75% of NRC recommendations), and an overnutrition (ON) group (150% of NRC recommendations); the nutritional treatments were introduced from day 1 of gestation to farrowing. RESULTS: By comparison, the small intestine weight (+33%; P < 0.01) and the ratio of small intestine weight to length (+25%; P < 0.05) for the ON group were higher than those for the control group. Meanwhile, the maternal ON treatment had significant effect on the activity of brush border lactase in the jejunum (+125%; P < 0.05) at birth. Expression of mRNA for SGLT1, GLUT2, PEPT1, and GLP2R were significantly increased (+1.7∼9.6-fold; P < 0.05) in jejunum of newborn or weaned piglets from the ON group; whereas maternal UN significantly affected small intestine weight (-28%; P < 0.01), length, and the ratio of weight to length (-11% and -19%; P < 0.05, respectively), and villus height (-29% and -22%; P < 0.05, respectively) in jejunum and ileum. Furthermore, maternal UN enhanced the activity of brush border lactase in the jejunum of newborn piglets (P < 0.05). CONCLUSIONS: Maternal ON enhanced intestinal function via up-regulating digestive enzyme activities and gene expression of transporters in both newborn and weaning piglets. Maternal UN impaired fetal intestinal development that could be partially compensated during the neonatal period.