【作者】 高大威；

【导师】 李青旺；

【作者基本信息】 燕山大学 ， 应用化学， 2006， 博士

【摘要】 糖尿病是世界范围内最常见的内分泌性疾病之一,其死亡率在发达国家仅次于心血管疾病、恶性肿瘤,成为第三位致死病因疾病。WHO调查材料显示,目前全世界至少有1.71亿糖尿病患者,预测到2030年将增至3.66亿,其发病率也将由目前的2.8%上升为4.4%。尽管有大量的降血糖药物在临床应用,但由于其副作用大、使用不便,疗效不稳定,目前仍未发现一种有效的治疗药物。胰岛素虽然是人类发现的有效降糖药物,但它只能缓解患者血糖的升高,并不能达到完全治愈的目的。天然抗糖尿病药物以其毒副作用小和不易产生耐药性普遍受到医学界的关注,西方发达国家正在加快天然药物开发,不断有源于天然产物的新药问世。女贞子是我国传统的中药,通常用来治疗癌症、肝炎等疾病。在降血糖研究中,尽管也有少量报道,但仍然缺乏较为系统的深入研究。有关女贞子治疗糖尿病的机理方面目前国际上仍未见相关的报道。本研究采用多重结晶技术从植物女贞子中提取出活性物质——齐墩果酸(OA),通过分离、纯化,薄层色谱和质谱鉴定,进一步将其磨成粒径为200-700nm的粉末。以腹腔注射链尿佐菌素(STZ)和四氧嘧啶(Alloxan)构建大鼠和小鼠糖尿病模型,将其分为糖尿病对照组、齐墩果酸低剂量、高剂量治疗组和正常对照组。采用免疫组化、单细胞电泳、酶联免疫吸附、免疫印迹和RT-PCR等技术研究了齐墩果酸对糖类和脂类代谢相关基因PPARα、PPARγ、PKB和PKC的表达变化,以及对糖尿病鼠β细胞凋亡、抗氧化能力和糖代谢相关激素水平的影响,并获得如下研究结果。糖尿病大鼠经OA处理40 d后,低、高剂量组血糖分别下降了32.4%和46.4%;高剂量治疗组TG、TC、LDL-c水平明显下降,HDL-c水平则升高(P <0.05, 0.01),表明其具有显著的降脂功能。治疗组大鼠血清ALP、AST和ALT水平均接近于正常,揭示OA对肝脏具有明显的保护作用。此外,OA也升高了SOD和GSH-px水平(P <0.05, 0.01),增强了机体的抗氧化能力。采用HE染色、急毒性试验和单细胞电泳技术(SCGE)对OA的毒副作用进行检测,结果表明,OA能使胰岛数量及胰岛内细胞数量增加;同时它能够修复STZ诱导的糖尿病鼠的肝和肾的损伤。在急毒性试验中,小鼠灌服OA 1000 mg·kg-1剂量时,未见出现死亡。SCGE结果揭示,OA对淋巴细胞的DNA没有损伤。通过对糖尿病大鼠胰岛素和甲状腺素水平分析表明,OA能够明显增加糖尿病大鼠的胰岛素分泌(P <0.05),但对血清T3、T4和TSH水平没有明显影响。此外,OA对糖尿病大鼠的肝脏、肺、胰腺和肾脏等器官的重量没有明显影响,但能够显著增加胸腺的重量(P <0.05),这可能是OA通过作用于胸腺而增强机体的免疫功能。通过对细胞凋亡影响的研究表明,OA处理后,小鼠胰腺、肾组织的Bax mRNA表达均减少,而Bcl-2表达增加,Bcl-2/Bax比值接近正常对照组,与糖尿病组存在明显差异(P <0.05, 0.01)。说明OA对胰腺和肾脏细胞的凋亡可能具有明显的抑制作用。免疫组化分析表明,OA也改善了肝脏中Bax和Bcl-2蛋白的表达,恢复肝细胞的正常的糖、脂代谢功能,避免糖尿病并发症的发生。通过对细胞因子的表达进行分析,结果表明经OA治疗8 w后,糖尿病鼠的PPARα表达上调,但与对照组没有明显的不同(P >0.05);OA明显刺激了胰腺、肾脏PPARγmRNA的表达水平(P <0.05)。通过Westhern Blot分析表明,OA治疗的糖尿病鼠肾组织中PKB蛋白含量明显增加(P <0.05)。这些结果揭示OA降血糖和血脂作用可能是通过PKB/PPAR途径完成,即OA刺激胰岛素分泌,修复肝、肾和胰腺的损伤,而发挥作用。此外,OA对肝、肾组织PKC的表达未发生显著影响(P >0.05)。本文较系统地研究了女贞子提取物——齐墩果酸对糖尿病鼠的治疗作用,并从糖类和脂类相关激素和基因水平初步探讨了其作用机制。该研究为OA降糖作用与机理的进一步深入研究提供了科学依据和手段。更多还原

