【作者】 吕琳；

【导师】 吴曙光；

【作者基本信息】 南方医科大学 ， 药理学， 2010， 博士

【摘要】 糖尿病是一种由遗传或后天胰岛素分泌不足或胰岛素抵抗所致的以糖代谢紊乱为主的慢性综合性疾病。生活水平提高、体力劳动减少、人口老龄化及肥胖发生率的增加等多种因素,致使糖尿病的发病率有逐年上升的趋势,现已成为继肿瘤、心脑血管疾病之后的第三位严重危害人类健康的慢性疾病,被世界卫生组织称为“21世纪的灾难”。目前我国有糖尿病患者9200多万,成为全球第一糖尿病大国,其中约97%患者属于2型糖尿病(2DM)。更为严重的是糖尿病的发病有年轻化的趋势,甚至一些肥胖、超重的儿童、青少年也患上了糖尿病。目前,对于1型糖尿病(1DM)治疗的研究方向是开发给药方便、有效的胰岛素制剂及其代用品；对于治疗2DM的药物,根据药理作用的机制不同,分为胰岛素及其同类药物、胰岛素增敏剂、胰岛素促泌剂、减少碳水化合物调节剂等。但是传统的糖尿病治疗药物降糖疗效有限,且无法根本阻止胰岛β细胞的进一步坏死。随着人们对糖尿病发病机制认识的不断深入,许多治疗糖尿病的新型药物不断问世。研发药物的方向也从糖尿病发病机制的分子机理切入,寻找可能的药物作用靶点。其中主要的研究热点相对集中在胰岛素信号转导途径和糖尿病代谢途径中相关的蛋白和调控因子。糖尿病属于中医消渴症范畴,几千年的中医文化和丰富的中草药积累了丰富的中草药降糖经验。降血糖中药新药越来越受到关注,尤其是从中药中筛选出来的具有降糖作用的药物及有效成分的研究,已成为抗糖尿病药物的研究热点和开发方向。黄芪(Astragalus membranaceus)药用迄今已有2000多年的历史,现代药理学研究证实,黄芪含皂甙、蔗糖、多糖、多种氨基酸、叶酸及硒、锌、铜等多种微量元素；有增强机体免疫功能、保肝、利尿、抗衰老、抗应激、降压和广泛的抗菌作用。据相关报道,黄芪对糖尿病患者具有增加胰岛素敏感性和降低血糖的作用。黄芪可通过增加糖原合成酶活性、胰岛素受体底物活性、蛋白激酶B和蛋白激酶C活性,使骨骼肌细胞、心肌组织葡萄糖转运蛋白水平增加,使糖原合成酶活性增加而增加胰岛素敏感性,从而发挥降低血糖作用。本实验采用高脂饮食联合化学药物链脲左菌素(streptozotocin, STZ)诱导的C57BL/6J小鼠糖尿病模型,研究黄芪甲苷对糖尿病小鼠血糖、血脂以及胰岛素水平的作用。采用基因芯片方法研究黄芪甲苷降血糖作用可能的分子靶点。接下来我们选取糖代谢途径中的两个关键酶：肝糖原磷酸化酶和肝葡萄糖-6-磷酸酶,进行下一步研究；我们采用酶活性法、实时荧光定量RT-PCR和Westernblot法研究黄芪甲苷对这两个酶的影响,拟阐明其降血糖作用的分子机制。研究的主要方法和结果如下：一、采用高脂饮食联合STZ诱导C57BL/6J糖尿病小鼠模型,评价不同剂量黄芪甲苷对糖尿病小鼠血糖、体重、总胆固醇、甘油三酯和胰岛素水平的影响。雄性C57BL/6J小鼠,随机分成普通饲料组和高脂饮食(High fat diet, HFD)组,用普通饲料和高脂饲料分别喂养3周后,普通饲料组小鼠腹腔注射柠檬酸缓冲液一次,高脂饮食组小鼠给予STZ100mg/kg腹腔注射一次。注射2周后用血糖仪测定血糖,选取血糖水平在10.0-20.0mmol/L之间的小鼠作为糖尿病模型。继续用原饮食喂养3周后将小鼠分成正常空白对照组(Control groups),糖尿病模型组(HFD+STZ groups)和糖尿病黄芪甲苷治疗组(HFD+STZ+Geniposide 12.5mg/kg,25mg/kg,50mg/kg)。糖尿病模型组(HFD+STZ)小鼠的平均血糖明显升高,与正常空白对照组(Control groups)比较有显著差异(P<0.05)。分别用12.5mg/kg、25mg/kg和50mg/kg的黄芪甲苷给糖尿病小鼠灌胃,每日两次,连续灌胃3周之后用血糖仪测定小鼠血糖；分别用总胆固醇酶联免疫分析试剂盒、甘油三酯检测试剂盒、胰岛素检测试剂盒检测小鼠血中的总胆固醇、甘油三酯和胰岛素水平,观察不同剂量的黄芪甲苷对糖尿病小鼠的血糖、体重、血脂(甘油三酯和胆固醇)和胰岛素水平的作用。结果表明,黄芪甲苷25mg/kg和50mg/kg治疗组与糖尿病模型组小鼠比较,其血糖、体重、总胆固醇水平和胰岛素水平均显著降低(P<0.05),12.5mg/kg黄芪甲苷治疗组小鼠与糖尿病模型组小鼠比较血糖、体重、甘油三酯水平及胰岛素水平均无显著差异(P>0.05),黄芪甲苷12.5mg/kg、25mg/kg和50mg/kg治疗组与糖尿病模型组小鼠比较总胆固醇水平变化无显著性差异(P>0.05)。结果说明黄芪甲苷能降低糖尿病小鼠的血糖、体重、甘油三酯及胰岛素水平,对总胆固醇水平影响不大。二、基因芯片法研究黄芪甲苷对高脂饮食联合STZ糖尿病小鼠相关糖尿病基因的影响使用SuperArray的RT2 Profiler TM PCR芯片,Prism 7900测试系统进行实时荧光PCR检测。芯片上包含了与糖尿病发生、发展和恶化相关的84个基因,根据基因的功能分为受体、转运及通道蛋白、核受体、代谢酶、分泌因子、信号转导、转录因子及其他共七大类基因组。结果发现50mg/kg黄芪甲苷治疗组下调了六大类19个基因水平,上调了六大类24个基因水平。下调基因包括：受体、转运、通道蛋白类的突触结合蛋白、囊泡膜蛋白、白介素4受体、癌胚抗原相关细胞粘附分子、胰高血糖素受体；代谢酶类的聚合ADP核糖、肝糖原磷酸酶、甘油-3-磷酸脱氢酶、葡萄糖-6-磷酸酶、果糖二磷酸酶、二肽激肽酶4；分泌因子类的胰岛素、血管上皮生长因子、白介素-12b；信号转导类的磷脂酰基醇-3-激酶；转录因子类的转录因子1、叉头框G1、固醇调节元件结合因子；核受体类的过氧化物酶增殖活化受体。上调基因包括：受体、转运、通道蛋白类的细胞粘附分子、肿瘤坏死因子受体超家族a、肿瘤坏死因子受体超家族b、RAS癌基因家族、选择素；代谢酶类的血管紧张素转化酶Ⅰ、超氧化物歧化酶、葡萄糖激酶、一氧化氮合酶和血红素氧化酶1；分泌因子类的血管紧张素、趋化因子受体2、肿瘤坏死因子、抵抗素、白介素-6、白介素-10；信号转导类的叉头框C2、胰岛素启动因子1；其他类的非偶联蛋白和丝氨酸肽酶抑制剂。三、采用酶活性法、荧光实时定量聚合酶链式反应(real time RT-PCR)法和Western blot法研究黄芪甲苷对与糖代谢相关的肝糖原磷酸化酶和肝葡萄糖-6-磷酸酶的作用。在上面的实验中,我们从黄芪甲苷诱导下调表达的基因中,选取2个与糖代谢密切相关的基因,即GP和G6Pase的基因Pygl和G6pc;采用酶活性法、荧光定量RT-PCR法和Western blot法对两个酶的活性、mRNA水平和蛋白表达进行下一步的实验。