Functional Identification of HhUGT74AG11-A Key Glycosyltransferase Involved in Biosynthesis of Oleanane-Type Saponins in Hedera helix.

Hedera helix is a traditional medicinal plant. Its primary active ingredients are oleanane-type saponins, which have extensive pharmacological effects such as gastric mucosal protection, autophagy regulation actions, and antiviral properties. However, the glycosylation-modifying enzymes responsible for catalyzing oleanane-type saponin biosynthesis remain unidentified. Through transcriptome, cluster analysis, and PSPG structural domain, this study preliminarily screened four candidate UDP-glycosyltransferases (UGTs), including Unigene26859, Unigene31717, CL11391.Contig2, and CL144.Contig9. In in vitro enzymatic reactions, it has been observed that Unigene26859 (HhUGT74AG11) has the ability to facilitate the conversion of oleanolic acid, resulting in the production of oleanolic acid 28-O-glucopyranosyl ester. Moreover, HhUGT74AG11 exhibits extensive substrate hybridity and specific stereoselectivity and can transfer glycosyl donors to the C-28 site of various oleanane-type triterpenoids (hederagenin and calenduloside E) and the C-7 site of flavonoids (tectorigenin). Cluster analysis found that HhUGT74AG11 is clustered together with functionally identified genes AeUGT74AG6, CaUGT74AG2, and PgUGT74AE2, further verifying the possible reason for HhUGT74AG11 catalyzing substrate generalization. In this study, a novel glycosyltransferase, HhUGT74AG11, was characterized that plays a role in oleanane-type saponins biosynthesis in H. helix, providing a theoretical basis for the production of rare and valuable triterpenoid saponins.

Effect of the Combination of Clostridium butyricum and Mycelium of Phellinus igniarius on Intestinal Microbiota and Serum Metabolites in DSS-Induced Colitis.

Clostridium butyricum (CB) and Phellinus igniarius (PI) have anti-inflammatory, immune regulation, anti-tumor, and other functions. This study aimed to explore the therapeutic effect of CB and mycelium of PI (MPI) alone and in combination on colitis mice induced by dextran sodium sulfate (DSS). Mice were randomly assigned to five groups: (1) control (CTRL), (2) DSS, (3) CB, (4) MPI, and (5) CB + MPI (CON). The weight of the mice was recorded daily during the experiment, and the length of the colon was measured on the last day of the experiment. The colons were collected for hematoxylin and eosin staining, colon contents were collected for intestinal flora analysis, and serum was collected for metabolite analysis. The results showed that compared with the DSS group, CB, MPI, and CON treatments inhibited the weight loss and colon length shortening caused by DSS, significantly increased the concentrations of interleukin (IL)-4, IL-10, and superoxide dismutase, and significantly decreased the concentrations of IL-6, tumor necrosis factor-α, and myeloperoxidase. Gene sequence analysis of 16S rRNA showed that CB, MPI, and CON treatments changed the composition and structure of intestinal microorganisms. Metabolome results showed that CB, MPI, and CON treatments changed serum metabolites in DSS-treated mice, including dodecenoylcarnitine, L-urobilinogen, and citric acid. In conclusion, CB, MPI, and CON treatments alleviated DSS-induced colitis in mice by regulating intestinal flora and metabolites, with the CON group having the best effect.

Colonic phosphocholine is correlated with Candida tropicalis and promotes diarrhea and pathogen clearance.

Diarrhea is characterized by alterations in the gut microbiota, metabolites, and host response to these changes. Studies have focused on the role of commensal bacteria in diarrhea; however, the effect of fungi on its pathogenesis remains unexplored. Here, using post-weaned piglets with or without diarrhea, we found an unexpected decrease in the abundance of Candida tropicalis in diarrheal piglets. We also observed increased accumulation of reactive oxygen species (ROS) and the formation of neutrophil extracellular traps (NETs) in the colonic tissues of diarrheal piglets. Using dectin-1-knockout mice, we found that the over-accumulation of ROS killed C. tropicalis by promoting NET formation, which was dependent on dectin-1. The decreased abundance of C. tropicalis resulted in reduced phosphocholine consumption. Then, colonic phosphocholine accumulation drives water efflux by increasing cAMP levels by activating adenylyl cyclase, which promotes the clearance of pathogenic bacteria. Collectively, we demonstrated that phosphocholine is correlated with colonic C. tropicalis and promotes diarrhea and pathogen clearance. Our results suggest that mycobiota colonizing the colon might be involved in maintaining intestinal metabolic homeostasis through the consumption of certain metabolites.

