RESUMO
BACKGROUND: The increase in circulating insulin levels is associated with the onset of type 2 diabetes (T2D), and the levels of branched-chain amino acids and aromatic amino acids (AAAs) are altered in T2D, but whether AAAs play a role in insulin secretion and signaling remains unclear. OBJECTIVES: This study aimed to investigate the effects of different AAAs on pancreatic function and on the use of insulin in finishing pigs. METHODS: A total of 18 healthy finishing pigs (Large White) with average body weight of 100 ± 1.15 kg were randomly allocated to 3 dietary treatments: Con, a normal diet supplemented with 0.68% alanine; Phe, a normal diet supplemented with 1.26% phenylalanine; and Trp, a normal diet supplemented with 0.78% tryptophan. The 3 diets were isonitrogenous. There were 6 replicates in each group. RESULTS: Herein, we investigated the effects of tryptophan and phenylalanine on pancreatic function and the use of insulin in finishing pigs and found that the addition of tryptophan and phenylalanine aggravated pancreatic fat deposition, increased the relative content of saturated fatty acids, especially palmitate (C16:0) and stearate (C18:0), and the resulting lipid toxicity disrupted pancreatic secretory function. We also found that tryptophan and phenylalanine inhibited the growth and secretion of ß-cells, downregulated the gene expression of the PI3K/Akt pathway in the pancreas and liver, and reduced glucose utilization in the liver. CONCLUSIONS: Using fattening pigs as a model, multiorgan combined analysis of the insulin-secreting organ pancreas and the main insulin-acting organ liver, excessive intake of tryptophan and phenylalanine will aggravate pancreatic damage leading to glucose metabolism disorders, providing new evidence for the occurrence and development of T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Triptofano , Suínos , Animais , Fenilalanina , Fosfatidilinositol 3-Quinases , Dieta , Insulina , Ração Animal/análiseRESUMO
Intensive breeding of broilers met the increasing demands of human for broiler products, but it raised their increased susceptibility to various stressors resulting in the disorder of lipid metabolism. Pterostilbene, the methoxylated analogue of resveratrol, exhibits astonishing functions of antioxidant, anti-inflammatory and glycolipid regulatory. The study aimed to elucidate the protective effects of pterostilbene on broiler liver and to explore the potential mechanisms. A total of 480 one-day-old male Arbor Acres (AA) broilers were randomly divided into four groups: the control group (basal diet) and pterostilbene groups (PT200, PT400, and PT600 feeding with basal diet containing 200, 400 and 600 mg/kg pterostilbene, respectively). The results showed that the dietary pterostilbene supplementation significantly improved the ADG of broilers. Dietary pterostilbene supplementation regulated the expression levels of the genes Sirt1 and AMPK and the downstream genes related to lipid metabolism to protect liver function and reduce lipid accumulation in broilers. Dietary pterostilbene supplementation upregulated the expression levels of the Nrf2 gene and its downstream antioxidant genes (SOD, CAT, HO-1, NQO-1, GPX) and phase II detoxification enzyme-related genes (GST, GCLM, GCLC). Collectively, pterostilbene was confirmed the positive effects as a feed additive on lipid metabolism and antioxidant via regulating Sirt1/AMPK and Nrf2 signalling pathways in broilers.
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Ração Animal , Antioxidantes , Galinhas , Dieta , Suplementos Nutricionais , Metabolismo dos Lipídeos , Fígado , Estilbenos , Animais , Masculino , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Dieta/veterinária , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Estilbenos/farmacologia , Estilbenos/administração & dosagemRESUMO
BACKGROUND: The antimicrobial peptide LL37 is produced by white blood cells (mainly neutrophils) and various epithelial cells, and has the outstanding advantages of participating in immune regulation, causing chemotaxis of immune cells and promoting wound healing. However, the central domain of LL37 needs to be improved in terms of antimicrobial activity. RESULTS: In this study, the amino acid substitution method was used to improve the antimicrobial activity of the LL37 active center, and a dimeric design with a better selection index was selected. A flexible linker was selected and combined with the 6 × His-SUMO tag and LG was successfully expressed using Pichia pastoris as a host. Recombinant LG displayed strong antimicrobial activity by destroying the cell membrane of bacteria but had low hemolytic activity. In addition, compared with monomeric peptide FR, rLG had improved ability to tolerate salt ions. CONCLUSION: This research provides new ideas for the production of modified AMPs in microbial systems and their application in industrial production.
