Curcumin alleviated lipopolysaccharide-induced lung injury via regulating the Nrf2-ARE and NF-κB signaling pathways in ducks.

BACKGROUND: Under the intensive modern poultry farming system, the lung of duck is one of the main target organs for various bacterial and viral infections. Curcumin is a kind of natural polyphenol compound for which various beneficial biological functions exist, including being an anti-inflammatory, antioxidant, and antiviral. The aim of this work was to investigate the mechanism of curcumin-alleviated lipopolysaccharide (LPS)-induced lung damage by the nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant reaction element (ARE) and nuclear factor kappa B (NF-κB) signaling pathway in ducks. RESULTS: In total, 450 one-day-old male specific pathogen-free ducks were randomly assigned into three dietary treatments: CON, basal diet; LPS, basal diet + LPS treatment; LPS + CUR, basal diet + LPS + 500 mg kg-1 of curcumin. At the end of the experiment (21 days), ducks in LPS treatment were challenged with 5 mg LPS per kilogram of body weight and the other two treatments were injected with the same dose of phosphate-buffered saline solution. The results showed that LPS caused acute inflammation, oxidation stress, and lung injury. Dietary addition of curcumin significantly relieved the oxidation stress and inflammation parameters. Moreover, the results showed that remission may be through the signaling pathways of both Nrf2-ARE and NF-κB. CONCLUSION: In conclusion, dietary supplementation of 500 mg kg-1 of curcumin exhibited a lung-protective effect in ducks. This experiment broadens the mode of metabolism actions of curcumin in the target organs and provides an insight for the application of curcumin in waterfowl feed. © 2022 Society of Chemical Industry.

Biochemical property and membrane-peptide interactions of de novo antimicrobial peptides designed by helix-forming units.

Typical peptides composed of Phe, Ile, and Arg residues have not been reported, and the effect of the helix-forming unit (HFU) composed of the tripeptide core on biological activity remains unclear. In this study, multimers of the 3-residue HFU were designed to investigate the structure-function relationships. The in vitro biological activities of the peptides were determined. We used synthetic lipid vesicles and intact bacteria to assess the interactions of the peptides with cell membranes. The well-studied peptide melittin was chosen as a control peptide. The results showed that the antimicrobial and hemolytic activities of the peptides increased with the number of HFUs. HFU3 had optimal cell selectivity as determined by the therapeutic index. HFU3 and HFU4 exhibited strong resistance to salts, pH, and heat. CD spectra revealed that the peptides except HFU2 displayed α-helix-rich secondary structures in the presence of SDS or trifluoroethanol (TFE). The peptides interacted weakly with zwitterionic phospholipids (mimicking mammalian membranes) but strongly with negatively charged phospholipids (mimicking bacterial membranes), which corresponds well with the data for the biological activities. There was a correlation between the cell selectivity of the peptides and their high binding affinity with negatively charged phospholipids. Cell membrane permeability experiments suggest that the peptides targeted the cell membrane, and HFU3 showed higher permeabilization of the inner membrane but lower permeabilization of the outer membrane than melittin. These findings provide the new insights to design antimicrobial peptides with antimicrobial potency by trimers.

Dietary Grape Seed Proanthocyanidin Alleviates the Liver Injury Induced by Long-Term High-Fat Diets in Sprague Dawley Rats.

In mammals, the liver is the most important organ that plays a vital function in lipid metabolism. Grape seed proanthocyanidin (GSPE) is a kind of natural polyphenolic compound primarily obtained from grape skin and seeds. Recent research found it had high bioavailability in defending against obesity, hyperlipidemia, inflammatory, oxidative stress, and targeting liver tissue. However, the mechanism of GSPE in regulating obesity induced by dietary high-fat (HF) was not fully understood, particularly the influences on liver functions. Therefore, this study aimed to investigate the effects of GSPE supplementation on the liver function and lipid metabolic parameters in rats fed HF diets long-term. A total of 40 healthy female Sprague Dawley rats were selected. After 8 weeks of obesity model feeding, the rats were randomly divided into four treatments: NC, standard diet; NC + GSPE, standard diet + 500 mg/kg body weight GSPE; HF, high-fat diet; HG + GSPE, high fat diet + 500 mg/kg body weight GSPE. Results indicated that long-term HF feeding caused severe liver problems including megalohepatia, steatosis, inflammation, and hepatocyte apoptosis. The supplementation of GSPE alleviated these symptoms. The results of the current experiment confirmed that GSPE addition up-regulated the expression of the Wnt3a/ß-catenin signaling pathway, thereby restraining the liver cell endoplasmic reticulum stress and hepatocyte apoptosis. Furthermore, the microRNA-103 may play a role in this signal-regulated pathway. In summary, GSPE had a protective effect on the liver and the current experiment provided a reference for the application of GSPE in animal nutrition as a kind of natural feed additive.