Antioxidant and Anti-Inflammatory Effects of Different Zinc Sources on Diquat-Induced Oxidant Stress in a Piglet Model.

Zinc (Zn) plays a crucial role in reducing oxidative stress and diarrhea in postweanling piglets. This study is aimed at comparing the effects of zinc chelate of 2-hydroxy-4 methyl-thio butanoic acid (HMZn) and ZnSO4 on the oxidative stress in weaned piglets. A total of 32 piglets were randomly divided into 4 treatments: CON: basal diet+80 mg/kg Zn as ZnSO4; DIQ: basal diet+80 mg/kg Zn as ZnSO4; HMZn: basal diet+200 mg/kg Zn as HMZn; and ZnSO4: basal diet+200 mg/kg Zn as ZnSO4. On day 15, the DIQ, HMZn, and ZnSO4 groups were injected intraperitoneally with diquat except for the CON group. The trial lasted 21 days. The results showed that zinc sources did not influence the growth performance during the first 14 days. But HMZn increased activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and total antioxidant capacity (T-AOC) in serum (P < 0.05). After diquat injection, the fecal score was decreased in the HMZn group. Both HMZn and ZnSO4 increased the activities of GPX and T-AOC in serum and the relative mRNA expressions of hepatic and renal Nrf2, SOD1, and GPX compared with the DIQ group (P < 0.05). Moreover, the relative mRNA expression of inflammatory factors in the small intestine, liver, and kidney was downregulated; the phosphorylation of NF-κB protein was inhibited in the HMZn group compared with the DIQ and ZnSO4 groups (P < 0.05). In general, HMZn showed notable advantage over ZnSO4 in reducing diarrhea and improving antioxidant and anti-inflammatory ability in piglets challenged with diquat.

Sulfur-containing amino acid supplementation to gilts from late pregnancy to lactation altered offspring's intestinal microbiota and plasma metabolites.

Maternal nutrition during late pregnancy and lactation is highly involved with the offspring's health status. The study was carried out to evaluate the effects of different ratios of methionine and cysteine (Met/Cys: 46% Met, 51% Met, 56% Met, and 62% Met; maintained with 0.78% of total sulfur-containing amino acids; details in "Materials and methods") supplements in the sows' diet from late pregnancy to lactation on offspring's plasma metabolomics and intestinal microbiota. The results revealed that the level of serum albumin, calcium, iron, and magnesium was increased in the 51% Met group compared with the 46% Met, 56% Met, and 62% Met groups. Plasma metabolomics results indicated that the higher ratios of methionine and cysteine (0.51% Met, 0.56% Met, and 0.62% Met)-supplemented groups enriched the level of hippuric acid, retinoic acid, riboflavin, and δ-tocopherol than in the 46% Met group. Furthermore, the 51% Met-supplemented group had a higher relative abundance of Firmicutes compared with the other three groups (P < 0.05), while the 62% Met-supplemented group increased the abundance of Proteobacteria compared with the other three groups (P < 0.05) in piglets' intestine. These results indicated that a diet consisting with 51% Met is the optimum Met/Cys ratio from late pregnancy to lactation can maintain the offspring's health by improving the serum biochemical indicators and altering the plasma metabolomics profile and intestinal gut microbiota composition, but higher proportion of Met/Cys may increase the possible risk to offspring's health.

Biological Effects and Applications of Chitosan and Chito-Oligosaccharides.

The numerous functional properties and biological effects of chitosan and chito-oligosaccharides (COS) have led to a significant level of interest, particularly with regard to their potential use in the agricultural, environmental, nutritional, and pharmaceutical fields. This review covers recent studies on the biological functions of COS and the impacts of dietary chitosan and COS on metabolism. The majority of results suggest that the use of chitosan as a feed additive has favorable biological effects, such as antimicrobial, anti-oxidative, cholesterol reducing, and immunomodulatory effects. The biological impacts reviewed herein may provide a new appreciation for the future use of COS.

