几种大西洋来源海藻多糖及其衍生物抗2型糖尿病活性的比较研究
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摘要
1前言
糖类与蛋白质及核酸称为生物体内三大活性分子。糖类广泛分布于所有生命体,不仅是细胞组成成分,更重要的是细胞间信息传递物质。细胞膜表面寡糖序列的多样化,及其对蛋白、脂类等分子的“修饰”作用,使其隐含着巨大信息而值得我们去破译。糖类物质提供了多种生物学功能,如从协助折叠新生蛋白质到保证淋巴细胞和粒细胞顺利转移和流动等过程均有糖的参与。因此,从事糖类的研究具有重要的意义。酸性多糖如糖胺聚糖(GAGs)广泛存在于脊椎动物组织中,通过与各种蛋白质,包括生长因子、酶、细胞粘附分子等作用而发挥重要生物学功能。海洋来源的多糖,往往带有聚阴离子基团(如糖醛酸、硫酸基),性质类似于糖胺聚糖。自然界中的GAGs类分子由于其显著的抗病毒、抗氧化和抗凝血活性被广泛应用在食品和制药业。最近的研究资料显示,聚阴离子的海洋多糖,可作为非常重要的信号分子,参与了植物与微生物的相互作用,以及从昆虫的防卫反应到脊椎动物的免疫反应活动等。许多海藻物种已被开发用作传统食品、海洋蔬菜以及中草药,越来越多的研究资料显示,各种海藻不仅有营养作用,而且有利于各种疾病的防治、保持健康。海藻多糖不同于陆地上的植物多糖,有其独特的结构组成,由于其硫酸酯的结构而类似于哺乳动物糖胺聚糖。越来越多的科研工作者研究了海藻硫酸多糖及其寡糖在活体实验和动物模型中以及一些临床研究中的生物活性,显示了良好的前景。
基于上述信息,开发安全的海藻食品及添加剂,探讨其对人体健康的影响,开发以海藻活性成分为基础的天然营养保健品成为极具吸引力的课题。
1.1海藻多糖结构多样性及分析手段
1.1.1海藻多糖结构多样性
海藻主要包括红藻(Red Algae),绿藻(Green Algae),褐藻(Brown Algae),和微藻(Microalgae)等。海藻种类不同所含有多糖的种类和结构类型也不同。
红藻多糖(如卡拉胶、琼胶等)由于其自身的凝胶特性,作为胶凝剂和增稠剂已经广泛应用于食品行业中。红藻硫酸多糖主要分为琼胶和卡拉胶两种,前者是由L-半乳糖及其衍生物构成,而后者则含有D-半乳糖和3,6-内醚L-半乳糖[3]。此外,多数红藻多糖同时含有D-和L-构型半乳糖,被称为DL-hybrids [4].红藻多糖主要由带硫酸根的半乳糖组成,同时可能含有少量木糖,甘露糖,丙酮酸酮化及甲基化半乳糖等[5]。
石莼胶(Ulvan)是绿藻中主要的水溶性多糖,含有硫酸根,鼠李糖,木糖,艾杜糖醛酸和葡萄糖醛酸等。根据Lahaye课题组的报道[6], Ulvan结构复杂,但主要具有两种重复二糖结构单元：
一种是[→4)-β-D-Glc-(1→4)-a-L-Rhap3S-(1→]n;
一种是[→4)-a-L-Idop-(1→4)-a-L-Rhap3S-(1→]n
除了葡萄糖醛酸-木糖-鼠李糖聚合物之外,绿藻中还含有木糖-半乳糖-阿拉伯糖聚合物,主要存在于刺松藻(Codium fragile)属中[7]。
褐藻多糖主要分为三种,褐藻胶(Alginate),岩藻聚糖硫酸酯(Fucoidan)和褐藻淀粉(Laminarin)。其中,Fucoidan由于其多样的生物活性,近年来引起了科研工作者们广泛的关注。早期提取于Fucus vesiculosus的岩藻聚糖结构被认为主要为(1→2)连接的4-O-硫酸-岩藻聚糖[8]。但随着科技不断发展,Chevolot课题组认为其结构应该是二位带有岩藻糖取代的(1→3)/(1→4)连接的4-O-硫酸-岩藻聚糖[9]。近年,科学家们倾向于将褐藻中岩藻聚糖根据结构和来源分为两大类：一类主要来源于海带(Laminaria saccharina)、掌状海带(三.digitata),松藻(Analipus japonicus),枝管藻(Cladosiphon okamuranus),和绳藻(Chorda filum)等,主要含有(1→3)连接-α-L-岩藻糖；另一类来源于泡叶藻(Ascophyllum nodosum)和墨角藻属(Fucus spp),主要含有(1→3)/(1→4)-连接的α-L-岩藻糖[10]。然而,岩藻聚糖结构复杂,不仅连接方式多样,所含单糖组成也呈现多种,主要有岩藻糖,还含有少量的葡萄糖,半乳糖,甘露糖,木糖,葡糖糖醛酸,甚至含有乙酰基等[11,12]。
1.1.2多糖结构分析手段
完整的多糖分子结构表征需要提供许多信息,包括分子量、单糖组成、异头碳构型、糖环状形状和大小、糖残基的连接顺序、连接位置、非糖基取代物的位置及其高级结构等。虽然海藻多糖结构复杂,但解析其一级和高级结构,对于明确其生物学功能具有重要的意义。
甲基化分析在确定海藻多糖的糖残基连接顺序和硫酸根取代位置方面具有十分重要的作用。通过将多糖中的游离羟基全部甲基化,进而水解、还原、乙酰化,可以得到甲基化的糖醇乙酰衍生物。各水解产物用气相色谱(GC)进行定性和定量分析,可确定组成糖链各种单糖的种类和比例。再用气质联用(GC/MS)结合标准谱图的对照分析,可进行各种甲基化单糖衍生物的归属,从而确定各单糖的连接位置,即糖苷键的位置,但甲基化分析无法确定异头碳糖苷键构型和糖链中单糖残基的顺序信息[13]。
核磁共振波谱(NMR)和质谱(MS)是近年来用于多糖结构分析的二种强有力的手段。结合1D和2D-NMR技术,多级质谱技术可以提供更详细的多糖结构序列信息,包括单糖组成,连接顺序,异头碳构型,硫酸根和支链的取代位置等[14]。高分辨MS除了能提供准确的寡糖分子量信息外,由于所需样品量少,分析时间短等优点,逐渐被用于细胞膜微量寡糖的结构分析中[15]。
此外,通过红外光谱(IR),气质联用(GC/MS),高效离子色谱(HPAEC-PAD),毛细管电泳(CE),二维和多维液相色谱(2D/3D-HPLC),凝集素亲和层析和糖芯片(Glycoarrays)技术等多种技术方法的选择使用和巧妙配合中,可得到来自各个角度的信息和数据,从而推断糖链一级和二级结构。
1.2海藻多糖的生物活性及其应用
海藻硫酸多糖为硫酸酯基团连接在多糖残基上的多糖,如褐藻中的岩藻聚糖,红藻中的硫酸半乳聚糖,绿藻中的硫酸鼠李聚糖或其它聚糖。由于海藻硫酸多糖与哺乳动物的糖胺聚糖如肝素、硫酸软骨素等结构类似,以生物活性及潜在用途作为治疗剂或其他保健产品开发海藻多糖已有60多年的历史。很多研究报道表明,硫酸多糖具有抗凝血[16,17]、抗病毒[18,19]、抗氧化[20-22]、降血脂[23-25]、免疫调节[26-28]等多种生物活性。因多糖结构复杂,吸收性差,质量不易控制等限制,其低聚糖的制备及生物活性和安全性评价分析有重要的意义。
1.3糖芯片技术开发与应用
随着基因组学和蛋白组学研究的发展,大分子芯片技术作为一种多功能的生物分析手段逐渐发展起来。糖芯片技术根据糖与其它生物分子之间的特异性结合,将微量糖类化合物以共价或非共价作用固定于芯片上,进而对蛋白质、活体细胞、微生物等进行检测和分析。因其具有用量少、通量高、灵敏快速及信息量大等优点,糖芯片逐渐成为糖组学研究强有力的工具。
2002年,Wang课题组发表了将48种碳水化合物(包括多糖和糖蛋白)通过物理吸附作用固定在硝酸纤维薄膜芯片上制备碳水化合物芯片的论文(这是第一篇有关糖芯片的应用文章)[29]。在硝酸纤维素膜芯片上成功制做多糖芯片以来,糖芯片技术的制备和应用取得了一定的发展进步。常用的芯片固相载体有硅胶片、玻璃片、聚偏氟乙烯、硝酸纤维薄膜及微孔板,还有应用相对较少的微球芯片等。根据载体基质表面活性的不同,糖芯片固定技术一般可以分成共价固定和非共价固定两种[30,31]。多糖或者糖复合物可以通过非特异性结合直接固定在具有空间网状结构的纤维薄膜覆盖的玻璃板上。而单糖和寡糖由于分子量小,水溶性好,一般需要通过偶联疏水非极性化合物制成拟糖酯,来提高其疏水性,从而提高其在固体介质上的保留率[32,33]。共价固定碳水化合物制备的糖芯片是目前研究最广泛并被科学家们大量使用的一类方法,其利用共价键将糖类物质固定在芯片基质材料的表面。由于其结合稳定性强,糖分子在芯片基质上排列规则,与待测分子结合牢靠,共价固定技术适合分子量不大,分子结构简单的单糖和寡糖等,但其制备过程较为繁琐,要求较高,芯片保存也较困难[34]。
糖芯片制备好之后,固定在基片上的糖类及其复合物与蛋白相互作用,通过一些特殊实验装置信号转化成分析处理的图像数据。经过多年的研究,糖芯片检测系统主要方法有荧光标记检测法、底物沉降检测法,化学发光检测法,表面基质共振技术、质谱法以及同位索标记检测法等[35-37]。
糖类物质及其复合物在生物发育、肿瘤发生、转移、细胞黏附和免疫识别等生命过程中发挥着无法替代的作用。糖芯片技术运用高通量筛选糖结合蛋白,研究糖类与蛋白作用的构效关系,在鉴定未知多糖的结构、筛选糖类结合蛋白的抑制剂、寻找新的治疗靶点及研究生命过程中糖类及其复合物所扮演的角色等方面有很广阔的前景[38,39]。
1.4海藻多糖与糖尿病的关系
