【作者】 赵蕊；

【导师】 李青旺；

【作者基本信息】 燕山大学 ， 应用化学， 2007， 博士

【摘要】 世界各国糖尿病的发病率均在上升,WHO预计到2010年全世界糖尿病的人数将达到2.21亿,其中90%-95%是非胰岛素依赖型糖尿病(NIDDM)。糖尿病肾病(DN)是NIDDM的一种常见而严重的并发症,也是糖尿病患者致死的重要原因。天然抗糖尿病药物以其毒副作用小和不产生耐药性已引起了人们的高度关注,特别是在发展中国家得到了广泛的发展和利用。枸杞多糖(LBP)是从传统的中草药枸杞子中提取出的活性成分,已经发现LBP具有抗癌、抗氧化和降糖的生物活性。但是,关于其治疗NIDDM胰岛素抵抗(IR)和DN作用机理的研究很少有人报道。本研究采用水提醇沉法从宁夏枸杞中提取出LBP,并对其进行分离、纯化得主要活性成分LBP-4a。采用高糖高脂饮食结合小剂量腹腔注射链脲佐菌素(STZ)建立NIDDM大鼠模型,并将其分为模型对照组、LBP-4a低、高剂量组和罗格列酮治疗组,同时设立正常对照组。应用单细胞凝胶电泳、免疫组化、反转录PCR、酶联免疫吸附试验、免疫沉淀及Western blot等先进技术,从细胞因子、信号转导等方面对LBP-4a降糖、改善IR及治疗DN的作用机理进行了较为深入系统的研究,获得如下研究结果。(1)宁夏枸杞经烘干、脱脂、脱色、脱小分子糖、乙醇沉淀、有机溶剂洗涤、聚酰胺色谱柱除蛋白、真空干燥后,得粉末状枸杞粗多糖LBP(2.58士0.14(g/100 g))。用DEAE-Cellulose柱层析,并经NaCl梯度洗脱、透析、冻干等步骤,获得4个枸杞多糖级分LBP-1、LBP-2、LBP-3、LBP-4。采用Sephadex G-200柱层析法对级分LBP-4活性成分进一步分离,获得相应的枸杞多糖亚级分LBP-4a。SDS-PAGE银染显示LBP-4a为单一的一条带,冻干品为白色或略显黄色的絮状固体。LBP-4a的得率为1.07%。(2)LBP-4a处理4周(w),高剂量组可以明显减轻模型大鼠的体重(P<0.01),并显著降低内脏脂肪含量(P<0.05)和空腹血浆胰岛素水平(P<0.01)。LBP-4a不仅对NIDDM大鼠具有降低血糖、血脂和增加抗氧化性的功能,还具有修复DNA损伤的作用。(3)病理形态学结果显示,LBP-4a对NIDDM大鼠胰腺、肝脏和肾脏有一定的保护作用。光镜下,LBP-4a能使胰岛数量及胰岛内细胞数量增多。电镜下,LBP-4a高剂量组肝细胞核膜完整、清楚,常、异染色质和糖原颗粒分布均匀,粗面内质网结构完整;线粒体肿胀、空泡化和脂肪滴沉积现象减轻。LBP-4a处理组肾小球滤过膜结构基本完整,足突增粗,但排列整齐;基底膜增厚和系膜细胞增生现象明显改善。(4)对LBP-4a改善NIDDM大鼠IR的机制研究表明,LBP-4a能使NIDDM大鼠骨骼肌细胞的GLUT4进行转位,且这种作用与增加PKB蛋白磷酸化表达有关。(5)应用Western blot和免疫沉淀方法检测了LBP-4a对NIDDM大鼠骨骼肌p38MAPK蛋白表达的影响。结果表明,LBP-4a能通过增加NIDDM大鼠骨骼肌p38MAPK的活性,从而影响胰岛素的信号转导通路。(6)对NIDDM大鼠肾功能及肾脏抗氧化活性进行分析,揭示LBP-4a能改善NIDDM大鼠的肾损伤,并能提高NIDDM大鼠肾脏抗氧化酶的活性。(7)对改善DN功能机制的研究表明,LBP-4a能使NIDDM大鼠肾组织细胞膜PKC活性显著降低(P<0.01),PPAR-γ蛋白含量和PPAR-γmRNA表达显著增加(P<0.01)。免疫沉淀结果显示,LBP-4a对高糖引起的肾组织p38MAPK活性增加有明显的对抗作用。本文较系统地研究了枸杞多糖LBP-4a亚级分对NIDDM大鼠IR和DN的治疗作用,并多角度地探讨了其作用机制,为进一步深入开展枸杞作为天然抗糖尿病药物的研究提供了科学的理论依据。更多还原