Assuntos
Substituição de Aminoácidos/genética , Peptídeos Catiônicos Antimicrobianos/genética , Expressão Gênica , Pichia/genética , Proteínas Recombinantes/genética , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/classificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Proteínas Recombinantes/farmacologia , CatelicidinasRESUMO
PR-FO is a novel α-helical hybrid antimicrobial peptide (AMP) with strong antimicrobial activities and high stability, and the potential to develop into a new generation of antimicrobial agents. In this study, the encoded gene sequence of SMT3-PR-FO was designed and transformed into B. subtilis WB800N. Fusion proteins with concentrations of 16 mg L-1 (SPamyQ) and 23 mg L-1 (SPsacB) were obtained after purification by a Ni-NTA resin column. A total of 3 mg (SPamyQ) and 4 mg (SPsacB) of PR-FO with a purity of 90% was obtained from 1 L fermentation cultures. Recombinant PR-FO exhibited high inhibition activities against both gram-negative bacteria and gram-positive bacteria, and low haemolytic activity against human red blood cells. These results indicated that the rSMT3-PR-FO could be expressed under the guidance of SPamyQ and SPsacB, and the maltose-induced expression strategy might be a safe and efficient method for the soluble peptides production in B. subtilis.
Assuntos
Antibacterianos , Bacillus subtilis/química , Expressão Gênica , Bactérias Gram-Negativas/crescimento & desenvolvimento , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes de Fusão , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Humanos , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/isolamento & purificação , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Plectasin, the first fungus defensin, is especially efficient against Gram-positive bacteria. To explore an effective approach for expressing plectasin in Bacillus subtilis, the sequence encoding plectasin fused with the small ubiquitin-like modifier (SUMO) gene, the 6 × His gene and the signal peptide of SacB were cloned into an E. coli-B. subtilis shuttle vector pGJ148 in which the maltose utilization operon promoter Pglv directed the expression. The fusion protein successfully secreted in culture and approximately, 41 mg of the recombinant fusion protein SUMO-plectasin was purified per liter of culture supernatant. After purification by Ni-NTA resin column and digestion by SUMO protease, 5.5 mg of plectasin with a purity of 94 % was obtained from 1 L fermentation culture. Recombinant plectasin was found inhibition activity against S. pneumoniae, S. aureus and S. epidermidis. These results indicate that the maltose-induced expression system may be a safe and efficient way for the large-scale production of soluble peptides in B. subtilis.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Expressão Gênica/efeitos dos fármacos , Maltose/farmacologia , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Escherichia coli/genética , Vetores Genéticos/genética , Maltose/metabolismo , Testes de Sensibilidade Microbiana , Peptídeo Hidrolases/metabolismo , Peptídeos/genética , Peptídeos/isolamento & purificação , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Solubilidade , Staphylococcus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
The antimicrobial peptide PMAP-36 is a highly cationic and amphipathic α-helical peptide. PRW4 is a truncated analog that replaces paired lysine residues with tryptophan along the N-terminal and deletes the C-terminal hydrophobic tail of PMAP-36. Studies on the two peptides have already been performed. However, whether there is a synergistic effect with antibiotics has not been investigated, and the study of the antibacterial mechanism of the peptides is inadequate. In this study, antibiotic-peptide combinations were tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, and the confocal laser scanning microscopy (LSCM) and DNA gel retardation were measured. The results indicated synergy between the peptides and gentamicin when tested against E. coli [fractional lethal concentration (FLC) < 