氨基单糖—葡萄糖胺在内皮细胞活化调节中的作用
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摘要
近年来,众多的临床及病理学的研究改变了研究人员对动脉粥样硬化发病过程的认识。尤其是免疫组织化学方法的应用,明确了作为脂肪斑或粥样斑块的主组成分的泡沫细胞主要是巨噬细胞,而不是一直以来认为的平滑肌细胞;而且各时期动脉硬化斑块组织中均观察到活化T细胞,这使研究者们着眼于动脉粥样硬化发生过程中的炎症细胞的作用。虽然动脉粥样硬化斑块组织中可见脂质块,动脉粥样硬化不再是单纯的脂质的异常堆积,而是伴有内皮细胞的损伤及炎症的免疫紊乱状态。内皮细胞的活化可诱导细胞黏附分子(如ICAM-1)和趋化因子(如MCP-1),内皮细胞表面表达的黏附分子可诱导单核细胞和T淋巴细胞等与内皮细胞的结合,这就是炎症的起始阶段,也是动脉粥样硬化发生的关键性的一步。与内皮细胞结合的炎性细胞在单核细胞趋化因子和氧化低密度脂蛋白(LDL)的趋化因子的作用下浸润至内皮下,进而转化成成熟的巨噬细胞并表达清道夫(scavenger)受体,吞噬修饰脂蛋白,进一步变成泡沫细胞。促炎症细胞因子例如肿瘤坏死因子(TNF-α)白介素-1β(IL-1β)等也常在动脉粥样硬化灶中表达,而且早期动脉粥样硬化病人以血清TNF-α浓度增加为特点。研究证明这些细胞因子可改变细胞形状及活力,并影响炎症部位的血管渗透性;还可以诱导内皮细胞表达表面黏附分子,调节血管张力、血栓形成及单核细胞的浸润等。
Cationic antibacterial protein of 18 kDa,因为它的序列由两个亮氨酸开始并由37个氨基酸组成,所以也称为LL-37。值得关注的是,最近发现动脉粥样硬化灶的巨噬细胞、内皮细胞表达LL-37,而且可诱导内皮细胞的ICAM-1和MCP-1,还可诱发血管平滑肌细胞的死亡。
葡萄糖胺是可在生理条件下自然生成的氨基单糖,是氨基葡聚糖生物合成的底物,已在欧洲作为变形性关节炎的治疗药物应用20余年,在美国和日本则以辅助治疗剂广泛应用。一些短期和长期临床实验证实葡萄糖胺可显著改善变形性关节炎的症状并不伴有明显的副作用。近年来,研究者们开始探索葡萄糖胺对其他细胞的作用及其作用机制,发现葡萄糖胺可抑制巨噬细胞的一氧化氮合酶的诱导产生;并抑制中性粒细胞的过氧化物的产生、吞噬、颗粒酶释放及趋化。Chen等人研究证明葡萄糖胺还可抑制视网膜色素上皮细胞的ICAM-1的表达,提出葡萄糖胺缓解眼部炎症的可能性。这些结果均提示葡萄糖胺的潜在的抗炎症作用。基于这些实验结果,我们将葡萄糖胺对内皮细胞活化的作用为研究对象,观察了葡萄糖胺对由TNF-α或LL-37(一种表达在动脉粥样硬的抗菌肽)刺激的内皮细胞ICAM-1和MCP-1表达的作用。
方法
1、细胞实验
(1)细胞的培养及处理
人脐带静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)培养在内皮细胞培养基(EGM-2)中。50-60%融合密度的HUVECs先用不同浓度的葡萄糖胺孵育两个小时,随后用4μM LL-37或0.5ng/ml TNF-α刺激24小时(mRNA及蛋白质的测定)或10分钟(信号转导分子磷酸化的分析及测定)。
(2)实时逆转录聚合酶链反应检测ICAM-1、MCP-1、GAPDH的mRNA水平的表达情况,评价葡萄糖胺对LL-37或TNF-α诱导产生的ICAM-1和MCP-1的mRMA的影响。
(3)ELISA法测定内皮细胞表达产生的MCP-1蛋白质。
(4)Western blot法分析ICAM-1蛋白质的表达情况。
(5)Western blot法分析葡萄糖胺对HUVECs蛋白质O-GlcNAc修饰的影响,并用Alloxan,O-GlcNAc修饰抑制剂分析该修饰与ICAM-1、MCP-1蛋白质的表达的相关性。
(6)Western blot法研究葡萄糖胺对LL-37或TNF-α诱导活化的p38MAPK、NF-κB、ERK活化即磷酸化的影响。
2、动物实验
(1)动物分组:8周龄B6.KOR.Apoe~(sh1)小鼠(高胆固醇血症,动脉粥样硬化自然发生小鼠模型,雌性),并作为对照组同时购C57BL/6(8周,雌性)。小鼠在24±3℃,相对湿度55±15%,7:00AM-7:00PM照明条件下饲养。B6.KOR.Apoe~(sh1)小鼠随机分为3组即葡萄糖胺非处理组、300mg/kg和1000mg/kg投药组(10只/组),对照组为5只C57BL/6。
(2)动物的处理及样品的制备在乙醚麻醉下,腹腔大动脉穿刺取血(使用肝素),血液样品离心(12,000rpm,4℃)5min,取上清即血浆在-80℃保存。采血后的小鼠用20%福尔马林/PBS灌流、固定、并摘取心脏。
(3)心脏的Oil Red O染色法分析葡萄糖胺处理对主动脉瓣处的动脉粥样硬化病灶大小的影响。
