Changes in health-promoting metabolites associated with high-altitude adaptation in honey.

The levels of metabolites in honey are influenced by floral origin, production region, and bee species. However, how environmental factors affect honey quality remains unclear. Based on untargeted metabolomics and using UPLC Q-Orbitrap MS, we analyzed 3596 metabolites in 51 honey samples from Yunnan and Shennongjia. Comparative analysis revealed that geniposidic acid, kynurenic acid and caffieine accumulated at significantly different levels between Shennongjia and Yunnan honey. Based on cluster structure analysis, 36 Yunnan honey samples were divided into two distinct groups by altitude. Notably, quercetin, hyperoside, taxifolin, rutin, tryptophan, astragalin and phenylalanine were higher levels in high-altitude honey (>1700 m), whereas abscisic acid was higher levels in low-altitude honey (≤1700 m). Among these, significantly elevated levels of hyperoside, taxfolin, astragalin, and tryptophan were observed in honey collected from high-altitude areas in Shennongjia. Our findings highlight the effect of altitude on honey health-promoting components, providing valuable insights into honey quality.

Traceability of chemicals from Tripterygium Wilfordii Hook. f. in raw honey and the potential synergistic effects of honey on acute toxicity induced by celastrol and triptolide.

The object of this study was to trace TwHf-derived toxins in raw honey and clarify their acute toxic effect related to the addition of honey or sugars. TwHf flowers, raw honey from TwHf planting base and from beekeepers in high-risk area were detected using LC-MS/MS. The results revealed five target toxins were detected in TwHf flowers; only celastrol was detected in one raw honey sample, as a food safety risk factor, celastrol had been traced back to TwHf flowers from raw honey. In a series of acute toxic tests on zebrafish, toxification effects were observed when honey, mimic honey or sugar was mixed with toxins. The degree of toxicity varied among various sugar-based solutions. At the same mass concentration, they follow this order: raw honey/mimic honey > glucose > fructose. The main toxic target organs of triptolide and celastrol with honey were the heart and liver.

Salmonella Phages Affect the Intestinal Barrier in Chicks by Altering the Composition of Early Intestinal Flora: Association With Time of Phage Use.

Phages show promise in replacing antibiotics to treat or prevent bacterial diseases in the chicken breeding industry. Chicks are easily affected by their environment during early growth. Thus, this study investigated whether oral phages could affect the intestinal barrier function of chicks with a focus on the cecal microbiome. In a two-week trial, forty one-day-old hens were randomly divided into four groups: (1) NC, negative control; (2) Phage 1, 109 PFU phage/day (days 3-5); (3) Phage 2, 109 PFU phage/day (days 8-10); and (4) AMX, 1 mg/mL amoxicillin/day (days 8-10). High-throughput sequencing results of cecal contents showed that oral administration of phages significantly affected microbial community structure and community composition, and increased the relative abundance of Enterococcus. The number of different species in the Phage 1 group was much higher than that in the Phage 2 group, and differences in alpha and beta diversity also indicated that the magnitude of changes in the composition of the cecal microbiota correlated with the time of phage use. Particularly in the first stage of cecal microbiota development, oral administration of bacteriophages targeting Salmonella may cause substantial changes in chicks, as evidenced by the results of the PICRUSt2 software function prediction, reminding us to be cautious about the time of phage use in chicks and to avoid high oral doses of phages during the first stage. Additionally, the Phage 2 samples not only showed a significant increase in the relative abundance of Bifidobacterium and Subdoligranulum, but also improved the intestinal morphology (jejunum) and increased the mRNA expression level of occludin and ZO-1. We concluded that phages do not directly interact with eukaryotic cells. The enhancement of intestinal barrier function by phages in chicks may be related to changes in the intestinal flora induced by phages. This implies that phages may affect intestinal health by regulating the intestinal flora. This study provides new ideas for phage prevention of intestinal bacterial infections and promotes large-scale application of phages in the poultry industry.

