【作者】 王立俊；

【导师】 汪翼；

【作者基本信息】 山东大学 ， 儿科学， 2009， 博士

【摘要】 研究背景和目的:近年来研究证实血管内皮细胞(Vascular endothelial cell,VEC)损伤及内皮功能障碍(endothelial dysfunction,ED)是启动动脉粥样硬化(Atherosclerosis,AS)发生的早期事件,故研究内皮细胞功能及代谢的变化对探讨AS的发病机制有重要意义。而随着对AS自然病程认识的深入,已经明确VEC的完整性和功能性损害可始于儿童期,AS是一种发生于青少年、发病于成人阶段的慢性渐进性损害过程。儿童期糖尿病(Diabetes Mellitus,DM)绝大多数属于胰岛素依赖型糖尿病(Insulin-DependentDiabetes Mellitus,IDDM),其发病早、病程长,远期并发症尤其是糖尿病血管病变对成人期的危害,已引起人们的广泛关注。其中,糖尿病大血管病变是患者致死、致残的主要原因,其病理实质是血管AS。同样,糖尿病儿童血管内皮功能障碍亦是启动AS的早期关键环节,其血管内皮损伤和功能障碍尚属可逆性病理改变。所以,早期检测、早期干预血管内皮功能障碍对于预防糖尿病血管病变的发生、改善青少年糖尿病病人生活质量至关重要。近年来研究证实,高血糖造成的病理损害主要是通过诱导胞内氧化应激导致。糖尿病病理状态下,氧化应激过强,机体和血管局部抗氧化能力的降低是糖尿病血管内皮功能障碍的主要原因。作为氧化应激的重要产物,氧化低密度脂蛋白(Oxidizedlow-density lipoprotein,oxLDL)被认为是诱发血管内皮功能紊乱的一个关键因子。内皮细胞摄取oxLDL后,oxLDL的脂质成份可以通过降低血管舒张功能、诱导血栓形成和炎症反应等损伤VEC的功能。Sawamura研究证实,oxLDL的内皮毒性作用主要是通过其特异性内皮细胞表面受体-植物血凝素样氧化低密度脂蛋白受体1(Lectin-likeOxLDL receptor-1,LOX-1)介导。OxLDL通过与LOX-1的结合,可以激活核转录因子(Nuclear Factor,NF)-κB,在转录水平上诱导内皮细胞间粘附分子1(intercellularadhesion molecule-1,ICAM-1)、血管细胞粘附分子1(vascular cell adhesionmolecule-1,VCAM-1)、单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)等的表达,趋化粒细胞、单核巨噬细胞粘附于内皮,影响内皮源性一氧化氮(Nitric oxide,NO)的生成和利用,使内皮依赖的血管舒张功能受损,启动AS过程。既往已有研究证实LOX-1在高脂喂养AS兔中的表达和重要作用;而对1型糖尿病早期oxLDL/LOX-1在血管内皮功能障碍中的表达和作用,国内外研究甚少。LOX-1是一种在内皮细胞上表达的单链跨膜蛋白,在结构上属于C型血凝素家族,其基因的5`区域含有一个NF-κB样的序列及结合位点。生理情况下,LOX-1在内皮细胞的表达量极少,研究证实oxLDL及其他氧化应激产物均可诱导内皮细胞LOX-1的表达。而NF-κB通路是一个重要的氧化应激信号转导途径,体外证实其在oxLDL/LOX-1诱导的内皮损伤中发挥重要作用。体外研究还证实,内皮细胞LOX-1的表达被其自身配基oxLDL浓度依赖性诱导,抗LOX-1抗体和抗氧化剂能阻断oxLDL的这种诱导作用。而且,近几年在体实验亦发现诸如他汀类、ACEI类等药物均可不同程度的实现对oxLDL/LOX-1体系的调控进而产生抗AS作用,这种调控作用是依赖其胞内抗氧化特性,而非本身的降脂、降压作用。这些结果证实oxLDL/LOX-1系统的调控为一氧化还原敏感模式,这为深入研究某些疾病的发病机制及药物治疗提供了锲机。在AS病变的发生发展中,发生于儿童与青少年期脂质条纹形成之前的病变包括内皮功能障碍,是一可逆的过程,早期干预可逆转其向纤维斑块甚或粥样硬化斑块的进展,而适宜干预措施的制定重点在于考察其有效性和安全性。临床上对1型糖尿病仍以胰岛素控制血糖为基本治疗;同时近年来,抗氧化治疗成为改善糖尿病病情、减少糖尿病并发症发生的又一个热点。牛磺酸(taurine,TAU)又名2-氨基乙磺酸,是内源性抗氧化系统的重要组成部分,在兴奋性较高组织如神经、肌肉、晶状体、肾小球及心血管组织含量最丰富,具有抗炎、抗氧化、抗血小板聚集、调节渗透压及钙离子平衡等多种生理作用。近年来研究证实在1型糖尿病患者或动物模型,血浆及局部组织中牛磺酸水平显著下降,饮食中添加牛磺酸治疗对糖尿病心肌病、周围神经病变及视网膜病变等的改善具有促进作用。而体外研究亦表明牛磺酸对内皮细胞功能具有保护作用,但这些作用的具体分子机制尚不清楚。作为一种内源性抗氧化剂,在体情况下牛磺酸对于糖尿病早期oxLDL/LOX-1系统和血管内皮功能障碍的干预作用及其可能的机制值得研究。本研究以链脲佐菌素(streptozotocin,STZ)诱导1型糖尿病大鼠模型,在造模成功后即给予牛磺酸干预,并同胰岛素治疗做比较,通过在体直接检测糖尿病大鼠早期血管内皮的超微结构改变和功能状态改变,同时从分子生物学水平检测循环中或主动脉血管内皮oxLDL、LOX-1、NF-κBp65及ICAM-1的表达水平,初步探讨oxLDL/LOX-1系统及NF-κB信号通路在糖尿病早期血管内皮功能障碍中的作用,以及牛磺酸对糖尿病血管内皮的保护作用与可能机制。研究方法:1.实验模型的建立及分组:选用8周龄SPF级雄性Wistar大鼠65只,体重180～200g,随机取出16只作为正常对照组(NC组),其余给予一次性腹腔注射STZ 60mg/kg诱导1型糖尿病模型,注射STZ 72h后尾静脉取血检测血糖≥16.7mmol/L、尿糖+++以上者表示造模成功。从造模成功大鼠中随机取40只分为3组:糖尿病未干预组(DM组,n=20),胰岛素治疗组(In组,n=10),牛磺酸干预组(TAU组,n=10)。于造模成功后第二天开始,胰岛素治疗组每天下午5点给予颈背部皮下注射一次超长效胰岛素来得时(甘精胰岛素锌)3～10u,次晨9点左右检测血糖,根据血糖结果调整胰岛素用量,使非空腹血糖控制在3.9～10mmol/L;牛磺酸干预组每日饮用水中添加1%牛磺酸。整个实验期间所有大鼠均喂标准饲料,自由饮水。NC、DM组于实验2周末、6周末,各干预组于6周末分别随机处死8只大鼠取材和检测。2.检测指标及方法①体重和血糖:每周测量一次体重和空腹血糖,血糖采用One TouchⅡ血糖仪检测。②外周血指标的检测:外周血甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)水平采用全自动生化分析仪检测;血清可溶性ICAM-1(sICAM-1)、oxLDL水平采用Elisa方法检测;血清NO水平采用硝酸还原酶比色法检测;血浆内皮素(endothelin,ET)含量采用放射免疫法检测。③离体胸主脉环内皮依赖性血管舒张反应:大鼠处死取血后,立即游离胸主动脉,剪取最上段约3～5mm主动脉环检测不同浓度乙酰胆碱(acetylcholine,Ach)(1nM～100μM)诱导的内皮依赖性血管舒张反应,计算并绘制log Ach浓度依赖的血管舒张曲线,Rmax代表Ach诱导的血管环舒张最大百分比,直线回归方程计算EC50(血管环达到50%最大舒张时所需Ach浓度)。④血管内皮超微结构观察:胸主动脉组织依次经戊二醛、锇酸固定、脱水、包埋,甲苯胺兰超薄切片后行透射电镜铀—铅双重染色,观察主动脉血管内皮细胞超微结构的改变。⑤主动脉壁LOX-1、NF-κBp65、ICAM-1蛋白的组织表达:冰冻切片免疫荧光染色法定性检测胸主动脉血管壁LOX-1蛋白的表达;石蜡切片免疫组化SP法检测胸主动脉血管壁NF-κBp65、ICAM-1蛋白的表达。⑥Western blot方法定量分析LOX-1、NF-κBp65在主动脉壁的蛋白含量:提取主动脉组织标本的蛋白质样品,经变性、电泳、转膜及封闭后,先后加入稀释的LOX-1、NF-κBp65一抗及辣根过氧化物酶标记的二抗室温孵育,经ECL显影、凝胶成像分析系统扫描蛋白条带进行光密度分析。以β-actin作为内参,以目的蛋白/β-actin蛋白条带吸光度值作为蛋白表达强度。