Cell selectivity and interaction with model membranes of Val/Arg-rich peptides.

Antimicrobial peptides are major components of the innate self-defence system and a large number of peptides have been designed to study the mechanism of action. In the present study, a small combinatorial library was designed to study whether the biological activity of Val/Arg-rich peptides is associated with targeted cell membranes. The peptides were produced by segregating hydrophilic residues on the polar side and hydrophobic residues on the opposite side. The peptides displayed strong antimicrobial activity against Gram-negative and Gram-positive bacteria, but weak haemolysis even at a concentration of 256 µM. CD spectra showed that the peptides formed α-helical-rich structure in the presence of negatively charged membranes. The tryptophan fluorescence and quenching experiments indicated that the peptides bound preferentially to negatively charged phospholipids over zwitterionic phospholipids, which corresponds well with the biological activity data. In the in vivo experiment, the peptide G6 decreased the bacterial counts in the mouse peritoneum and increased survival after 7 days. Overall, a high binding affinity with negatively charged phospholipids correlated closely with the cell selectivity of the peptides and some peptides in this study may be likely candidates for the development of antibacterial agents.

Expression and characterization of recombinant gallinacin-9 and gallinacin-8 in Escherichia coli.

Gallinacins (Gal) are antimicrobial peptides that play significant roles in innate immunity in chickens. Two Gal genes--Gal-8 and Gal-9--were cloned and sequenced from chicken liver and tongue, respectively, by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, the mRNA expression of these genes has been demonstrated across a panel of chicken tissues. It was demonstrated that Gal-9 mRNA was highly expressed in the tongue and small intestine and moderately expressed in the chicken proventriculus, lung, liver, heart, spleen, and thymus. However, Gal-8 mRNA was highly expressed in the chick small intestine and liver, and moderately expressed in the chick tongue, and lung. The recombinant fusion proteins containing Gal-9 or Gal-9 and Gal-8, namely rGal-9 and rGal-9-Gal-8, were produced and purified, respectively. Both rGal-9 and rGal-9-Gal-8 were expressed as insoluble bodies and exhibited the expected antimicrobial activity against Escherichia coli and pathogenic Streptococci suis CAB strain, as determined by the measurement of the inhibition zone and a liquid growth inhibition assay.

Selenium Deficiency Affects the mRNA Expression of Inflammatory Factors and Selenoprotein Genes in the Kidneys of Broiler Chicks.

The aim of this study was to investigate the influence of Se deficiency on the transcription of inflammatory factors and selenoprotein genes in the kidneys of broiler chicks. One hundred fifty 1-day-old broiler chicks were randomly assigned to two groups fed with either a low-Se diet (L group, 0.033 mg/kg Se) or an adequate Se diet (C group, 0.2 mg/kg Se). The levels of uric acid (UA) and creatinine (Cr) in the serum and the mRNA levels of 6 inflammatory factors and 25 selenoprotein genes in the kidneys were measured as the clinical signs of Se deficiency occurred at 20 days old. The results indicated that the contents of UA and Cr in the serum increased in L group (p < 0.05), and the mRNA levels of the inflammatory factors (NF-κB, iNOS, COX-2, and TNF-α) increased in L group (p < 0.05). Meanwhile, the mRNA levels of PTGEs and HO-1 were not changed. In addition, 25 selenoprotein transcripts displayed ubiquitous expression in the kidneys of the chicks. The mRNA levels of 14 selenoprotein genes (Dio1, Dio2, GPx3, Sepp1, SelH, SelI, SelK, Sepn1, SelO, SelW, Sep15, SelT, SelU, and SelS) decreased, and 9 selenoprotein genes (GPx1, GPx2, GPx4, SelPb, Txnrd1, Txnrd2, Txnrd3, SPS2, and SelM) increased in L group (p < 0.05), but the Dio3 and Sepx1 mRNA levels did not change. The results indicated that Se deficiency resulted in kidney dysfunction, activation of the NF-κB pathway, and a change in selenoprotein gene expression. The changes of inflammatory factor and selenoprotein gene expression levels were directly related to the abnormal renal functions induced by Se deficiency.