Macleaya cordata extract alleviated oxidative stress and altered innate immune response in mice challenged with enterotoxigenic Escherichia coli.

This study examines the effects of dietary Macleaya cordata extract (MCE) on bacterial burden and resistance to enterotoxigenic Escherichia coli (ETEC) in ICR mice. ICR mice were randomly distributed into one of the following groups: (i) basal diet; (ii) basal diet with 200 mg kg-1 MCE; (iii) basal diet challenged with ETEC; and (iv) basal diet with 200 mg kg-1 MCE and challenged with ETEC. Following a 7-day period of pre-treatment, CTRL-ETEC and MCE-ETEC mice were subjected to oral infection using 5×108E. coli SEC 470. The results showed dietary 200 mg kg-1 MCE markedly reduced intestinal ETEC burden (P < 0.05) and the disease-associated mortality was significantly alleviated in the MCE treated group (P < 0.05). In addition, dietary MCE markedly alleviated ETEC-induced oxidative stress, evidenced by the lowered methane dicarboxylic aldehyde (MDA) abundance and enhanced activities of catalase and glutathione peroxidase (P < 0.05). Furthermore, MCE mice exhibited higher immune activity, which might have further mediated ETEC infection. These results indicate MCE plays a preventative role with respect to ETEC infection. Future research should aim to develop MCE as a therapeutic approach to the promotion of intestinal health and a safeguard against ETEC infection.

Involvement of calcium-sensing receptor activation in the alleviation of intestinal inflammation in a piglet model by dietary aromatic amino acid supplementation.

Ca2+-sensing receptor (CaSR) represents a potential therapeutic target for inflammatory bowel diseases and strongly prefers aromatic amino acid ligands. We investigated the regulatory effects of dietary supplementation with aromatic amino acids - tryptophan, phenylalanine and tyrosine (TPT) - on the CaSR signalling pathway and intestinal inflammatory response. The in vivo study was conducted with weanling piglets using a 2 × 2 factorial arrangement in a randomised complete block design. Piglets were fed a basal diet or a basal diet supplemented with TPT and with or without inflammatory challenge. The in vitro study was performed in porcine intestinal epithelial cell line to investigate the effects of TPT on inflammatory response using NPS-2143 to inhibit CaSR. Dietary supplementation of TPT alleviated histopathological injury and decreased myeloperoxidase activity in intestine challenged with lipopolysaccharide. Dietary supplementation of TPT decreased serum concentration of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-12, granulocyte-macrophage colony-stimulating factor, TNF-α), as well as the mRNA abundances of pro-inflammatory cytokines in intestine but enhanced anti-inflammatory cytokines IL-4 and transforming growth factor-ß mRNA levels compared with pigs fed control diet and infected by lipopolysaccharide. Supplementation of TPT increased CaSR and phospholipase Cß2 protein levels, but decreased inhibitor of NF-κB kinase α/ß and inhibitor of NF-κB (IκB) protein levels in the lipopolysaccharide-challenged piglets. When the CaSR signalling pathway was blocked by NPS-2143, supplementation of TPT decreased the CaSR protein level, but enhanced phosphorylated NF-κB and IκB levels in IPEC-J2 cells. To conclude, supplementation of aromatic amino acids alleviated intestinal inflammation as mediated through the CaSR signalling pathway.

The Anticancer Effect of Sanguinarine: A Review.

In vitro and in vivo studies have revealed that Sanguinarine has antioxidant, anti-inflammatory, proapoptotic, and growth inhibitory effects on tumor cells of a variety of cancers. Previous research showed that sanguinarine induced apoptosis (cell death) and/or antiproliferative while reducing tumor cell antiangiogenic and anti-invasive properties. This paper describes various sanguinarine anti-cancer mechanisms, including inhibition of erroneously-activated signal transduction pathways, apoptosis, and tumor cell proliferation inhibition.

Implications of Antioxidant Systems in Inflammatory Bowel Disease.

