Chemical Space Charting of Different Parts of Inula nervosa Wall.: Upregulation of Expression of Nrf2 and Correlated Antioxidants Enzymes.

The edible and medicinal part of Inula nervosa Wall. (Xiaoheiyao) is confined to its root without sufficient phytochemical and biological investigation. In this study, the secondary metabolites of root, stem, leaf, and flower of I. nervosa Wall. were visualized using Global Natural Products Social Molecular Networking (GNPS), MolNetEnhancer, XCMS(xcmsonline.scripps.edu) analysis, and `ili mapping based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) data to reveal their chemical differences. Among the 11 kinds of chemical repertoires annotated by MolNetEnhancer and 16 hits against the GNPS library, 10-isobutyryloxy-8,9-epoxythymol isobutyrate (1) was revealed as the most dominant and responsible marker between the roots and the other parts. Moreover, a battery of unique MS features as well as differential markers were discovered from different parts of the plant. The chemical differences contribute to the bioactivity differences, which presented in the 2,2-diphenyl-1-picryl-hydrazyl (DPPH)assay and H2O2-insulted HepG2 cells and were in significant correlations with the contents of 1. real-time reverse transcription polymerase chain reaction (RT-PCR)results demonstrated that I. nervosa Wall. extracts upregulated the mRNA expression of nuclear factor E2-related factor 2(Nrf2), heme oxygenase 1(HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), manganese superoxide dismutase (MnSOD), and glutamate-cysteine ligase catalytic subunit (GCLC) actors involved in antioxidative response in H2O2-challenged HepG2 cells. These findings support the roots of I. nervosa Wall. as active parts of Xiaoheiyao, and also indicate the potential antioxidant activities of other parts.

Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora.

AIM: To demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) to exert immunomodulatory functions in the mouse small intestine. METHODS: Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro. RESULTS: TOTO-1 iodide staining confirmed existence of eDNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, suggesting the predominance for maintaining immune homeostasis of the gut. CONCLUSION: Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.

Structure-based approach for the study of thyroid hormone receptor binding affinity and subtype selectivity.

Thyroid hormone (TH) possesses the ability to lower cholesterol and improve cardiac performance, which have prompted the efforts to design analogs that can utilize the cholesterol-lowering property without adversely affecting heart function. In order to gain insights into the interaction mechanism for agonists at the active site of thyroid hormone receptor ß (TRß), quantitative structure-activity relationship (QSAR) models have been developed on TRß agonists, significant statistical coefficients were obtained (CoMFA, R(2)cv, .732), (CoMSIA, R(2)cv, .853), indicating the internal consistency of the models, the obtained models were further validated using the test set, the acquired R(2)pred values .7054 and .7129 were in good agreement with the experimental results. The key amino acids affecting ligand binding were identified by molecular docking, and the detailed binding modes of the compounds with different activities were also determined. Furthermore, molecular dynamics (MD) simulations were conducted to assess the reliability of the derived models and the docking results. Moreover, TH exerts significant physiological effects through modulation of the two human thyroid hormone receptor subtypes. Because TRß and TRα locate in different target cells, selective TR ligands would target specific tissues regulated by one receptor without affecting the other. Thus, the 3D information was analyzed to reveal the most relevant structural features involved in selectivity. The findings serve as the basis for further investigation into selective TRß/TRα agonists.

High-fat-diet-induced obesity is associated with decreased antiinflammatory Lactobacillus reuteri sensitive to oxidative stress in mouse Peyer's patches.

OBJECTIVE: Diet-induced inflammation in the small intestine may represent an early event that precedes and predisposes to obesity and insulin resistance. This is related to decrease of lactobacilli in Peyer's patches (PP) revealed in our previous study. The present study aimed to clarify specific changes of PP Lactobacillus on the strain level and related biological activity. METHODS: C57 BL/6 J male mice were fed with either low-fat diet (control [CT]; 10% calories from fat) or high-fat diet (HFD; 50% calories from fat) for 25 wk, and the HFD-fed mice were classified into obesity prone (OP) or obesity resistant (OR) on the basis of their body weight gain. Lactobacillus was isolated from PP using a selective medium. Oxidative resistance and cytokine-inducing effect were analyzed in vitro. RESULTS: We obtained 52, 18, and 22 isolates from CT, OP, and OR mice, respectively. They belonged to 13 different types according to enterobacterial repetitive intergenic consensus sequence-PCR analysis. Lactobacillus reuteri was the most abundant strain, but its abundance in OP mice was much lower than that in CT and OR mice. This strain includes eight subgroups according to genotyping. L. reuteri L3 and L. reuteri L8 were the specific strains found in CT and OP mice, respectively. Oxidative-resistant L. reuteri was much higher in HFD-fed mice. When co-cultured with PP cells, L8 induced higher production of proinflammatory cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factor-α, whereas L3 induced higher production of an anti-inflammatory cytokine (IL-10). CONCLUSION: HFD may induce oxidative stress that drives strain selection of Lactobacillus strains, resulting in decreased anti-inflammatory response in PP.