⑦逆转录—聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)定量检测LOX-1、NF-κBp65、ICAM-1 mRNA在主动脉血管壁的表达水平:提取各组主动脉总RNA,将mRNA逆转录成cDNA后行PCR扩增。PCR产物经琼脂糖凝胶电泳、成像分析。分别以LOX-1,NF-κBp65,ICAM-1与β-actin扩增条带吸光度比值作为其mRNA表达强度。3.统计分析:采用SPSS 11.0统计软件包,统计资料用(?)±s描述,组间数据分析之前先进行方差齐性检验,方差齐性指标进行单因素方差分析(one-way ANOVA)及两两比较Q检验,方差不齐的指标进行独立样本秩和检验及两两比较;部分变量间相关性分析采用Person相关性分析与Partial相关分析(控制干扰因素后进行相关分析)。P＜0.05认为差异有统计学意义。研究结果:1.一般情况及体重、血糖的变化:整个实验期间,DM组大鼠死亡一只,死亡原因考虑为高血糖衰竭和严重感染。与正常对照组比较,所有糖尿病大鼠血糖明显升高,并一直维持于高水平状态,且体重增长缓慢,明显低于正常对照组大鼠(P均＜0.01);胰岛素治疗组大鼠血糖控制理想,体重增长理想,同正常对照组比较差异不明显;牛磺酸干预对糖尿病大鼠体重、血糖无明显改善。2.血脂水平的变化:同正常对照组比较,糖尿病组大鼠2周时血脂变化不明显,6周时出现血TC、TG、LDL的明显升高(P均＜0.01);胰岛素治疗组血脂指标明显改善,TC、TG、LDL降至正常水平(同正常对照组比较,P＞0.05),牛磺酸干预组大鼠TC、TG水平明显下降(同糖尿病组比较,P均＜0.01),但与正常对照组比较仍有差异(P＜0.05)3.外周血oxLDL、NO、ET及sICAM-1水平的变化:糖尿病组2周时,大鼠血清oxLDL、NO和血浆ET水平出现升高(P均＜0.05),6周时血清oxLDL、sICAM-1及血浆ET水平明显升高,同时伴随着血清NO水平及NO/ET值的明显降低,同正常对照组比较差异均有统计学意义(P均＜0.01);同糖尿病组比较,胰岛素治疗组和牛磺酸干预组血清oxLDL水平明显降低(P均＜0.01),但与正常对照组比较仍有差异(P均＜0.05)。4.胸主动脉血管内皮细胞超微结构的变化:正常大鼠主动脉内膜表面平整,内皮细胞扁平状,排列规则,细胞器形态数量正常,胞核规则;糖尿病2周时,内皮超微结构变化不明显,6周时内皮超微结构变化明显,出现内皮细胞肿胀、部分脱落,基底板部分断裂,内皮细胞微绒毛消失、线粒体嵴突模糊,高尔基体、内质网肿胀、核周池扩张、核皱缩变形等改变。5.离体血管Ach诱导的内皮依赖性舒张反应的变化:糖尿病组大鼠2周时,主动脉环对Ach诱导的内皮依赖性血管舒张反应迟钝,EC50大于正常对照组(P＜0.05),而最大舒张百分比Rmax变化不明显;6周时DM组Ach诱导的离体胸主动脉环内皮依赖性血管舒张功能明显受损,EC50升高,Rmax明显降低(P均＜0.01);与糖尿病组相比,胰岛素治疗组和牛磺酸干预组内皮依赖性血管舒张功能损伤明显减轻,EC50降低,Rmax升高(P均＜0.01),其中胰岛素组改善最明显,EC50能恢复到正常组水平(P＞0.05)。6.主动脉血管壁LOX-1蛋白的表达:6.1免疫荧光染色法定性检测主动脉血管壁LOX-1蛋白的组织表达:正常对照组及糖尿病2周时大鼠主动脉血管壁基本未见或极少LOX-1蛋白的阳性表达(亮绿色荧光染色),而6周时糖尿病大鼠主动脉内皮层可见明显的亮绿色荧光染色信号;胰岛素和牛磺酸干预组大鼠主动脉内皮层的亮绿色荧光染色信号均明显减弱减少。6.2 Western Blot定量分析主动脉血管壁LOX-1蛋白表达量:ECL显影结果及以β-actin蛋白表达为参照,图像系统定量分析结果显示,各组腹主动脉壁LOX-1在蛋白水平上均有表达,正常对照组和糖尿病2周时LOX-1蛋白表达水平极低,而糖尿病6周时LOX-1蛋白表达水平明显高于正常对照组(P＜0.01);胰岛素、牛磺酸干预组LOX-1蛋白表达水平明显降低,同糖尿病组比较差异明显(P均＜0.01),其中胰岛素组LOX-1蛋白水平下降最明显,但与正常对照组比较仍有差异(P＜0.05)。7.主动脉血管壁NF-κBp65蛋白的表达:7.1免疫组织化学SP法检测NF-κBp65蛋白的组织表达:NF-κBp65在所有大鼠主动脉血管内皮层及肌层均有阳性表达,正常对照组和2周时糖尿病组表达很弱,仅极少内皮和平滑肌细胞浆内有稀疏淡染棕黄色染色颗粒;6周时糖尿病大鼠主动脉血管内皮及平滑肌细胞内均可见有密集的棕黄色阳性染色颗粒,内皮层细胞核内亦出现棕色阳性染色颗粒;胰岛素治疗组和牛磺酸干预组内皮层及平滑肌细胞均着色较浅,细胞核内阳性表达信号亦明显减弱减少。7.2 Western Blot定量分析胸主动脉血管壁NF-κBp65蛋白的表达水平:显影结果及图像系统定量分析显示,各组胸主动脉壁NF-κBp65蛋白均有表达,正常对照组和糖尿病2周时蛋白条带显影很弱,蛋白表达水平很低;糖尿病6周时蛋白条带显影较强,蛋白表达水平明显高于正常对照组(P＜0.01);胰岛素治疗、牛磺酸干预组NF-κBp65蛋白表达水平明显降低,同糖尿病组比较差异有统计学意义(P均＜0.01),其中胰岛素治疗组NF-κBp65蛋白表达水平降至接近正常水平(同NC组比较,P＞0.05)。8.胸主动脉血管壁ICAM-1蛋白的表达水平免疫组织化学SP法检测ICAM-1蛋白在主动脉壁的表达部位,计算机图像分析系统计算内皮细胞阳性染色平均积分光密度值作为ICAM-1蛋白表达水平显示:ICAM-1主要表达于大鼠胸主动脉血管内皮层细胞。正常对照组和2周时DM组ICAM-1蛋白阳性表达极少,只有少数内皮细胞胞浆内有淡染的颗粒;DM组内皮层可见连续、浓染棕色颗粒,ICAM-1蛋白表达水平明显高于正常对照组大鼠(P＜0.01);胰岛素和牛磺酸干预组,内皮细胞ICAM-1蛋白阳性表达明显减弱,同糖尿病组比较差异有统计学意义(P均＜0.01),胰岛素治疗组ICAM-1蛋白表达水平降低最明显,但与正常对照组比较仍有差异(P＜0.05)。9.RT-PCR定量检测主动脉血管壁LOX-1,NF-κBp65,ICAM-1 mRNA表达水平:正常对照组和2周时糖尿病大鼠胸主动脉血管壁LOX-1,NF-κBp65,ICAM-1 mRNA表达极微弱;6周时,糖尿病组LOX-1,NF-κBp65,ICAM-1 mRNA表达明显增强,表达水平明显高于正常对照组(P均＜0.01);同糖尿病组比较,胰岛素和牛磺酸干预组LOX-1,NF-κBp65,ICAM-1 mRNA在主动脉壁的表达均明显受抑制(P均＜0.01),其中胰岛素组抑制作用最明显,NF-κBp65,ICAM-1 mRNA与正常对照组比较无显著差异(P均＞0.05)。10.相关性分析:糖尿病大鼠血清oxLDL与NO水平、Rmax、NO/ET比值负相关,与血浆ET、血清sICAM-1水平正相关;主动脉内皮最大舒张Rmax与ET、oxLDL、sICAM-1水平负相关,与NO、NO/ET正相关,二者均与血糖水平无明显相关性。控制体重和血脂干扰行Partial相关分析显示,以上相关性仍具有统计学意义。同时,主动脉血管壁LOX-1mRNA表达水平与血清oxLDL水平及血管壁NF-κBp65和ICAM-1mRNA表达水平亦显著正相关,与Rmax负相关。研究结论:1.糖尿病早期即可出现血管内皮功能及超微结构损伤,内皮依赖性血管舒张反应是最直接最敏感的内皮功能损伤检测指标;外周血NO、ET、NO/ET和sICAM-1水平可作为筛查内皮功能障碍的早期间接指标。2.糖尿病早期存在过强的LDL氧化修饰,oxLDL是诱导糖尿病早期血管内皮功能障碍的独立危险因子;外周血oxLDL水平可作为糖尿病血管内皮功能障碍的早期预测指标。3.糖尿病早期血管内皮功能损伤,同步发生了主动脉血管壁LOX-1蛋白和基因表达上调,且与血清oxLDL水平密切相关,表明oxLDL与LOX-1相互作用介导血管内皮损伤,oxLDL/LOX-1系统是诱导糖尿病早期血管内皮功能障碍的重要分子因素。4.糖尿病早期主动脉壁NF-κBp65表达明显增强,并伴有明显核转位,且其基因表达与LOX-1,ICAM-1mRNA表达正相关,提示糖尿病大鼠早期主动脉壁NF-κB激活,NF-κB信号通路活化诱导内皮黏附因子ICAM-1表达参与了oxLDL/LOX-1诱导的内皮损伤过程。