摘要
前言
Ghrelin是近年来发现的生长激素促分泌物质受体(growth hormonesecretagogue receptor,GHSR)的内源性配体,其在心血管系统中的保护作用已成为人们关注的焦点之一。Ghrelin在人体及动物体内具有强大的刺激生长激素释放的作用,并可调节食欲及胃肠功能。近年来研究发现,心肌及血管内皮处均有编码ghrelin及其受体的mRNA的表达。健康志愿者和充血性心力衰竭患者静脉注射ghrelin,可在不影响心率的情况下显著降低平均动脉压及外周血管阻力,增加心输出量及心脏指数。体外研究结果证实,ghrelin可明显改善缺血再灌注损伤时的左心功能,缩小心肌梗死的面积。Ghrelin亦被证实可通过降低基础状态下及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的趋化因子的表达,改善内皮功能。因此,ghrelin可能通过改善内皮细胞功能,在动脉粥样硬化(atherosclerosis,AS)的防治中起重要作用。
全球吸烟者的人数已超过十亿,吸烟引起的死亡率正在逐年升高。流行病学调查研究已经证实,吸烟是AS的独立危险因素,烟雾中的尼古丁(nicotine)被认为是引起心血管疾病的主要物质之一。尼古丁可通过成纤维细胞生长因子及转化生长因子β1,诱导平滑肌细胞增殖;通过活化蛋白激酶C(protein kinase C,PKC)、p38丝裂原活化蛋白激酶(p38 mitogen activated protein kinase,p38 MAPK)及核转录因子-κB(nuclear transcription factor-kappa B,NF-κB)途径诱导细胞粘附分子的表达,引起内皮细胞功能紊乱,诱发并加速AS的发生和发展。
最近一项研究数据显示,吸烟与非吸烟个体血中ghrelin的浓度无统计学差异,但是,非吸烟个体短期吸烟后,血中ghrelin浓度下降,而吸烟个体血中ghrelin浓度无显著变化。这一研究结果引起人们的注意,吸烟个体体内的ghrelin是否会代偿性升高以拮抗长期吸烟的致AS作用有待于进一步的研究和证实。
本实验拟观察尼古丁对ghrelin及其受体表达的影响,并观察尼古丁对ghrelin预处理的人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)及白介素-8(interleukin-8,IL-8)表达的变化,证实ghrelin是否会影响尼古丁诱导的血管内皮细胞功能紊乱。本实验通过观察ghrelin对尼古丁所致HUVEC VCAM-1及IL-8表达变化过程中PKC、p38 MAPK及NF-κ3的活性变化,并应用PKC、p38 MAPK及NF-κB的特异性抑制剂chelerytherine chloride、SB203580、PDTC,探讨ghrelin阻断尼古丁引起内皮细胞功能紊乱致AS作用的信号转导机制。
材料与方法
1、人脐静脉内皮细胞培养,尼古丁和/或ghrelin共同孵育细胞,收集内皮细胞及细胞培养液;分别加入PKC、p38 MAPK及NF-κB的特异性抑制剂chelerytherine chloride、SB203580、PDTC进行干预,收集内皮细胞及细胞培养液,用于指标检测。
2、采用Western blot法检测人脐静脉内皮细胞VCAM-1和p38 MAPK蛋白的表达。
3、采用ELISA法检测细胞培养液中IL-8蛋白表达的水平。
4、采用实时定量PCR技术检测细胞ghrelin mRNA的表达。
5、应用PepTag~(?)非放射性蛋白激酶检测系统检测细胞PKC的活性。
6、应用TransAM~(TM)NF-κB p50活性检测试剂盒检测细胞NF-κB的活性。
7、采用逆转录PCR法检测细胞GHSR-1a及GHSR-1b mRNA的表达。
8、各组实验数据均用(?)±S表示。利用SPSS11.5统计软件分析,组间差异比较采用单因素方差分析。P＜0.05为有统计学差异。
结果
1、尼古丁组与空白对照组相比VCAM-1蛋白表达明显升高(0.94±0.04 vs0.52±0.07,n=4,P＜0.05);IL-8蛋白表达亦明显升高(7.03±0.22vs0.58±0.13,n=3.p＜0.05)。
2、尼古丁组ghrelin mRNA表达较空白对照组明显下降(P＜0.05)。
3、不同浓度1ng/ml、10ng/ml、100ng/ml的ghrelin预处理内皮细胞组,较尼古丁组VCAM-1蛋白表达明显降低,且随ghrelin浓度的升高,VCAM-1蛋白表达的降低更加明显;ghrelin对尼古丁诱导VCAM-1表达的抑制作用亦呈时间依赖性。
4、不同浓度1ng/ml、10ng/ml、100ng/ml ghrelin预处理内皮细胞组,较尼古丁组IL-8蛋白表达明显降低,且随ghrelin浓度的升高,IL-8蛋白表达的降低更加明显;ghrelin对尼古丁诱导IL-8蛋白表达的抑制作用亦呈时间依赖性。