Dyslipidemic high-fat diet affects adversely bone metabolism in mice associated with impaired antioxidant capacity.

OBJECTIVE: The present study examined impacts of dyslipidemic high-fat diet on the bone antioxidant system and bone metabolism in growing mice. Furthermore, the relationship was studied between them. METHODS: Male C57BL/6 mice (4 wk old) were fed with normal diet, high-fat diet (HFD), or HFD supplemented with 0.1% antioxidant lipoic acid (LA). After 13-wk feeding, the markers of plasma lipids status, bone metabolism in plasma and in urine, and femora oxidative stress were measured. To provide molecular evidence for abnormal bone metabolism affected by HFD, bone cell-specific mRNA levels were tested by real-time quantitative polymerase chain reaction. Moreover, insulin-like growth factor I and tumor necrosis factor-alpha in plasma and their mRNA levels in femur were measured. RESULTS: The feeding dyslipidemic HFD induced both inhibitory bone formation reactions and enhancement of bone resorption reactions, accompanied by impaired bone antioxidant system, low levels of insulin-like growth factor I in plasma and in bone, and high levels of tumor necrosis factor-alpha in plasma but not in bone. In contrast, these alternatives were prevented completely or partially in mice fed LA supplement. Further, plasma propeptide of І collagen C-propeptide as a marker of bone formation was positively correlated with both total antioxidant capacity (r=0.683, P<0.001) and reduced glutathione/oxidized glutathione ratio (r=0.565, P<0.003) of bone. Cross-linked N-telopeptides of bone type І collagen as a marker of bone resorption was negatively correlated with both total antioxidant capacity (r=-0.753, P<0.001) and glutathione/oxidized glutathione ratio (r=-0.786, P<0.001). CONCLUSION: Dyslipidemia induces impaired bone antioxidant system. Oxidative stress could be an important mediator of hyperlipidemia-induced bone loss.

Antioxidative peptides derived from enzyme hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection.

This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen.

Effects of duodenal redox status on calcium absorption and related genes expression in high-fat diet-fed mice.

OBJECTIVE: This study investigated whether duodenal redox imbalance induced by high-fat diet (HFD) influenced expression of genes involved in transcellular calcium absorption, thus leading to reduced intestinal calcium absorption. METHODS: Male C57BL/6 mice were randomly assigned to one of four groups with eight mice in each group. The control group consumed an ordinary diet (4.9% fat, w/w). The other three groups were fed a HFD (21.2% fat), the HFD plus 0.1% lipoic acid, or the HFD plus an additional 0.9% calcium supplement. After 9 wk, plasma and duodenal oxidative stress biomarkers including malondialdehyde, superoxide dismutase, catalase, total antioxidant capacity, reduced glutathione/oxidized glutathione ratio, and reactive oxygen species were examined. The intestinal calcium absorption state was evaluated through examining the calcium balance, bone mineral density, and calcium metabolism biomarkers. Furthermore, quantitative reverse transcription-polymerase chain reaction was carried out to analyze the changes in expression of transcellular calcium absorption-related genes. RESULTS: The HFD induced marked decreases in intestinal calcium absorption and bone mineral density of the whole body, accompanied by redox imbalance and increased oxidative damage in duodenum; duodenal expression of calbindin-D(9K), plasma membrane calcium ATPase (PMCA(1b)), and sodium-calcium exchanger was significantly down-regulated by 1.9-, 2.7-, and 1.5-fold, respectively. Furthermore, duodenal glutathione and oxidized glutathione (GSH/GSSG) ratios were strongly positively correlated with the apparent calcium absorption rate and the expression of PMCA(1b) and Calbindin-D(9K), whereas reactive oxygen species levels were negatively correlated with them. CONCLUSION: Our results demonstrated that a HFD-induced duodenal oxidation state could significantly down-regulate expression of calbindin-D(9K), PMCA(1b), and sodium-calcium exchanger, thus causing an inhibitory effect on intestinal calcium absorption.

Reactive oxygen species serve as signals mediating glucose-stimulated somatostatin secretion from cultured rat gastric primary D-cells.