5.胰岛素严格控制血糖对糖尿病早期内环境紊乱和血管内皮功能障碍有不同程度的缓解,对LDL的氧化修饰及LOX-1的表达亦有明显阻抑作用,但胰岛素治疗并未能完全阻滞早期糖尿病血管内皮功能障碍的发生。6.牛磺酸早期干预虽不能控制血糖,但可缓解oxLDL/LOX-1诱导的糖尿病早期血管内皮功能障碍,其内皮保护作用可能与其抗氧化、抗炎特性阻断LDL的氧化修饰、抑制oxLDL/LOX-1及NF-κB活化、下调ICAM-1的内皮表达有关。创新和意义:1.在体直接、动态地观察了1型糖尿病早期血管内皮功能障碍的发生与外周血oxLDL及内皮源性舒缩因子、黏附分子指标的变化,并通过相关分析和偏相关分析发现oxLDL与糖尿病早期血管内皮功能障碍的密切关系,为临床发现及早期预测糖尿病血管病变的发生提供参考依据。2.首次在体证实糖尿病早期主动脉血管壁同步出现LOX-1、NF-κB、ICAM-1蛋白和基因的过度表达,相关分析证实LOX-1mRNA表达水平与外周血oxLDL水平、内皮依赖性血管舒张功能、NF-κB和ICAM-1mRNA表达密切相关。提示oxLDL/LOX-1系统的表达是诱导糖尿病血管内皮功能障碍的早期关键分子事件,NF-κB信号通路活化参与了oxLDL/LOX-1介导的内皮损伤过程,早期干预和调控oxLDL/LOX-1、NF-κB的表达及活化,阻断其相互协同作用为临床早期干预糖尿病血管AS的发生提供了重要线索。3.首次对比研究发现在糖尿病早期药物干预中,牛磺酸早期干预虽不能有效控制血糖,但能同胰岛素一样明显减轻糖尿病血管内皮功能的损伤,并同步出现外周血oxLDL水平及组织LOX-1、NF-κB、ICAM-1蛋白和基因表达水平的降低,提示牛磺酸的血管内皮保护作用与其抗氧化和抗炎作用有关,在糖尿病血管病变的早期防治中,牛磺酸不失为一很好的药物选择。更多还原

【Abstract】 Backgroud and objectives:In recent years,many investigations have demonstrated that vascular endothelial cells（VEC） injury and endothelium dysfunction（ED） is the initiating and early event in the development of atherosclerosis（AS）.So it’s very important to study the early changes of endothelial function for the pathogenesy of AS.At the same time,as more is learned about the natural course of the development of AS,it is clear that AS is a pathological process that progressed slowly but steadily,in which the damage of endothelium cellular integrity and function begins in childhood,while becomes symptomatic during the adults.Diabetes mellitus（DM） in childhood mostly belongs to insulin-dependent diabetes mellitus（IDDM）, which is easily complicated diabetes vasculopathy because of its early onset and long course.Of the total,diabetic macroangiopathy is the main reason of death and diasability for diabetes patients,which pathological change is vescular AS in essence.Equally,early vascular ED in children with diabetes also is the early key step for priming AS process. While,injury of VEC and ED appear reversible pathological change.Therefore,it is crucial to detect and prevent vascular endothelial dysfunction earlier for the prevention of diabetic vasculopathy and improve the live quality of young diabetes patients.In DM,hyperglycaemia and dyslipidemia increased oxidative stress and decreased antioxidative activity of body or partial tissue,which is viewed the main cause of vascular endothelium dysfunction in diabetic conditions.As one important product of oxidative stress,oxidized low-denstity lipoprotein（oxLDL） was a key factor to cause endothelium dysfunction.The endothelial phagocytosis of oxLDL damages the function of VEC by decreasing vascular diastolic function,inducing thrombogenesis and inflammatory reaction. On the other hand,more and more studies have confirmed that endothelial cells internalize and degrade the oxidized LDL mainly by its specificially endothelial cell surface receptor, lectin-like oxLDL receptor-1（LOX-1）.The binding of oxLDL to LOX-1 can activate nuclear factor-KappaB（NF-κB）,then further induce the expression of intercellular adhesion molecule-1（ICAM-1） and vascular cell adhesion molecule-1（VCAM-1） as well as monocyte chemoattractant protein-1（MCP-1） in transcriptional levels,which facilitate the adhesion of neutrophil and monocytes to endothelium,and affect the product of endothelium-derived NO and diminish endothelium-dependent vasorelaxation,initiating atherosclerosis.The previous study have confired that oxLDL/LOX-1 was highly expressed in AS rabbits induced by high-cholesterol die.But it was very few about study in vivo on the expression and role as well as