A Cocktail of Three Virulent Phages Controls Multidrug-Resistant Salmonella Enteritidis Infection in Poultry.

Salmonella enterica is not only the most common pathogen of poultry and poultry-derived products but is also a significant foodborne pathogen. In recent years, many S. enterica isolates have exhibited multi-drug resistance, which places huge pressure on global economy and health. Since phages are an attractive alternative to biocontrol pathogens, we isolated a total of 15 Salmonella phages from sewage effluent, sediment, and chicken manure. The GRNsp1, GRNsp3, GRNsp6, GRNsp21, GRNsp27, GRNsp30, GRNsp50, and GRNsp51 phages exhibited a wide host range against S. enterica serovars Enteritidis and Typhimurium in vitro. In particular, GRNsp51 exerted highly efficient lytic effects against a large proportion of S. Enteritidis and S. Typhimurium strains isolated from different regions of China. Meanwhile, GRNsp8 expanded the host range of GRNsp6 and GRNsp51. Based on their host ranges and lytic capacities, GRNsp6, GRNssp8, and GRNsp51 were selected for further investigation. Morphology, one-step growth curves, and stability assays revealed that GRNsp6, GRNsp8, and GRNsp51 all belong to the Caudovirales order and display relatively short latency periods with broad pH and thermal stability. Genomic analysis indicated that the genomes of these three phages contained no genes related to virulence, antibiotic resistance, or lysogeny. In addition, we tested the effectiveness of a cocktail composed of these three phages against S. Enteritidis in a chicken model. Treatment with the oral phage cocktail 24 h before or alongside Salmonella challenge significantly reduced colonization of the intestinal tract and decreased the mRNA expression of IL-6, IFN-γ, and IL-1ß in the duodenum. Together, these findings indicate that a cocktail of the GRNsp6, GRNsp8, and GRNsp51 phages could serve as an effective antimicrobial therapeutic agent against multidrug-resistant Salmonella in animal production to mitigate infections by multiple zoonotic Salmonella species.

Effects of A-type oligomer procyanidins on protein glycation using two glycation models coupled with spectroscopy, chromatography, and molecular docking.

Non-enzymatic glycation of proteins can produce advanced glycation end products (AGEs) and important intermediates (α-dicarbonyl compounds such as methylglyoxal), which have potential health risks due to their association with diabetes complications. Therefore, it is of great significance to search for effective inhibitors on protein glycation. In this study, the anti-glycation potential of different kinds of A-type procyanidins was investigated by spectroscopy, chromatography and molecular docking. The results revealed that different kinds of A-type procyanidins could potently suppress the protein glycation in BSA-Glc and BSA-MGO models. Notably, these procyanidins exhibited the most potent AGEs inhibitory potential with IC50 values of 41.50, 37.00, 53.99, and 34.99 µg/mL in BSA-Glc models, respectively. A comparison analysis showed that these procyanidins with different structures have various inhibitory effects on the production of AGEs, which may be related to their spatial configuration, polymerization degree and bond connection mode. The inhibitory effect of A-type procyanidins on protein glycation may be attributed to their specific binding with some amino acid residues in BSA, inhibition on the formation of fructosamine and capture of MGO. Moreover, when 600 µg/mL of these procyanidins were incubated with MGO, 91.30%, 80.25%, 75.07%, and 70.82% MGO decrease were observed, respectively. And the formed three new types of A-type trimer procyanidin-MGO adducts adducts were verified by Orbitrap LC-MS/MS analysis. These results indicate that A-type procyanidins may be used as inhibitors of glycation and provide the theoretical basis for their application.

Identification and safety assessment of Enterococcus thailandicus TC1 isolated from healthy pigs.