Somatostatin plays an important role in glucose homeostasis. It is normally secreted in response to glucose and ATP generation is believed to be the key transduction signal of glucose-stimulated somatostatin secretion (GSSS). However, in the present study, in cultured rat gastric primary D-cells, GSSS was accompanied by increases in cellular reactive oxygen species (ROS). GSSS is dependent on the cellular ROS and independently of the ATP production linked to glucose metabolism. The antioxidant, alpha-lipoic acid or catalase inhibitor, 3-aminotriazole can influence the intracellular calcium concentration and abolish or further elevate GSSS. It is suggested that ROS production may serve as a signal modulating the necessary Ca(2+) recruitment for GSSS. Since somatostatin is thought to exert broad regulatory functions on gastrointestinal physiology and nutrient intake, the interaction with ROS may lead to potential targets for mediating nutrition and energy homeostasis.

Effect of somatostatin analog on high-fat diet-induced metabolic syndrome: involvement of reactive oxygen species.

Oxidative stress plays an important role in overnutrition-induced metabolic syndrome. Somatostatin (SST) inhibits a wide variety of physiologic functions in the gastrointestinal tract, which may in turn control the levels of reactive oxygen species (ROS) derived from ingestion of macronutrients. In this study, the involvement of SST in the progression of metabolic syndrome in response to a high-fat diet (HFD) was investigated. Male C57BL/6 mice were fed either a normal diet (4.89% fat) or a high-fat diet (21.45% fat) for 4 weeks. The SST analog octreotide (20 microg/kg/day) was then administered intraperitoneally to half of the HFD mice throughout the 10-day experimental period. Body weight, adipose tissue weight, gastric acidity, total bile acid, and lipase activity were measured. Plasma lipid, glucose, insulin, SST, the levels of ROS and GSH/GSSG, and lipid peroxidation in the stomach, small intestine, pancreas, and liver were also evaluated. Following HFD intake for 38 days, a decrease in the plasma levels of SST and GSH/GSSG ratio was observed, while there was an increase in body weight, adipose tissue weight, plasma glucose, triglyceride, and levels of ROS and lipid peroxidation of the stomach, small intestine, pancreas, and liver. However, simultaneous administration of SST analog octreotide to HFD-fed mice significantly reduced ROS production of the digestive system and resulted in the improvement of all the aforesaid adverse changes, suggesting the involvement of SST in the progression of HFD-induced metabolic syndrome.

Microarray analysis of high-glucose diet-induced changes in mRNA expression in jejunums of C57BL/6J mice reveals impairment in digestion, absorption.

Long term intake of high-glucose diet (HGD) may induce many diseases such as dyslipidemia, fatty liver and diabetes disease. Most of the research for molecular mechanisms of the association between HGD and the above diseases focus on the metabolism of glucose and lipid. However, there are few studies on molecular mechanism of the effect of HGD on digestion and absorption. We used HGD (containing 20% glucose) to feed C57BL/6J mice for 4 weeks, detected the expressions of 13,098 genes in jejunums of C57BL/6J mice with DNA microarray. Microarray analysis showed the expression of genes related to digestive enzyme, gastrointestinal peptide and nutrient transporters were significantly changed, which indicated that HGD induced the suppression of digestive enzyme gene expression, attenuation of alimentary tract movement and nutrient transportation. In one word, the microarray analysis suggested that HGD impaired the function of digestion and absorption in jejunum of C57BL/6J mice. We validated our microarray findings by conducting real-time RT-PCR assays on selected genes and detecting the activities of disaccharidases such as lactase, maltase and sucrase in jejunum of C57BL/6J mice.

Design and analysis of structure-activity relationship of novel antimicrobial peptides derived from the conserved sequence of cecropin.

We have de novo designed four antimicrobial peptides AMP-A/B/C/D, the 51-residues peptides, which are based on the conserved sequence of cecropin. In the present study, the four peptides were chemically synthesized and their activities assayed. Their secondary structure, amphipathic property, electric field distribution and transmembrane domain were subsequently predicted by bioinformatics tools. Finally, the structure-activity relationship was analyzed from the results of activity experiments and prediction. The results of activity experiments indicated that AMP-B/C/D clearly possessed excellent broad-spectrum activity against bacteria, whereas AMP-A was almost inactive against most of the bacterial strains tested. AMP-B/C/D showed more potent activity against Gram-positive bacteria than against Gram-negative bacteria. By utilizing bioinformatics analysis tools, we found that the secondary structure of the four cation peptides was mainly alpha-helix, and the result of CD spectrum also displayed that all the peptides had considerable alpha-helix in the presence of either 50% TFE or SDS micelles. AMP-C showed much better activity than other peptides against most of the bacteria tested, owing to its remarkable cation property and the amphipathic character of its N-terminal. The study of structure-activity relationship of the designed peptides confirmed that amphipathic structure and high net positive charge were prerequisites for maintaining their activities.

