AP-1 and SP1 trans-activate the expression of hepatic CYP1A1 and CYP2A6 in the bioactivation of AFB1 in chicken

Dietary exposure to aflatoxin B1(AFB1)is harmful to the health and performance of domestic animals.The hepatic cytochrome P450s(CYPs),CYP1A1 and CYP2A6,are the primary enzymes responsible for the bioactivation of AFB1to the highly toxic exo-AFB1-8,9-epoxide(AFBO)in chicks.However,the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1metabolism remains unknown.Dual-luciferase reporter assay,bioinformatics and site-directed mutation results indicated that specificity protein 1(SP1)and activator protein-1(AP-1)motifs were located in the core region-1,063/-948,-606/-541 of the CYP1A1 promoter as well as-636/-595,-503/-462,-147/-1 of the CYP2A6 promoter.Furthermore,overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6.Moreover,bioactivation of AFB1to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression,which was trans-activated owing to the upregulation of AP-1,rather than SP1,stimulated by AFB1-induced reactive oxygen species.Additionally,nano-selenium could reduce ROS,downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6,thus alleviating the toxicity of AFB1.In conclusion,AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1to AFBO in chicken liver,which could provide novel targets for the remediation of aflatoxicosis in chicks.

Selenium deficiency-induced multiple tissue damage with dysregulation of immune and redox homeostasis in broiler chicks under heat stress

Broiler chicks are fast-growing and susceptible to dietary selenium(Se) deficiency. This study sought to reveal the underlying mechanisms of how Se deficiency induces key organ dysfunctions in broilers. Day-old male chicks(n=6 cages/diet, 6 chicks/cage) were fed with a Se-deficient diet(Se-Def, 0.047 mg Se/kg) or the Se-Def+0.3 mg Se/kg(Control, 0.345 mg Se/kg) for 6weeks. The serum, liver, pancreas, spleen, heart, and pectoral muscle of the broilers were collected at week 6 to assay for Se concentration, histopathology, serum metabolome, and tissue transcriptome. Compared with the Control group, Se deficiency induced growth retardation and histopathological lesions and reduced Se concentration in the five organs. Integrated transcriptomics and metabolomics analysis revealed that dysregulation of immune and redox homeostasis related biological processes and pathways contributed to Se deficiency-induced multiple tissue damage in the broilers. Meanwhile, four metabolites in the serum, daidzein, epinephrine, L-aspartic acid and 5-hydroxyindoleacetic acid, interacted with differentially expressed genes with antioxidative effects and immunity among all the five organs, which contributed to the metabolic diseases induced by Se deficiency. Overall, this study systematically elucidated the underlying molecular mechanisms in the pathogenesis of Se deficiency-related diseases, which provides a better understanding of the significance of Se-mediated heath in animals.

Translocation of gut microbes to epididymal white adipose tissue drives lipid metabolism disorder under heat stress

Heat stress induces multi-organ damage and serious physiological dysfunction in mammals,and gut bacteria may translocate to extra-intestinal tissues under heat stress pathology.However,whether gut bacteria translocate to the key metabolic organs and impair function as a result of heat stress remains unknown.Using a heat stress-induced mouse model,heat stress inhibited epididymal white adipose tissue(eWAT)expansion and induced lipid metabolic disorder but did not damage other organs,such as the heart,liver,spleen,or muscle.Microbial profiling analysis revealed that heat stress shifted the bacterial community in the cecum and eWAT but not in the inguinal white adipose tissue,blood,heart,liver,spleen,or muscle.Notably,gut-vascular barrier function was impaired,and the levels of some bacteria,particularly Lactobacillus,were higher in the eWAT,as confirmed by catalyzed reporter deposition fluorescence in situ hybridization(CARD-FISH)staining when mice were under heat stress.Moreover,integrated multi-omics analysis showed that the eWAT microbiota was associated with host lipid metabolism,and the expression of genes involved in the lipid metabolism in eWAT was upregulated under heat stress.A follow-up microbial supplementation study after introducing Lactobacillus plantarum to heat-stressed mice revealed that the probiotic ameliorated heat stress-induced loss of eWAT and dyslipidemia and reduced gut bacterial translocation to the eWAT by improving gut barrier function.Overall,our findings suggest that gut bacteria,particularly Lactobacillus spp.,play a crucial role in heat stress-induced lipid metabolism disorder and that there is therapeutic potential for using probiotics,such as Lactobacillus plantarum.

Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

Deoxynivalenol(DON)is an inevitable contaminant in animal feed and can lead to liver damage,then decreasing appetite and causing growth retardation in piglets.Although many molecular mechanisms are related to hepatoxicity caused by DON,few studies have been done on cytochrome P450(CYP450)enzymes and DNA methylation.To explore the role of CYP450 enzymes and DNA methylation in DONinduced liver injury,male piglets were fed a control diet,or diet containing 1.0 or 3.0 mg/kg DON for 4 weeks.DON significantly raised the activity of aspartate aminotransferase(AST),alanine aminotransferase(ALT),and glutamyl transpeptidase(GGT)(P<0.01),leading to liver injury.In vivo study found that DON exposure increased the expression of CYP450 enzymes(such as CYP1A1,CYP2E1,CYP3A29)(P<0.05),and disturbed the expression of nicotinamide N-methyltransferase(NNMT),galanin-like peptide(GALP)and insulin-like growth factor 1(IGF-1)(P<0.05),in which DNA methylation affected the expression of these genes.In vitro study(human normal hepatocytes L02)further proved that DON elevated the expression of CYP1A1,CYP2E1 and CYP3A4(P<0.05),and inhibited cell growth in a dose-dependent manner,resulting in cell necrosis.More importantly,knockdown of CYP1A1 or CYP2E1 could alleviate DON-induced growth inhibition by promoting IGF-1 expression.Taken together,increased CYP450 enzymes expression was one of the mechanisms of hepatoxicity and growth inhibition induced by DON,suggesting that the decrease of CYP450 enzymes can antagonize the hepatoxicity in animals,which provides some value for animal feed safety.