糖尿病(Diabetes Mellitus, DM)是由胰岛素分泌缺陷及(或)其生物学作用障碍引起的以高血糖、高血脂为特征的全身代谢紊乱性疾病。根据其病理生理机制,糖尿病主要分为两类：1型糖尿病(胰岛素依赖性糖尿病,IDDM)是由胰岛细胞破坏,造成胰岛素分泌不足而致病,需要依赖外源性胰岛素；2型糖尿病(胰岛素非依赖性糖尿病,NIDDM)病因复杂,以胰岛素抵抗为主要特征,伴随明显的糖脂代谢紊乱[40]。糖尿病患病率和糖尿病患者的数量增长速度飞快,其并发症对患者的生命和生活质量威胁极大,给个人和社会带来了沉重的经济负担。目前,糖尿病已成为与心脑血管病、肿瘤并列的危害人类健康的三大慢性疾病。因此,深入研究和探讨糖尿病的发病机制,对于防治糖尿病、降低糖尿病的发病率、提高人们健康水平都有着重大意义。
2型糖尿病发病是由胰岛素抵抗和胰岛素分泌受损引发而至。胰岛素抵抗是由遗传和环境因素导致的,表现为机体对胰岛素生理作用的反应性降低,即胰岛素敏感组织(如肌肉、脂肪组织)对葡萄糖的摄取量减少,抑制肝葡萄糖输出的作用减弱,从而导致代偿性胰岛素分泌增多,引起机体一系列病理生理变化,最终导致各种代谢疾病的发生和发展[41,42]。胰岛素抵抗主要发生在脂肪、肝脏和骨骼肌等含有大量的胰岛素受体的组织细胞中,其结合胰岛素并在调节葡萄糖代谢稳态上发挥重要作用。
根据其作用机制和结构,目前治疗糖尿病的药物主要分为双胍类、α-葡糖苷酶抑制剂、胰岛素增敏剂、胰岛素促泌剂、糖原异生抑制剂和醛糖还原酶抑制剂等多种类型[43]。各种口服药物的作用机制不同,其药效和毒副作用也不尽相同。然而,现有的抗2型糖尿病药物大多存在各种缺陷,无法满足患者的需求。因此,研制高效低毒的新型抗糖尿病药物,对控制血糖、防治糖尿病及其并发症的发生发展,提高患者的生活质量具有十分重要的意义。
近年来,海藻多糖抗糖尿病活性的报道引起了科学工作者们的广泛关注,其主要作用表现为降血脂和降血糖等[24,25]。在高血脂大鼠模型中,岩藻聚糖被发现通过增加脂蛋白脂肪酶(LPL)、脂解酵素(HL)和卵磷脂胆固醇酰基转移酶(LCAT)的活性而能降低血清的总胆固醇(TCH)、低密度脂蛋白(LDL-CH)和甘油三酯(TG)的含量,而升高高密度脂蛋白(HDL-CH)含量[25]。低分子量石莼胶(Ulvan)喂养的高胆固醇大鼠模型被发现血清中总胆固醇和低密度脂蛋白的含量也有所降低[24]。另有国内外多份报道表明,来源于海藻的多糖及寡糖能够降低四氧嘧啶诱导的糖尿病小鼠的高血糖症,促进RIN-5F细胞分泌胰岛素,改善糖尿病患者的糖耐量,降低血糖、尿糖,并可使糖尿病患者其它症状减轻或消失[44-46]。此外,研究发现铬在生理状态下与羟基形成的配位复合物能在人体内发挥重要的生理功能[47,48],抗糖尿病方面的主要表现为纠正低血糖症,维持机体正常的糖代谢；调节脂肪代谢,降低糖尿病人血清中胆固醇、甘油三脂的水平,升高高密度脂蛋白胆固醇水平。Hao等发现海藻寡糖铬配合物能够通过激活胰岛素信号通路和AMPK通路来调节糖脂代谢,增加胰岛素敏感性,从而改善胰岛素抵抗[49]。海洋独特的环境使其拥有众多具有特异功能的活性物质,因此开发海洋来源的糖类药物意义重大。
2本课题研究背景、思路及研究方法
本课题以海洋藻类为研究对象,从事海藻糖类化合物的提取分离、结构鉴定、分子修饰及构效关系研究,发现糖类药物先导化合物,研制开发海洋创新药物、现代海洋中药及海洋生物功能制品,为海洋糖工程药物的研究开发提供理论与技术支撑。
加拿大新斯科舍省和圣劳伦斯地区被认为是世界上海藻资源和种类最丰富的地区之一。此地盛产的皱波角叉菜(爱尔兰海藻)是加拿大所有海藻中商业上最重要的卡拉胶原料；泡叶藻和海带也是此区域丰富的海藻资源,用来作为海藻酸钠的工业来源。结合大西洋加拿大沿岸丰富的海藻资源,制备不同类型、结构及来源的多糖,并运用不同的荧光试剂标记不同结构类型的海藻多糖,按照多糖芯片制备方法,运用海藻多糖芯片研究与蛋白的相互作用。制备海藻岩藻聚糖硫酸酯及其一系列衍生物,并对其进行2型糖尿病活性筛选,以期望发现抗糖尿病的新型化合物。
主要研究思路为：以6种不同类型的加拿大大西洋沿岸来源的海藻为实验对象,制备不同类型及结构的多糖,分离纯化褐藻中的岩藻聚糖,并制备其不同化学结构的衍生物。结合多糖的理化特性,选择荧光标记方法,分离纯化后对其进行荧光和糖含量分析,再利用微阵列技术将多种具有确定结构的多糖荧光标记物固定于Whatman硝酸纤维素薄膜,用建立的ELISA方法研究多糖与不同蛋白分子的相互作用,筛选出与糖尿病相关蛋白有关系的海藻多糖。同时,制备岩藻聚糖硫酸酯及其一系列衍生物,包括脱硫酸化、过硫酸酯化、磷酸化衍生物及系列寡糖。将各样品及所制备的衍生物运用3T3-L1细胞系筛选,结合糖芯片筛选结果,发现具有抗糖尿病作用的化合物,并对其抗糖尿病机理进行研究。
研究的难点和创新点：由于不同类型的海藻含有不同结构类型的多糖,对其进行提取分离纯化和结构分析,是一项艰巨的任务。运用不同的荧光标记方法,制备出一系列具有荧光稳定,并保持其生物活性的衍生物,提高多糖芯片上多糖固定率,建立完整的海洋来源的多糖荧光标记是本研究的难点之一。建立海藻多糖分离纯化、结构修饰、多糖芯片、糖与蛋白相互作用平台,并在细胞水平对活性化合物进行评价,获得其初步构效关系,将是本研究的创新点。目前虽然已有报道糖类化合物应用在糖尿病的防治方面,但是海洋来源的多糖抗糖尿病活性的机理仍有待于进一步研究。
3实验内容及结果
3.1海藻多糖的制备
从加拿大大西洋海域采集或购买了6种资源丰富的海藻,包括三种红藻(Polysiphonia lanosa, Furcellaria lumbricalis和Palmaria palmata),两种褐藻(Ascophyllum nodosum和Fucus evanescence)，及一种绿藻(Ulva lactuca)。海藻原料先用甲醇提取,以去除色素和其它非多糖成分,然后分别用冷水、热水、2%Na2CO3溶液和0.5M NaOH溶液在不同温度下提取。提取的粗多糖用80%乙醇沉淀,透析后冻干,得到24种不同来源和结构的海藻多糖。
理化性质分析结果表明,这些海藻多糖得率从19.71%到49.20%不等；多糖含量从15.42%到91.39%不等；硫酸根含量从0到17.55%不等。结构分析表明,红藻多糖主要由硫酸半乳聚糖(Polysiphonia lanosa和Furcellaria lumbricalis)或木聚糖(Palmaria palmata)组成,褐藻多糖主要由糖醛酸及硫酸化岩藻聚糖组成,而绿藻多糖(Ulva lactuca)则主要由葡萄糖醛酸、木糖、鼠李糖及葡萄糖成。
3.2岩藻聚糖及其衍生物的制备
利用CaCl2及乙醇沉淀的方法,将岩藻聚糖从褐藻多糖中分离出来,经透析除盐后得到8种岩藻聚糖。与Sigma公司岩藻聚糖(FC)相比,所制备的岩藻聚糖分子量均较大,约150～360kDa；多糖含量大约在60%左右；硫酸根含量在20%左右；蛋白含量在2-20%；岩藻糖占单糖组成百分比均在50%以上。
运用多聚磷酸-三正丁胺的方法制备了岩藻聚糖磷酸酯；运用三氧化硫-吡啶的方法制备了岩藻聚糖过硫酸酯；运用二甲基亚砜-甲醇-吡啶的方法制备了岩藻聚糖脱硫衍生物；运用0.1M三氟乙酸降解的方法制备了一系列低分子量岩藻聚糖。通过对9种岩藻聚糖的结构改造和化学降解,共制备了45种岩藻聚糖衍生物,通过红外光谱和理化性质分析,结果表明这些衍生物均成功获得。
3.3海藻多糖的抗2型糖尿病活性筛选
根据多糖的理化性质和结构特征,分别运用fluoresceinisothiocyanate (FITC)或5-(((2-(carbohydrazino)methyl)thio)acetyl)aminofluorescein(AF)荧光试剂成功标记了24种海藻多糖及8种岩藻聚糖,同时对市售岩藻聚糖(FC)、肝素(HEP)、褐藻胶(Alg)和卡拉胶(KCA)等也进行了荧光标记。将标记好的多糖分别固定于醋酸纤维素薄膜包被的玻璃芯片上,制备成糖芯片,用于多糖抗糖尿病活性的筛选。通过与人胰岛淀粉样蛋白多肽(IAPP)的结合实验结果发现,红藻多糖FL3和PP3能够有效地结合IAPP,表明其具有潜在的拮抗胰淀素蛋白沉积、提高胰岛素敏感性从而抵抗2型糖尿病的能力。
α-葡萄糖苷酶抑制剂可以延缓肠道对葡萄糖的吸收而达到控制血糖,尤其是餐后血糖过高的目的。通过对α-葡萄糖苷酶抑制活性的筛选发现,大多数褐藻多糖及部分岩藻聚糖均具有很好的抑制α-葡萄糖苷酶的作用。其中,AN1, AN4和FVl,FV2抑制能力最高,其IC50分别为2.62,21.32,3.15和44.37μg/mL。
3.4海藻多糖抗2型糖尿病活性的机制研究