The global incidence of inflammatory bowel disease (IBD), a group of chronic gastrointestinal disorders, has been rising. The preponderance of evidence demonstrates that oxidative stress (OS) performs a critical function in the onset of IBD and the manner of its development. The purpose of this review is to outline the generation of reactive oxygen species and antioxidant defense mechanisms in the gastrointestinal tract and the role played by OS in marking the onset and development of IBD. Furthermore, the review demonstrates the various ways through which OS is related to genetic susceptibility and the mucosal immune response. The experimental results suggest that certain therapeutic regimens for IBD could have a favorable impact by scavenging free radicals, reducing cytokine and prooxidative enzyme concentrations, and improving the antioxidative capabilities of cells. However, antioxidative activity characterized by a high level of specificity may be fundamental for the development of clinical therapies and for relapsing IBD patients. Therefore, additional research is required to clarify the ways through which OS is related to the pathogenesis and progression of IBD.

L-Glutamine and L-arginine protect against enterotoxigenic Escherichia coli infection via intestinal innate immunity in mice.

Dietary glutamine (Gln) or arginine (Arg) supplementation is beneficial for intestinal health; however, whether Gln or Arg may confer protection against Enterotoxigenic Escherichia coli (ETEC) infection is not known. To address this, we used an ETEC-infected murine model to investigate the protective effects of Gln and Arg. Experimentally, we pre-treated mice with designed diet of Gln or Arg supplementation prior to the oral ETEC infection and then assessed mouse mortality and intestinal bacterial burden. We also determined the markers of intestinal innate immunity in treated mice, including secretory IgA response (SIgA), mucins from goblet cells, as well as antimicrobial peptides from Paneth cells. ETEC colonized in mouse small intestine, including duodenum, jejunum, and ileum, and inhibited the mRNA expression of intestinal immune factors, such as polymeric immunoglobulin receptor (pIgR), cryptdin-related sequence 1C (CRS1C), and Reg3γ. We found that dietary Gln or Arg supplementation decreased bacterial colonization and promoted the activation of innate immunity (e.g., the mRNA expression of pIgR, CRS1C, and Reg3γ) in the intestine of ETEC-infected mice. Our results suggest that dietary arginine or glutamine supplementation may inhibit intestinal ETEC infection through intestinal innate immunity.

N-Acetyl-L-cysteine Protects the Enterocyte against Oxidative Damage by Modulation of Mitochondrial Function.

The neonatal small intestine is susceptible to damage caused by oxidative stress. This study aimed to evaluate the protective role of antioxidant N-acetylcysteine (NAC) in intestinal epithelial cells against oxidative damage induced by H2O2. IPEC-J2 cells were cultured in DMEM-H with NAC and H2O2. After 2-day incubation, IPEC-J2 cells were collected for analysis of DNA synthesis, antioxidation capacity, mitochondrial respiration, and cell apoptosis. The results showed that H2O2 significantly decreased (P < 0.05) proliferation rate, mitochondrial respiration, and antioxidation capacity and increased cell apoptosis and the abundance of associated proteins, including cytochrome C, Bcl-XL, cleaved caspase-3, and total caspase-3. NAC supplementation remarkably increased (P < 0.05) proliferation rate, antioxidation capacity, and mitochondrial bioenergetics but decreased cell apoptosis. These findings indicate that NAC might rescue the intestinal injury induced by H2O2.

Dietary Chitosan Supplementation Increases Microbial Diversity and Attenuates the Severity of Citrobacter rodentium Infection in Mice.

C57BL/6 mice were tested in order to investigate the effects of dietary chitosan (COS) supplements on intestinal microflora and resistance to Citrobacter rodentium infection. The findings reveal that, after consuming a 300 mg/kg COS diet for 14 days, microflora became more diverse as a result of the supplement. Mice receiving COS exhibited an increase in the percentage of Bacteroidetes phylum and a decrease in the percentage of Firmicutes phylum. After Citrobacter rodentium infection, the histopathology scores indicated that COS feeding resulted in less severe colitis. IL-6 and TNF-α were significantly lower in colon from COS-feeding mice than those in the control group. Furthermore, mice in COS group were also found to experience inhibited activation of nuclear factor-kappa B (NF-κB) in the colonic tissue. Overall, the findings revealed that adding 300 mg/kg COS to the diet changed the composition of the intestinal microflora of mice, resulting in suppressed NF-κB activation and less production of TNF-α and IL-6; and these changes led to better control of inflammation and resolution of infection with C. rodentium.