【Abstract】 The incidence of diabetes mellitus is rapidly increasing in all parts of the world. The World Health Organization has estimated that diabetes mellitus will affect 221 million people worldwide by 2010, and most cases (90-95%) are non-insulin dependent diabetes mellitus (NIDDM). Diabetic nephropathy (DN) is a common but serious complication of NIDDM, and it is a main cause of death in diabetic patients. Recently, the increasing interest has been attracted for development and utilization of antidiabetic plants due to their less side effects and no-drug-resistance, especially in developing countries. Lycium barbarum polysaccharide (LBP), extracted from the traditional Chinese herb Lycium barbarum, is found to have bioactivities such as anticancer, antioxidant and hypoglycemic activities. However, very little is reported about the effect and mechanism of LBP in treating insulin resistance (IR) and DN in NIDDMLBP was extracted from Ningxia-Lycium barbarum by water extraction and ethanol precipitation technique. And the ingredient of LBP-4a was obtained by separation and purification. The animal model of NIDDM was made by feeding high-glucose and high-fat diet and subjecting to.i.p.little-dose streptozotocin (STZ). The rats of NIDDM model were divided into four groups, including model group, LBP-4a low-dose and high-dose groups, rosiglitazone group and normal control group. In order to research the effect of LBP-4a on reducing blood glucose, ameliorating IR and treating DN in NIDDM rats, a lot of techniques were used such as Single Cell Gel Electrophoresis, immunohistochemical staining, ELISA, immunoprecipitation and Western blot et al. We studyed systematically the effect of LBP-4a on IR and DN in NIDDM rats and researched the mechanism from the point of view of cytokine and signal transduction. The main results were as follows.(1)LBP was extracted by the following procedure: baking, defatting, discoloration, removing small molecular sugar, deposition by ethanol, cleaning by organic solvent, removing protein by Sephadex G-200 column and vacuum drying, and so on. LBP was powder and the extraction rate was 2.58±0.14(g/100 g). Four LBP fractions, LBP-1, LBP-2, LBP-3 and LBP-4, were got by DEAE-Cellulose column with eluent of NaCl、 dialysis and freeze drying respectively. One sub-fraction LBP-4a was got from the corresponding active LBP-4 fractions by Sephadex G-200 column with water as eluent. LBP-4a was identified to be homogeneous by SDS-PAGE, which showed a single band after staining with Ag. LBP-4a was a white or buff cotton-like solid. The extraction rate of LBP-4a was 1.07%.(2)After LBP-4a (high-dose) treatment for 4 weeks, body weight and the content of visceral fat mass were evidently decreased (P<0.01, P<0.05) and the level of fasting blood insulin was also significantly decreased (P<0.01). LBP-4a had glucose-lowering and lipid-lowering effects on NIDDM rats. Furthermore, LBP-4a had antioxidative function and repairing DNA damage on NIDDM rats.(3)The morphologic changes suggested that LBP-4a had protective function on pancreas, liver and kidney in NIDDM rats. It showed that the amount of pancreatic islets and cells was increased by light microscope. An electron micrograph of hepatocyte in high-dose LBP-4a treatment rats showed the nuclear membrane was distinct; the disposition of euchromatin, heterochromatin and glycogen particles is uniform. The rough surfaced endoplasmic reticulum appeared normal. The structural changes in mitochondria with severe swelling, extensive vacuolization and lipid droplets were ameliorated associated with LBP-4a. Ultrastructure of glomerulus was observed by electron microscope. The result displayed that the structure of filter membrane was intact and the foot process was thickening, but their arrangement was regularity after LBP-4a treatment Furthermore, LBP-4a could improve the phenomena of hyperplasia of intercapillary cells and thickening of glomerular basement membrane.(4)In the present study, the effective mechanism of LBP-4a on insulin resistance was investigated in NIDDM rats. It showed that LBP-4a could induce GLUT4 translocation in skeletal muscle of NIDDM rats, and the effect was related to the increase of hyper-phosphorylated PKB.(5)By Western blot and immunoprecipitation, the expression and activity of p38MAPK were detected in the skeletal muscle of NIDDM rats. It indicated that administration with LBP-4a could enhance the activity of p38MAPK in skeletal muscle of NIDDM rats, and so, LBP-4a affected the insulin signal transduction passageway. (6)The effect of LBP-4a on renal function and antioxidative activity of kidney was examined in NIDDM rats. LBP-4a significantly ameliorated diabetic renal damage and enhanced the activities of antioxidant enzymes.(7)The effective mechanism of LBP-4a on DN was investigated. The results indicated that LBP-4a treatment for 4 weeks, the membrane PKC activity was significantly decreased (P<0.01). The expression of PPAR-γmRNA and the content of PPAR-γwere significantly increased in NIDDM rats kidney treated with LBP-4a (P<0.01). By immunoprecipitation, it showed that LBP-4a could confront the increased activity of p38MAPK induced by hyperglycemia in renal tissue.In this study, the effects of sub-fraction LBP-4a on IR and DN were investigated systematically in NIDDM rats and the action mechanism was discussed from much points of view. It will provide a comprehensive and scientific evidence for development of Lycium barbarum as a suitable natural antidiabetic agent.更多还原