小鼠3T3-L1前脂肪细胞在体外经诱导后可分化为成熟的脂肪细胞,可以较好的模拟脂肪细胞分化和活体脂肪组织的功能,成为筛选胰岛素增敏剂类抗2型糖尿病的体外模型之一。通过对3T3-L1成熟脂肪细胞积累的脂滴多少以及细胞内甘油三酯含量的高低,对海藻多糖及其衍生物进行了抗糖尿病活性筛选。结果表明,PL2,PL3,FL3,PP2,PP3和多数褐藻能抑制3T3-L1细胞内脂滴的积累,而FL2,FL3,AN2和FVl处理的细胞中甘油三酯含量较低。同时发现,大多数岩藻聚糖均能抑制3T3-L1细胞分化过程。通过对脂肪细胞分化前期细胞转录因子的表达分析,发现岩藻聚糖通过抑伟(PPARΥ及其相关调控因子C/EBPa(?)(?)C/EBPP而抑制3T3-L1细胞的分化过程。
综上所述,本课题以6种大西洋加拿大沿岸的海藻为研究对象,制备了不同结构类型的海藻多糖及其衍生物共69种(24+45)。运用不同荧光试剂对其进行标记,制备了海藻多糖芯片,研究了各海藻多糖与IAPP和的相互作用。采用细胞模型对各化合物进行了2型糖尿病活性筛选,并对有效成分进行作用机理研究。
4创新点
本论文的创新点主要有以下3个方面：
1)首次对大西洋加拿大沿岸6种海藻所含多糖结构进行了研究,成功制备了69种海藻多糖及其衍生物。
2)首次运用多糖芯片技术,结合细胞模型等对海藻多糖进行了抗2型糖尿病活性筛选。通过糖芯片手段,发现两种红藻多糖能较有效的与胰岛淀粉样蛋白相结合。通过酶化学方法,获得了具有能抑制α-糖苷酶活性的褐藻多糖化合物。通过细胞模型,发现泡叶藻来源的岩藻聚糖能调节脂肪代谢,抑制脂肪细胞分化过程。
3)在细胞水平上对岩藻聚糖抗2型糖尿病活性进行了机制研究,首次发现泡叶藻来源的岩藻聚糖能够通过抑制PPARy的活性,激活C/EBPa和PPARa的活性,而增强脂代谢,改善胰岛素抵抗,为2型糖尿病药物开发提供了理论基础。
5下一步工作计划
岩藻聚糖是一类从褐藻中分离得到的硫酸多糖,主要是由L-岩藻相组成,同时可能含有少量木糖,半乳糖和甘露糖等。跟据论文目前的研究结果,下一步我们以A. nosodum来源的岩藻聚糖为研究对象,继续开展以下2方面工作：
1)在分子水平上探讨泡叶藻岩藻聚糖及其衍生物与IAPP作用关系,获得结构明确的寡糖片段,并从事其信号通路研究。
2)针对本研究发现的岩藻聚糖具有调节脂代谢,抑制脂肪细胞分化的作用,在动物水平上进一步评价其抗2型糖尿病作用效果,并对其作用机制进行探讨。
Diabetes mellitus is a group of diseases characterized by chronic hyperglycemia due to deficiency of insulin action which is a common basis and leads to characteristic abnormalities in the metabolism of carbohydrate, lipid, and protein. As a world wisely spread diseases, diabetes mellitus is mainly divided into type-1diabetes (Insulin dependent, IDDM) and type-2diabetes (Non-insulin dependent, NIDDM). Type-2diabetes is the most common form and has escalated to epidemic proportions. Main factors of type-2diabetes are related with a genetic disposition, overweight or obesity, lack of physical activity, consumption of high-fat, low-fiber diets, oxidative stress, and possibly deficiencies in certain minerals. Insulin resistance is an initial mechanism to reduce type-2diabetes. Adipose tissue stores excess energy in the form of triglyceride, and releases free fatty acids in response to energy requirements. It is one of the insulin sensitive peripheral organizations and plays a critical role in insulin resistance and type-2diabetes. Oral agents including insulin sensitizers, secretagogue, bile acid sequestrant, alpha-glucosidase inhibitor, and DPP-IV inhibitor are applied for improving glucose production, carbohydrate intake, insulin secretion, and peripheral uptake associated with type-2diabetes. However, the disadvantages of these agents provide more challenges for anti-diabetes research. Seaweeds species have been used traditionally as food, vegetable, as well as herbal drugs, and many investigations revealed that seaweeds have not only nutritional effects but also beneficial properties to cure various diseases and maintain good health. It has been reported that polysaccharides from marine sources may possess anti-diabetic potential. The objective of this project is to develop natural polysaccharides and their derivatives from marine algae for anti-diabetes studies.
Polysiphonia lanosa, Furcellaria lumbricalis, Palmar ia palmata, Ascophyllum nodosum, Fucus vesiculosus and Ulva lactuca, were collected from Atlantic Canadian coast where is one of the richest sources of seaweeds in the world. Algal polysaccharides were prepared under four conditions, cold water, hot water,2% and0.5M NaOH solutions; and twenty four polysaccharides were successfully obtained, respectively. Their chemical properties, including contents of total sugar, protein and sulphate groups, and structural informations, including molecular weight, monosaccharides compositions and characteristic groups, were studied by chemical methods and1H-NMR and FTIR spectra analysis. Fucoidan is a sulfated polysaccharide from brown seaweeds, which has been reported with various bioactivities. We isolated and purified fucoidan fractions from extracts of Ascophyllum nodosum and Fucus vesiculosus. Their phosphorylated, oversulphated, desulphated and degraded derivatives were also prepared and the derivatives were indicated successfully processed on FT-IR spectra and characteristic chemical analysis.