0.5]; partial synergy was observed between the peptides and gentamicin against S. aureus (0.5 < FLC < 1); and streptomycin showed no reaction with the peptides against E. coli and S. aureus (1 < FLC < 4). LSCM and DNA binding suggest that PMAP-36 was able to translocate across the bacterial membranes and interact with intracellular DNA, but PRW4 presented no DNA-binding ability. These results indicate that the combination of PMAP-36 and PRW4 with aminoglycosides may provide useful information for clinical application, and the antibacterial mechanism of peptides likely does not solely involve cytoplasmic-membrane permeabilization.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Proteínas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Microscopia Confocal , Ligação ProteicaRESUMO
To ameliorate the diminished antimicrobial efficiency and physiological stability associated with monomeric antimicrobial peptides (AMPs) molecules, future research will focus on the artificial design of self-assembling peptides to replace monomeric entities, aiming to combat the antibiotic resistance crisis caused by microbial infections. In this study, the "bola" structure was used as the foundational architecture driving molecular self-assembly, with hydrophobic amino acids at the termini to anchor and finely adjust the sequence, thereby organizing a range of novel multidomain peptides (MDPs) templates into an ABA block motif. The results indicate that FW2 (GMSI = 53.94) exhibits the highest selectivity index among all MDPs and can form spherical micelles in an aqueous medium without the addition of any exogenous additives. FW2 exhibited high stability in vitro in the presence of physiological salt ions, serum, and various pH conditions. It exhibited excellent biocompatibility and efficacy both in vivo and in vitro. Furthermore, FW2 strongly interacts with the lipid membrane and employs various synergistic mechanisms, such as reactive oxygen species (ROS) accumulation, collectively driving cellular apoptosis. This study demonstrates a straightforward strategy for designing self-assembling peptides and promotes the advancement of peptide-based biomaterials integration progress with nanotechnology.
RESUMO
The intestine defends against pathogenic microbial invasion via the secretion of host defense peptides (HDPs). Nutritional immunomodulation can stimulate the expression of endogenous HDPs and enhance the body's immune defense, representing a novel non-antibiotic strategy for disease prevention. The project aims to explore the regulatory mechanism of protegrin-1 (PG-1) expression using sodium phenylbutyrate (PBA) by omics sequencing technology and further investigate the role of key regulatory genes on intestinal health. The results showed that PBA promoted PG-1 expression in intestinal epithelial cells based on cell density through epidermal growth factor receptor (EGFR) and G protein-coupled receptor (GPR43). Transcriptome sequencing and microRNA sequencing revealed that C-X-C motif chemokine receptor 2 (CXCR2) exhibited interactions with PG-1. Pre-treatment cells with a CXCR2 inhibitor (SB225002) effectively suppressed the induction of PG-1 by PBA. Furthermore, SB225002 significantly suppressed the gene expression of HDPs in the jejunum of mice without influencing on the morphology, number of goblet cells, and proliferation of the intestine. CXCR2 inhibition significantly reduced the expression of HDPs during E. coli infection, and resulted in the edema of jejunal epithelial cells. The 16S rDNA analysis of cecal contents showed that the E. coli and SB225002 treatments changed gut microbiota diversity and composition at different taxonomic levels. Correlation analysis suggested a potential regulatory relationship between gut microbiota and HDPs. To that end, a gene involved in the HDP expression, CXCR2, has been identified in the study, which contributes to improving intestinal immune function. PBA may be used as a functional additive to regulate intestinal mucosal function, thereby enhancing the health of the intestinal and host.
