Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-kappaB signaling

Immunology. 2008 Mar;123(3):348-57. doi: 10.1111/j.1365-2567.2007.02683.x. Epub 2008 Jan 18.

Abstract

Glucosamine (GlcN) has been reported to possess several biomedical properties, and currently a great deal of attention has been focused on improving the functional properties of GlcN for different applications. Therefore, this study was conducted to introduce a carboxybutyryl functional group to GlcN and to find out the inhibitory mechanism of a novel GlcN derivative, carboxybutyrylated GlcN (CGlcN), on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in bacterial lipopolysaccharide (LPS)-induced mouse macrophages (RAW264.7 cells). In the initial experiments, the production of NO and prostaglandin E(2) (PGE(2)) was inhibited by CGlcN pretreatment and suggested the possibility of down-regulating their respective genes, iNOS and COX-2. Reverse transcription-polymerase chain reaction and Western blot analysis revealed that CGlcN can affect both transcriptional and translational levels of iNOS and COX-2 expression. The data from the nuclear factor-kappaB (NF-kappaB) promoter gene transfection experiment supported the idea that inhibition of iNOS and COX-2 is caused by the down-regulation of their transcription factor, NF-kappaB. Following stimulation with LPS, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) present upstream of NF-kappaB signaling were also inhibited by CGlcN treatment. However, the protein level of another MAPK, extracellular signal-regulated kinase (ERK), remained unaffected. Moreover, following treatment with CGlcN, the protein expression of I-kappaB kinase (IKK) clearly confirmed that its down-regulation directly inhibited the degradation of IkappaB and release of NF-kappaB. Therefore, it can be concluded that CGlcN is capable of inhibiting iNOS and COX-2 expression in LPS-induced RAW264.7 cells via attenuation of NF-kappaB signaling by p38 MAPK and JNK, but not by ERK.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Butyrates / pharmacology
  • Cell Survival / drug effects
  • Cells, Cultured
  • Cyclooxygenase 2 / immunology*
  • Cyclooxygenase 2 Inhibitors / pharmacology
  • Dinoprostone / biosynthesis
  • Dose-Response Relationship, Drug
  • Down-Regulation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation / drug effects
  • Glucosamine / pharmacology*
  • Lipopolysaccharides / immunology
  • Mice
  • Mitogen-Activated Protein Kinases / physiology
  • NF-kappa B / physiology
  • Nitric Oxide / biosynthesis
  • Nitric Oxide Synthase Type II / antagonists & inhibitors*
  • Nitric Oxide Synthase Type II / genetics
  • RNA, Messenger / genetics
  • Signal Transduction / drug effects

Substances

  • Butyrates
  • Cyclooxygenase 2 Inhibitors
  • Enzyme Inhibitors
  • Lipopolysaccharides
  • NF-kappa B
  • RNA, Messenger
  • Nitric Oxide
  • Nitric Oxide Synthase Type II
  • Ptgs2 protein, mouse
  • Cyclooxygenase 2
  • Mitogen-Activated Protein Kinases
  • Dinoprostone
  • Glucosamine