Glycoarrays technologies, an important tool in glycomics, have been developed as a top-leading technology to study the interactions of glycan-binding proteins with their glycan ligands. Islet-amyloid polypeptide (IAPP) is an important player in the formation of amyloid deposits and the progression of type2diabetes. Exploring a successful anti-amyloidogenic agent is another option to prevent diabetes mellitus. Fluoresceinisothiocyanate (FITC) or5-(((2-(Carbohydrazino)methyl)thio)acetyl)aminofluorescein (AF) labeled polysaccharides were printed on a nitrocellulose coated glass platform to create the glycoarrays and their IAPP binding abilities were screened as a high-throughput manner. FL3from F. lumbricalis and PP3from P. palmate showed the highest binding abilities on glycoarrays which suggested that these algal polysaccharides might show the optional application against type-2diabetes.
Alpha-glucosidase is a key enzyme for carbohydrate digestion associated with type-2diabetes mellitus. Algal polysaccharides and their derivatives were then invested the inhibitory effects against a-glucosidase by spectrophotometrical method. Among them, AN1and AN4from A. nodosum, FV1and FV4from F. vesiculosus showed highest inhibition. Further studies showed that the inhibitory effects were caused by the polysacchardes not their binding proteins.
Adipocyte tissue is the main target organ in response of insulin and plays a critical role in insulin resistance and type-2diabetes.3T3-L1preadipocyte cell lines are important in use to study the pathogenesis mechanism of insulin action and lipid-lowing in vitro. Algal polysaccharides and their derivatives were next studied on lipid accumulation in cultured3T3-L1adipocytes by measuring triglyceride (TG) contents and Oil Red O (ORO) staining as indicators. Treatment with PL2, PL3(from P. lanosa), FL3(from F. lumbricalis), PP2, PP3(from P. palmata) and most of fucoidan fractions decreased the lipid accumulation by ORO staining while FL2, FL3(from F. lumbricalis), AN2(from A. nodosum), and FV1(from F. vesiculosus) could reduce the TG content in3T3-L1adipocytes.
Two families of transcription factors, CCAAT/enhancer binding protein (C/EBP) and peroxisome proliferator-activated receptor-Υ (PPARy), are induced early during adipocyte differentiation. By enzyme-lined immunosorbent assay (ELISA), algal polysaccharides FL3(from F. lumbricalis), AN2(from A. nodosum) and two fucoidan fractions, AN3F and FV3F, were invested to decrease the expression of PPARy protein.
According to these results, we next focused on the effect of FL3, PP3, AN1, AN4, FV1, FV2, AN3F and FV3F against the early stage of3T3-L1adipogenesis study. AN3F showed the strongest suppression on PPARy and C/EBPβ,and the promotion on PPARa and C/EBPa in a dose-dependent manner, which suggested that it decreased the lipid accumulation in3T3-L1adipocytes through the effects on these PPARs and C/EBPs transcription factors and its anti-diabetic acitivity could be related to the MAPK signal involving many cytokines.
In a conclusion, we successfully prepared24algal polysacchardes and45fucoidan derivatives from six kinds of seaweeds collected from Atlantic Canadian coast (including the commercial fucoidan and its derivatives). Their anti-diabetic activities were screened by glycoarrays technologies, enzymatic spectrophotomctrical assay, enzyme-lined immunosorbent assay and western blot analysis. Among them, AN3F showed the potential anti-type-2diabetes affection by suppression the transcription factors in adipogenesis. Although, further molecular mechanism of AN3F and animal studies remains to be elucidated, AN3F provided potential of a new target for anti-diabetes research.