【Abstract】 Diabetes mellitus (DM) is one of the most common endocrine diseases. The mortality is only inferior to cardiovascular disease and malignancy in the developed country, and becoming the third fatal pathogeny. The World Health Organization has estimated that the total number of diabetic patients is not less than 171 million presently, and expected that it will rise to 366 million in 2030. The incidence of diabetes will be increased to 4.4% in 2030 from 2.8% presently. In despite of the application of plentiful hypoglycemic agents in clinic, there is not effective drug for diabetes due to the severe toxicity and side effects, inconvenience and instable efficacy. Insulin is an effective hypoglycemic drug, but it only alleviates hyperglycemia of diabetic patient. And diabetes can not be cured completely by insulin. Natural anti-diabetic pharmaceutical are attracting attention in the medicinal field by the less toxicity and no-drug-resistance. The natural drugs are exploited rapidly in the developed country. Some new agents that were rooted in natural products frequently appeared. Ligustrum lucidum Ait has been used in traditional Chinese medicine due to its antitumor and hepatoprotective properties. There are a few reports on hypoglycemia of Ligustrum lucidum Ait. However, the treated mechanisms of Ligustrum lucidum Ait in diabetes are not reported yet. In order to develop new anti-diabetic pharmaceutical, the systematic and deep research on hypoglycemia of Ligustrum lucidum Ait is highly desired.In this paper, the principal active compound-oleanolic acid (OA) was extracted from Ligustrum lucidum Ait by multi-crystal method. The extract was isolated, purified and analyzed by thin layer chromatography (TLC) and mass spectra (MS) technology. OA was ground to powder with 200-700 nm diameters. Diabetic rats and mice were induced with STZ or alloxan intraperitoneally. Diabetic animals were randomly divided into normal control group (NC), diabetic control group (DM), DM+OA low dose group (DM+OA LD) and DM+OA high dose group (DM+OA HD). The genes of PPARα, PPARγ, PKB and PKC were relative with metabolizing of saccharide and lipid. The expression of PPARα, PPARγ, PKB and PKC in the OA-treated animals was analyzed by immunohistochemical stain, single cell gel electrophoresis, ELISA, Westhern Blot and RT-PCR and so on. Meanwhile, the effect of OA was studied systematically on cell apoptosis, antioxidant ability and hormone level which are related with saccharide metabolizing. The results were listed as follows.When diabetic rats were treated with OA for 40 days, the decreasing rates of the plasma glucose levels were 32.4% (in DM+OA LD group) and 46.4% (in DM+OA HD group), respectively. The levels of TG, TC, LDL-c in DM+OA HD group were significantly lower, while their level of HDL-c increased significantly (P <0.05, 0.01), which implied that OA has the function of hypolipidemia. The levels of serum ALP, AST and ALT of OA-treated rats approached to normal value. The results indicated that OA could protect the liver of the diabetic rats. Furthermore, OA also strengthened the antioxidant ability by increasing the activities of SOD and GSH-px.The toxicity and side effect of OA was examined by HE stain, acute toxic test and single cell gel electrophoresis. Results showed that OA could increase the quantity of the pancreatic islets and the cells in pancreatic islet; OA has the ability to amend the impairment on liver and kidney of STZ-induced diabetic rats. In acute toxic test, the mice were fed OA 1000 mg/kg, as a result, there is not death of mouse. Moreover, the result of single cell gel electrophoresis indicated that OA did not destroy DNA of the lymphocytes basically.The levels of insulin and thyroid hormones of the diabetic rats were analyzed, and the results indicated that OA could increase significantly the insulin secretion of diabetic rats (P <0.05), but the changes of serum T3, T4 and TSH levels were insignificant. Furthermore, OA did not affect significantly the weight of liver, lung, kidney and pancreas of the diabetic rats (P >0.05), but the weight of the thymus was significantly higher (P < 0.05). It is tempting to speculate that OA might stimulate the growth of thymus, consequently increasing the immune function of animals.Effect of OA on apoptosis was studied; the results showed that expression of Bax mRNA in pancreas and kidney tissues was reduced after OA-treated, while expression of Bcl-2 was enhanced. The ratio of Bcl-2/Bax approached to normal level, and there was significant difference with diabetes control mice (P <0.05, 0.01). The research indicated that OA might restrain the apoptosis of pancreas and kidney. By immunohistochemical analysis, the results implied that OA also ameliorated expression of Bax and Bcl-2 protein in liver, and restore the function of saccharide and lipid metabolizing, consequently, which avoid diabetic complication.The mechanisms of OA on anti-diabetes were investigated by determining expression of some cytokines. The results indicated that PPARαlevel was up-regulated in pancreas and kidney of OA-treated diabetic mice after OA treatment for 8 weeks, but the difference was insignificant with control mice (P >0.05). The expression of PPARγmRNA was significantly up-regulated in OA-treated diabetic mice kidney and pancreas tissue (P >0.05). Westhern Blot determining indicated that the expression of PKB protein was increased significantly in OA-treated diabetic mice kidney (P <0.05). The present study provided further evidence in support of OA on stimulating secretion of insulin, amending the impairment on liver, kidney and pancreas of STZ-induced diabetic rats, hypoglycemic and hypolipidemic activity. The study also found that OA did not affect the expression of PKC in diabetic mice liver and kidney (P >0.05).In the study, the effects of extract of Ligustrum lucidum Ait-oleanolic acid on diabetic rats were investigated systematically, and the action mechanisms were discussed on hormone, protein and molecule levels. It will provide a scientific proof for development of Ligustrum lucidum Ait as a suitable natural anti-diabetic agent.更多还原