Assuntos
Homeostase , Mucosa Intestinal , Receptores de Interleucina-8B , Animais , Humanos , Masculino , Camundongos , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/genética , Infecções por Escherichia coli/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Receptores Acoplados a Proteínas G , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
Food contamination from microbial deterioration requires the development of potent antimicrobial peptides (AMPs). The deployment of approved AMPs as dietary preservatives is limited due to barriers such as instability, toxicity, and high synthetic costs. This exploration utilizes the primary structural elements of the Trp-pocket backbone to engineer a series of ß-hairpin AMPs (XWRWRPGXKXXR-NH2, X representing I, V, F, and/or L). Peptides WpLF, with Phe as X and Leu arranged at the 11th position, demonstrated exceptional selectivity index (SI = 123.08) and sterilization effects both in vitro and in vivo. WpLF consistently exhibited stable bacteriostasis, regardless of physiological salts, serum, and extreme pH. Mechanistic analysis indicated that the peptide penetrates microbial cell membranes, inducing membrane disruption, thereby impeding drug resistance evolution. Conclusively, AMPs engineered by the Trp-pocket skeleton hold substantial potential as innovative biological preservatives in food preservation, providing valuable insights for sustainable and safe peptide-based food preservatives.
RESUMO
The objective of the present study was to investigate the effect of vitamin E on laying performance, egg quality, egg fatty acid composition, antioxidant capacity, and several biochemical parameters of laying hens fed corn distillers dried grains with solubles (DDGS) during the laying period (40 to 63 wk of age). A total of 360 Hy-Line Variety Brown hens were randomly assigned to 6 groups, consisting of 6 replicates with 10 hens each. Hens were allocated to diets 1 through 6 in a 3 × 2 factorial design. The dietary treatments included 3 levels of DDGS (0, 10, and 20%) and 2 levels of vitamin E (0 and 200 mg/kg). The results indicated that yolk color and eggshell thickness increased with increasing DDGS (P < 0.05). However, increasing DDGS to 20% in laying hen diets significantly reduced feed conversion (P < 0.05). Supplementation with 200 mg/kg of vitamin E significantly improved egg production and yolk percentage (P < 0.05). Increasing the dietary levels of vitamin E caused a decrease in cholesterol and an increase in the α-tocopherol concentration of the egg yolk and serum (P < 0.05). Diets supplemented with DDGS decreased the proportion of saturated fatty acids (P < 0.05) and increased the proportion of unsaturated fatty acids in egg yolk (P < 0.05). Supplementation with high levels of vitamin E decreased malondialdehyde and increased glutathione peroxidase and total superoxide dismutase concentrations of the egg yolk and serum (P < 0.05). In conclusion, our results showed that DDGS was successfully fed to laying hens at levels up to 10% without adverse effects on laying performance. Additionally, vitamin E supplementation improved egg production and egg quality and provided health benefits to laying hens.
Assuntos
Ração Animal/análise , Galinhas/fisiologia , Suplementos Nutricionais/análise , Zea mays/química , alfa-Tocoferol/administração & dosagem , Fenômenos Fisiológicos da Nutrição Animal , Animais , Antioxidantes/metabolismo , Dieta/veterinária , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Feminino , Óvulo/química , Óvulo/efeitos dos fármacos , Óvulo/fisiologia , Distribuição Aleatória , Reprodução/efeitos dos fármacosRESUMO
Antimicrobial peptides (AMPs) have attracted attention in the field of food preservatives due to their favorable biosafety and potential antimicrobial activity. However, high synthetic cost, systemic toxicity, a narrow antimicrobial spectrum, and poor antimicrobial activity become the main bottlenecks for their practical applications. To address these questions, a set of derived nonapeptides were designed based on a previously discovered ultra-short peptide sequence template (RXRXRXRXL-NH2) and screened to identify an optimal peptide-based food preservative with excellent antimicrobial properties. Among these nonapeptides, the designed peptides 3IW (RIRIRIRWL-NH2) and W2IW (RWRIRIRWL-NH2) presented a membrane-disruptive and reactive oxygen species (ROS) accumulation mechanism to execute potent and rapid broad-spectrum antimicrobial activity without observed cytotoxicity. Moreover, they exhibited favorable antimicrobial stability regardless of high ionic strength, heat, and excessive acid-base conditions, retaining potent antimicrobial effects for chicken meat preservation. Collectively, their ultra-short sequence length and potent broad-spectrum antimicrobial capacity may be beneficial for the further development of green and safe peptide-based food preservatives.