糖类与蛋白质及核酸称为生物体内三大活性分子。糖类广泛分布于所有生命体,不仅是细胞组成成分,更重要的是细胞间信息传递物质。细胞膜表面寡糖序列的多样化,及其对蛋白、脂类等分子的“修饰”作用,使其隐含着巨大信息而值得我们去破译。糖类物质提供了多种生物学功能,如从协助折叠新生蛋白质到保证淋巴细胞和粒细胞顺利转移和流动等过程均有糖的参与。因此,从事糖类的研究具有重要的意义。酸性多糖如糖胺聚糖(GAGs)广泛存在于脊椎动物组织中,通过与各种蛋白质,包括生长因子、酶、细胞粘附分子等作用而发挥重要生物学功能。海洋来源的多糖,往往带有聚阴离子基团(如糖醛酸、硫酸基),性质类似于糖胺聚糖。自然界中的GAGs类分子由于其显著的抗病毒、抗氧化和抗凝血活性被广泛应用在食品和制药业。最近的研究资料显示,聚阴离子的海洋多糖,可作为非常重要的信号分子,参与了植物与微生物的相互作用,以及从昆虫的防卫反应到脊椎动物的免疫反应活动等。许多海藻物种已被开发用作传统食品、海洋蔬菜以及中草药,越来越多的研究资料显示,各种海藻不仅有营养作用,而且有利于各种疾病的防治、保持健康。海藻多糖不同于陆地上的植物多糖,有其独特的结构组成,由于其硫酸酯的结构而类似于哺乳动物糖胺聚糖。越来越多的科研工作者研究了海藻硫酸多糖及其寡糖在活体实验和动物模型中以及一些临床研究中的生物活性,显示了良好的前景。
基于上述信息,开发安全的海藻食品及添加剂,探讨其对人体健康的影响,开发以海藻活性成分为基础的天然营养保健品成为极具吸引力的课题。
1.1海藻多糖结构多样性及分析手段
1.1.1海藻多糖结构多样性
海藻主要包括红藻(Red Algae),绿藻(Green Algae),褐藻(Brown Algae),和微藻(Microalgae)等。海藻种类不同所含有多糖的种类和结构类型也不同。
红藻多糖(如卡拉胶、琼胶等)由于其自身的凝胶特性,作为胶凝剂和增稠剂已经广泛应用于食品行业中。红藻硫酸多糖主要分为琼胶和卡拉胶两种,前者是由L-半乳糖及其衍生物构成,而后者则含有D-半乳糖和3,6-内醚L-半乳糖[3]。此外,多数红藻多糖同时含有D-和L-构型半乳糖,被称为DL-hybrids [4].红藻多糖主要由带硫酸根的半乳糖组成,同时可能含有少量木糖,甘露糖,丙酮酸酮化及甲基化半乳糖等[5]。
石莼胶(Ulvan)是绿藻中主要的水溶性多糖,含有硫酸根,鼠李糖,木糖,艾杜糖醛酸和葡萄糖醛酸等。根据Lahaye课题组的报道[6], Ulvan结构复杂,但主要具有两种重复二糖结构单元：
一种是[→4)-β-D-Glc-(1→4)-a-L-Rhap3S-(1→]n;
一种是[→4)-a-L-Idop-(1→4)-a-L-Rhap3S-(1→]n
除了葡萄糖醛酸-木糖-鼠李糖聚合物之外,绿藻中还含有木糖-半乳糖-阿拉伯糖聚合物,主要存在于刺松藻(Codium fragile)属中[7]。
褐藻多糖主要分为三种,褐藻胶(Alginate),岩藻聚糖硫酸酯(Fucoidan)和褐藻淀粉(Laminarin)。其中,Fucoidan由于其多样的生物活性,近年来引起了科研工作者们广泛的关注。早期提取于Fucus vesiculosus的岩藻聚糖结构被认为主要为(1→2)连接的4-O-硫酸-岩藻聚糖[8]。但随着科技不断发展,Chevolot课题组认为其结构应该是二位带有岩藻糖取代的(1→3)/(1→4)连接的4-O-硫酸-岩藻聚糖[9]。近年,科学家们倾向于将褐藻中岩藻聚糖根据结构和来源分为两大类：一类主要来源于海带(Laminaria saccharina)、掌状海带(三.digitata),松藻(Analipus japonicus),枝管藻(Cladosiphon okamuranus),和绳藻(Chorda filum)等,主要含有(1→3)连接-α-L-岩藻糖；另一类来源于泡叶藻(Ascophyllum nodosum)和墨角藻属(Fucus spp),主要含有(1→3)/(1→4)-连接的α-L-岩藻糖[10]。然而,岩藻聚糖结构复杂,不仅连接方式多样,所含单糖组成也呈现多种,主要有岩藻糖,还含有少量的葡萄糖,半乳糖,甘露糖,木糖,葡糖糖醛酸,甚至含有乙酰基等[11,12]。
1.1.2多糖结构分析手段
完整的多糖分子结构表征需要提供许多信息,包括分子量、单糖组成、异头碳构型、糖环状形状和大小、糖残基的连接顺序、连接位置、非糖基取代物的位置及其高级结构等。虽然海藻多糖结构复杂,但解析其一级和高级结构,对于明确其生物学功能具有重要的意义。
甲基化分析在确定海藻多糖的糖残基连接顺序和硫酸根取代位置方面具有十分重要的作用。通过将多糖中的游离羟基全部甲基化,进而水解、还原、乙酰化,可以得到甲基化的糖醇乙酰衍生物。各水解产物用气相色谱(GC)进行定性和定量分析,可确定组成糖链各种单糖的种类和比例。再用气质联用(GC/MS)结合标准谱图的对照分析,可进行各种甲基化单糖衍生物的归属,从而确定各单糖的连接位置,即糖苷键的位置,但甲基化分析无法确定异头碳糖苷键构型和糖链中单糖残基的顺序信息[13]。
核磁共振波谱(NMR)和质谱(MS)是近年来用于多糖结构分析的二种强有力的手段。结合1D和2D-NMR技术,多级质谱技术可以提供更详细的多糖结构序列信息,包括单糖组成,连接顺序,异头碳构型,硫酸根和支链的取代位置等[14]。高分辨MS除了能提供准确的寡糖分子量信息外,由于所需样品量少,分析时间短等优点,逐渐被用于细胞膜微量寡糖的结构分析中[15]。
此外,通过红外光谱(IR),气质联用(GC/MS),高效离子色谱(HPAEC-PAD),毛细管电泳(CE),二维和多维液相色谱(2D/3D-HPLC),凝集素亲和层析和糖芯片(Glycoarrays)技术等多种技术方法的选择使用和巧妙配合中,可得到来自各个角度的信息和数据,从而推断糖链一级和二级结构。
1.2海藻多糖的生物活性及其应用
海藻硫酸多糖为硫酸酯基团连接在多糖残基上的多糖,如褐藻中的岩藻聚糖,红藻中的硫酸半乳聚糖,绿藻中的硫酸鼠李聚糖或其它聚糖。由于海藻硫酸多糖与哺乳动物的糖胺聚糖如肝素、硫酸软骨素等结构类似,以生物活性及潜在用途作为治疗剂或其他保健产品开发海藻多糖已有60多年的历史。很多研究报道表明,硫酸多糖具有抗凝血[16,17]、抗病毒[18,19]、抗氧化[20-22]、降血脂[23-25]、免疫调节[26-28]等多种生物活性。因多糖结构复杂,吸收性差,质量不易控制等限制,其低聚糖的制备及生物活性和安全性评价分析有重要的意义。
1.3糖芯片技术开发与应用
随着基因组学和蛋白组学研究的发展,大分子芯片技术作为一种多功能的生物分析手段逐渐发展起来。糖芯片技术根据糖与其它生物分子之间的特异性结合,将微量糖类化合物以共价或非共价作用固定于芯片上,进而对蛋白质、活体细胞、微生物等进行检测和分析。因其具有用量少、通量高、灵敏快速及信息量大等优点,糖芯片逐渐成为糖组学研究强有力的工具。
2002年,Wang课题组发表了将48种碳水化合物(包括多糖和糖蛋白)通过物理吸附作用固定在硝酸纤维薄膜芯片上制备碳水化合物芯片的论文(这是第一篇有关糖芯片的应用文章)[29]。在硝酸纤维素膜芯片上成功制做多糖芯片以来,糖芯片技术的制备和应用取得了一定的发展进步。常用的芯片固相载体有硅胶片、玻璃片、聚偏氟乙烯、硝酸纤维薄膜及微孔板,还有应用相对较少的微球芯片等。根据载体基质表面活性的不同,糖芯片固定技术一般可以分成共价固定和非共价固定两种[30,31]。多糖或者糖复合物可以通过非特异性结合直接固定在具有空间网状结构的纤维薄膜覆盖的玻璃板上。而单糖和寡糖由于分子量小,水溶性好,一般需要通过偶联疏水非极性化合物制成拟糖酯,来提高其疏水性,从而提高其在固体介质上的保留率[32,33]。共价固定碳水化合物制备的糖芯片是目前研究最广泛并被科学家们大量使用的一类方法,其利用共价键将糖类物质固定在芯片基质材料的表面。由于其结合稳定性强,糖分子在芯片基质上排列规则,与待测分子结合牢靠,共价固定技术适合分子量不大,分子结构简单的单糖和寡糖等,但其制备过程较为繁琐,要求较高,芯片保存也较困难[34]。
糖芯片制备好之后,固定在基片上的糖类及其复合物与蛋白相互作用,通过一些特殊实验装置信号转化成分析处理的图像数据。经过多年的研究,糖芯片检测系统主要方法有荧光标记检测法、底物沉降检测法,化学发光检测法,表面基质共振技术、质谱法以及同位索标记检测法等[35-37]。
糖类物质及其复合物在生物发育、肿瘤发生、转移、细胞黏附和免疫识别等生命过程中发挥着无法替代的作用。糖芯片技术运用高通量筛选糖结合蛋白,研究糖类与蛋白作用的构效关系,在鉴定未知多糖的结构、筛选糖类结合蛋白的抑制剂、寻找新的治疗靶点及研究生命过程中糖类及其复合物所扮演的角色等方面有很广阔的前景[38,39]。
1.4海藻多糖与糖尿病的关系
