【作者】 陈金龙；

【导师】 吴祖泽；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2007， 博士

【摘要】 糖尿病是一种以高血糖为特征的胰岛素缺乏或相对缺乏导致的内分泌代谢疾病，并发的多种慢性合并症是病人致残或致死的主要原因。已有的口服降糖药均不能完全有效的改善糖尿病的病理异常，因此，寻找预防和治疗糖尿病的有效药物已成为世界范围内的一个重大课题。二苯乙烯类化合物E2是我室从大黄根茎中提取纯化的一类结构全新的具有自主知识产权的降糖药物，作用机制不明。为此，我们开展了E2的降糖以及相关并发症的改善作用实验研究，系统的研究其可能的作用机制，同时对糖尿病发病机理进行了探索性研究。目的是评价E2的降糖作用，阐明其降糖作用机制，包括E2对多种糖尿病模型的降糖效果，对靶细胞的生物学效应和分子调控，为开发新型降糖药物提供理论基础；并在国内外首次进行鞘氨醇激酶(sphingosine kinase，SPK)在糖代谢调控中的作用研究，探讨其作为调控糖代谢新靶标的可能性，为糖尿病发病机制研究提供实验基础。本文首先采用5种不同类型的糖尿病实验动物模型系统研究了E2对糖尿病及其并发症的治疗效果，研究结果表明：E2能明显降低不同糖尿病动物的血糖血脂水平，显著改善葡萄糖耐量的异常，血清生化指标检测证实E2不能促进胰岛素分泌，病理形态学结果显示E2对胰岛细胞具有保护作用，血液动力学和电镜观察超微结构结果表明E2对受损的心脏、肾脏、肝脏和脾脏的结构具有修复和保护作用；E2能显著增加糖尿病大鼠肝脏和心肌组织的糖原合成；血流动力学结果表明，E2能有效裂解糖基化形成的终产物AGEs从而能显著提高大鼠心输出量，降低总外周阻力，提高全身动脉顺应性；Western Blot结果显示E2能明显提高肝脏胰岛素受体含量，活化肝脏和心肌组织Akt、GSK-3β信号通路。在上述药效学实验的基础上，我们对E2降糖降脂改善并发症的细胞和分子机理进行了较为系统的探索研究。首先对调控糖代谢的靶细胞进行了筛选，发现E2能明显的促进高糖状态下的肝L0-2细胞的葡萄糖消耗，而对肌母C2C12细胞作用不明显，提示E2作用的靶细胞可能是肝细胞。选用βTC3胰岛细胞观察E2是否具有促胰岛分泌作用，结果证实，E2不能促进胰岛细胞分泌胰岛素。其次我们对E2调控糖代谢的分子机理进行了探讨，研究结果表明：E2在体外可以激活Akt、GSK-3β激酶磷酸化，促进肝细胞的葡萄糖调控。同时我们也首次发现胰岛素和E2能明显增强SPK激酶活性，SPK和磷脂酰肌醇-3激酶(Phosphoinositide 3-kinase,PI-3K)同属脂质激酶，而PI-3K激酶是调控糖代谢的一个关键分子，结合脂质激酶在糖代谢中的重要地位，我们大胆推测，SPK很有可能是糖代谢调控的另一个新的关键分子。为此，我们又对鞘氨醇激酶SPK在糖代谢调控中的作用进行了探索性研究，试图阐明SPK是否是除PI-3K外的另一个与糖代谢密切相关的分子。首先用腺病毒介导的SPK1基因转染肝L0-2细胞和C2C12肌母细胞，结果表明，SPK1基因转染可以直接刺激细胞的葡萄糖吸收，也能明显促进胰岛素刺激的细胞对糖的吸收，SPK阻断剂DMS抑制SPK1表达及其酶活性，能够导致细胞摄取葡萄糖的能力减弱，并且也使细胞在胰岛素刺激下的葡萄糖吸收显著地降低。其次用腺病毒介导的SPK1基因转染原发2型糖尿病KK-Ay小鼠，在整体动物水平观察并确定SPK对糖代谢的调控作用，研究结果发现，SPK1能够明显降低2型糖尿病KK-Ay小鼠的血糖；改善胰岛素抵抗，SPK1在肝组织、心肌组织、骨骼肌组织中都有较高的表达，这些结果提示SPK1是参与糖代谢调控的一个关键分子，有望成为糖尿病防治的新靶点。通过以上结果，我们可以得出：(1)E2能明显的降低糖尿病模型的血糖和血脂，显著改善心功能，逆转血管硬化，对肝脏、脾脏和肾脏损伤具有较为明显的保护作用，因此，E2是预防和治疗糖尿病及其相关并发症的一种新的行之有效的药物(2)E2的作用机制是：①不刺激胰岛细胞分泌胰岛素，对胰岛细胞具有保护作用；②增加肝脏胰岛素受体数目；③通过激活Akt、GSK-3β激酶磷酸化促进肝糖原和心肌糖原合成；④促进肝细胞的葡萄糖消耗；⑤通过裂解糖基化终产物AGEs改善血管硬化；⑥活化鞘氨醇激酶(sphingosine kinase，SPK)，提高对糖代谢的调控；(3)SPK可能是除PI-3K外的另一个与糖代谢密切相关的脂质激酶。更多还原