its related signal pathway of oxLDL/LOX-1 in early vascular endothelial dysfunction of diabetes.LOX-1 is a single transmembrane protein expressed on endothelial cells,and it belongs to the C-type lectin family in structure,which includes a NF-κBgene-like sequence and a AP-1 binding site in its 5’domain.In the physiological conditions,the basal expression of LOX-1 in endothelial cells is very low,while it can be induced in vitro by ox-LDL and other oxidant species.More importantly,LOX-1 expression is highly upregulated in cultured endothelial cells by its ligand,oxidized LDL,while this upregulation of LOX-1 was blocked by LOX-1 antibody and antioxidant.Recent studies in vivo or vitro showed that many drugs such as stains and ACEI（angiotensin converting enzyme inhibitor） may prevent the development of AS by regulating oxLDL/LOX-1 system,and this effect of regulation relys on their intracellular antioxidant abilities rather than the original hypolipidemic or hypotensive activities.All these results indicate that redox-sensitive regulation of oxLDL/LOX-1 actually occurs in vivo,which gives us a deep insight into the pathogenesis of some cardiovascular diseases and provides a potential selective therapeutic approach for clinical physicians. During the development of AS,the early pathological changes including endothelium dysfunction before lipids streak in young patients are reversible,and early intervention can reversed the tendency of development into fibrous or atherosclerotic plaque.While the drug choice of fit intervention focus on it’s availability and safety.At present,chronic administration of insulin to control hyperglycaemia is still the basically clinical treatment for IDDM.At the same time,recent studies showed that anti-oxidative treatment is also an effective therapy to obtain a better control of diabetic complication.Taurine,also called 2-aminoethane sulfonic acid,acts as an important endogenous antioxidant,and its content is abundant in organs or tissues of high excitability,such as nerves,muscle,crystalline lens, glomerulus and cardiovascular system.Taurine exhibits a diversity of actions such as an anti-oxidative,anti-inflammatory,antiplatelet activity,osmoregulator and intracellular Ca²⁺ regulator et al.Recent studies confirmed that the taurine levels of plasma and tissues were dereased in type 1 diabetes conditions,and taurine- supplemented diet had beneficial effect to ameliorate diabetic cardiomyopathy,diabetic polyneuropathy and retinopathy.Also,in vitro it had been manifested that taurine could protect against endothelial dysfunction induced by oxLDL.However,the exact molecule mechanisms of these actions are not very clear.As an endogenous antioxidant,the intervention effects of taurine in vivo on diabetes-induced early vascular dysfunction and the possible mechamism involved oxLDL/LOX-1 are worthwhile to study.In the present study,type 1 diabetes model was induced by STZ and given intervetion of insulin or taurine at once after the onset of diabetes.Then,the damages of endothelial ultramicrostructure and function,the expression of LOX-1、NF-κBp65 and ICAM-1 on aorta as well as the circulating levels of oxLDL were examed,aiming to find the effects of oxLDL/LOX-1 and NF-κB signal pathway on early vascular endothelium dysfunction as well as the endothelial protective effects of taurine and the possible mechanism in type 1 diabetes.Methods:1.The establishment of type 1 diabetes model:8-week-aged male Wistar rats （180-200g） were divided randomly into control