结果发现黄芪甲苷能降低糖原磷酸化酶和葡萄糖-6-磷酸酶的活性,减少其mRNA及蛋白的表达。本论文得出如下结论：一、我们使用高脂饮食联合STZ诱导的C57BL/6J小鼠成功造成了糖尿病小鼠模型,模型小鼠的血糖升高,与文献报导一致。二、黄芪甲苷能降低高脂饮食联合STZ诱导的C57BL/6J糖尿病小鼠的血糖、甘油三酯和胰岛素水平,但对总胆固醇水平无明显影响。三、用包含有与糖尿病发生、发展与恶化相关的84个基因的第二代基因芯片检测结果提示,黄芪甲苷能上调24个基因,下调19个基因。黄芪甲苷下调与糖代谢相关的酶基因肝糖原磷酸化酶2.74倍,葡萄糖-6-磷酸酶45.20倍。四、肝酶活性实验结果表明,黄芪甲苷能明显抑制糖尿病小鼠肝糖原磷酸化酶和肝葡萄糖-6-磷酸酶的活性,实时荧光RT-PCR法和Western blot实验验证了黄芪甲苷能显著抑制糖尿病小鼠血清中GP和G-6-Pase的mRNA和蛋白的表达。综上所述,我们的实验首次发现黄芪甲苷降血糖作用的机制是通过抑制肝脏的糖原磷酸化酶和肝葡萄糖-6-磷酸酶的活性,抑制肝糖原的分解和葡萄糖的氧化来实现的。我们的研究为阐明黄芪甲苷降血糖作用的分子机制提供了有意义的实验依据。更多还原

【Abstract】 Diabetes is a metabolic disease characterized by the absolute or relative deficiency of insulin. Accumulating evidences demonstrate that diabetes becomes one of the most important diseases threatening human health after cancer and cardiovascular diseases. The morbidity of diabetes in the world bursts out because of the combination of elevated living standards, decreased physical work, increased unhealthy lifestyle and enlarged number of the elderly nowadays. China has becoming the largest "diabetic country" and 97% of the patients belong to type 2 diabetes. Moreover, a tendency of younger onset is rapidly increasing.It’s very important to develop new drugs or find new therapeutic targets, for diabetes and its complications are now worldwide problems. The present drugs against diabetes consist of drugs promoting the secretion of insulin, drugs increasing the tissue sensitivity to insulin, drugs inhibiting glycosidase a as well as insulin and its homologs. With the increased recognization on the pathogenesis of diabetes, more and more drugs are designed based on the molecular mechanism of diabetes.Astragaloside IV us a glycoside of cycloartane-type triterpene isolated from the astragalus memebranaceus, (Fisch) Bunge which has long been used widely in traditional Chinese medicine. This compound has been shown to possess many pharmacological activities, antivirus, antiischemic injury, and antiinflammation. AstragalosideⅣwas first reported to have protective effect against the progression of peripheral neuropathy in STZ-induced diabetic mice. A possible mechanism for this protection is that this compound induced a decrease in blood glucose concentration and an increase in plasma insulin levels. However, the underlying mechanisms responsible for the hypoglycemic activity of astragalosideⅣremain unknown, and there is little evidence on how astragalosideⅣregulated hepatic glucose regulating enzymes.Thus the goal of this study was to investigate the effects of astragalosideⅣon blood glucose, triglyceride (TG), total cholesterol (TC), as well as the insulin levels, and to investigate the possible mechanisms involved in the hypoglycemic effect of