糖尿病(Diabetes Mellitus, DM)是由胰岛素分泌缺陷及(或)其生物学作用障碍引起的以高血糖、高血脂为特征的全身代谢紊乱性疾病。根据其病理生理机制,糖尿病主要分为两类：1型糖尿病(胰岛素依赖性糖尿病,IDDM)是由胰岛细胞破坏,造成胰岛素分泌不足而致病,需要依赖外源性胰岛素；2型糖尿病(胰岛素非依赖性糖尿病,NIDDM)病因复杂,以胰岛素抵抗为主要特征,伴随明显的糖脂代谢紊乱[40]。糖尿病患病率和糖尿病患者的数量增长速度飞快,其并发症对患者的生命和生活质量威胁极大,给个人和社会带来了沉重的经济负担。目前,糖尿病已成为与心脑血管病、肿瘤并列的危害人类健康的三大慢性疾病。因此,深入研究和探讨糖尿病的发病机制,对于防治糖尿病、降低糖尿病的发病率、提高人们健康水平都有着重大意义。
2型糖尿病发病是由胰岛素抵抗和胰岛素分泌受损引发而至。胰岛素抵抗是由遗传和环境因素导致的,表现为机体对胰岛素生理作用的反应性降低,即胰岛素敏感组织(如肌肉、脂肪组织)对葡萄糖的摄取量减少,抑制肝葡萄糖输出的作用减弱,从而导致代偿性胰岛素分泌增多,引起机体一系列病理生理变化,最终导致各种代谢疾病的发生和发展[41,42]。胰岛素抵抗主要发生在脂肪、肝脏和骨骼肌等含有大量的胰岛素受体的组织细胞中,其结合胰岛素并在调节葡萄糖代谢稳态上发挥重要作用。
根据其作用机制和结构,目前治疗糖尿病的药物主要分为双胍类、α-葡糖苷酶抑制剂、胰岛素增敏剂、胰岛素促泌剂、糖原异生抑制剂和醛糖还原酶抑制剂等多种类型[43]。各种口服药物的作用机制不同,其药效和毒副作用也不尽相同。然而,现有的抗2型糖尿病药物大多存在各种缺陷,无法满足患者的需求。因此,研制高效低毒的新型抗糖尿病药物,对控制血糖、防治糖尿病及其并发症的发生发展,提高患者的生活质量具有十分重要的意义。
近年来,海藻多糖抗糖尿病活性的报道引起了科学工作者们的广泛关注,其主要作用表现为降血脂和降血糖等[24,25]。在高血脂大鼠模型中,岩藻聚糖被发现通过增加脂蛋白脂肪酶(LPL)、脂解酵素(HL)和卵磷脂胆固醇酰基转移酶(LCAT)的活性而能降低血清的总胆固醇(TCH)、低密度脂蛋白(LDL-CH)和甘油三酯(TG)的含量,而升高高密度脂蛋白(HDL-CH)含量[25]。低分子量石莼胶(Ulvan)喂养的高胆固醇大鼠模型被发现血清中总胆固醇和低密度脂蛋白的含量也有所降低[24]。另有国内外多份报道表明,来源于海藻的多糖及寡糖能够降低四氧嘧啶诱导的糖尿病小鼠的高血糖症,促进RIN-5F细胞分泌胰岛素,改善糖尿病患者的糖耐量,降低血糖、尿糖,并可使糖尿病患者其它症状减轻或消失[44-46]。此外,研究发现铬在生理状态下与羟基形成的配位复合物能在人体内发挥重要的生理功能[47,48],抗糖尿病方面的主要表现为纠正低血糖症,维持机体正常的糖代谢；调节脂肪代谢,降低糖尿病人血清中胆固醇、甘油三脂的水平,升高高密度脂蛋白胆固醇水平。Hao等发现海藻寡糖铬配合物能够通过激活胰岛素信号通路和AMPK通路来调节糖脂代谢,增加胰岛素敏感性,从而改善胰岛素抵抗[49]。海洋独特的环境使其拥有众多具有特异功能的活性物质,因此开发海洋来源的糖类药物意义重大。
2本课题研究背景、思路及研究方法
本课题以海洋藻类为研究对象,从事海藻糖类化合物的提取分离、结构鉴定、分子修饰及构效关系研究,发现糖类药物先导化合物,研制开发海洋创新药物、现代海洋中药及海洋生物功能制品,为海洋糖工程药物的研究开发提供理论与技术支撑。
加拿大新斯科舍省和圣劳伦斯地区被认为是世界上海藻资源和种类最丰富的地区之一。此地盛产的皱波角叉菜(爱尔兰海藻)是加拿大所有海藻中商业上最重要的卡拉胶原料；泡叶藻和海带也是此区域丰富的海藻资源,用来作为海藻酸钠的工业来源。结合大西洋加拿大沿岸丰富的海藻资源,制备不同类型、结构及来源的多糖,并运用不同的荧光试剂标记不同结构类型的海藻多糖,按照多糖芯片制备方法,运用海藻多糖芯片研究与蛋白的相互作用。制备海藻岩藻聚糖硫酸酯及其一系列衍生物,并对其进行2型糖尿病活性筛选,以期望发现抗糖尿病的新型化合物。
主要研究思路为：以6种不同类型的加拿大大西洋沿岸来源的海藻为实验对象,制备不同类型及结构的多糖,分离纯化褐藻中的岩藻聚糖,并制备其不同化学结构的衍生物。结合多糖的理化特性,选择荧光标记方法,分离纯化后对其进行荧光和糖含量分析,再利用微阵列技术将多种具有确定结构的多糖荧光标记物固定于Whatman硝酸纤维素薄膜,用建立的ELISA方法研究多糖与不同蛋白分子的相互作用,筛选出与糖尿病相关蛋白有关系的海藻多糖。同时,制备岩藻聚糖硫酸酯及其一系列衍生物,包括脱硫酸化、过硫酸酯化、磷酸化衍生物及系列寡糖。将各样品及所制备的衍生物运用3T3-L1细胞系筛选,结合糖芯片筛选结果,发现具有抗糖尿病作用的化合物,并对其抗糖尿病机理进行研究。
研究的难点和创新点：由于不同类型的海藻含有不同结构类型的多糖,对其进行提取分离纯化和结构分析,是一项艰巨的任务。运用不同的荧光标记方法,制备出一系列具有荧光稳定,并保持其生物活性的衍生物,提高多糖芯片上多糖固定率,建立完整的海洋来源的多糖荧光标记是本研究的难点之一。建立海藻多糖分离纯化、结构修饰、多糖芯片、糖与蛋白相互作用平台,并在细胞水平对活性化合物进行评价,获得其初步构效关系,将是本研究的创新点。目前虽然已有报道糖类化合物应用在糖尿病的防治方面,但是海洋来源的多糖抗糖尿病活性的机理仍有待于进一步研究。
3实验内容及结果
3.1海藻多糖的制备
从加拿大大西洋海域采集或购买了6种资源丰富的海藻,包括三种红藻(Polysiphonia lanosa, Furcellaria lumbricalis和Palmaria palmata),两种褐藻(Ascophyllum nodosum和Fucus evanescence)，及一种绿藻(Ulva lactuca)。海藻原料先用甲醇提取,以去除色素和其它非多糖成分,然后分别用冷水、热水、2%Na2CO3溶液和0.5M NaOH溶液在不同温度下提取。提取的粗多糖用80%乙醇沉淀,透析后冻干,得到24种不同来源和结构的海藻多糖。
理化性质分析结果表明,这些海藻多糖得率从19.71%到49.20%不等；多糖含量从15.42%到91.39%不等；硫酸根含量从0到17.55%不等。结构分析表明,红藻多糖主要由硫酸半乳聚糖(Polysiphonia lanosa和Furcellaria lumbricalis)或木聚糖(Palmaria palmata)组成,褐藻多糖主要由糖醛酸及硫酸化岩藻聚糖组成,而绿藻多糖(Ulva lactuca)则主要由葡萄糖醛酸、木糖、鼠李糖及葡萄糖成。
3.2岩藻聚糖及其衍生物的制备
利用CaCl2及乙醇沉淀的方法,将岩藻聚糖从褐藻多糖中分离出来,经透析除盐后得到8种岩藻聚糖。与Sigma公司岩藻聚糖(FC)相比,所制备的岩藻聚糖分子量均较大,约150～360kDa；多糖含量大约在60%左右；硫酸根含量在20%左右；蛋白含量在2-20%；岩藻糖占单糖组成百分比均在50%以上。
运用多聚磷酸-三正丁胺的方法制备了岩藻聚糖磷酸酯；运用三氧化硫-吡啶的方法制备了岩藻聚糖过硫酸酯；运用二甲基亚砜-甲醇-吡啶的方法制备了岩藻聚糖脱硫衍生物；运用0.1M三氟乙酸降解的方法制备了一系列低分子量岩藻聚糖。通过对9种岩藻聚糖的结构改造和化学降解,共制备了45种岩藻聚糖衍生物,通过红外光谱和理化性质分析,结果表明这些衍生物均成功获得。
3.3海藻多糖的抗2型糖尿病活性筛选
根据多糖的理化性质和结构特征,分别运用fluoresceinisothiocyanate (FITC)或5-(((2-(carbohydrazino)methyl)thio)acetyl)aminofluorescein(AF)荧光试剂成功标记了24种海藻多糖及8种岩藻聚糖,同时对市售岩藻聚糖(FC)、肝素(HEP)、褐藻胶(Alg)和卡拉胶(KCA)等也进行了荧光标记。将标记好的多糖分别固定于醋酸纤维素薄膜包被的玻璃芯片上,制备成糖芯片,用于多糖抗糖尿病活性的筛选。通过与人胰岛淀粉样蛋白多肽(IAPP)的结合实验结果发现,红藻多糖FL3和PP3能够有效地结合IAPP,表明其具有潜在的拮抗胰淀素蛋白沉积、提高胰岛素敏感性从而抵抗2型糖尿病的能力。
α-葡萄糖苷酶抑制剂可以延缓肠道对葡萄糖的吸收而达到控制血糖,尤其是餐后血糖过高的目的。通过对α-葡萄糖苷酶抑制活性的筛选发现,大多数褐藻多糖及部分岩藻聚糖均具有很好的抑制α-葡萄糖苷酶的作用。其中,AN1, AN4和FVl,FV2抑制能力最高,其IC50分别为2.62,21.32,3.15和44.37μg/mL。
3.4海藻多糖抗2型糖尿病活性的机制研究
小鼠3T3-L1前脂肪细胞在体外经诱导后可分化为成熟的脂肪细胞,可以较好的模拟脂肪细胞分化和活体脂肪组织的功能,成为筛选胰岛素增敏剂类抗2型糖尿病的体外模型之一。通过对3T3-L1成熟脂肪细胞积累的脂滴多少以及细胞内甘油三酯含量的高低,对海藻多糖及其衍生物进行了抗糖尿病活性筛选。结果表明,PL2,PL3,FL3,PP2,PP3和多数褐藻能抑制3T3-L1细胞内脂滴的积累,而FL2,FL3,AN2和FVl处理的细胞中甘油三酯含量较低。同时发现,大多数岩藻聚糖均能抑制3T3-L1细胞分化过程。通过对脂肪细胞分化前期细胞转录因子的表达分析,发现岩藻聚糖通过抑伟(PPARΥ及其相关调控因子C/EBPa(?)(?)C/EBPP而抑制3T3-L1细胞的分化过程。