(4)测定血浆中的HDL、胆固醇、过氧化脂质(lipid peroxide)等动脉粥样硬化评价指标的含量,观察葡萄糖胺对这些物质浓度的影响,并分析与动脉粥样硬化病灶面积的相关性。
结果
1、葡萄糖胺在mRNA和蛋白水平抑制了LL-37和TNF-α诱导的内皮细胞的ICAM-1和MCP-1表达。
2、葡萄糖胺诱导了蛋白的O-GlcNAc修饰,这与对表达的抑制作用一致。
3、O-GlcNAc修饰的抑制剂alloxan恢复了葡萄糖胺对ICAM-1和MCP-1的抑制作用。
4、葡萄糖胺抑制了LL-37和TNF-α诱导活化的p38MAPK、NF-κB、ERK磷酸化,O-GlcNAc抑制剂Alloxan可部分恢复葡萄糖胺的抑制作用。
5、葡萄糖胺处理组与对照组相比,明显抑制了动脉粥样硬化病灶的形成(300mg/kg.day葡萄糖胺组,P＜0.05)。虽然观察不到剂量依存性效果,1000mg/kg.day葡萄糖胺组也有抑制动脉粥样硬化病灶形成的趋势。
6、葡萄糖胺虽然没有影响血浆中的HDL和胆固醇浓度,但是显著抑制了过氧化(LPO)脂质的形成。
讨论
动脉粥样硬化是动脉内壁表达黏附分子并诱导单核淋巴细胞黏附,并在单核细胞趋化因子的作用下进一步浸润至内膜下的一种慢性炎症过程。一些促炎症因子,例如TNF-α、IL-1β常表达在动脉粥样硬化病灶中,并诱导炎症。前期研究证明细胞因子可改变细胞形状和活性,改变炎症过程中的血管通透性;还可诱导产生内皮细胞的表面黏附分子和单核趋化因子等。我们在本次实验中观察了葡萄糖胺对LL-37、TNF-α诱导的ICAM-1和MCP-1表达的影响。作为黏附分子和单核细胞趋化分子的一种,它们在动脉粥样硬化病变的发生过程中起到重要的作用。当动脉壁内膜中的内皮细胞受到炎症刺激,黏附分子(例如ICAM-1)的表达将升高,并使血液中的单核细胞结合到内皮细胞,并在趋化因子(例如MCP-1)浓度梯度的趋化下浸润到内膜下。内膜下的单核细胞表达清道夫受体,变成巨噬细胞,巨噬细胞可以吞噬修饰的脂蛋白(氧化LDL等)成为泡沫细胞,这些细胞聚集在动脉粥样硬化病灶中心,从而恶化病变。
葡萄糖胺是广泛应用于治疗变形性关节炎的一种可在体内自然合成的氨基单糖,除了治疗变形性关节炎之外,近年来的多个实验室的研究结果证明它同时具有一些抗炎作用,并参与调节信号转导系统。例如,葡萄糖胺抑制中性粒细胞的超氧化物的合成、颗粒释放及趋化;还可抑制血小板的凝集;通过抑制IL-1β诱导产生的NO、PGE_2、IL-8等,调节滑膜细胞的活化。Chen等人研究证明葡萄糖胺还可抑制人视网膜色素上皮细胞的ICAM-1的表达,同样提示着潜在的抗炎作用。
动脉粥样硬化形成过程中,内皮细胞的活化起着重要的作用,所以我们研究了葡萄糖胺对内皮细胞活化的影响。我们的结果说明葡萄糖胺而不是N-acetylglucosamine在mRNA和蛋白水平抑制了ICAM-1和MCP-1的表达。
研究证明多种胞浆和和蛋白质的丝氨酸/苏氨酸残基可被O-连接-N-acetylglucosamine修饰。哺乳动物只有一组调节此修饰的酶,O-GlcNActransferase(OGT)和O-N-acetylglucosaminidase(O-GlcNAcase),而且基因敲除技术研究说明这种修饰对早期胚胎的发育及细胞功能是不可缺少的。用O-GlcNAcase抑制剂-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate(PUGNAc)灌流分离的心脏,可观察到所诱导的O-GlcNAc修饰保护缺氧/再灌流损伤,而OGT抑制剂-alloxan阻断其保护作用。另一个研究也得出相似的结论,即葡萄糖胺和PUGNAC对分离培养的心肌细胞缺氧/再给氧也起着保护作用。还有研究报道称,葡萄糖胺可增强trauma/hemorrhage大鼠模型的心脏功能,而此过程与O-GlcNAc修饰相关。上述研究结果提示着在不同疾病条件下,葡萄糖胺可阻止各种细胞组织的过度活化,相关的可能机制为蛋白质的O-GlcNAc修饰。有趣的是,O-GlcNAc修饰与O-phosphate拥有很多共同点:它们都由特异性酶调节其动态平衡,并对细胞外刺激做出迅速的反应,都可修饰蛋白质的丝氨酸/苏氨酸残基等。这说明O-GlcNAc修饰可能直接修饰磷酸化的位点或周围氨基酸而影响磷酸化,进而影响蛋白质的功能。
我们用单克隆抗体分析了葡萄糖胺对HUVEC蛋白质O-GlcNAc修饰的影响,得出以下结论:葡萄糖胺而非N-acetylglucosamine诱导蛋白质的O-GlcNAc修饰,这个结果与对ICAM-1和MCP-1表达的影响一致,即表现出抑制作用的是葡萄糖胺,而不是N-acetylglucosamine。这使我们联想到葡萄糖胺诱导的蛋白质的O-GlcNAc修饰与其对表达的抑制作用的某种联系。所以,我们进一步用alloxan,OGT抑制剂分析了这两者的关系,我们发现alloxan可有效抑制葡萄糖胺诱导的蛋白质的O-GlcNAc修饰。葡萄糖胺(1mM)可有效诱导O-GlcNAc修饰,同时也抑制ICAM-1和MCP-1的表达;alloxan有效抑制此修饰,同时也恢复了葡萄糖胺对蛋白表达的抑制作用。这些结果提示着葡萄糖胺对表达的抑制是可能介导蛋白质的O-GlcNAc修饰。