Lithium chloride's inhibition of 3T3-L1 cell differentiation by regulating the Wnt/ß-catenin pathway and enhancing villin 2 expression.

The aim of this study is to reveal the relation among villin 2, Wnt/ß-catenin, and adipogenesis by adding appropriate lithium chloride (LiCl). The study comprises three parts: the selection of LiCl concentration, the effect of LiCl on adipocyte differentiation during and after differentiation induction. By comprehensively analyzing the results of the experiments, we proved that LiCl can inhibit adipocyte differentiation and enhance villin 2 and ß-catenin expressions not only during differentiation induction but also after it. Moreover, villin 2 has a significant impact on ß-catenin. We suggest that villin 2 may participate in Wnt/ß-catenin signaling.

Effects of dietary selenium deficiency or excess on gene expression of selenoprotein N in chicken muscle tissues.

Previous studies have determined the effects of dietary selenium (Se) supplementation on selenoprotein N (SelN, SEPN1), selenophosphate synthetase-1 (SPS1), and selenocysteine-synthase (SecS) mRNA abundance in chicken skeletal and cardiac muscles. To investigate collective responses of these genes to dietary Se concentrations ranging from deficiency to moderately high level in muscle tissues of chicken, 1-day-old chickens were exposed to a diet of deficient Se and supplemented with Se (0.15 mg Se/kg and 1.50 mg Se/kg) as sodium selenite in the feed for 35 days. Muscle tissues (flight, breast, leg, and cardiac muscles) were collected and examined for Se content and mRNA levels of SelN on days 1, 15, 25, and 35 days, respectively. Moreover, SPS1 and SecS mRNA levels were analyzed. The results showed that the expression of SelN gene in cardiac muscle responded to dietary Se concentrations. SelN gene was downregulated in the Se deficiency group (L group), and upregulated in the Se excess group (H group) compared with the moderate Se group (M group) (P < 0.05) in cardiac muscle. Se deficiency mainly unregulated SelN mRNA level in skeletal muscles compared with M group. Excess dietary Se mainly resulted in the upregulation of SelN mRNA level in skeletal muscles compared with the M group. SecS mRNA levels responded to dietary Se concentrations showed a similar change compared with SelN in cardiac muscle. SPS1 mRNA levels responded to dietary Se concentrations showed a downregulation in L group and upregulation in H group. However, SelN mRNA levels displayed a different expression pattern in different skeletal and cardiac muscles. Moreover, Se also regulated the levels of SPS1 and SecS mRNAs. In summary, Se regulated the expression of SelN gene and affected the mRNA levels of SecS and SPS1. The level of Se in the feed may regulate SelN biosynthesis by affecting the levels of SPS1 and SecS mRNA.

The influence of isoleucine and arginine on biological activity and peptide-membrane interactions of antimicrobial peptides from the bactericidal domain of AvBD4.

In this study, the influence of isoleucine and arginine on the biological activity and peptide-membrane interactions of linear avian ß-defensin-4 (RL38) analogs was investigated. Results of biological activities showed that the antimicrobial activities of AvBD-4 analogs were closely related to hydrophobicity and amphipathicity. The peptide GLI19 with high hydrophobicity value and amphipathicity displayed broad spectrum antimicrobial activity against both gram-negative and gram-positive, whereas GLR19 with increasing multiple charges only exhibited activity against gram-negative. The interaction between peptides and the liposome membrane demonstrated that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which supported the antimicrobial activity data. The outer membranes assay further demonstrated that GLI19 had a greater capacity than the other tested peptides to penetrate the cell membrane at a low concentration. Collectively, the peptides derived from the bactericidal domain of linear ß- defensins by truncation and hydrophobic amino acid substitution may be effective high-potential antibacterial agents.

Influence of truncation of avian ß-defensin-4 on biological activity and peptide-membrane interaction.