Enterococci have the dual characteristics of being opportunistic pathogens and promising probiotics. The isolation from patients of CDC PNS-E2, a newly described Enterococcus species Enterococcus sanguinicola, may pose potential hazards. Enterococcus thailandicus from fermented sausage is a senior subjective synonym of E. sanguinicola. In this study, Enterococcus thailandicus TC1 was first isolated in healthy pigs in Tongcheng, China and identified by phenotypic analysis and 16S rRNA-based techniques. To evaluate the strain safety, an approach including virulence factors, antibiotic resistance, and animal experiments was adopted. The results show that cylA, gelE, esp, agg, ace, efaAfm, efaAfs, ptsD genes were undetected, and that the strain was sensitive or poorly resistant to some clinically relevant antibiotics. However, the isolated strain demonstrated ß-hemolytic activity in rabbit blood agar plates. Analysis of animal experiments revealed that the isolated strain had no adverse effect on translocation and the internal organ indices, though significant differences in histology (villi height, crypts height) of ileum were observed. The data acquired suggest that E. thailandicus TC1 may be associated with a potential health risk.

Porcine interleukin-6 enhances the expression of CYP2C33 through a constitutive androstane receptor/retinoid X receptor-mediated pathway.

Cytochrome P450, which is expressed in humans and other animals, is a superfamily of drug-metabolizing enzymes that play important roles in the metabolism of endogenous and xenobiotic substrates via oxidation, peroxidation and reduction. Of endogenous substrates, interleukin (IL)-6 is a crucial cytokine involved in inflammation in the liver. The present study aims to elucidate the mechanisms through which IL-6 modulates cytochrome P450 expression. CYP2C33 expression was found to be increased in HepLi cells and primary porcine hepatocytes treated with IL-6 in a concentration-dependent manner. IL-6 treatment also increased the expression of the transcriptional regulators, constitutive androstane receptor (CAR) and pregnane X receptor. Overexpression of CAR promoted CYP2C33 expression at the mRNA and protein levels, whereas knockdown of CAR by small interfering RNA reduced CYP2C33 expression. Luciferase assays showed that IL-6 treatment of HepLi cells and primary porcine hepatocytes increased CYP2C33 promoter activity. Co-immunoprecipitation and western blotting demonstrated that CAR and RXR could form heterodimers. IL-6 affects CYP2C33 expression through CAR/retinoid X receptor (RXR) heterodimers.

Relationship between CYP3A29 and pregnane X receptor in landrace pigs: Pig CYP3A29 has a similar mechanism of regulation to human CYP3A4.

The objective of this study was to provide evidence of the validity of utilizing pigs as a model to study the regulation of human CYP3A4, with special emphasis on drug-drug interactions. We determined the mRNA expression and distribution of CYP3A and metabolic nuclear receptors in different tissues isolated from landrace pigs. Our results showed that CYP3A and metabolic nuclear receptor mRNAs were most highly expressed in liver tissues. The expression of the metabolic nuclear receptor pregnane X receptor (PXR) had a significant correlation with expression of CYP3A29, an analog of human CYP3A4. The correlation between their transcriptional levels was further demonstrated using LPS and TNF-α. The mRNA and protein expression of CYP3A29 and PXR in HepLi cells was significantly reduced by LPS and TNF-α treatment. CYP3A29 promoter activity was dramatically elevated by PXR over expression, whereas LPS and TNF-α treatment inhibited the enhanced CYP3A29 promoter activity that was induced by PXR; presumably through inhibition of PXR promoter activity. Furthermore, the inhibition of CYP3A29 promoter activity by LPS and TNF-α treatment was blocked by knockdown of PXR or retinoid X receptor (RXR). These data suggest high similarity in the regulation mechanism of pig CYP3A29 and human CYP3A4. Our research provided a significant evaluation to determine whether pigs are suitable as an experimental animal model.

Development of immunochromatographic test strips for rapid, quantitative detection of H9AIV antibodies.