5、尼古丁干预内皮细胞5min、10min、30min、60min、120min后检测细胞PKC的活性,5min时PKC活性明显增强;10ng/ml ghrelin预处理的内皮细胞在5min时PKC活性较尼古丁组降低。5min时间点时尼古丁组PKC活性较空白对照组升高1.32倍(P＜0.05);ghrelin及PKC抑制剂预处理组PKC活性较尼古丁组明显下降。
6、尼古丁干预内皮细胞5min、10min、30min、60min、120min后检测细胞p38 MAPK的活性,30min时p38 MAPK活性明显增强;10ng/ml ghrelin预处理的内皮细胞在30min时间点p38 MAPK活性较尼古丁组降低。30min时间点时尼古丁组p38 MAPK活性较空白对照组升高2.57倍(P＜0.05);ghrelin及p38 MAPK抑制剂预处理组p38 MAPK活性较尼古丁组明显下降;PKC抑制剂预处理组p38MAPK活性较尼古丁组无明显差异。
7、尼古丁组NF-κB活性较空白对照组明显升高(P＜0.05);ghrelin预处理组NF-κB活性较尼古丁组明显下降;PKC、p38 MAPK、NF-κB抑制剂预处理组NF-κB活性较尼古丁组明显下降。
8、PKC、p38 MAPK、NF-κB抑制剂可明显抑制尼古丁诱导的VCAM-1及IL-8的蛋白表达。
9、Ghrelin可升高内皮细胞GHSR-1b mRNA的表达,但对GHSR-1a mRNA的表达无明显影响。
讨论
VCAM-1在AS斑块区的内皮细胞上表达明显升高,介导单核细胞及白细胞向血管内皮细胞的迁移和粘附,在AS发展的初期阶段起重要作用。本实验结果显示,尼古丁组VCAM-1的表达较空白对照组明显升高,进一步证实尼古丁可诱导内皮细胞粘附分子的表达。既往研究发现,尼古丁可激活p38 MAPK信号转导通路,从而活化NF-κB,诱导VCAM-1的表达,PKC亦可介导VCAM-1的表达,但加入PKC抑制剂2h后对尼古丁诱导的NF-κB活化无明显影响,却明显抑制了尼古丁诱导的核转录因子激活蛋白-1(activator protein-1,AP-1)的活化,结果提示,尼古丁通过PKC、p38 MAPK信号转导通路,活化NF-κB及AP-1,从而诱导VCAM-1的表达。在本实验中,尼古丁诱导VCAM-1表达的过程中PKC、p38MAPK及NF-κB活性升高,并且PKC、p38 MAPK及NF-κB特异性抑制剂可不同程度阻断尼古丁诱导的VCAM-1的表达,这一结果与既往实验结果相一致;PKC及p38 MAPK抑制剂预处理组加入尼古丁后6h,我们观察到PKC及p38MAPK抑制剂均可抑制这一过程中NF-κB的活性,考虑观察NF-κB活性时间点的不同是本实验研究结果不同于既往实验结果的主要原因;本实验研究结果表明,尼古丁可通过激活PKC及p38 MAPK信号转导通路,触发NF-κB的活化,从而诱导VCAM-1的表达,破坏内皮功能,促进AS的发生和发展。
IL-8属于CXC类趋化因子,诱发单核细胞的聚集和粘附,在AS的发生发展中起重要作用。近来有研究结果显示,香烟烟雾的提取物可诱导小鼠肺树突状细胞表达IL-8升高,且这一过程可被抗氧化剂N乙酰半胱氨酸阻断;尼古丁可使内毒素刺激下的人肺泡巨噬细胞MAPK磷酸化、IL-8表达增加;体内研究证实,尼古丁可通过N乙酰胆碱能受体,刺激产生过氧化亚硝酸盐,继而活化NF-κB,从而升高中性粒细胞IL-8的表达,引起吸烟个体的白细胞增多症。本实验观察了尼古丁对人脐静脉内皮细胞IL-8表达的影响,结果显示,尼古丁组人脐静脉内皮细胞IL-8的表达较空白对照组明显升高,同时PKC、p38 MAPK及NF-κB活性升高,并且PKC、p38 MAPK及NF-κB特异性抑制剂可不同程度阻断尼古丁诱导的IL-8的表达,PKC及p38 MAPK抑制剂亦可抑制这一过程中NF-κB的活性。本实验研究结果表明,尼古丁可通过激活人脐静脉内皮细胞PKC及p38 MAPK信号转导通路,触发NF-κB的活化,从而诱导IL-8的表达,在AS的发生和发展起重要作用。
近年来关于ghrelin调节血管内皮细胞功能的研究发现,10ng/ml ghrelin即可明显抑制TNF-α诱导的人脐静脉内皮细胞NF-κB的活化及趋化因子IL-8、单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)的表达,拮抗过氧化所致的内皮细胞IL-8水平的升高,并抑制TNF-α诱发的单核细胞与内皮细胞的粘附;另有实验结果显示,ghrelin在0.1nmol/l至1.35nmol/l(约为0.4ng/ml至4.5ng/ml)浓度范围内升高VCAM-1的表达,但不影响白介素-1β(interleukin-1β,IL-1β)诱发的粘附分子的表达及单核细胞的粘附。本实验研究结果显示,ghrelin在1ng/ml至100ng/ml浓度范围内剂量依赖性的抑制尼古丁诱导的人脐静脉内皮细胞的VCAM-1及IL-8的表达,并且这一抑制作用呈明显的时间依赖性。这一实验结果提示,ghrelin可抑制尼古丁引起的内皮细胞功能紊乱,改善内皮功能,在尼古丁所致AS的防治中起重要作用,并且这一保护作用与ghrelin的浓度直接相关。