TNF-α可激活NF-κB和p38 MAPK等各种信号转导通路,并诱导一系列的炎症反应,例如黏附分子、趋化因子、促炎症细胞因子等的表达。我们利用抑制剂证明了TNF-α介导NF-κB和p38 MAPK途径刺激ICAM-1和MCP-1的表达。这些信号转导分子的活化需要特异氨基酸的磷酸化,我们首先研究了葡萄糖胺是否影响这些信号转导分子的磷酸化。我们的结果说明,用葡萄糖胺预孵育HUVEC可抑制TNF-α诱导的NF-κB和p38 MAPK分子的活化,提示葡萄糖胺可能通过影响这些信号转导途径而抑制ICAM-1和MCP-1的表达。
我们在研究中使用B6.KOR.Apoe~(sh1)小鼠(高胆固醇血症,动脉粥样硬化自然发生小鼠模型),用经口投药方式给0mg/kg.day,300mg/kg.day和1000mg/kg.day三种剂量。实验结果说明,葡萄糖胺虽然对总胆固醇和高密度脂蛋白无明显的影响,但是对过氧化脂质有明显的抑制作用,过氧化脂质是受损细胞膜的不饱和脂肪酸氧化而形成,可对周围组织细胞(例如动脉内壁)造成继发性损害,这提示着葡萄糖胺具有抗动脉粥样硬化发生的作用。心脏标本实际动脉粥样硬化病变面积(主动脉瓣膜处)的测量的结果也说明300mg/kg.day的剂量有效抑制了病变的发生,而1000mg/kg.day剂量虽然没有统计学意义,也有抑制病变发生的倾向。
总之,葡萄糖胺可抑制HUVEC的LL-37和TNF-α诱导的ICAM-1、MCP-1的表达,其可能机制为介导蛋白质O-GlcNAc修饰的对p38 MAPK和NF-κB的信号转导分子磷酸化的抑制。使用动物模型的in vivo实验也证明了葡萄糖胺具有抗动脉粥样硬化的作用。
葡萄糖胺是非处方类药物,而且已经长期应用于变形性关节炎的治疗,虽然长期使用无明显副作用,但是对其他慢性代谢性疾病的作用还是有待研究。最近也有不少实验室研究葡萄糖胺与动脉粥样硬化的关系,Duan等人证明葡萄糖胺可能通过诱导细胞外基质perlecan HSPG(heparin sulfate proteoglycan)抑制平滑肌细胞的增生,因而起到抗动脉粥样硬化的作用,并用apo-E敲除鼠证明了葡萄糖胺明显抑制了主动脉瓣的动脉粥样硬化病变的发生。但是也有实验室利用LDL受体缺损小鼠模型得出相反的结果,即葡萄糖胺恶化早期动脉粥样硬化的发生,但是对晚期的发展无明显影响的报导。作者后来又提出人类的动脉粥样硬化的早期发生在儿童时期,服用葡萄糖胺的人群是高龄人,所以实际上葡萄糖胺应该没有促进动脉硬化的作用。这些不同的实验结果可能是因为使用的动物模型种类、投药方式、投药时间的长短不同而造成的。通常用于人变形性关节炎治疗的葡萄糖胺的剂量为1.5或3g/天,这时人血清中的葡萄糖胺浓度可达到0.02-0.03 mM,我们在本次研究中In vitro使用的浓度为0.01-1mM,虽然在生理浓度下没有统计学意义,但是在LL-37刺激下0.01mM也表现出抑制的倾向。我们的各种实验结果均表明葡萄糖胺可能通过抑制ICAM-1和MCP-1的表达和减少血浆中过氧化脂质,从而起到动脉粥样硬化发生过程中的抗炎作用。
结论
1、葡萄糖胺抑制LL-37和TNF-α诱导产生的黏附分子ICAM-1和MCP-1的表达,从而调节内皮细胞的活化。
2、葡萄糖胺诱导的蛋白O-GlcNAc修饰与其对表达的抑制作用相关,而且O-GlcNAc修饰的抑制剂alloxan恢复了葡萄糖胺对蛋白表达的抑制作用,说明此修饰是葡萄糖胺调节内皮细胞活化的一种可能机制之一。
3、葡萄糖胺可抑制LL-37和TNF-α诱导活化的p38MAPK、NF-κB、ERK等信号转导分子的磷酸化,说明葡萄糖胺可能介导这些信号转导分子调节ICAM-1和MCP-1的表达。
4、葡萄糖胺处理组的小鼠饮水量及体重较对照组有所增加。
5、葡萄糖胺抑制了动脉粥样硬化病变的发展。
Introduction
In 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.
Methods
1、cell experiment
(1) cell culture and treatment
Human 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.Apoe~(sh1) 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.
Results