【Abstract】 Diabetes is characterized by hyperglycemia, which results from impaired insulin secretion and/or insulin action. It is also the leading cause of many clinical chronic complications, which lead to a markedly increased morbidity and mortality. Drugs currently used in clinical practices could not correct the abnormality of diabetes with satisfaction. Therefore, it is still an important mission in the world to find a more effective agent in the treatment and prophylaxis of diabetes.Stilbenoids E2, a derivate from rheum officinal rootstock, is a kind of glucose-lowering agent owned by our laboratory. However, we know little about its glucose-lowering mechanism and complication improving effects. In order to evaluate its glucose-lowering effects and clarify the underlying mechanisms, we performed a systemic research using several experimental diabetic animal models.. Meantime, we investigated the role of SPK in the regulation of glucose metabolism, and tested the possibility of taking it as a new target in managing the diabetes mellitus..First, we observed the functions of Stilbenoids E2 on glucose-lowering and complication controlling by using 5 kind experimental animal models The results showed that Stilbenoids E2 could decrease the serum glucose, triglyceride and cholesterol levels, and could markedly improve the oral glucose tolerance of the diabetic animals. Stilbenoids E2 could not promote the secretion of insulin but protect the islet cells. Stilbenoids E2 showed a potent ability to protect and reconstruct many organs, such as the heart, kidney, liver and spleen. Glucogen synthesis in the rat liver and heart was accelerated after the administration of Stilbenoids E2. Stilbenoids E2 could also improve the rat’s heart output through the decomposing glycosylating terminal products-AGEs, decreasing the distal resistance of blood vessels, and improving the compliances of the arteries. Western Blot results showed that stilbenoids E2 could increase the expression of insulin receptor in the liver and activate the signaling pathways of Akt、GSK-3βin the liver and heart tissues. we then probed the molecular and cellular mechanisms of stilbenoids E2 for its abilities to decrease the serum levels of triglyceride and cholesterol, and its controlling on the related complications. Stilbenoids E2 enhanced the consumption of hepatin in the L0-2 liver cells but not in the C2C12 muscle cells, impling that the liver cells might be a target of stilbenoids E2. We found that stilbenoids E2 could not induce insulin secretion of theβTC3 islet cells, but could active the phosphorylation of Akt and GSK-3βin the SMMC-7721 hepatoma cells. We also found that stilbenoids E2 could activate the SPK, another important lipid kinase as PI3K. To clarify whether SPK is really a key molecule involved in the glucose metabolism, we prepared the Ad-SPK1, the recombinant adenovirus harboring SPK1 gene, then transfected the SMMC-7721 hepatoma cells and C2C12 myoblasts with Ad-SPK1. We found that SPK1 gene tranfer could significantly increase both basal and insulin-induced glucose uptake, and DMS, a potent inhibitor of SPK1, inhibited both basal and insulin-induced glucose uptake. We further investigated the role of SPK1 in glucose metabolism in the spontaneous KK-Ay type 2 diabetic mice model using a adenoviral-mediated gene transfer approach. It was found that the adenoviral-mediated gene transfer of SPK1 markedly decreased blood glucose levels and alleviated their insulin-resistance of the animals. All these findings strongly suggest that SPK1 is a key molecule in modulating the glucose metabolism, and stimulating activation of SPK may be an important mechanism of the anti-diabetic action of stilbenoids E2.In conclusion, our results demonstrate that: (1) Stilbenoids E2 can decrease the blood glucose and lipid levels, improve the structures and functions of the heart, liver, spleen and kidney, so it is a new and effective agent in the treatment of diabete. (2) The glucose-lowering mechanism of Stilbenoids E2 are:①Protect the islet cells but do not stimulate the secretion of insulin;②Increase the expression of insulin receptors;③Accelerate the glycogen synthesis in the liver and the heart through activating Akt and GSK-3βsignaling;④stimulating activation of SPK is an important mechanism of the anti-diabetic action of stilbenoids E2. (3) SPK is a another lipid kinase involved in the regulation of glucose metabolism and homeostasis.更多还原