title: Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin.
abstract: BACKGROUND
  Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1(-/-) and PPAR-α(-/-) knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis.
  
  
  RESULTS
  Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters.
  
  
  CONCLUSIONS
  The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.
authors.affiliation: Department of Basic Medical Sciences, University of Missouri-Kansas City, 2411 Holmes Street, Kansas City, MO 64108, USA. qureshia@umkc.edu
authors.forename: Asaf A
authors.initials: AA
authors.lastname: Qureshi
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Tan
authors.forename: Julia C
authors.initials: JC
authors.lastname: Reis
authors.forename: Mostafa Z
authors.initials: MZ
authors.lastname: Badr
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Papasian
authors.forename: David C
authors.initials: DC
authors.lastname: Morrison
authors.forename: Nilofer
authors.initials: N
authors.lastname: Qureshi
mesh_terms: D000367:Age Factors; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D019587:Dietary Supplements; D020869:Gene Expression Profiling; D007249:Inflammation / Q000188:drug therapy / Q000276:immunology; D016899:Interferon-beta / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011794:Quercetin / Q000494:pharmacology* / Q000627:therapeutic use; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014810:Vitamin E / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D015430:Weight Gain / Q000187:drug effects
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D014810:Vitamin E; C082097:tocotrienol, delta; D009569:Nitric Oxide; D016899:Interferon-beta; D003907:Dexamethasone; D007371:Interferon-gamma; D011794:Quercetin; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse