【作者】 郝翠；

【导师】 于广利；

【作者基本信息】 中国海洋大学 ， 药物化学， 2011， 博士

【摘要】 糖尿病(Diabetes Mellitus, DM)是一种常见的代谢性疾病,其发病率在发达国家和发展中国家都在逐年增加,中国已经成为世界糖尿病发病人数最多的国家。糖尿病主要分为1型(胰岛素依赖性糖尿病,IDDM)和2型(胰岛素非依赖性糖尿病,NIDDM)。其中2型糖尿病占糖尿病患者的90%以上,为典型的多基因疾病。2型糖尿病主要与两种生理缺陷有关,分别是胰岛素分泌缺陷和胰岛素抵抗。胰岛素抵抗主要发生在脂肪、肝脏和骨骼肌中,这些组织的细胞中含有大量的胰岛素受体,它们能结合胰岛素从而在调节葡萄糖代谢稳态上发挥重要作用,尤其骨骼肌中的胰岛素抵抗是2型糖尿病的主要致病因素。防治2型糖尿病胰岛素抵抗所致的代谢异常,及改善胰岛素抵抗或增加胰岛素敏感性药物的研发具有重要意义。然而,现有的抗2型糖尿病药物多存在着继发性失效、副作用大及容易诱发低血糖等缺陷,因此研发低毒且能改善胰岛素抵抗的长效药物十分必要。海洋独特的环境造就了众多结构多样和功能独特的活性物质,海洋糖类化合物以其资源丰富、低毒和活性广泛而备受关注。大量研究表明,各种有机铬配合物具有改善糖代谢和调节血脂的作用。在前期研究基础上,本研究将各种海洋寡糖与铬离子配合,制备了系列海洋寡糖铬衍生物,通过筛选获得一种具有较好增加胰岛素敏感性的寡糖铬化合物OM2,并对其抗2型糖尿病作用的分子机理进行研究。首先运用酸降解方法,以褐藻胶和卡拉胶为原料,成功制备了不同聚合度的褐藻寡糖和卡拉胶寡糖,再将其与Cr3+配合,制备了系列海洋寡糖铬衍生物。利用正交实验对甘露糖醛酸寡糖铬配合物和卡拉胶寡糖铬配合物的制备条件进行了优化,确定其最佳反应条件。通过紫外全波长扫描和红外光谱等分析手段对所得寡糖铬合物进行了结构表征。利用C2C12骨骼肌细胞模型和硫磺素荧光法对各种海洋寡糖衍生物进行活性筛选。发现海洋酸性寡糖铬配合物OM2不仅能够在体外阻断胰淀素纤维化还能够激活AMPK信号通路,具有潜在的改善胰岛素抵抗活性。在此基础上,利用遗传性糖尿病转基因db/db小鼠模型进一步评价其在体内抗2型糖尿病效果。研究表明,海洋寡糖OM2无急性降糖作用,不存在一过性血糖降低的危险；不仅能够有效降低血糖、改善血脂代谢、减轻小鼠胰岛素抵抗,而且具有一定的增强胰岛素敏感性的作用。在确定OM2具有抗2型糖尿病作用基础上,进一步利用C2C12细胞和db/db小鼠模型分别在细胞和动物整体水平上对OM2提高胰岛素敏感性的作用机制进行了系统研究。结果发现,OM2能够显著增加胰岛素刺激的葡萄糖转运,且效果优于阳性对照药物二甲双胍。ELISA和实时定量RT-PCR实验结果表明,OM2不仅能够通过激活胰岛素信号通路中的关键蛋白IR、Akt和PI3K的的磷酸化来增加胰岛素受体及GLUT4的数量,还能够增加AMPK信号通路中关键蛋白AMPK和ACC的磷酸化水平,调节脂肪代谢,从而发挥其胰岛素增敏作用。免疫印迹法研究发现,OM2还能够在肝脏中通过激活胰岛素信号通路来调节糖原合成和糖异生过程,从而减轻小鼠的高血糖症状。将OM2进行FITC荧光标记后,利用活细胞成像技术研究发现,OM2可以进入C2C12细胞并定位于线粒体,说明其发挥作用可能与提高线粒体功能有关。综上所述,本研究成功获得了在体内外都具有较好抗2型糖尿病作用的海洋寡糖铬配合物OM2；并通过作用机制研究阐明其通过激活胰岛素信号通路和AMPK信号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,为将其开发为新型抗2型糖尿病海洋药物提供了理论依据。更多还原

【Abstract】 Diabetes mellitus is the most common metabolic disease and its prevalence is increasing in both developed and developing countries year by year. China has become the country with the largest number of diabetes prevalence all over the world. Diabetes mellitus can mainly be divided into type 1 diabetes (insulin dependent, IDDM) and type 2 diabetes (non insulin dependent, NIDDM). More than 90% of diabetes patients suffer from non-insulin-dependent diabetes mellitus (NIDDM, type 2 diabetes) which is a typical polygenic disease. Type 2 diabetes is mainly associated with two principal physiological defects:resistance to the action of insulin and deficiency in insulin secretion. Insulin resistance mainly occurs in fat tissue, liver and skeletal muscles, where cells contain many insulin receptors that can bind insulin to regulate the steady-state of glucose metabolism. Resistance to the actions of insulin in skeletal muscle is a major pathogenic factor in diabetes mellitus. So the pharmaceutical researches on the prevention of metabolic disorder caused by insulin resistance and improvement of insulin sensitivity are very important for the development of anti-type 2 diabetes drugs. However, current drugs for type 2 diabetes therapies often have many defects such as secondary failure, side effects and risk of hypoglycemia, so it is important to develop low toxic and long acting anti-diabetes drugs which can improve insulin resistance. The unique environment of ocean creates many active substances with special structures and functions, especially for the marine polysaccharides which attract much attention for their wide variety of sources, low toxicity and broad activity. Many researches indicated that different kinds of organic chromium complexes can improve glucose metabolism and regulate blood lipid. Based on the former research, series of marine oligosaccharide-chromium derivatives were prepared by complexing oligosaccharides with chromium ion in this research, and one of them named OM2 was found to be able to effectively increase the insulin sensitivity, and its molecular mechanisms of anti-type 2 diabetes were also investigated.Alginate oligosaccharides and carrageenan oligosaccharides with different degrees of polymerization were firstly prepared by using acid degradation of alginate and carrageenan, and then series of marine oligosaccharide-chromium derivatives were prepared by complexing oligosaccharides with chromium (Ⅲ) ion. Moreover, the orthogonal experiments were used to optimize the preparation conditions of oligomannuronate-chromium complexes and carrageenan oligosaccharide-chromium complexes, and the optimal reaction conditions were determined in this research. The structure characterization of prepared marine oligosaccharide-chromium derivatives was performed by using full wavelength UV scanning and IR analysisSkeletal muscle C2C12 cell model and Thioflavin T fluorescence assay were then used to perform activity screening of different kinds of marine oligosaccharide derivatives. The results indicated that the marine acidic oligosaccharide-chromium complex OM2 could not only inhibit amylin fibrosis in vitro but also activate AMPK signal pathway, which suggested OM2 has potential activity to improve insulin resistance. Based on these results, the hereditary diabetes transgenic db/db mice were used to evaluate the anti-diabetes effect of marine acidic oligosaccharide-chromium complex OM2 in vivo. The results showed that OM2 has no acute hypoglycemic effect and no risk of transient lower blood sugar. OM2 could not only effectively decrease the blood sugar; improve blood lipid metabolism and attenuate insulin resistance in db/db mice, but also have the activity to increase insulin sensitivity to some extent in vivo.Based on the results that OM2 has good anti-type 2 diabetes effects, the mechanisms of improving insulin sensitivity by marine acidic oligosaccharide OM2 were then systematically investigated in vitro and in vivo by using skeletal muscle C2C12 cells and db/db mice. The results indicated that the marine acidic oligosaccharide-chromium complex OM2 could significantly increase insulin induced glucose transport, and the actions are more effective than metformin, the positive control drug for type 2 diabetes therapy. Moreover, OM2 was found to be able to activate the phosphorylation of key protein IR, Akt and PI3K in insulin signal pathway to increase the production of IR and GLUT4 by using ELISA and real time quantitative RT-PCR analysis. OM2 could also increase the phosphorylation of AMPK and ACC in AMPK signal pathway to regulate lipid metabolism. So OM2 can activate both the insulin signal pathway and AMPK signal pathway to increase insulin sensitivity. Furthermore, by using western blot assay, marine oligosaccharide OM2 was also found to be able to activate the insulin signal pathway to regulate the glycogen synthesis and gluconeogenesis in liver, and can attenuate the hyperglycemic symptom in diabetes mice. Moreover, fluorescence labeled marine acidic oligosaccharide OM2 was found to be able to localize to the mitochondria after its internalization into skeletal muscle C2C12 cells by using living cell imaging, which suggested that the anti-diabetes effect of marine acidic oligosaccharide OM2 might be related to its improving the functions of mitochondria in skeletal muscle cells.In conclusion, marine oligosaccharide chromium complex OM2 which having good anti-diabetes effects in vivo and in vitro were successfully prepared in this research. The marine oligosaccharide OM2 could activate both the insulin signal and AMPK signal pathways to regulate the glucose and lipid metabolism, and increase insulin sensitivity to improve the insulin resistance. Moreover, the researches about improving insulin resistance mechanisms of marine oligosaccharide OM2 can provide the theoretical basis for developing it into new type anti-type 2 diabetes marine drugs.更多还原