Nonopsonic phagocytosis of Lactobacilli by mice Peyer's patches' macrophages.

The ingestion of lactobacilli is of great importance for the probiotic effect of host gut Peyer's patches (PPs) macrophages. The present study is in time focus on the investigation of the factors determining the ingestion of lactobacilli by PPs macrophages. Physicochemical properties of cell surface and adhesive property of nine Lactobacillus strains were examined in the present work. The association of the bacteria with PPs macrophage was checked with macrophage monolayers on coverslips. The influence of lactobacilli on macrophages phagocytic capacity was also investigated with a neutral red uptake assay in vitro. The results show that the macrophages could ingest lactobacilli in a strain dependent manner, and the most ingested strain is L. plantarum Lp6 compared to other tested strains, which displayed strain specific enhancement on the phagocytic activity of PPs macrophages. And there is no correlation between the physicochemical or adhesive properties of the cell surface and the ingestion. The association of L. plantarum Lp6 with PPs macrophage could be decreased by Protease K treatment. Surface proteins of L. plantarum Lp6 could promote the ingestion of fluorescent latex beads by PPs macrophages. In conclusion, the hydrophobicity of the cell surface might not be the key factor determining the association of lactobacilli with PPs macrophages. Cell surface proteins are the media for the binding L. plantarum Lp6 to macrophages.

Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein.

Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively.

[Antibacterial spectrum of antibacterial peptides from Musca domestica larvae and synergic interaction between the peptides and antibiotics].

In vitro antibacterial activities of three antibacterial peptides, alone and in combination with the antibiotics were measured by MIC and antibacterial spectrum. Synergy was observed when the peptides were combined with penicillin, streptomycin by FIC. The results show that antibacterial peptides have stronger inhibition of pathogenic bacterium, and antibacterial spectrum are very different. The interaction in antibacterial peptides, antibiotics and bacterium is extraordinary complex, except synergic action, there are antagonism and indifference.

Direct spectrophotometric measurement of angiotensin I-converting enzyme inhibitory activity for screening bioactive peptides.

A direct, extraction-free spectrophotometric assay was developed for determination of angiotensin I-converting enzyme activity (ACE) in the presence of ACE inhibitors using hippuryl-l-histidyl-l-leucine (HHL) as the ACE-specific substrate. This method relies on previously published spectrophotometric determination of hippuric acid (HA) content in the urine, the method of which was based on the specific colorimetric reaction of HA with benzene sulfonyl chloride (BSC) in the presence of quinoline. The proposed ACE inhibition assay was applied to the measurement of the ACE inhibitory activity of Captopril. IC(50) value of Captopril corresponded well with literature data. Furthermore, Alcalase hydrolysates of mung bean and rice protein isolates were assessed for ACE inhibitory activity by this method. These two hydrolysates showed high ACE inhibitory activity. This method proposed here was shown to be direct, sensitive, accurate, reproducible, and less expensive without separation of HA from ACE reaction mixture, and can be used for the screening of ACE inhibitory peptides derived from food proteins.

Distinct immune response induced by peptidoglycan derived from Lactobacillus sp.

AIM: To analyze the distinct immune responses induced by Lactobacillus peptidoglycan (PG). METHODS: BALB/c mice were intraperitoneally injected with PG once a day for three consecutive days. Peritoneal macrophage and splenocyte mRNA was extracted and the gene expression profile was studied using high-density oligonucleotide microarrays. Inhibitory effects of Lactobacillus PG on colon tumor tissue were studied in vitro and in vivo. RESULTS: The gene expression profiles revealed that the TLR-NF-kappaB; and Jak-STAT signaling pathways were highly activated. An inflammatory phenotype was induced when peritoneal macrophages were initially exposed to Lactobacillus PG and switched to a more complex phenotype when BALB/c mice were treated with three doses of Lactobacillus PG. A protective physiological inflammatory response was induced after three consecutive days of PG treatment. It was tending toward Th1 dominant immune response. Lactobacillus PG also appeared to induce a significant in vivo anti-colon tumor effect. CONCLUSION: Lactobacillus PG is responsible for certain immune responses induced by Lactobacilli. Anti-tumor effects of Lactobacilli are likely to attribute to the activation of macrophages by PG expressed on the bacterial cell surface.