Assuntos
Anti-Infecciosos , Conservantes de Alimentos , Conservantes de Alimentos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Antibacterianos/farmacologia , Antibacterianos/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Sequência de Aminoácidos , Testes de Sensibilidade MicrobianaRESUMO
Recently, much emphasis has been placed on solving the intrinsic defects of antimicrobial peptides (AMPs), especially their susceptibility to protease digestion for the systemic application of antibacterial biomaterials. Although many strategies have increased the protease stability of AMPs, antimicrobial activity was severely compromised, thereby substantially weakening their therapeutic effect. To address this issue, we introduced hydrophobic group modifications at the N-terminus of proteolysis-resistant AMPs D1 (AArIIlrWrFR) through end-tagging with stretches of natural amino acids (W and I), unnatural amino acid (Nal) and fatty acids. Of these peptides, N1 tagged with a Nal at N-terminus showed the highest selectivity index (GMSI=19.59), with a 6.73-fold improvement over D1. In addition to potent broad-spectrum antimicrobial activity, N1 also exhibited high antimicrobial stability toward salts, serum and proteases in vitro and ideal biocompatibility and therapeutic efficacy in vivo. Furthermore, N1 killed bacteria through multiple mechanisms, involving disruption of bacterial membranes and inhibition of bacterial energy metabolism. Indeed, appropriate terminal hydrophobicity modification opens up new avenues for developing and applying high-stability peptide-based antibacterial biomaterials. STATEMENT OF SIGNIFICANCE: To improve the potency and stability of proteolysis-resistant antimicrobial peptides (AMPs) without increasing toxicity, we constructed a convenient and tunable platform based on different compositions and lengths of hydrophobic end modifications. By tagging an Nal at the N-terminal, the obtained target compound N1 exhibited strong antimicrobial activity and desirable stability under multifarious environments in vitro (proteases, salts and serum), and also showed favorable biocompatibility and therapeutic efficacy in vivo. Notably, N1 exerted its bactericidal effect by damaging bacterial cell membranes and inhibiting bacterial energy metabolism in a dual mode. The findings provide a potential method for designing or optimizing proteolysis-resistant AMPs thus promoting the development and application of peptide-based antibacterial biomaterial.
Assuntos
Anti-Infecciosos , Peptídeos Antimicrobianos , Proteólise , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Sais , Anti-Infecciosos/farmacologia , Bactérias , Antibacterianos/farmacologia , Antibacterianos/química , Peptídeo Hidrolases/farmacologia , Aminoácidos , Testes de Sensibilidade MicrobianaRESUMO
Mangroves as typical blue carbon ecosystems exhibit a high level of heavy metal accumulation capability. In this study, we investigated how extreme rainstorm effects the spatial variability and pollution risk of sediment heavy metals (i.e., Fe, Mn, Cr, Cu, Zn, Cd, Pb, As and Hg) at different compartments of a typical tidal flat, including the bare mudflat, mangrove zone, and tidal creek in Shenzhen Bay, China. The results showed that the extreme rainstorm can change the sediment particle size, which further regulated the spatial distribution, and source-sink pattern of heavy metals. Due to the strong rainstorm flushing, the concentrations of most heavy metals increased toward the sea and the comprehensive pollution level increased by 8.3 % after the extreme rainstorm. This study contributes to better understanding of how extreme rainstorm regulates heavy metal behavior in mangrove sediments to achieve sustainable development of mangroves under the pressures of extreme weather events.