本实验中,ghrelin预处理组人脐静脉内皮细胞PKC的活性较尼古丁组明显下降,提示尼古丁诱导的PKC通路的活化可被ghrelin明显抑制,之前实验已经证实尼古丁可通过PKC途径诱导VCAM-1及IL-8的表达,ghrelin可拮抗尼古丁诱导的VCAM-1及IL-8的表达,实验结果提示,ghrelin可通过阻断尼古丁激活的PKC信号转导通路,抑制尼古丁诱导的VCAM-1及IL-8的表达。另有实验发现,在人结肠上皮细胞中,ghrelin可通过PKC信号转导通路活化NF-κB,升高IL-8基因的表达;并可通过PKC途径促进鼠脑垂体生长激素分泌细胞的增殖。这些实验结果提示ghrdin在不同的细胞和组织中,通过不同的调节方式,在改善机体功能过程中扮演不同的角色。
P38 MAPK是参与粘附分子及趋化因子表达的另一个重要信号转导通路。本实验结果发现,ghrelin组p38 MAPK的活性较尼古丁组明显抑制,提示ghrelin可能通过抑制p38 MAPK的活性,从而抑制尼古丁诱导的VCAM-1及IL-8的表达;另外本实验中,PKC的抑制剂对p38 MAPK的活性无明显影响,提示在尼古丁诱导VCAM-1及IL-8表达的过程中PKC对p38 MAPK通路可能无调节作用。亦有研究发现,ghrelin可刺激鼠巨噬细胞中p38 MAPK的活化,从而抑制前炎症因子IL-1β的表达,明显增加抗炎症因子白介素-10(interleukin-10,IL-10)的表达,并且这一作用呈浓度依赖和时间依赖性。Ghrelin对p38 MAPK活性的不同影响提示,ghrelin在不同的细胞和组织中可能通过不同的方式调节p38 MAPK的活性来起到抗炎的保护作用。
NF-κB是调节基因转录的关键因子之一,它参与许多基因特别是与机体防御功能及炎症反应有关的早期应答基因的表达调控。已经证实,在动脉粥样斑块区和纤维化斑块区均检测到激活的NF-κB,并对多种促炎症细胞因子、粘附分子、趋化因子和生长因子等的表达起着关键的调控作用。本实验结果显示,ghrelin组NF-κB的活性较尼古丁组明显抑制;之前实验结果已经证实,尼古丁可通过激活人脐静脉内皮细胞PKC及p38 MAPK信号转导通路,触发NF-κB的活化,从而诱导VCAM-1及IL-8的表达;ghrlein可拮抗尼古丁诱发的PKC及p38 MAPK的活化,抑制尼古丁诱导的VCAM-1及IL-8的表达。所以本研究结果提示,ghrelin可能通过抑制尼古丁诱导的PKC及p38 MAPK通路的激活,抑制NF-κB的活化,从而抑制尼古丁诱导的VCAM-1及IL-8的表达。
总之,本实验结果证实,ghrelin可通过抑制尼古丁诱导的PKC、p38 MAPK及NF-κB的活化,抑制尼古丁诱导的粘附分子及趋化因子的表达,为ghrelin抗AS机制研究提供新的依据。目前国内外对ghrelin在AS中的研究尚待进一步深入,ghrelin有可能成为内皮功能紊乱及AS防治对策中一种新的治疗药物。
结论
1、尼古丁可诱导人脐静脉内皮细胞VCAM-1及IL-8的表达,破坏内皮细胞功能。
2、尼古丁短时间抑制内皮细胞ghrelin的表达,可能为尼古丁损伤内皮功能的又一理论依据。
3、Ghrelin可抑制尼古丁诱导的人脐静脉内皮细胞VCAM-1及IL-8的表达,对尼古丁引起的内皮功能紊乱起保护作用。
4、PKC、p38 MAPK及NF-κB信号转导通路调节血管内皮细胞VCAM-1及IL-8的表达。
5、尼古丁通过激活PKC、p38 MAPK信号转导通路,活化NF-κB,从而引起VCAM-1及IL-8表达的升高,促进AS的发生发展。
6、Ghrelin可能依赖于PKC、p38 MAPK信号转导通路及NF-κB途径,拮抗尼古丁诱发的内皮功能损伤,起到抗AS的作用。
7、Ghrelin对血管内皮细胞功能的调节作用可能由GHSR-1b受体介导。
Introduction
Ghrelin 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 methods
1、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.
Results
1、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.
Discussion
VCAM-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.
Conclusions
1、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.
引文
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