group（NC,n=16） and diabetic models group.Diabetic models were induced by a single intraperitoneal injection of STZ 60 mg/kg and blood glucose level was measured after 72 hours of the injection.Rats with blood glucose（BG）≥16.7mmol/L and urine glucose≥3+ were indicated as diabetes. Then randomly picked 40 diabetic rats and subdivided into three groups:diabetes group （DM group,n=20）,insulin-treated diabetes group（In group,n=10） and taurine-treated diabetes group（TAU group,n=10）.Insulin-treated rats were injected subcutaneously with long acting insulin once daily at 17 o ’clock and then measured the non-fasting BG at about 9 o’clock of the second day,the dose of insulin（3～10unit/rat） was adjusted for individual animals to keep the non-fasting BG between 3.9～10mmol/L.While the rats in TAU group were given freely drinking water containing 1%taurine.The whole study lasted for 6 weeks.8 rats were randomly picked and killed in NC and DM rats at the end of week 2 and week 6,while 8 rats of other groups were killed at the end of week 6 respectively,then blood and tissues were collected.2.Measurement Methods:①Body weight and BG:The body weight and fasting BG of every rat were measured every week during the study.BG was determined by blood glucose meter OneTouch Ultra.②Determination of circulating indexes:the levels of total cholesterol（TC）, low-density lipoprotein（LDL）,high-density lipoprotein（HDL） and triglyceride（TG） in serum were determined by Olympus AU-1000 biochemical instrument.The levels of serum oxLDL and sICAM-1 were measured by enzyme-labeled immunosorbent assay（ELISA） kit, and the level of serum nitric oxide（NO） as well as plasma endothelin（ET） were determined by nitrate reductase indirect assay and radioimmunity homogeneous phase competition assay respectively.③Functional assessment of isolated thoracic aortas:After the rats were killed and blood samples were collected,the thoracic aortas were rapidly isolated at once.Segments of the suprema aortas were rapidly cut into rings of 3～5mm length,and the endothelium-dependent vasodilator responses induced by acetylcholine（Ach） were determined.Then the percentage of accumulative concentration-vasodilatator response to aeetylcholine（1nM～100μM） were calculated.Rmax was the Ach-induced maximum relaxation of arteries,and EC50 represents the Ach concentration needed to induce 50%maximum relaxation calculated by linear regression. ④Ultrastructure observation of vascular endothelium:Ultrastructure changes of aorta endothelium were observed by double staining transmission electron microscope.⑤Determination of LOX-1,NF-κBp65 and ICAM-1 protein expression on aortic tissues:Expression of LOX-1 and NF-κBp65 as well as ICAM-1 protein on aortic endotheilium were qualitatively determined by immunofluorescent staining with frozen section and by SP immunostaining kit with paraffin sections respectively.⑥The protein content of LOX-1 and NF-κBp65 on aortic tissues were determined by Western blotting assay:freshly frozen samples of partial aortic tissues were subsequently homogenized and lysed in lysis buffer,then protein samples were resolved and extracted. Immunoblotting was performed using diluted LOX-1 or NF-κBp65 antibody.The membranes were subsequently probed with horseradish peroxidase-conjugated secondary antibody and developed by chemiluminescence.The membranes were then exposed to X-ray film and subsequently developed.Densitometry was performed with gel imaging system and levels of protein expression were measured as the ratio toβ-actin protein.