Assuntos
Mercúrio , Metais Pesados , Ecossistema , Carbono , ChinaRESUMO
The biggest application bottleneck of antimicrobial peptides (AMPs) is the low oral bioavailability caused by the poor stability of digestive enzymes in the gastrointestinal tract. However, the research methods and evaluation criteria of available studies about anti-proteolytic strategies are not uniform and far from the actual environment in vivo. Here, we developed a research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving the protease stability of AMPs on the same platform for the first time. After a comprehensive analysis, Dab modification is identified as the most effective strategy to improve the trypsin stability of AMPs. By further modulating the proteolytic resistance optimization motif (DabW)n, U1-2WD is obtained with ideal stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which forms amorphous aggregates in the bacteria environment to trigger the agglutination of bacterial cells to prevent bacterial escape. It then kills bacteria by disrupting bacterial membranes and inhibiting bacterial energy metabolism. Overall, our work has led to a new understanding of the effectiveness of proteolytic resistance strategies and accelerated the development of anti-proteolytic AMPs to combat multidrug-resistant bacterial infections. STATEMENT OF SIGNIFICANCE: We developed research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving protease stability of AMPs on the same platform for the first time. we found effective strategies to resist trypsin hydrolysis: modification with backbone (ß-Arg), D-enantiomer (D-Arg) and L-2,4-diaminobutanoic acid (Dab). Further, the proteolytic resistance optimization motif (DabW)n was designed. When n=3, derivative U1-2WD was obtained with desirable stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which can self-aggregate into amorphous aggregates in the bacteria environment to mediate the agglutination and sedimentation of bacterial cells to prevent bacterial escape, and then kill bacteria by destroying bacterial membranes and inhibiting bacterial energy metabolism.
Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Tripsina/farmacologia , Bactérias , Antibacterianos/farmacologia , Antibacterianos/química , Anti-Infecciosos/farmacologia , Peptídeo Hidrolases/farmacologia , Aglutinação , Testes de Sensibilidade MicrobianaRESUMO
Tryptophan has drawn wide attention due to its involvement in improving intestinal immune defense directly and indirectly by regulating metabolic pathways. The study aims to elucidate the potential modulating roles of tryptophan to protect against intestinal inflammation and elucidate the underlying molecular mechanisms. The protective effects of tryptophan against intestinal inflammation are examined in the lipopolysaccharide (LPS)-induced inflammatory model. We first found that tryptophan markedly (p < 0.01) inhibited proinflammatory cytokines production and nuclear factor κB (NF-κB) pathway activation upon LPS challenge. Next, we demonstrated that tryptophan (p < 0.05) attenuated LPS-caused intestinal mucosal barrier damage by increasing the number of goblet cells, mucins, and antimicrobial peptides (AMPs) in the ileum of mice. In addition, tryptophan (p < 0.05) inhibited LPS-induced autophagic flux through the AMP-activated protein kinase (AMPK)-sirtuin 1 (SIRT1) pathway in the intestinal systems to maintain autophagy homeostasis. Meanwhile, tryptophan also reshaped the gut microbiota composition in LPS-challenge mice by increasing the abundance of short-chain fatty acid (SCFA)-producing bacteria such as Acetivibrio (0.053 ± 0.017 to 0.21 ± 0.0041%). Notably, dietary tryptophan resulted in the activation of metabolic pathways during the inflammatory response. Furthermore, exogenous treatment of tryptophan metabolites kynurenine (Kyn) and 5-HT in porcine intestinal epithelial cells (IPEC-J2 cells) reproduced similar protective effects as tryptophan to attenuate LPS-induced intestinal inflammation through regulating the AMPK-SIRT1-autophagy. Taken together, the present study indicates that tryptophan exhibits intestinal protective and immunoregulatory effects resulting from the activation of metabolic pathways, maintenance of gut mucosal barrier integrity, microbiota composition, and AMPK-SIRT1-autophagy level.