astragalosideⅣ, diabetes-related genes was screened by a gene chip. Two genes with close relationship with glucose metabolism, glycogen phosphorylase (GP) and glucose-6-phosphatase (G6Pase) were selected and verified by real time RT-PCR and western blot. Finally, the enzymatic activities of hepatic GP and G6Pase were tested.Methods1. Measurement of astragalosideⅣon blood glucose, body weight, TG, TC and insulin levels of diabetic mice.The C57BL/6J male mice were randomly divided into 2 groups, fed with normal diet (normal control group) or high fat diet (HFD) separately. After exposure to the respective diets for 3 weeks, the HFD groups were injected i.p with a single dose of STZ (100 mg/kg body weight). Blood samples were collected through tail vein and blood glucose levels were measured by glucometer. The mice with blood glucose levels between 10.0mmol/L～20.0mmol/L were used for study. At week 3 after STZ injection, the diabetic mice were divided randomly into 4 groups (n=10 each), orally treated with astragalosideⅣ(12.5,25 and 50 mg.kg-1day-1) or without astragalosideⅣ. The body weights and blood glucose levels of mice were determined weekly. At week 2 after drug administration, the animals were euthanized and blood was collected into EDTA-coated tubes. Plasma TG, TC levels were measured using LabassayTM kits according to the manufacture’s protocols. Plasma insulin was detected by an ELISA kit.2. Analysis of diabetes-related genes by a gene chipTo investigate the possible mechanisms involved in the hypoglycemic activity of astragalosideⅣ, genes were screened by a gene chip analysis. SuperArray RT2 Profiler TM PCR gene chip containing diabetes-related genes was adopted for hybridization. The chip was rinsed and detected by the chemiluminescence method. The result was analyzed by the software of Prism 7900, provided by manufacture.3、Measurement of the activity of G6Pase and GPThe G6Pase activity was determined through the use fo the glucose-6-phosphate dehydrogenase (G6PD)-coupled reaction. Total GP activity, including a and b forms of the enzyme, was also determined by using G6PD-coupled reaction. And determination of the mRNA expression of screened genes by real time RT-PCR, the protein expression of screened genes by Western Blot4. Statistical AnalysisAll data are expressed as mean±SD. Continuous variables between groups at each time point were compared using one-way ANOVA followed by the Student-Newman-Keuls procedure or Dunnett’s T3 procedure when the assumption of equal variances did not hold. Two-tailed P values<0.05 were considered statistically significant. Statistical analyses were conducted with SPSS 13.0.Results 1. Effects of astragaloasieⅣon plasma glucose, body weight, TC, TG and insulin levelsAll control mice gained body