1、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.
Discussion
Atherosclerosis 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,PGE_2, 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.
Conclusions
1、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.
Cationic antibacterial protein of 18 kDa,因为它的序列由两个亮氨酸开始并由37个氨基酸组成,所以也称为LL-37。值得关注的是,最近发现动脉粥样硬化灶的巨噬细胞、内皮细胞表达LL-37,而且可诱导内皮细胞的ICAM-1和MCP-1,还可诱发血管平滑肌细胞的死亡。
葡萄糖胺是可在生理条件下自然生成的氨基单糖,是氨基葡聚糖生物合成的底物,已在欧洲作为变形性关节炎的治疗药物应用20余年,在美国和日本则以辅助治疗剂广泛应用。一些短期和长期临床实验证实葡萄糖胺可显著改善变形性关节炎的症状并不伴有明显的副作用。近年来,研究者们开始探索葡萄糖胺对其他细胞的作用及其作用机制,发现葡萄糖胺可抑制巨噬细胞的一氧化氮合酶的诱导产生;并抑制中性粒细胞的过氧化物的产生、吞噬、颗粒酶释放及趋化。Chen等人研究证明葡萄糖胺还可抑制视网膜色素上皮细胞的ICAM-1的表达,提出葡萄糖胺缓解眼部炎症的可能性。这些结果均提示葡萄糖胺的潜在的抗炎症作用。基于这些实验结果,我们将葡萄糖胺对内皮细胞活化的作用为研究对象,观察了葡萄糖胺对由TNF-α或LL-37(一种表达在动脉粥样硬的抗菌肽)刺激的内皮细胞ICAM-1和MCP-1表达的作用。
方法
1、细胞实验
(1)细胞的培养及处理
人脐带静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)培养在内皮细胞培养基(EGM-2)中。50-60%融合密度的HUVECs先用不同浓度的葡萄糖胺孵育两个小时,随后用4μM LL-37或0.5ng/ml TNF-α刺激24小时(mRNA及蛋白质的测定)或10分钟(信号转导分子磷酸化的分析及测定)。
(2)实时逆转录聚合酶链反应检测ICAM-1、MCP-1、GAPDH的mRNA水平的表达情况,评价葡萄糖胺对LL-37或TNF-α诱导产生的ICAM-1和MCP-1的mRMA的影响。
(3)ELISA法测定内皮细胞表达产生的MCP-1蛋白质。
(4)Western blot法分析ICAM-1蛋白质的表达情况。
(5)Western blot法分析葡萄糖胺对HUVECs蛋白质O-GlcNAc修饰的影响,并用Alloxan,O-GlcNAc修饰抑制剂分析该修饰与ICAM-1、MCP-1蛋白质的表达的相关性。
(6)Western blot法研究葡萄糖胺对LL-37或TNF-α诱导活化的p38MAPK、NF-κB、ERK活化即磷酸化的影响。
2、动物实验
(1)动物分组:8周龄B6.KOR.Apoe~(sh1)小鼠(高胆固醇血症,动脉粥样硬化自然发生小鼠模型,雌性),并作为对照组同时购C57BL/6(8周,雌性)。小鼠在24±3℃,相对湿度55±15%,7:00AM-7:00PM照明条件下饲养。B6.KOR.Apoe~(sh1)小鼠随机分为3组即葡萄糖胺非处理组、300mg/kg和1000mg/kg投药组(10只/组),对照组为5只C57BL/6。
(2)动物的处理及样品的制备在乙醚麻醉下,腹腔大动脉穿刺取血(使用肝素),血液样品离心(12,000rpm,4℃)5min,取上清即血浆在-80℃保存。采血后的小鼠用20%福尔马林/PBS灌流、固定、并摘取心脏。
(3)心脏的Oil Red O染色法分析葡萄糖胺处理对主动脉瓣处的动脉粥样硬化病灶大小的影响。
(4)测定血浆中的HDL、胆固醇、过氧化脂质(lipid peroxide)等动脉粥样硬化评价指标的含量,观察葡萄糖胺对这些物质浓度的影响,并分析与动脉粥样硬化病灶面积的相关性。
结果
1、葡萄糖胺在mRNA和蛋白水平抑制了LL-37和TNF-α诱导的内皮细胞的ICAM-1和MCP-1表达。
2、葡萄糖胺诱导了蛋白的O-GlcNAc修饰,这与对表达的抑制作用一致。
3、O-GlcNAc修饰的抑制剂alloxan恢复了葡萄糖胺对ICAM-1和MCP-1的抑制作用。
4、葡萄糖胺抑制了LL-37和TNF-α诱导活化的p38MAPK、NF-κB、ERK磷酸化,O-GlcNAc抑制剂Alloxan可部分恢复葡萄糖胺的抑制作用。
5、葡萄糖胺处理组与对照组相比,明显抑制了动脉粥样硬化病灶的形成(300mg/kg.day葡萄糖胺组,P＜0.05)。虽然观察不到剂量依存性效果,1000mg/kg.day葡萄糖胺组也有抑制动脉粥样硬化病灶形成的趋势。