综上所述,本课题以6种大西洋加拿大沿岸的海藻为研究对象,制备了不同结构类型的海藻多糖及其衍生物共69种(24+45)。运用不同荧光试剂对其进行标记,制备了海藻多糖芯片,研究了各海藻多糖与IAPP和的相互作用。采用细胞模型对各化合物进行了2型糖尿病活性筛选,并对有效成分进行作用机理研究。
4创新点
本论文的创新点主要有以下3个方面：
1)首次对大西洋加拿大沿岸6种海藻所含多糖结构进行了研究,成功制备了69种海藻多糖及其衍生物。
2)首次运用多糖芯片技术,结合细胞模型等对海藻多糖进行了抗2型糖尿病活性筛选。通过糖芯片手段,发现两种红藻多糖能较有效的与胰岛淀粉样蛋白相结合。通过酶化学方法,获得了具有能抑制α-糖苷酶活性的褐藻多糖化合物。通过细胞模型,发现泡叶藻来源的岩藻聚糖能调节脂肪代谢,抑制脂肪细胞分化过程。
3)在细胞水平上对岩藻聚糖抗2型糖尿病活性进行了机制研究,首次发现泡叶藻来源的岩藻聚糖能够通过抑制PPARy的活性,激活C/EBPa和PPARa的活性,而增强脂代谢,改善胰岛素抵抗,为2型糖尿病药物开发提供了理论基础。
5下一步工作计划
岩藻聚糖是一类从褐藻中分离得到的硫酸多糖,主要是由L-岩藻相组成,同时可能含有少量木糖,半乳糖和甘露糖等。跟据论文目前的研究结果,下一步我们以A. nosodum来源的岩藻聚糖为研究对象,继续开展以下2方面工作：
1)在分子水平上探讨泡叶藻岩藻聚糖及其衍生物与IAPP作用关系,获得结构明确的寡糖片段,并从事其信号通路研究。
2)针对本研究发现的岩藻聚糖具有调节脂代谢,抑制脂肪细胞分化的作用,在动物水平上进一步评价其抗2型糖尿病作用效果,并对其作用机制进行探讨。
Diabetes mellitus is a group of diseases characterized by chronic hyperglycemia due to deficiency of insulin action which is a common basis and leads to characteristic abnormalities in the metabolism of carbohydrate, lipid, and protein. As a world wisely spread diseases, diabetes mellitus is mainly divided into type-1diabetes (Insulin dependent, IDDM) and type-2diabetes (Non-insulin dependent, NIDDM). Type-2diabetes is the most common form and has escalated to epidemic proportions. Main factors of type-2diabetes are related with a genetic disposition, overweight or obesity, lack of physical activity, consumption of high-fat, low-fiber diets, oxidative stress, and possibly deficiencies in certain minerals. Insulin resistance is an initial mechanism to reduce type-2diabetes. Adipose tissue stores excess energy in the form of triglyceride, and releases free fatty acids in response to energy requirements. It is one of the insulin sensitive peripheral organizations and plays a critical role in insulin resistance and type-2diabetes. Oral agents including insulin sensitizers, secretagogue, bile acid sequestrant, alpha-glucosidase inhibitor, and DPP-IV inhibitor are applied for improving glucose production, carbohydrate intake, insulin secretion, and peripheral uptake associated with type-2diabetes. However, the disadvantages of these agents provide more challenges for anti-diabetes research. Seaweeds species have been used traditionally as food, vegetable, as well as herbal drugs, and many investigations revealed that seaweeds have not only nutritional effects but also beneficial properties to cure various diseases and maintain good health. It has been reported that polysaccharides from marine sources may possess anti-diabetic potential. The objective of this project is to develop natural polysaccharides and their derivatives from marine algae for anti-diabetes studies.
Polysiphonia lanosa, Furcellaria lumbricalis, Palmar ia palmata, Ascophyllum nodosum, Fucus vesiculosus and Ulva lactuca, were collected from Atlantic Canadian coast where is one of the richest sources of seaweeds in the world. Algal polysaccharides were prepared under four conditions, cold water, hot water,2% and0.5M NaOH solutions; and twenty four polysaccharides were successfully obtained, respectively. Their chemical properties, including contents of total sugar, protein and sulphate groups, and structural informations, including molecular weight, monosaccharides compositions and characteristic groups, were studied by chemical methods and1H-NMR and FTIR spectra analysis. Fucoidan is a sulfated polysaccharide from brown seaweeds, which has been reported with various bioactivities. We isolated and purified fucoidan fractions from extracts of Ascophyllum nodosum and Fucus vesiculosus. Their phosphorylated, oversulphated, desulphated and degraded derivatives were also prepared and the derivatives were indicated successfully processed on FT-IR spectra and characteristic chemical analysis.
