The Effect And Mechanism Of Ghrelin On Nicotine-induced VCAM-1 And IL-8 Expression In HUVEC

IntroductionGhrelin is a recently discovered peptide hormone and has been shown to be a natural ligand of the orphan growth hormone secretagogue receptor type 1 a(GHSR-1 a). Ghrelin strongly stimulates the release of growth hormone in animals and humans,and in addition modulates appetite and gastrointestinal function.Expression of mRNA that encodes ghrelin and its receptor has been observed in the heart and vasculature,and increasing evidence supports a role of ghrelin in the direct regulation of cardiovascular function.Intravenous administration of ghrelin in humans can significantly decrease mean arterial pressure without changing heart rate,and can increase coronary perfusion pressure in rat hearts.In addition,ghrelin inhibits pro-inflammatory interleukins such as interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α).Moreover,ghrelin also improves endothelial function by inhibiting basal and TNF-α-induced chemotactic cytokines secretion.There are more than one billion smokers in the world,and the mortality of smoking is increasing annually.Epidemiological studies have identified that cigarette smoke is a major risk factor for accelerated atherosclerosis,and nicotine is a major constituent of tobacco.Nicotine is found to induce smooth muscle cell proliferation, mediated through basic fibroblast growth factor and transforming growth factor-β1. Moreover,it have been reported that nicotine promotes the expression of cell adhesion molecules by activating protein kinase C(PKC),p38 mitogen-activated protein kinase (p38 MAPK) and the nuclear transcription factor-κB(NF-κB).Although there are many well-known health risks due to smoking,it remains commonplace,and preventing smoking-induced disease is an important goal.A recent study showed that fasting plasma ghrelin concentration was not different between male smokers and nonsmokers,but after short-term smoking the ghrelin level was decreased in nonsmokers,and no changes were observed in smokers.Ghrelin levels may be increased in long-term smokers to antagonize the effects of smoking on atherosclerosis development.However,there are few studies on the interaction between ghrelin and nicotine,especially the possible inhibiting effect of ghrelin on nicotine-induced atherosclerosis.We hypothesize that ghrelin might have a protective effect on nicotine-induced endothelium dysfunction.The aim of the present study was to study the effect of nicotine on ghrelin and the growth hormone secretagogue receptor(GHSR) expression, the effect of ghrelin on vascular cell adhesion molecule-1(VCAM-1) and interleukin-8 (IL-8) expression in nicotine incubated human umbilical vein endothelial cell (HUVEC),and the possible signaling transduction mechanisms associated with PKC, p38 MAPK and NF-κB.Materials and methods1,We cultured human umbilical vein endothelial cell line,and examined the effect of nicotine and ghrelin on cells.We also examined the effect of chelerytherine chloride,SB203580 and PDTC on nicotine incubated cells.2,The expression of VCAM-1 and p38 MAPK activity was quantified by using western blot analysis.3,The expression of IL-8 was determined by specific enzyme-linked immuno-sorbent assay(ELISA) method.4,The expression of ghrelin mRNA was determined with QuantiTect SYBR Green RT-PCR Assay.5,The PKC activity of HUVEC was detected with PepTag(?) Protein Kinase C Activity Assay. 6,The NF-κB activity of HUVEC was detected with TransAM^TM NF-κB p50 Activity Assay.7,The expression of GHSR mRNA was determined by reverse transcriptase PCR method.8,All experiments were performed at least in triplicate.Values were expressed as means±SD.Statistical analysis was performed by using one-way ANOVA.P＜0.05 were considered statistically significant.Results1,VCAM-1 and IL-8 expression were significant increased by 100nM nicotine.2,Ghrelin mRNA expression was significantly decreased by nicotine.3,We pretreated the cells with different concentrations of ghrelin(1,10,and 100ng/ml) for 1h,then exposed HUVECs to 100nM nicotine for 24h.Ghrelin inhibited nicotine-induced