6、葡萄糖胺虽然没有影响血浆中的HDL和胆固醇浓度,但是显著抑制了过氧化(LPO)脂质的形成。
讨论
动脉粥样硬化是动脉内壁表达黏附分子并诱导单核淋巴细胞黏附,并在单核细胞趋化因子的作用下进一步浸润至内膜下的一种慢性炎症过程。一些促炎症因子,例如TNF-α、IL-1β常表达在动脉粥样硬化病灶中,并诱导炎症。前期研究证明细胞因子可改变细胞形状和活性,改变炎症过程中的血管通透性;还可诱导产生内皮细胞的表面黏附分子和单核趋化因子等。我们在本次实验中观察了葡萄糖胺对LL-37、TNF-α诱导的ICAM-1和MCP-1表达的影响。作为黏附分子和单核细胞趋化分子的一种,它们在动脉粥样硬化病变的发生过程中起到重要的作用。当动脉壁内膜中的内皮细胞受到炎症刺激,黏附分子(例如ICAM-1)的表达将升高,并使血液中的单核细胞结合到内皮细胞,并在趋化因子(例如MCP-1)浓度梯度的趋化下浸润到内膜下。内膜下的单核细胞表达清道夫受体,变成巨噬细胞,巨噬细胞可以吞噬修饰的脂蛋白(氧化LDL等)成为泡沫细胞,这些细胞聚集在动脉粥样硬化病灶中心,从而恶化病变。
葡萄糖胺是广泛应用于治疗变形性关节炎的一种可在体内自然合成的氨基单糖,除了治疗变形性关节炎之外,近年来的多个实验室的研究结果证明它同时具有一些抗炎作用,并参与调节信号转导系统。例如,葡萄糖胺抑制中性粒细胞的超氧化物的合成、颗粒释放及趋化;还可抑制血小板的凝集;通过抑制IL-1β诱导产生的NO、PGE_2、IL-8等,调节滑膜细胞的活化。Chen等人研究证明葡萄糖胺还可抑制人视网膜色素上皮细胞的ICAM-1的表达,同样提示着潜在的抗炎作用。
动脉粥样硬化形成过程中,内皮细胞的活化起着重要的作用,所以我们研究了葡萄糖胺对内皮细胞活化的影响。我们的结果说明葡萄糖胺而不是N-acetylglucosamine在mRNA和蛋白水平抑制了ICAM-1和MCP-1的表达。
研究证明多种胞浆和和蛋白质的丝氨酸/苏氨酸残基可被O-连接-N-acetylglucosamine修饰。哺乳动物只有一组调节此修饰的酶,O-GlcNActransferase(OGT)和O-N-acetylglucosaminidase(O-GlcNAcase),而且基因敲除技术研究说明这种修饰对早期胚胎的发育及细胞功能是不可缺少的。用O-GlcNAcase抑制剂-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate(PUGNAc)灌流分离的心脏,可观察到所诱导的O-GlcNAc修饰保护缺氧/再灌流损伤,而OGT抑制剂-alloxan阻断其保护作用。另一个研究也得出相似的结论,即葡萄糖胺和PUGNAC对分离培养的心肌细胞缺氧/再给氧也起着保护作用。还有研究报道称,葡萄糖胺可增强trauma/hemorrhage大鼠模型的心脏功能,而此过程与O-GlcNAc修饰相关。上述研究结果提示着在不同疾病条件下,葡萄糖胺可阻止各种细胞组织的过度活化,相关的可能机制为蛋白质的O-GlcNAc修饰。有趣的是,O-GlcNAc修饰与O-phosphate拥有很多共同点:它们都由特异性酶调节其动态平衡,并对细胞外刺激做出迅速的反应,都可修饰蛋白质的丝氨酸/苏氨酸残基等。这说明O-GlcNAc修饰可能直接修饰磷酸化的位点或周围氨基酸而影响磷酸化,进而影响蛋白质的功能。
我们用单克隆抗体分析了葡萄糖胺对HUVEC蛋白质O-GlcNAc修饰的影响,得出以下结论:葡萄糖胺而非N-acetylglucosamine诱导蛋白质的O-GlcNAc修饰,这个结果与对ICAM-1和MCP-1表达的影响一致,即表现出抑制作用的是葡萄糖胺,而不是N-acetylglucosamine。这使我们联想到葡萄糖胺诱导的蛋白质的O-GlcNAc修饰与其对表达的抑制作用的某种联系。所以,我们进一步用alloxan,OGT抑制剂分析了这两者的关系,我们发现alloxan可有效抑制葡萄糖胺诱导的蛋白质的O-GlcNAc修饰。葡萄糖胺(1mM)可有效诱导O-GlcNAc修饰,同时也抑制ICAM-1和MCP-1的表达;alloxan有效抑制此修饰,同时也恢复了葡萄糖胺对蛋白表达的抑制作用。这些结果提示着葡萄糖胺对表达的抑制是可能介导蛋白质的O-GlcNAc修饰。
TNF-α可激活NF-κB和p38 MAPK等各种信号转导通路,并诱导一系列的炎症反应,例如黏附分子、趋化因子、促炎症细胞因子等的表达。我们利用抑制剂证明了TNF-α介导NF-κB和p38 MAPK途径刺激ICAM-1和MCP-1的表达。这些信号转导分子的活化需要特异氨基酸的磷酸化,我们首先研究了葡萄糖胺是否影响这些信号转导分子的磷酸化。我们的结果说明,用葡萄糖胺预孵育HUVEC可抑制TNF-α诱导的NF-κB和p38 MAPK分子的活化,提示葡萄糖胺可能通过影响这些信号转导途径而抑制ICAM-1和MCP-1的表达。