Low Dosage of Chitosan Supplementation Improves Intestinal Permeability and Impairs Barrier Function in Mice.

The purpose of this study was to explore relationships between low dose dietary supplementation with chitosan (COS) and body weight, feed intake, intestinal barrier function, and permeability in mice. Twenty mice were randomly assigned to receive an unadulterated control diet (control group) or a dietary supplementation with 30 mg/kg dose of chitosan (COS group) for two weeks. Whilst no significant differences were found between the conditions for body weight or food and water intake, mice in the COS group had an increased serum D-lactate content (P < 0.05) and a decreased jejunal diamine oxidase (DAO) activity (P < 0.05). Furthermore, mice in COS group displayed a reduced expression of occludin and ZO-1 (P < 0.05) and a reduced expression of occludin in the ileum (P < 0.05). The conclusion drawn from these findings showed that although 30 mg/kg COS-supplemented diet had no effect on body weight or feed intake in mice, this dosage may compromise intestinal barrier function and permeability. This research will contribute to the guidance on COS supplements.

Macleaya cordata Extract Decreased Diarrhea Score and Enhanced Intestinal Barrier Function in Growing Piglets.

Macleaya cordata extract is of great scientific and practical interest to researchers, due to its antimicrobial and anti-inflammatory responses within experimental animals. This study was designed to determine the diarrhea score and innate immunity of growing piglets after they had received Macleaya cordata extract supplements. A total of 240 growing pigs were randomly assigned to one of three dietary treatments, with 8 replicates per treatment and 10 piglets per replicate. All pigs received a basal diet containing similar amounts of nutrients. The three treatments were a control (no additive), an antibiotic (200 mg/kg colistin), and the Macleaya cordata extract supplement group (40 mg/kg Macleaya cordata extract). The diarrhea score was calculated after D 28. The jejunal samples were obtained from five piglets selected randomly from each treatment on D 28. In comparison with the control group, the dietary Macleaya cordata extract and colistin group demonstrated a substantially decreased diarrhea score. The introduction of Macleaya cordata extract supplements to the diet significantly increased volumes of ZO-1 and claudin-1, particularly in comparison with the pigs in the control group (P < 0.05). The findings indicate that Macleaya cordata extract does enhance intestinal barrier function in growing piglets and that it could be used as a viable substitute for antibiotics.

Analysis of the Impact of Isoquinoline Alkaloids, Derived from Macleaya cordata Extract, on the Development and Innate Immune Response in Swine and Poultry.

Medicinal extract has been chronicled extensively in traditional Chinese medicine. Isoquinoline alkaloids, extract of Macleaya cordata (Willd.) R. Br., have been used as feed additive in both swine and poultry. Dietary supplementation with isoquinoline alkaloids increases feed intake and weight gain. In addition, recent researches have demonstrated that isoquinoline alkaloids can regulate metabolic processes, innate immune system, and digestive functioning in animals. This review summarizes the latest scientific researches on isoquinoline alkaloids which are extracted from Macleaya cordata (Willd.) R. Br. This review specifically focuses on its role as a feed supplement and its associated impact on growth performance and innate immune system, as well as its capacity to act as a substitute for oral antibiotics.

Chitosan Modulates Inflammatory Responses in Rats Infected with Enterotoxigenic Escherichia coli.

This study aims to investigate the effects of dietary chitosan (COS) on gastrointestinal pathogen resistance in mice model. For two weeks, a control group of ICR mice received a basal diet whilst the intervention group received the basal diet supplemented with 300 mg/kg COS. After two weeks, the mice fed the supplemented diet had a lower body weight. Then enterotoxigenic Escherichia coli (E. coli) was administered to the mice through oral gavage, with each mouse receiving 108 CFU. At day 7 after infection, the bacterial load in the jejunum and faeces was significantly lower in the COS group than that in the control group. Moreover, the mRNA and protein levels of IL-1ß, IL-6, IL-17, IL-18, and TNF-α were significantly lower in the group of mice receiving the COS diet; also the jejunal production of toll-like receptor-4 (TLR-4) was suppressed in the COS group. These results indicate the intervention influenced inflammation and controlled E. coli infection.