VCAM-1 expression in a concentration-dependent way.In addition, when HUVECs were preincubated in the presence of ghrelin(10ng/ml) for 1,6,12,and 24h,the expression of VCAM-1 was significantly decreased at lh,and this downregulation became further enhanced when the culture periods were extended.4,Similar to the effect of ghrelin on nicotine-induced VCAM-1 expression, ghrelin inhibited nicotine-induced IL-8 expression in concentration and time-dependent ways.5,The activity of PKC was significantly increased at 5min by nicotine,but there were no obvious changes in the HUVECs incubated by ghrelin.The nicotine-induced PKC activation was inhibited by PKC inhibitor CC.Then HUVECs were pretreated with ghrelin(10ng/ml) for lh before exposed to nicotine,PKC activation was inhibited by ghrelin to 86%of nicotine group.6,The cells were incubated with 100nM nicotine for 5,10,30,60,and 120min, then the activity of p38 MAPK was examined.Nicotine-induced p38 MAPK activation was observed at a significant level at 30min incubation.In contrast,there was no difference of p38 MAPK activation among groups incubated with ghrelin.And ghrelin inhibited nicotine-induced p38 MAPK activation at 30min.The activation of p38 MAPK increased by nicotine was inhibited by ghrelin and SB203580,in contrast no exchanges by CC.7,Assay showed that nicotine increased activation of NF-κB,which was markedly attenuated by CC and SB203580.Ghrelin and PDTC strongly decreased nicotine-induced NF-κB activity.8,Nicotine-induced VCAM-1 and IL-8 expression were blocked by the PKC inhibitor CC,p38 MAPK inhibitor SB203580 and NF-κB inhibitor PDTC.9,Ghrelin enhanced the GHSR-1b mRNA expression,but there were no effect on the GHSR-1 a mRNA expression.DiscussionVCAM-1,an important adhesion molecule,has been found to be expressed in endothelial cells in atherosclerotic plaques.It mediates leukocyte migration across the endothelium and plays an important role in the initiation of the atherosclerotic process in mice.In the present study,we demonstrated that nicotine increased expression of VCAM-1 in HUVECs.And nicotine-induced VCAM-1 expression was blocked by the inhibitors of PKC,p38 MAPK and NF-κB,and inhibitors of PKC and p38 MAPK blocked NF-κB activation at 6h.This result is consistent with a previous study.In which study,inhibitors of PKC did not blocked NF-κB activation after 2h later incubation of nicotine in HUVECs,which was the different with our study.Our data are consistent with the hypothesis that nicotine increased VCAM-1 expression through PKC and p38 MAPK-mediated activation of NF-κB.Induction of chemotactic cytokines such as IL-8 is thought to play a key role in monocyte recruitment and adhesion to endothelial cells in atherosclerosis.In the recent study,cigarette smoke extract augmented the generation of IL-8 in human dendritic cells,and the generation of IL-8 conditioned with cigarette smoke extract was suppressed by the anti-oxidant n-acetyl cysteine;cigarette smoke extract also increased the phosphorylation of MAPK and the production of IL-8.Nicotine stimulates neutrophil-IL-8 production via nAChR by generating peroxynitrite and subsequent NF-κB activation,and the IL-8 appears to contribute to leukocytosis in smokers.In our study,we demonstrated that nicotine increased IL-8 expression in HUVECs.And nicotine-induced IL-8 expression was blocked by the inhibitors of PKC,p38 MAPK and NF-κB.The result indicated nicotine increased VCAM-1 expression through PKC and p38 MAPK-mediated activation of NF-κB.Moreover,we showed that ghrelin significantly inhibited nicotine-induced VCAM-1 and IL-8 expression in a concentration-dependent at concentration of ghrelin from 1ng/ml to 100ng/ml and time-dependent manner.Other research showed exogenic ghrelin inhibited TNF-α-induced release of IL-8 and monocyte chemottractant protein-1 in HUVECs,with significant inhibition observed at a concentration of ghrelin as low as 10ng/ml.A recent study has reported that ghrelin produced increases in VCAM-1 expression at a concentration of ghrelin 0.1nmol/1 to 1.35nmol/1(0.4ng/ml to 4.5ng/ml,approximately),and ghrelin did not affect either the