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‐κB signaling

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.