Defensins are important components in host defense systems. The therapeutic use of ß-defensins is limited by their innate toxicity and high cost due to the size and complex disulfide pairing. In this study, we used linear avian ß- defensin-4 (RL38) without disulfide bonds as model peptide to derive two peptides by the truncation. GL23 is the C-terminal truncated sequence of RL38, and GLI23 is the derivative of GL23 by the replacement of cysteines with isoleucines. Results showed that these peptides exhibited strong antibacterial activity against gram-negative and gram-positive bacteria. An exception was that GL23 showed weak antimicrobial activity against gallinaceous pathogenic bacteria Salmonella Pullorum C79-13. Two truncated peptides GL23 and GLI23 displayed no or weak hemolysis, which was in accordance with little blue shifts of the peptides in the presence of synthetic eukaryotic membranes. CD spectroscopy demonstrated that these peptides appeared to be unfolded in aqueous solution but acquire structure in the presence of membrane- mimicking phospholipids. GLI23 kept the antibacterial activity at high concentrations of NaCl or low concentration of divalent cations (Mg2+ and Ca2+). The peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. The outer and inner membranes assay displayed that GLI23 killed bacteria by targeting the cell membrane. These results suggest the peptides derived by truncation of linear ß-defensins may be a promising candidate for future antibacterial agent.

Biochemical property and in vivo efficacies of novel Val/Arg-rich antimicrobial peptide.

A novel α-helical antimicrobial peptide G6 rich in Val/Arg residues has been screened previously. In this study, we further evaluated the biochemical stability, interaction with whole bacteria, and in vivo therapeutic or prophylactic role of the peptide in Salmonella typhimurium-infected mice. The results showed that G6 exhibited strong resistance to pH, heat, and salts. But G6 lost the antimicrobial activity when treated with proteolytic enzymes. G6 had no toxicity on mammalian cell. An intraperitoneal model of sepsis caused by Salmonella typhimurium was established in mice. G6 was administered intraperitoneally 1 h before or after mice were infected with Salmonella typhimurium. For the mice given peptide post-bacterial infection, the mortality of the mice and the peritoneal bacterial counts were significantly lower in the groups that were administered 2.5 mg/kg BW and 5.0 mg/kg BW of G6 (P < 0.05) compared to the PBS-treated group. Similar trend was obtained when G6 was given 1 h prior to Salmonella typhimurium infection. Peptide-membrane experiments showed that G6 was effective in permeabilizing the outer and inner membrane in a dose dependent manner, indicating that the peptide targets the cell membrane. Taken together, the results revealed that the peptide G6 may provide a useful alternative to antibiotic agents to treat or prevent bacterial infections.

Novel design of short antimicrobial peptides derived from the bactericidal domain of avian ß-defensin-4.

Short antimicrobial peptides were designed and synthesized by C-terminal truncation and residue substitution of avian ß-defensin-4. The biological activity of these peptides was examined to elucidate the quantitative structure-activity relationships and find a lead peptide for the development of a novel antimicrobial peptide. The results showed that the truncation of the avian ß-defensin-4 eliminated the hemolysis of the peptide. The GLI13 derivative, developed by substituting the Cys of the truncated peptide with Ile, led to increased antimicrobial activity. These results suggest that the peptides with antimicrobial activity can be derived by truncating the avian ß-defensin-4. We further developed the GLI13 derivative with an increased net charge by residue substitution. The results showed that the GLI13-5 with 5 net charges had the highest cell selectivty. An increase in the net charge from 6 to 8 did not result in the improvement of antimicrobial potency. Membrane-simulating experiments showed that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. A membrane depolarization assay showed that GLI13-5 killed bacteria by targeting the cytoplasmic membrane. These results suggest that the short peptide developed by truncation of linear ß-defensin may be a promising candidate for future antibacterial agents.

The effects of Leu or Val residues on cell selectivity of α-helical peptides.

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.

Fusion expression of bovine lactoferricin in Escherichia coli.

The drug resistance problem has been growing with the utilization of current antibiotics in feed and medical industries. LfcinB, a 25-amino acid antibacterial peptide derived from bovine lactoferrin, is one of potential alternatives of antibiotics. According to the bias of codon utilization of Escherichia coli, a fragment encoding LfcinB has been chemically synthesized, inserted into vector pGEX-4T-2 and expressed in E. coli. The antibacterial peptide was fused with GST with a protease cleavage site located between them. Two constructs with different cleavage sites were made. One construct, pGEX-Th-LfcinB, contains a thrombin cleavage site carried by the vector, and the other, pGEX-Th-Xa-LfcinB, contains a Factor Xa cleavage site which was introduced after the thrombin cleavage site. Fusion protein GST-Th-LfcinB protein was efficiently cleaved by thrombin, yielding recombinant LfcinB showing antibacterial activity. However, fusion protein GEX-Th-Xa-Lfcin B containing Factor Xa recognition site could not be cleaved by Factor Xa at the conditions tried in this study. Successful expression of LfcinB in E. coli provides a possible method to produce LfcinB in large amounts.