⑦Reverse Transcription-Polymerase Chain Reaction（RT-PCR）:Total RNA of aortic tissues were isolated using Trizol reagent and single stranded-cDNA were synthesized according to the manufactures instruction.Then Polymerase chain was reacted.Each specific mRNA band was normalized with a band of relative internal referenceβ-actin mRNA.Relative intensity of band of interest was analyzed by gel imaging system and the levels of mRNA were expressed as the ratio to the optical density ofβ-actin band.3.Statistical analysisAll data are expressed as mean±S.D and analyzed using statistical package SPSS 11.0 for Windows.One-way analysis of variance（ANOVA） was used to determine statistically significant differences among different groups.Some data were performed with Pearson correlation analysis and Partial correlations analysis.A level of P<0.05 was considered statistically significant.Results:1.General health state or changes of body weight and blood glucose（BG）:During the whole experimental period,one rat in DM group died.The BG of all diabetic rats were kept on high levels until the end of experiment.The body weight failed off and were largely lower than normal control rats（P<0.01）.But the blood glucose and body weight levels of diabetic rats were normalized by insulin treated,while there were no obvious improvement by taurine treated.2.Serum lipid levels:At 6 weekend of experiment,the levels of serum TC,TG and LDL in DM group were markedly higher than NC group（P<0.01）,while insulin-treatment restored all these changes almost to normal levels（P>0.05） and taurine intervention also markedly decreased the serum levels of TC and TG（P<0.01）.3.Circulating levels of oxLDL,NO,ET and sICAM-1:at 2 weekend of experiment, the levels of serum oxLDL,NO and plasma ET were all higher in DM group than those in NC group（P<0.05）.Also at 6 weekend,the serum levels of oxLDL,sICAM-1 and plasma ET were markedly increased,and the serum NO levels were largely decreased（all P<0.01）. Insulin and taurine intervention markedly reversed these changes,but there still had differences with NC group.4.Ultrastructural changes of aortic endothelial cell:in NC group,aortic tunica intima was very smooth,and the arrangement or shape of endothelial cells or organella were all normal.At 2 weekend of diabetic course,the changes were not very obvious.But at 6 weekend,the changes became obvious,presenting swelling of cell and organella,partially showing amotic of endothelial cells or breaked of base plate and vanishing of microvilli, bluring of crista mitochondriales as well as dilation of perinuclear cisterna.5.Endothelium-dependent relaxation of isolated aortic rings:At 2 weekend of diabetic course,the Ach-induced endothelium-dependent relaxations became blunted in DM groups,but the difference of Rmax between DM and NC groups wasn’t obvious. While at 6 weekend,compared with normal control,the Ach induced concentration-dependent vasorelaxation of diabetic rats were markedly blunted,Rmax was markedly decreased and EC50 was largely increased（P<0.01）.The function of endothelium-dependent relaxation were all markedly improved by insulin and taurine intervention（P<0.01）,and the effect of insulin was extremely obvious,showing the completely restoration of EC50 to normal level（P>0.05）.6.Expression of LOX-1 protein on aorta tissues: Immunofluorescent staining:almost no LOX-1 immunostaining were found on the whole layers of aortas in NC group.On the other hand,significantly positive LOX-1 immunostaining（bright green fluorescent