To improve viral antibody detection and disease control, laboratories need faster and simpler methods for direct detection of H9AIV in clinical samples. In this study, test strips were developed for rapid detection of H9AIV antibodies in poultry serum by a sandwich method with double-line detection. The hemagglutinin protein was labeled with colloidal gold as a detection reagent and was blotted on the test lines, while goat anti-rabbit IgG was utilized on the control line of the nitrocellulose membrane. The test strips have high specificity, sensitivity, and stability, with a correlation coefficient of 0.9656 and coefficients of variation below 10%. Application of the kit to quantitative detection of H9AIV antibodies in 504 samples collected from chickens showed a coincidence rate of 80.56% with previously run HI assays.

Cecropin B Represses CYP3A29 Expression through Activation of the TLR2/4-NF-κB/PXR Signaling Pathway.

Cecropins are peptide antibiotics used as drugs and feed additives. Cecropin B can inhibit the expression of CYP3A29, but the underlying mechanisms remain unclear. The present study was designed to determine the mechanisms responsible for the effects of cecropin B on CYP3A29 expression, focusing on the Toll-like receptors (TLRs) and NF-κB pathways. Our results indicated that the CYP3A29 expression was inhibited by cecropin B, which was regulated by pregnane X receptor (PXR) in a time- and dose-dependent manner. Cecropin B-induced NF-κB activation played a pivotal role in the suppression of CYP3A29 through disrupting the association of the PXR/retinoid X receptor alpha (RXR-α) complex with DNA sequences. NF-κB p65 directly interacted with the DNA-binding domain of PXR, suppressed its expression, and inhibited its transactivation, leading to the downregulation of the PXR-regulated CYP3A29 expression. Furthermore, cecropin B activated pig liver cells by interacting with TLRs 2 and 4, which modulated NF-κB-mediated signaling pathways. In conclusion, cecropin B inhibited the expression of CYP3A29 in a TLR/NF-κB/PXR-dependent manner, which should be considered in future development of cecropins and other antimicrobial peptides.

IFN-γ regulates cytochrome 3A29 through pregnane X receptor in pigs.

1. The expression and the activity of cytochromes P450 (CYPs) can be elevated by the activation of nuclear receptors. The pregnane X receptor (PXR, or nuclear receptor NR1I2) is a ligand-activated transcription factor that mediates responses to diverse xenobiotics and endogenous chemicals. Here we investigated the regulatory role of PXR in IFN-γ-mediated CYP3A29 expression in pig liver microsomes, primary porcine hepatocytes, and a cultured hepatocyte cell line. 2. IFN-γ significantly up-regulated CYP3A29 and PXR expressions at mRNA and protein levels in a dose-dependent manner. IFN-γ treatment significantly increased the metabolism of nifedipine. PXR and IFN-γ treatments significantly enhanced the activity of CYP3A29 promoter and the upstream region from -1473 to -1021 of CYP3A29 might be PXR-binding site. Moreover, the IFN-γ-induced CYP3A29 expression was blocked by PXR knockdown, whereas CYP3A29 mRNA and protein expression levels were dramatically elevated by PXR overexpression. 3. The regulatory effect of IFN-γ on CYP3A29 expression is mediated via PXR.

Pregnane X receptor is required for IFN-α-mediated CYP3A29 expression in pigs.

Pregnane X receptor (PXR) has been identified as a central mediator for coordinate responses to xenobiotic and drug metabolism, and is the major transcriptional regulator of cytochrome P-450 (CYP). Interferon (IFN)-α is known to induce antiviral mechanisms and exert immune regulatory capacity in various cell types. Here, we used primary porcine hepatocytes and a cultured hepatocyte cell line to identify the metabolic role of PXR in IFN-α-mediated CYP3A29 expression. We found that IFN-α could activate PXR in both time- and dose-dependent manners in pigs. Activation of PXR significantly increased CYP3A29 mRNA and protein expression. Meanwhile, the expression of CYP3A29 induced by IFN-α occurred after the increase of PXR expression in porcine hepatocytes. In addition, the IFN-α-induced CYP3A29 expression was blocked by PXR knockdown. The PXR-overexpressed cells (transfected with porcine PXR) increased CYP3A29 mRNA and protein expression. Furthermore, in animal experiments, we found that IFN-α increased both CYP3A29 mRNA and protein levels. Collectively, our results suggest that PXR plays an important role in IFN-α-mediated CYP3A29 expression in porcine hepatocytes.

Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle.

A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM(2)), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM(2)/SDZ/SQX-bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T(1), T(2), and T(3), respectively. With this method, the cutoff values for the three test lines were achieved at 80 µg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 µg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods.

Development of an immunochromatographic lateral-flow test strip for rapid detection of sulfonamides in eggs and chicken muscles.

A rapid immunochromatographic lateral-flow test strip was developed in the competitive reaction format for the detection of sulfonamides in eggs and chicken muscle. A monoclonal antibody against the common structure of sulfonamides was conjugated to colloidal gold particles as the detection reagent and an N-sulfanilyl-4-aminobenzoic acid (SUL)-bovine serum albumin (BSA) conjugate was immobilized to a nitrocellulose membrane as the capture reagent to prepare the test strip. With this method, it required only 15 min to accomplish the semiquantitative or quantitative detection of sulfonamides. The sensitivity to sulfonamides (sulfamonomethoxine, sulfamethoxydiazine, sulfadimethoxine, and sulfadiazine) was at least 10 ng/mL, as determined with an optical density scanner. By eye measurement, the sensitivity was 20 ng/mL for sulfamonomethoxine, sulfamethoxydiazine, and sulfadimethoxine and 40 ng/mL for sulfadiazine. On the basis of a sulfamonomethoxine standard curve, recoveries were from 89.5 to 95.6% for sulfamonomethoxine, from 89.5 to 95.1% for sulfamethoxydiazine, from 85.0 to 95.6% for sulfadimethoxine, and from 44.8 to 60.9% for sulfadiazine in egg and chicken muscle samples. A parallel analysis of 27 egg samples and 28 chicken muscle samples from the animal experiment showed that the differences between test strips and high-performance liquid chromatography (HPLC) were from 0.8 to 11.2% for egg samples and from 2.2 to 34% for chicken muscle samples for the quantitative detection, and the agreement rates between test strips and HPLC were 100%, based on the maximum allowed residue level of sulfadiazine (100 ng/g) established by the European Union and China. In conclusion, the method is rapid and accurate for the detection of sulfonamides in eggs and chicken muscles.

Development and validation of an immunochromatographic assay for rapid detection of sulfadiazine in eggs and chickens.

A rapid immunochromatographic assay (ICA) was developed and validated for the detection of sulfadiazine in eggs and chickens. Based on the competitive reaction mechanism, the competitor of sulfadiazine (sulfadiazine-BSA conjugate) was immobilized to the defined detection zone on a nitrocellulose membrane which acted as the capture reagent, and the monoclonal antibody against sulfadiazine was conjugated to colloidal gold particles which served as the detection reagent for the preparation of the immunochromatographic strips to test sulfadiazine. With this method, the semi-quantitative detection of sulfadiazine was accomplished in less than 15min, with high sensitivity to sulfadiazine (5ng/g) and low cross-reactivities with other sulfonamides. With experimental egg and chicken samples spiked with sulfadiazine at concentrations of 10, 20, and 100ng/g, recoveries were demonstrated to be from 71% to 97% in egg samples and 71% to 95% in chicken samples. This method was compared with the enzyme-linked immunosorbent assay by testing 52 egg samples from the animal experiment, and compared with the high-performance liquid chromatographic method by testing 56 chicken samples, with an agreement rate of 100% for both comparisons, by using the maximum allowed residue of sulfadiazine (i.e. 100ng/g) as the cut-off level as set by the European Union and China. The accuracy of ICA was also confirmed in an initial study with marketed egg and chicken samples. In conclusion, the method is rapid and accurate for the detection of sulfadiazine in eggs and chickens.