我们在研究中使用B6.KOR.Apoe~(sh1)小鼠(高胆固醇血症,动脉粥样硬化自然发生小鼠模型),用经口投药方式给0mg/kg.day,300mg/kg.day和1000mg/kg.day三种剂量。实验结果说明,葡萄糖胺虽然对总胆固醇和高密度脂蛋白无明显的影响,但是对过氧化脂质有明显的抑制作用,过氧化脂质是受损细胞膜的不饱和脂肪酸氧化而形成,可对周围组织细胞(例如动脉内壁)造成继发性损害,这提示着葡萄糖胺具有抗动脉粥样硬化发生的作用。心脏标本实际动脉粥样硬化病变面积(主动脉瓣膜处)的测量的结果也说明300mg/kg.day的剂量有效抑制了病变的发生,而1000mg/kg.day剂量虽然没有统计学意义,也有抑制病变发生的倾向。
总之,葡萄糖胺可抑制HUVEC的LL-37和TNF-α诱导的ICAM-1、MCP-1的表达,其可能机制为介导蛋白质O-GlcNAc修饰的对p38 MAPK和NF-κB的信号转导分子磷酸化的抑制。使用动物模型的in vivo实验也证明了葡萄糖胺具有抗动脉粥样硬化的作用。
葡萄糖胺是非处方类药物,而且已经长期应用于变形性关节炎的治疗,虽然长期使用无明显副作用,但是对其他慢性代谢性疾病的作用还是有待研究。最近也有不少实验室研究葡萄糖胺与动脉粥样硬化的关系,Duan等人证明葡萄糖胺可能通过诱导细胞外基质perlecan HSPG(heparin sulfate proteoglycan)抑制平滑肌细胞的增生,因而起到抗动脉粥样硬化的作用,并用apo-E敲除鼠证明了葡萄糖胺明显抑制了主动脉瓣的动脉粥样硬化病变的发生。但是也有实验室利用LDL受体缺损小鼠模型得出相反的结果,即葡萄糖胺恶化早期动脉粥样硬化的发生,但是对晚期的发展无明显影响的报导。作者后来又提出人类的动脉粥样硬化的早期发生在儿童时期,服用葡萄糖胺的人群是高龄人,所以实际上葡萄糖胺应该没有促进动脉硬化的作用。这些不同的实验结果可能是因为使用的动物模型种类、投药方式、投药时间的长短不同而造成的。通常用于人变形性关节炎治疗的葡萄糖胺的剂量为1.5或3g/天,这时人血清中的葡萄糖胺浓度可达到0.02-0.03 mM,我们在本次研究中In vitro使用的浓度为0.01-1mM,虽然在生理浓度下没有统计学意义,但是在LL-37刺激下0.01mM也表现出抑制的倾向。我们的各种实验结果均表明葡萄糖胺可能通过抑制ICAM-1和MCP-1的表达和减少血浆中过氧化脂质,从而起到动脉粥样硬化发生过程中的抗炎作用。
结论
1、葡萄糖胺抑制LL-37和TNF-α诱导产生的黏附分子ICAM-1和MCP-1的表达,从而调节内皮细胞的活化。
2、葡萄糖胺诱导的蛋白O-GlcNAc修饰与其对表达的抑制作用相关,而且O-GlcNAc修饰的抑制剂alloxan恢复了葡萄糖胺对蛋白表达的抑制作用,说明此修饰是葡萄糖胺调节内皮细胞活化的一种可能机制之一。
3、葡萄糖胺可抑制LL-37和TNF-α诱导活化的p38MAPK、NF-κB、ERK等信号转导分子的磷酸化,说明葡萄糖胺可能介导这些信号转导分子调节ICAM-1和MCP-1的表达。
4、葡萄糖胺处理组的小鼠饮水量及体重较对照组有所增加。
5、葡萄糖胺抑制了动脉粥样硬化病变的发展。
Introduction
In 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.
Methods
1、cell experiment
(1) cell culture and treatment
Human 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.Apoe~(sh1) 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.
Results
1、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.
Discussion
Atherosclerosis 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,PGE_2, 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.
Conclusions
1、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.
引文