Characterization and Regulation of the Amino Acid Transporter SNAT2 in the Small Intestine of Piglets.

The sodium-dependent neutral amino acid transporter 2 (SNAT2), which has dual transport/receptor functions, is well documented in eukaryotes and some mammalian systems, but has not yet been verified in piglets. The objective of this study was to investigate the characteristics and regulation of SNAT2 in the small intestine of piglets. The 1,521-bp porcine full cDNA sequence of SNAT2 (KC769999) from the small intestine of piglets was cloned. The open reading frame of cDNA encodes 506 deduced amino acid residues with a calculated molecular mass of 56.08 kDa and an isoelectric point (pI) of 7.16. Sequence alignment and phylogenetic analysis revealed that SNAT2 is highly evolutionarily conserved in mammals. SNAT2 mRNA can be detected in the duodenum, jejunum and ileum by real-time quantitative PCR. During the suckling period from days 1 to 21, the duodenum had the highest abundance of SNAT2 mRNA among the three segments of the small intestine. There was a significant decrease in the expression of SNAT2 mRNA in the duodenal and jejunal mucosa and in the expression of SNAT2 protein in the jejunal and ileal mucosa on day 1 after weaning (P < 0.05). Studies with enterocytes in vitro showed that amino acid starvation and supplementation with glutamate, arginine or leucine enhanced, while supplementation with glutamine reduced, SNAT2 mRNA expression (P < 0.05). These results regarding the characteristics and regulation of SNAT2 should help to provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine.

Effect of dietary selenium yeast supplementation on porcine circovirus type 2 (PCV2) infections in mice.

The present study was performed to determine the protective role of dietary selenium (Se) yeast supplementation in porcine circovirus type 2 (PCV2) infected mice. Forty-eight Kun Ming female mice were randomly assigned to Se yeast group (0.3%Se +basal diet, n = 24) and control group (basal diet, n = 24). After 3 days of adaptive feeding and 15 days treatment with the experimental feed, mice were challenged by intraperitioneal injection of PCV2 at the dosage of 2000 TCID50 (50% tissue culture infection dose, TCID50). Serum total superoxide dismutase (SOD) activity, malondialdehyde (MDA) level, tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP) and interleukin-1 beta (IL-1ß) levels were measured at 5, 10, 15, 20 days post infection (dpi). The PCV2 virus load in the liver, spleen and lung, and the microscopic lesions in the liver, spleen and lung also were determined on 5, 10, 15, and 20 dpi. Dietary Se yeast supplementation decreased (Pµ0.05) the serum levels of TNF-α, but had no significant effect on the activity of SOD and the levels of MDA, CRP and IL-1ß between experimental and control groups. Dietary Se yeast supplementation had little effect on the PCV2 virus load in the liver, spleen and lung. However, mice in the selenium yeast group showed a significant decrease in microscopic lesion scores in the lung and spleen compared with those in the control group (Pµ0.05). These data indicate Se yeast attenuated the PCV2 infection through altering the systemic inflammation and maintaining the normal organ morphology.

Purification and characterization of a novel serine protease from the mushroom Pholiota nameko.

A novel serine protease, with a molecular mass of 19 kDa and the N-terminal sequence of ARTPEAPAEV, was isolated from dried fruiting bodies of the mushroom Pholiota nameko. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, Q-Sepharose and SP-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on DEAE-cellulose and Q-Sepharose but adsorbed on SP-Sepharose. It exhibited an optimum temperature at 50°C, an optimum pH at pH 8.8, a Km of 5.64 mg/mL and a Vmax of 0.98 µmol/min/mL against substrate casein. A number of metal ions inhibited the enzyme including Pb(2+), Mn(2+), Ca(2+), Hg(2+), Zn(2+), Cu(2+), Co(2+), Fe(3+) and Al(3+), with the inhibition of the last two cations being the most potent. K(+) and Mg(2+) slightly enhanced, while Li(+) moderately potentiated the activity of the protease. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease.