| IntroductionIn recent years,many different clinical and pathological studies have altered the concepts for the pathogenesis of atherosclerosis.Although large lipid deposits can be seen in atheromatous lesions and the role of different lipoproteins are suggested in the pathogenesis,atherosclerosis is no longer considered to be a primary disorder of lipid accumulation.It is a state of disordered immunity in which there is dynamic interaction between endothelial dysfunction,inflammation and repeated cycles of "wound healing response".Expression of endothelial cell adhesion molecules such as intercellular adhesion molecule(ICAM-1),induces the binding of monocytes and lymphocytes,thus initiating an inflammatory process that ultimately leads to the formation of atherosclerotic plaque.The migration of inflammatory cells into the subendothelial space is facilitated by chemoattractants such as monocyte chemoattractant protein-1 (MCP-1) and oxidized low-density lipoprotein.Once migrated in the subendothelial space,monocytes mature into macrophages and express scavenger receptors to internalize modified lipoproteins,which gives rise to lipid-laden macrophages or foam cells.Pro-inflammatory cytokines,such as tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β),are found in atherosclerosis lesions,and have been known to contribute to the inflammatory process.Previous studies have shown that these cytokines can induce the expression of cell adhesion molecules,chemokines and genes in endothelial cells involved in the regulation of vessel tonicity,thrombosis and recruitment of leukocytes.Human cationic antibacterial protein of 18 kDa(hCAP18),also owes its name from its 37-amino acid peptide with the two leading leucine residues.Of importance,it has been recently revealed that LL-37 is expressed in the atherosclerotic plaques and involved in inflammatory responses in endothelial cells via the induction of ICAM-1 and MCP-1 expression,and vascular smooth muscle cell death.Glucosamine,a naturally occurring amino monosaccharide,is acting as a preferred substrate for biosynthesis of glycosaminoglycan,and is used for the treatment of osteoarthritis.Several short- and long-term clinical trials in osteoarthritis have shown the significant symptom-modifying effect of glucosamine with no side effects. Furthermore,glucosamine is shown to inhibit the expression of inducible nitric oxide synthase in macrophages,and neutrophil functions such as superoxide generation, phagocytosis,granule enzyme release,chemotaxis and CD11b expression,thereby possibly exhibiting an anti-inflammatory action.More recently,glucosamine has been revealed to inhibit ICAM-1 expression in human retinal pigment epithelial cells,which suggests a potential of glucosamine to attenuate inflammation in eyes.Based on these findings,we hypothesized that glucosamine may affect the endothelial cell activation. In this research,we investigated the effect of glucosamine on the activation of endothelial cells(expression of a monocyte chemoattractant factor MCP-1 and an adhesion molecule ICAM-1) induced by TNF-α,a cytokine and LL-37,an antimicrobial peptide expressed in the atherosclerotic lesions.Methods1,cell experiment(1) cell culture and treatmentHuman umbilical vein endothelial cells(HUVEC) were maintained in the endothelial cell culture medium-EGM-2.HUVECs(50-60%confluent) were preincubated with different concentrations of glucosamine for 2h,then stimulated by 4μM LL-37 or 0.5ng/ml TNF-αfor 24h(for mRNA and protein determination) or 10min(for signal transduction analysis). (2)The effect of glucosamine on LL-37 or TNF-αinduced ICAM-1,MCP-1mRNA expression was evaluated by real time RT-PCR.(3)MCP-1 protien concentrations were determined by ELISA using the supernatant of cultue medium.(4)ICAM-1 protein expression was analyzed by Western blot。(5)Protein O-GlcNAc modification in HUVECs induced by glucosamine was evaluated by Western blot.And we investigated the relationship between the O-GlcNAc modification and ICAM-1,MCP-1 protein expression using alloxan,an O-GlcNAc modification inhibitor.(6)The effect of glucosamine on LL-37 and TNF-α-induced p38MAPK,NF-κB, ERK phosphorylation was investigated by Western blot using phospho-specific antibodies.2,Animal experiment(1)B6.KOR.Apoesh1 mice(8 week old,high cholesterol and atherosclerosis naturally occurring animal modle) were divided into 3 groups(control group, glucosamine 300mg/kg.day group and 1000mg/kg.day group.(2)Sacrifice of animals and the sample preparations:mice were anesthetized with ether,blood samples were drawn from the celiac artery,then perfused with 20% formalin/PBS.The hearts were fixed and removed for future analysis.Blood samples were centrifuged for 5 min(12,000rpm,4℃),the supernatant(plasma fraction) was recovered and stored at -80℃.(3)We investigated formation of atherosclerosis lesions using Oil Red O staining, then evaluate the effect of glucosamine on atherosclerosis.We also determined atherosclerosis related indexes such as HDL,cholesterol and lipid peroxide concentration containing in the plasma,and analyze the effect of glucosamine on these indexes. Results1,Glucosamine inhibited ICAM-1 and MCP-1 expression both on mRNA and protein level.2,Glucosmaine induced protein O-GlcNAc modification in HUVEC,and this is parallel with the inhibitory effect of glucosamine on ICAM-1 and MCP-1 expression.3,Preincubation with alloxan,an O-GlcNAc modification inhibitor abrogated the suppression of ICAM-1 and MCP-1 expression induced by glucosamine.4,Glucosamine inhibited LL-37 and TNF-αinduced p38MAPK,NF-κB,ERK phosphorylation,O-GlcNAc inhibitor-alloxan also partially eliminated the suppression effect induced by glucosamine.5,Glucosamine treatment significantly decreased the area of atherosclerosis leasion(300mg/kg.day glucosamine treatment group,p＜0.05).Although we can't observe dose-dependant manner,1000mg/kg.day glucosamine treatment also showed inhibitory tendancy.6,Glucosamine showed no effect on HDL and cholesterol concentration,but significantly reduced lipid superoxide concentration.DiscussionAtherosclerosis is a chronic inflammatory disease that is characterized by infiltration of mononuclear leukocytes and lymphocytes into the intima through the expression of adhesion molecules on the arterial wall.Pro-inflammatory cytokines, such as tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β),are found in atherosclerosis lesions,and contribute to the inflammatory process.Previous studies have shown that these cytokines can induce the expression of cell adhesion molecules and chemokines,and lead to the recruitment of leukocytes.Here we have evaluated the effect of glucosamine on TNF-α-induced expression of ICAM-1 and MCP-1,which play important roles in the process of atherosclerotic plaque formation as an adhesion molecule and a chemotractant protein,respectively. Upon inflammatory activation,endothelial cells upregulate adhesion molecule expression(such as ICAM-1),and monocytes can bind to the endothelium through the adhesion molecules.Then,monocytes migrate into the arterial intima,and this process requires a chemoattractant(such as MCP-1) gradient.Once monocytes reside in the intima,they become intimal macrophages and internalize modified lipoproteins to become foam cells.Eventually,these macrophages congregate in a central core of atherosclerotic plaques.Glucosamine is used to treat osteoarthritis,and it also exhibits anti-inflammatory actions.In brief,glucosamine suppresses neutrophil functions such as superoxide generation,granule enzyme release and chemotaxis.It also inhibits the aggregation of platelet.In addition,glucosamine suppresses the activation(such as nitric oxide,PGE2, IL-8 production) of synoviocytes.Furthermore,glucosamine inhibits ICAM-1 expression in human retinal pigment epithelial cells.In this study,we have investigated the effect of glucosamine on the activation of endothelial cells,and revealed that glucosamine but not N-acetylglucosamine can inhibit the ICAM-1 and MCP-1 expression at both mRNA and protein levels.In separate experiments,we stimulated endothelial cells with another pro-inflammatory cytokine IL-1β.We confirmed that IL-1βalso induced the expression of MCP-1 and ICAM-1,and the expression was similarly suppressed by glucosamine as observed in the TNF-α-induced MCP-1 and ICAM-1 expression.TNF-αactivates a variety of signaling cascades(such as p38MAPK and NF-κB pathways) that leads to the induction of inflammatory response.We indicated using p38MAPK and IKK inhibitors that TNF-αinduced the expression of ICAM-1 and MCP-1 via p38MAPK and NF-κB pathways.Furthermore,we revealed that glucosamine suppressed not only the expression of MCP-1 and ICAM-1 but also the phosphorylation of p38MAPK and NF-κB.These observations suggest that glucosamine inhibits the endothelial cell activation(i.e.,MCP-1 and ICAM-1 expression) possibly via the suppression of the intracellular signaling including p38MAPK and NF-κB. It is now recognized that the addition of O-linked N-acetylglucosamine (O-GlcNAc) to target proteins could modulate cellular functions,such as nuclear transport,transcription,translation,cell signaling,apoptosis and cell shape.In this context,it has been recently found that glucosamine treatment protects rat heart from ischemia-reperfusion injury,accompanied with the increased O-GlcNAc levels and attenuated phosphorylation of p38MAPK.Of note,Western blotting using anti-O-GlcNAc monoclonal antibody revealed that glucosamine but not N-acetylglucosamine increased the O-GlcNAc levels in HUVEC,and the effect of glucosamine on the O-GlcNAc-modification was negatively correlated with that on the HUVEC activation(the phosphorylation of p38 MAPK and NF-κB,and the production of MCP-1 and ICAM-1).Mammalian cells contain O-GlcNAc transferase(OGT),an O-GlcNAc-modification forming enzyme and O-N-acetylglucosaminidase (O-GlcNAcase),an O-GlcNAc modification degrading enzyme.Thus,we further investigated the effect of alloxan,an OGT inhibitor.Alloxan considerably abrogated the glucosamine-induced not only O-GlcNAc-modification but also suppression of ICAM-1 expression,although alloxan only partially abolished the glucosamine-induced suppression of MCP-1.Together these observations suggest that the activation of endothelial cells is likely modulated by the glucosamine-induced O-GlcNAc modification of target proteins,which may affect the intracellular signaling, transcription and translation(as observed for the phosphorylation of p38MAPK and NF-κB,mRNA and protein expression of MCP-1 and ICAM-1).In conclusion,glucosamine can inhibit TNF-α-induced ICAM-1 and MCP-1 expression,possibly by affecting p38MAPK and NF-κB signal pathways.Furthermore, O-GlcNAc modification may be involved in the modulation.Thus,it is tempting to speculate that glucosamine affects endothelial cell activation,thereby possibly exhibiting anti-inflammatory action on atherosclerosis.Recently,it has been reported that glucosamine administration significantly reduced the atherosclerotic lesion in aortic root of apoE-deficient mice.In contrast,glucosamine supplementation reportedly accelerates the early but not late atherosclerosis in LDL receptor-deficient mice.Thus, the in vivo effect of glucosamine on atherosclerotic disorders should be carefully evaluated in animal models and patients in the future.Conclusions1,Glucosamine can modulate endothelial cell activation through suppressing LL-37 and TNF-αinduced ICAM-1 and MCP-1 expression,and plays anti-inflammatory roles.2,Glucosamine induced protein O-GlcNAc modificationv is parallel with the inhibitory effect of glucosamine.And alloxan,an O-GlcNAc modification inhibitor eliminated the suppression of expression induced by glucosamine.These results indicates that protein O-GlcNAc modification is one of the possible mechanism which involves in the modulation.3,Glucosamine inhibited LL-37 and TNF-αinduced phosphorylation of p38MAPK,NF-κB,ERK,these indicate that glucoamine can inhibit ICAM-1 and MCP-1 expression via the signal transduction molecules mentioned above.4,Glucosamine treatment increased water intake and body weight of the mice.5,Glucosamine reduced the atherosclerosis lesion area,indicating the potential of glucosamine in anti atherosclerosis.
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