Adjuvant effects mediated by the carbohydrate recognition domain ofAgrocybe aegerita lectin interacting with avian influenza H9N2 viral surface glycosylated proteins

Objective： To evaluate the potential adjuvant effect of Agrocybe aegerita lectin （AAL）, which was isolatedfrom mushroom, against a virulent H9N2 strain in vivo and in vitro. Methods： In trial 1, 50 BALB/c male mice （8 weeksold） were divided into five groups （n=10 each group） which received a subcutaneous injection of inactivated HgN2（control）, inactivated HgN2＋0.2% （w/w） alum, inactivated HgN2＋0.5 mg recombinant AAL/kg body weight （BW）, inac-tivated HgN2＋1.0 mg AAl_/kg BW, and inactivated H9N2＋2.5 mg AAL/kg BW, respectively, four times at 7-d intervals. Intrial 2, 30 BALB/c male mice （8 weeks old） were divided into three groups （n=10 each group） which received a sub-cutaneous injection of inactivated HgN2 （control）, inactivated HmN2＋2.5 mg recombinant wild-type AAL （AAL-wt）/kg BWand inactivated H9N2＋2.5 mg carbohydrate recognition domain （CRD） mutant AAL （AAL-mutR63H）/kg BW,respectively, four times at 7-d intervals. Seven days after the final immunization, serum samples were collectedfrom each group for analysis. Hemagglutination assay, immunogold electron microscope, lectin blotting, and co-immunoprecipitation were used to study the interaction between hAL and HgN2 in vitro. Results： IgG, IgG1, and IgG2aantibody levels were significantly increased in the sera of mice co-immunized with inactivated HgN2 and AAL whencompared to mice immunized with inactivated H^N2 alone. No significant increase of the IgG antibody level was de-tected in the sera of the mice co-immunized with inactivated HgN2 and AAL-mutR63H. Moreover, AAL-wt, but notmutant AAL-mutR63H, adhered to the surface of H9N2 virus. The interaction between AAL and the H9N2 virus wasfurther demonstrated to be associated with the CRD of AAL binding to the surface glycosylated proteins, hemagglutininand neuraminidase. Conclusions： Our findings indicated that AAL could be a safe and effective adjuvant capable ofboosting humoral immunity against HgN2 viruses in mice through its interaction with the viral surface glycosylatedproteins, hemagglutinin and neuraminidase.

Cloning,expression,and characterization of a porcine pancreatic α-amylase in Pichia pastoris

Pancreatic α-amylase(α-1, 4-glucan-4-glucanohydrolase, EC.3.2.1.1) plays a primary role in the intestinal digestion of feed starch and is often deficient in weanling pigs.The objective of this study was to clone,express, and characterize porcine pancreatic α-amylase(PPA).The full-length c DNA encoding the PPA was isolated from pig pancreas by RT-PCR and cloned into the pPICZαA vector.After the resultant pPICZαА-PPA plasmid was transferred into Pichia pastoris, Ni Sepharose affinity column was used to purify the over-expressed extracellular recombinant PPA protein(re PPA) that contains a His-tag to the C terminus and was characterized against the natural enzyme(α-amylase from porcine pancreas).The re PPA exhibited a molecular mass of approximately 58 kDa and showed optimal temperature(50℃),optimal pH(7.5), K_m(47.8 mg/mL), and V_(max)(2,783 U/mg) similar to those of the natural enzyme.The recombinant enzyme was stable at 40℃ but lost 60% to 90%(P < 0.05) after exposure to heating at≥50℃ for 30 min.The enzyme activity was little affected by Cu^(2+)or Fe^(3+), but might be inhibited(40% to 50%) by Zn^(2+)at concentrations in pig digesta.However, Ca^(2+)exhibited a dose-dependent stimulation of the enzyme activity.In conclusion, the present study successfully cloned the porcine pancreatic aamylase gene and over-expressed the gene in P.pastoris as an extracellular, functional enzyme.The biochemical characterization of the over-produced enzyme depicts its potential and future improvement as an animal feed additive.

Effective protective agents against the organ toxicity of T-2 toxin and corresponding detoxification mechanisms:A narrative review

T-2 toxin is one of the most widespread and toxic fungal toxins in food and feed.It can cause gastrointestinal toxicity,hepatotoxicity,immunotoxicity,reproductive toxicity,neurotoxicity,and nephrotoxicity in humans and animals.T-2 toxin is physicochemically stable and does not readily degrade during food and feed processing.Therefore,suppressing T-2 toxin-induced organ toxicity through antidotes is an urgent issue.Protective agents against the organ toxicity of T-2 toxin have been recorded widely in the literature,but these protective agents and their molecular mechanisms of detoxification have not been comprehensively summarized.In this review,we provide an overview of the various protective agents to T-2 toxin and the molecular mechanisms underlying the detoxification effects.Targeting appropriate targets to antagonize T-2 toxin toxicity is also an important option.This review will provide essential guidance and strategies for the better application and development of T-2 toxin antidotes specific for organ toxicity in the future.