increased adhesion molecule expression or monocyte adhesion due to interleukin-1β(IL-1β).These findings indicated that ghrelin could improve endothelial function in the process of atherosclerosis,and the anti-inflammatory effects depend on the concentration of ghrelin.In our study,nicotine induced VCAM-1 and IL-8 expression though with PKC pathway,and ghrelin modestly but significantly inhibited PKC activation in HUVECs incubated with nicotine.This result indicates that the PKC pathway be involved in downregulation of nicotine-induced VCAM-1 and IL-8 expression by ghrelin.Other studies have found that ghrelin stimulates IL-8 gene expression through the PKC-mediated NF-κB pathway in human colonic epithelial cells,and that it exerted a proliferative effect on a rat pituitary somatotroph cell line via the PKC pathway.All of the evidences suggest that ghrelin plays a variety of different roles in different cells and tissues in different ways.P38 MAPK is another important cytoplasmic signal molecule involved in the process of VCAM-1 and IL-8 expression.Another important finding of our study was that ghrelin inhibited nicotine-induced p38 MAPK activation,which is consistent with the hypothesis that ghrelin inhibited nicotine-induced VCAM-1 and IL-8 expression through the p38 MAPK pathway.In addition,in our study p38 MAPK activation was not inhibited by PKC inhibitor,which indicated the activation of p38 MAPK was not mediated by PKC in the process of nicotine-induced VCAM-1 and IL-8 expression.A recent study showed that ghrelin administration enhanced activation of p38 MAPK, thereby inhibiting the production of pro-inflammatory cytokines IL-1βand significantly augmenting the release of the anti-inflammatory cytokine interleukin-10(IL-10) in a dose-dependent and time-dependent fashion from lipopolysaccharides-stimulated murine macrophages.Taken together,these data indicate that ghrelin regulates p38 MAPK activity with different modalities in pathologic anti-inflammatory states.The promoter region for NF-κB has been localized on many proinflammatory cytokine genes and forms the basis of simultaneous release of those cytokines during inflammatory cascade.The transcription factor,NF-κB partly mediates the production of VCAM-1 in HUVECs treated with nicotine.Moreover,ghrelin inhibited TNF-α-induced activation of NF-κB in HUVECs.Consistent with those findings, ghrelin inhabited NF-κB activity in HUVECs treated by nicotine in the present study. Our research also showed that inhibitors of PKC and p38 MAPK partly blocked NF-κB activity,and ghrelin inhibited activation of PKC and p38 MAPK.Accordingly,these reports indicate that ghrelin inhibited NF-κB activity partly due to inhibited PKC and p38 MAPK activation.If there are any other mediated pathway need further research.In conclusion,we demonstrated for the first time that ghrelin inhibits nicotine-induced VCAM-1 and IL-8 expression,partly due to inhibited PKC and p38 MAPK activation and NF-κB activity.These findings suggest that ghrelin has a potent anti-inflammatory effect.Moreover,our findings suggest that exogenous ghrelin may be a possible therapeutical target for atherosclerosis in smokers.Further study of the protective effect of ghrelin on cardiovascular diseases will benefit from further research.Conclusions1,Nicotine induces expression of VCAM-1 and IL-8 in HUVECs to provoke endothelium dysfunction.2,The short-term exposure of nicotine inhibits the expression of ghrelin in endothelial cells,which may be the another theoretical basis for the injury of endothelial function by nicotine.3,Ghrelin inhibits nicotine-induced VCAM-1 and IL-8 expression to play an anti-inflammatory role.4,The expression of VCAM-1 and IL-8 are mediated through PKC,p38 MAPK and NF-κB pathways.5,Nicotine induces VCAM-1 and IL-8 expression through PKC and p38 MAPK-mediated activation of NF-κB,by which accelerates the development of cardiovascular diseases.6,Ghrelin improves endothelial function,and inhibits nicotine-induced atherosclerosis through PKC,p38 MAPK and NF-κB pathways.7,The regulating role of Ghrelin on endothelial cell function may be mediated by GHSR-1b.