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38 N. Comtesse, E. Maldener, E. Meese. Identification of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a beta-N-acetylglucosaminidase. Biochem. Biophys. Res. Commun. 2001; 283: 634-640.
39 V.S. Farook, C. Bogardus, M. Prochazka. Analysis of MGEA5 on 10q24.1-q24.3 encoding the beta-O-linked N-acetylglucosaminidase as a candidate gene for type 2 diabetes mellitus in Pima Indians. Mol. Genet. Metab. 2002; 77:189-193.
40 Y. Akimoto, L.K. Kreppel, H. Hirano, et al. Localization of the OGlcNAc transferase in rat pancreas. Diabetes. 1999; 48: 2407-2413.
41 Y. Akimoto, L.K. Kreppel, H. Hirano, et al. Localization of the O-linked N-acetylglucosamine transferase in rat pancreas. Diabetes. 1999; 48:2407-2413.
42 D.L. Arvanitis, L.D. Arvanitis, I.G. Panourias, et al. Mitochondria-rich normal, metaplastic, and neoplastic cells show overexpression of the epitope H recognized by the monoclonal antibody H. Pathol. Res. Pract. 2005; 201: 319-324.
43 N.E. Zachara, G.W. Hart. O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim. Biophys. Acta. 2004; 1673: 13-28.
44 D.C. Love, J.A. Hanover. The hexosamine signaling pathway: deciphering the "OGlcNAc code",.Sci. STYKE. 2005; 312: 13.
45 T. Nagy, V. Champattanachai, R.B. Marchase, et al. Glucosamine inhibits angiotensin II induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-GlcNAc. Am. J. Physiol. Cell Physiol. 2005; C57-C65.
46 Y. Pang, P. Bounelis, J.C. Chatham, et al. Hexosamine pathway is responsible for inhibition by diabetes of phenylephrineinduced inotropy. Diabetes. 2004; 53:1074-1081.
47 K.C. Sohn, K.Y. Lee, J.E. Park, et al. OGT functions as a catalytic chaperone under heat stress response: a unique defense role of OGT in hyperthermia. Biochem. Biophys. Res. Commun. 2004; 322:1045-1051.
48 E. Masson, N. Wiernsperger, M. Lagarde, S. El Bawab. Glucosamine induces cell-cycle arrest and hypertrophy of mesangial cells: implication of gangliosides. Biochem. J. 2005; 388: 537-544.
49 Z.T. Kneass, R.B. Marchase. Protein O-GlcNAc modulates motilityassociated signaling inter mediates in neutrophils. J. Biol. Chem. 2005; 280: 14579-14585.