weitht throughtout the 2 weeks of study. There was no significant variance in blood glucose in the normal control group. HFD and STZ treatment significantly increased body weight, blood glucose, insulin and TG, but not TC levels compared with the normal control group. AstragalosideⅣat a dose of 12.5mg/kg twice a day for 2weeks had no effect on the above parameters. When the dose of astragalosideⅣwas increased to 25mg/kg and 50mg/kg, a significant decrease in body weight, blood glucose insulin and TG levels was observed, and there were no significant changes in TC levels compared with the diabetic control groups at the end of the experiment.2. Effects of astragalosideⅣon diabetic-related genes in liverAstragaloside IV at the ndose of 25mg/kg and 50mg/kg were down-regulated mRNA expression of 19 genes, and up-regulated mRNA expression of 24 genes in diabetic mice.3. Effects of astragalosideⅣon GP and G6Pase activities.The activities of GP and G6Pase were significantly increased in the HFD-STZ diabetic mice compared with normal control animals (P<0.05). Astragaloside IV with 25mg/kg and 50mg/kg, the activities of both enzymes were significantly decreased in the drug-treated diabetic groups compared with the diabetic control group.4. Effects of astragalosideⅣon mRNA and protein expression of GP and G6Pase.The mRNA levels of GP and G6Pase were increased significantly in the HFD-STZ diabetic control mice compared with the normal control mice. Astragaloside IV at 25mg/kg and 50mg/kg twice a day for 2 weeks significantly reduced the mRNA expression of GP and G6Pase compared with HFD-STZ diabetic control group. The effect of astragalosideⅣon the proteion levels of GP and G6Pase was further examined. The GP and G6Pase were significantly increased in the HFD-STZ diabetic control mice compared with the normal control animals. AstragalosideⅣat the dose of 25mg/kg and 50mg/kg for 2 weeks significantly decreased the up-regulated protein expressions compared to HFD-STZ diabetic control mice (P<0.05).Conclusion1. AstragalosideⅣdecreases the blood glucose, body weight, TC, TG and insulin levels of diabetic mice.2. AstragalosideⅣdown-regulates 19 genes and up-regulates 24 genes at the dose of 25mg/kg and 50mg/kg in diabetic mice.3. AstragalosideⅣdecreases the activity of hepatic GP and G6Pase, as well as the mRNA and protein expression of GP and G6Pase, suggesting that the hypoglycemic activity of astragalosideⅣbe induced by inhibition of glycogenolysis and glucose oxidation.In conclusion, astragalosideⅣexhibited a hypoglycemic effect in HFD-STZ induced diabetic mice. The underlying mechanism responsible for the hypoglycemic effect induced by this xompound may be explained, in part, by its inhibition on hepatic GP and G6Pase activities.更多还原