staining） were observed in DM group at 6 weekend and mainly localized on the endothelial layers.While LOX-1 immunoreactivities were very weak in In and TAU groups,and the positive staining area and strength of LOX-1 were extremely weak in In group and also markedly weakened in TAU group.Western blot quantitative assay:the protein content of LOX-1 in aortas tissues were significantly enhanced in STZ-induced diabetic rats（P<0.01 vs control group）,while insulin and taurine treatment largely suppressed this enhancement of LOX-1 protein expression（P<0.01 vs DM group）,also the inhibition of LOX-1 expression was most significant in In group.7.Expression of NF-κBp65 protein on aortic tissues:Immunohistochemistry staining:In every group,NF-κBp65 protein were all expressed on aortic endothelium and muscular layers.But there were very weak staining in NC group and only presenting in partial intracytoplasm.But on diabetic aortas at 6 weekend,thickly brown positive staining could be seen in most endothelial and muscular cells,part cellular nucleus of endothelium showed positive staining.While the positive staining in cells or cellular nucleus were markedly weakened in In and TAU groups.Western blot quantitative assay:the protein content of NF-κBp65 in aortic tissues were significantly enhanced in STZ-induced diabetic rats（P<0.01 vs control group）,while insulin and taurine treatment largely suppressed this enhancement（P<0.01 vs DM group）, in which the NF-κBp65 protein in In group was reduced nearly to that of NC group（P>0.05）.8.Expression of ICAM-1 protein on aorta tissues:Immunohistochemical staining for ICAM-1 on aortas showed that seldom ICAM-1 immunostaining were found on the whole layers of aortas in NC group,but significantly positive ICAM-1 immunostaining（brown,continuous staining） were observed in DM group and mainly localized on the endothelial layer.While ICAM-1 immunoreactivities were very weak in In and TAU groups,and the optical density integration calculated by positive area and the strength of ICAM-1 positive stainings were markedly lower（P<0.01 vs DM group）.The reduction of ICAM-1 protein expression by insulin treated was mostly obvious, but there still had difference with NC group（P<0.05）.9.RT-PCR quantitative assay:Expression of LOX-1,NF-κBp65 and ICMA-1mRNA on aortas were very low in normal control rats,but all of them dramatically upregulated in diabetic rats（P<0.01 vs NC group）.While insulin and taurine treatment markedly inhibited this upregulation（P <0.01 vs DM group）.Also,there was no difference of NF-κBp65 and ICAM-1mRNA expression between In group and NC group（P>0.05）.10.Correlation analysis:In DM group,serum oxLDL level was negatively correlated with NO,NO/ET and Rmax,but positively correlated with serum sICAM-1 and plasma ET.Rmax was negatively correlated with ET,oxLDL and sICAM-1,but positively correlated with NO and NO/ET.Controlling the interference of body weight and blood lipid,partial correlation analysis showed these correlations still had statistical significance.Also,the level of LOX-1mRNA expression was positively correlated with the levels of serum oxLDL, NF-κBp65 and ICAM-1mRNA expression,but negatively correlated with Rmax.Conclusions:1.Vascular endothelium dysfunction and ultramicrostructure injury have been present in early diabetes.Endothelium-dependent vasodilation was the most direct and sensitive measurement,and circulating levels of NO,ET,NO/ET and sICAM-1 may be used as early indirect markers to screen endothelium dysfunction in diabetes.2.Excessive