50 K.I. Larsen, M. Falany, W. Wang, et al. Glucose is a key metabolic regulator of osteoclasts; glucose stimulated increases in ATP/ADP ratio and calmodulin kinase II activity. Biochem. Cell Biol. 2005; 83: 667-673.
51 W.G. Kelly, M.E. Dahmus, G.W. Hart. RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc. J. Biol. Chem. 1993; 268: 10416-10424.
52 C. Guinez, W. Morelle, J.C. Michalski, et al. O-GlcNAc glycosylation: a signal for the nuclear transport of cytosolic proteins? Int. J. Biochem. Cell Biol. 2005; 37: 765-774.
53 Fulop N., Zhang Z., Marchase RB., et al.Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation. Am J Physiol Heart Circ Physiol. 2007; 292: H2227-36.
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35 Y. Hu, D. Belke, J. Suarez, et al. Adenovirus-mediated overexpression of O-GlcNAcase improves contractile function in the diabetic heart. Circ. Res. 2005; 96: 1006-1013.
36 C. Slawson, N.E. Zachara, K. Vosseller, et al. Perturbations in O-linked beta-N-acetylglucosamine protein modification cause severe defects in mitotic progression and cytokinesis. J. Biol. Chem. 2005; 280: 32944-32956.
37 Y. Gao, L. Wells, F.I. Comer, et al. Dynamic O-glycosylation of nuclear and cytosolic proteins—Cloning and characterization of a neutral, cytosolic beta-N-acetylglucosam inidase from human brain. J. Biol. Chem. 2001; 276: 9838-9845.
38 N. Comtesse, E. Maldener, E. Meese. Identification of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a beta-N-acetylglucosaminidase. Biochem. Biophys. Res. Commun. 2001; 283: 634-640.
39 V.S. Farook, C. Bogardus, M. Prochazka. Analysis of MGEA5 on 10q24.1-q24.3 encoding the beta-O-linked N-acetylglucosaminidase as a candidate gene for type 2 diabetes mellitus in Pima Indians. Mol. Genet. Metab. 2002; 77:189-193.
40 Y. Akimoto, L.K. Kreppel, H. Hirano, et al. Localization of the OGlcNAc transferase in rat pancreas. Diabetes. 1999; 48: 2407-2413.
41 Y. Akimoto, L.K. Kreppel, H. Hirano, et al. Localization of the O-linked N-acetylglucosamine transferase in rat pancreas. Diabetes. 1999; 48:2407-2413.
42 D.L. Arvanitis, L.D. Arvanitis, I.G. Panourias, et al. Mitochondria-rich normal, metaplastic, and neoplastic cells show overexpression of the epitope H recognized by the monoclonal antibody H. Pathol. Res. Pract. 2005; 201: 319-324.
43 N.E. Zachara, G.W. Hart. O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim. Biophys. Acta. 2004; 1673: 13-28.
44 D.C. Love, J.A. Hanover. The hexosamine signaling pathway: deciphering the "OGlcNAc code",.Sci. STYKE. 2005; 312: 13.
45 T. Nagy, V. Champattanachai, R.B. Marchase, et al. Glucosamine inhibits angiotensin II induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-GlcNAc. Am. J. Physiol. Cell Physiol. 2005; C57-C65.
46 Y. Pang, P. Bounelis, J.C. Chatham, et al. Hexosamine pathway is responsible for inhibition by diabetes of phenylephrineinduced inotropy. Diabetes. 2004; 53:1074-1081.
47 K.C. Sohn, K.Y. Lee, J.E. Park, et al. OGT functions as a catalytic chaperone under heat stress response: a unique defense role of OGT in hyperthermia. Biochem. Biophys. Res. Commun. 2004; 322:1045-1051.
48 E. Masson, N. Wiernsperger, M. Lagarde, S. El Bawab. Glucosamine induces cell-cycle arrest and hypertrophy of mesangial cells: implication of gangliosides. Biochem. J. 2005; 388: 537-544.
49 Z.T. Kneass, R.B. Marchase. Protein O-GlcNAc modulates motilityassociated signaling inter mediates in neutrophils. J. Biol. Chem. 2005; 280: 14579-14585.
50 K.I. Larsen, M. Falany, W. Wang, et al. Glucose is a key metabolic regulator of osteoclasts; glucose stimulated increases in ATP/ADP ratio and calmodulin kinase II activity. Biochem. Cell Biol. 2005; 83: 667-673.
51 W.G. Kelly, M.E. Dahmus, G.W. Hart. RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc. J. Biol. Chem. 1993; 268: 10416-10424.
52 C. Guinez, W. Morelle, J.C. Michalski, et al. O-GlcNAc glycosylation: a signal for the nuclear transport of cytosolic proteins? Int. J. Biochem. Cell Biol. 2005; 37: 765-774.
53 Fulop N., Zhang Z., Marchase RB., et al.Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation. Am J Physiol Heart Circ Physiol. 2007; 292: H2227-36.