Assuntos
Sirtuína 1 , Triptofano , Suínos , Camundongos , Animais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Lipopolissacarídeos , Autofagia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Suplementos NutricionaisRESUMO
BACKGROUND: Antibiotics are very effective for treating diarrhea in weaned pigs, but the global prohibition of antibiotics makes it urgent to find an alternative to antibiotics. OBJECTIVE: An experiment was conducted to determine the antimicrobial activity of a linear trpzip-like ß-hairpin antimicrobial peptide WK3 in vivo and to assess its effects on growth performance and intestinal health. DESIGN: Thirty-two piglets were weaned at 21 days and housed in individual metabolic cages, which were randomly divided into four groups and were maintained on a corn-soybean meal-based basal diet. Group 1 included a blank group. Groups 2, 3, and 4 were orally infected by feeding with Enterotoxigenic Escherichia coli (ETEC) K88, which was followed by saline treatment (group 2), enrofloxacin injection at a dose of 2.5 mg/kg (group 3), and WK3 injection at a dose of 2 mg/kg (group 4). The experiment lasted for 6 days, and feed and water were provided ad libitum. RESULTS: Both WK3 and enrofloxacin effectively attenuated diarrhea and improved growth performance of piglets. Compared with the control group, WK3 significantly improved the villus height in the ileum (P < 0.05) but did not affect the villus height in the duodenum or jejunum. Additionally, we did not observe any obvious difference in crypt depth or villus height/crypt depth among the duodenum, jejunum and ileum (P > 0.05). WK3 also reduced the numbers of Enterococcus spp (P < 0.01) in the cecal contents, and the number of Enterobacterium spp tended to decrease (0.05 < P < 0.1). Moreover, the jejunal mucosa of the WK3 group exhibited lower interleukin-1α (IL-1a; P < 0.01), toll-like receptors-4 (TLR-4; P < 0.05), and myeloid differentiation primary response 88 (MyD88; P < 0.01) messenger ribonucleic acid (mRNA) expression levels. The jejunum of the WK3 group also exhibited an increased antioxidant capacity, reduced concentration of malondialdehyde (MDA; P < 0.05), and enhanced superoxide dismutase (SOD) activity (P < 0.05). CONCLUSIONS: WK3 has the potential to replace antibiotics as a new generation feed additive.
RESUMO
The antimicrobial peptide PMAP-36 is a cationic peptide derived from porcine myeloid. The N-terminally paired lysine of PMAP-36 was substituted with tryptophan, and the C-terminal hydrophobic tail was deleted, thereby obtaining the antimicrobial peptide PRW4. PRW4 is a α-helical antimicrobial peptide with broad-spectrum antimicrobial activity. In this study, PRW4 was fused to the 6× His-Trx, and the fusion protein was successfully expressed in Pichia pastoris GS115 from the vector pPICZαA. The maximal induction of recombinant protein occurred in the presence of 1% methanol after 96 h at pH 6.0. After purification by a Ni-NTA resin column and digestion by enterokinase protease, 15 mg of recombinant PRW4 with a purity of 90% was obtained from 1 L of fermentation culture. The results indicated that recombinant PRW4 had similar antimicrobial activity as synthetic PRW4 against bacteria such as Escherichia coli ATCC 25922, Escherichia coli UB 1005, Salmonella typhimurium C7731, Salmonella typhimurium 7913, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, and Streptococcus faecalis ATCC 29212. We have successfully expressed PRW4 in P. pastoris, and this work provides a reference for the production of modified antimicrobial peptides in P. pastoris.