oxidative modification of LDL was occurred in early diabetes,oxLDL was the key risk factor to induce early vascular endothelium dysfunction in diabetes condition.Circulating levels of oxLDL can be used as a early predictor of diabetic vascular dysfunction.3.Aortic expression of LOX-1 protein and gene were all up-regulated and also closely correlated with circulating oxLDL level,indicating that the interaction of oxLDL and LOX-1 mediated the damage of vascular endothelium.OxLDL/LOX-1 system was the important agent that induced early vascular dysfunction of diabetes. 4.Aortic expression of NF-κBp65 were markedly enhanced and existing nuclear translocation,at the meantime,its mRNA expression were possitively correlated with the expression of LOX-1 and ICAM-1mRAN,indicating that aortic NF-κB was activated in early diabetes.ICAM-1 expression induced by activation of NF-κB signal pathway may be involved in the endothelial toxic effect of oxLDL/LOX-1.5.Strictly control of blood glucose by insulin treatment could restore the dyslipidemia and vascular endothelium dysfunction to some extent,at the same time could greatly inhibit LDL oxidative modification and LOX-1 expression in early diabetes.However insulin treatment couldn’t completely restore the vascular dysfunction to the normal level.6.Although early intervention by taurine couldn’t control hyperglycemia in diabetic rats,it may relieve the vascular endothelium dysfunction induced by oxLDL/LOX-1.This endothelium protection were possibly involved in the blockage of oxidative modification of LDL,the inhibition of oxLDL/LOX-1 and NF-κB activation as well as downregulation of ICAM-1 expression on aortic endothelium,relying on its antioxidation and anti-inflammatory action.Innovations and meanings:1.This study dynamically and directedly investigated in vivo the early occurrence of vascular endothelium dysfunction in type 1 diabetes as well as the levels of circulating oxLDL and other endothelium-derived markers,which provided referenced evidence for early discovery and intervention of diabetic vascular endothelium dysfunction in clinical practice.2.This study firstly in vivo comfirmed that the level of circulating oxLDL was increased,at the same time the aortic LOX-1 and NF-κBp65 were accordingly overexpressed.Correlation analysis also comfirmed that the expression of LOX-1mRNA was closely correlated with the level of circulating oxLDL,endothelium-dependent vasodilation as well as expression of NF-κBp65 and ICAM-1mRNA.All these indicated that during the process of diabetic vasculopathy,the activation of NF-κB signal pathway was involved in the damage of endothelium induced by oxLDL/LOX-1 in diabete,which may also provide a new clue for prevention of diabetic vasculopathy by mediating the expression of NF-κB and blocking the interaction of oxLDL/LOX-1. 3.This study firstly study the protection of insulin and taurine treatment against the early injury of vascular endothelium,discovering that strict control of hyperglycaemia by insulin treatment couldn’t completely avoid the endothelium dysfunction in diabetes,and early intervention by taurine could significantly relieve the diabetic endothelium-dysfuntion,simultaneously decrease circulating oxLDL level and downregulate LOX-1,NF-κB,ICAM-1 protein as well as gene expression on aortas.All these indicated that endothelial-protection of taurine was related to its antioxidant and anti-inflammatory action,and taurine may be a good choice for the early prevention of diabetic vasculopathy.更多还原