Glycoarrays technologies, an important tool in glycomics, have been developed as a top-leading technology to study the interactions of glycan-binding proteins with their glycan ligands. Islet-amyloid polypeptide (IAPP) is an important player in the formation of amyloid deposits and the progression of type2diabetes. Exploring a successful anti-amyloidogenic agent is another option to prevent diabetes mellitus. Fluoresceinisothiocyanate (FITC) or5-(((2-(Carbohydrazino)methyl)thio)acetyl)aminofluorescein (AF) labeled polysaccharides were printed on a nitrocellulose coated glass platform to create the glycoarrays and their IAPP binding abilities were screened as a high-throughput manner. FL3from F. lumbricalis and PP3from P. palmate showed the highest binding abilities on glycoarrays which suggested that these algal polysaccharides might show the optional application against type-2diabetes.
Alpha-glucosidase is a key enzyme for carbohydrate digestion associated with type-2diabetes mellitus. Algal polysaccharides and their derivatives were then invested the inhibitory effects against a-glucosidase by spectrophotometrical method. Among them, AN1and AN4from A. nodosum, FV1and FV4from F. vesiculosus showed highest inhibition. Further studies showed that the inhibitory effects were caused by the polysacchardes not their binding proteins.
Adipocyte tissue is the main target organ in response of insulin and plays a critical role in insulin resistance and type-2diabetes.3T3-L1preadipocyte cell lines are important in use to study the pathogenesis mechanism of insulin action and lipid-lowing in vitro. Algal polysaccharides and their derivatives were next studied on lipid accumulation in cultured3T3-L1adipocytes by measuring triglyceride (TG) contents and Oil Red O (ORO) staining as indicators. Treatment with PL2, PL3(from P. lanosa), FL3(from F. lumbricalis), PP2, PP3(from P. palmata) and most of fucoidan fractions decreased the lipid accumulation by ORO staining while FL2, FL3(from F. lumbricalis), AN2(from A. nodosum), and FV1(from F. vesiculosus) could reduce the TG content in3T3-L1adipocytes.
Two families of transcription factors, CCAAT/enhancer binding protein (C/EBP) and peroxisome proliferator-activated receptor-Υ (PPARy), are induced early during adipocyte differentiation. By enzyme-lined immunosorbent assay (ELISA), algal polysaccharides FL3(from F. lumbricalis), AN2(from A. nodosum) and two fucoidan fractions, AN3F and FV3F, were invested to decrease the expression of PPARy protein.
According to these results, we next focused on the effect of FL3, PP3, AN1, AN4, FV1, FV2, AN3F and FV3F against the early stage of3T3-L1adipogenesis study. AN3F showed the strongest suppression on PPARy and C/EBPβ,and the promotion on PPARa and C/EBPa in a dose-dependent manner, which suggested that it decreased the lipid accumulation in3T3-L1adipocytes through the effects on these PPARs and C/EBPs transcription factors and its anti-diabetic acitivity could be related to the MAPK signal involving many cytokines.
In a conclusion, we successfully prepared24algal polysacchardes and45fucoidan derivatives from six kinds of seaweeds collected from Atlantic Canadian coast (including the commercial fucoidan and its derivatives). Their anti-diabetic activities were screened by glycoarrays technologies, enzymatic spectrophotomctrical assay, enzyme-lined immunosorbent assay and western blot analysis. Among them, AN3F showed the potential anti-type-2diabetes affection by suppression the transcription factors in adipogenesis. Although, further molecular mechanism of AN3F and animal studies remains to be elucidated, AN3F provided potential of a new target for anti-diabetes research.
引文
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