Assuntos
Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Saccharomycetales/genética , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Fermentação , Expressão Gênica , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Saccharomycetales/metabolismo , SuínosRESUMO
Cecropin AD (CAD) is a hybrid peptide composed of 37 amino acids with the characters of strong antibacterial, antitumor properties and no hemolytic activity, which was regarded as a promising antibiotic candidate. Thus, a safe method to produce Cecropin AD is necessary to be found. In the study, Bacillus subtilis WB800N was employed as host strain. The CAD coding sequence fused with the signal peptide of SPsacB, the 6 × His gene and the gene of small ubiquitin-like modifier were cloned into the maltose-inducible vector pGJ148. Under the induction by 6% maltose, the recombinant fusion protein was successfully expressed and detected in culture substrate. An optimized amount (26.4 mg/L) of the recombinant CAD was purified of culture supernatant. After purification and digestion, the recombinant CAD was harvested about 4.5 mg/L with a purity of 93%. Recombinant CAD exhibited similar antimicrobial activity with synthetic CAD. This shows that the production of CAD in maltose-induced Bacillus subtilis expression system is a relatively safe method, which is vital for the application of CAD in animal husbandry production.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Bacillus subtilis , Expressão Gênica/efeitos dos fármacos , Maltose/farmacologia , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteína SUMO-1RESUMO
Xylanase is a member of an important family of enzymes that has been used in many biotechnological processes. However, the overall cost of enzyme production has been the main problem in the industrial application of enzymes. To obtain maximum xylanase production, statistical approaches based on the Plackett-Burman design and response surface methodology were employed. The results of the statistical analyses demonstrated that the optimal conditions for increased xylanase production were the following: inoculum size, 3.8%; maize meal, 4.5%; histidine, 0.6%; methanol, 1%; culture volume, 20%; bean pulp, 30 g L-1; and Tween-80, 0.8%; and pH 5.0. Verification of the optimization demonstrated that 3273 U mL-1 xylanase was observed under the optimal conditions in shake flask experiments. SDS-PAGE results showed that the size of xylanase protein was about 23 kDa. The results showed that the xylanase produced by fermentation came from Aspergillus Niger by MALDI-TOF-MS. The optimized medium resulted in 2.1- and 1.4-fold higher the activity of xylanase compared with the unoptimized medium (the main nutrients are maize meal and bean pulp) and laboratory medium (the main nutrients are yeast extract and peptone), respectively. The optimization of fermentation conditions is an effective means to reduce production cost and improve xylanase activity.
RESUMO
Due to compromised immune system, fungal infection incidences have markedly increased in the last few decades. Pathogenic fungi have developed resistance to the clinically available antifungal agents. Antifungal resistance poses a great challenge to clinical treatment and has stimulated the demand for novel antifungal agents. A promising alternative to the treatment of fungal diseases is the use of antimicrobial peptides (AMPs). However, the antifungal activities of AMPs have not been fully determined. Therefore, this study aimed at designing and screening α-helical peptides with potential antifungal activities. The effects of key physicochemical parameters on antifungal activities were also investigated. A series of lengthened and residue-substituted derivatives of the template peptide KV, a hexapeptide truncated from the α-helical region of porcine myeloid antimicrobial peptide-36, were designed and synthesized. Enhancement of hydrophobicity by introducing aromatic hydrophobic amino acids (tryptophan and phenylalanine) significantly increased the efficacies of the peptides against Candida albicans strains, including fluconazole-resistant isolates. Increased hydrophobicity also elevated the toxic properties of these peptides. RF3 with moderate hydrophobicity exhibited potent anticandidal activities (GM = 6.96 µM) and modest hemolytic activities (HC10 > 64 µM). Additionally, repeated exposure to a subinhibitory concentration of RF3 did not induce resistance development. The antifungal mechanisms of RF3 were due to membrane disruptions and induction of reactive oxygen species production. Such a dual-targeted mechanism was active against drug-resistant fungi. These results show the important role of hydrophobicity and provide new insights into designing and developing antifungal peptides. Meanwhile, the successful design of RF3 highlights the potential utility of AMPs in preventing the spread of drug-resistant fungal infections.