【作者】 亓竹青；

【导师】 胡弼；

【作者基本信息】 南华大学 ， 药理学， 2010， 硕士

【摘要】 【研究背景与目的】糖尿病是威胁人类健康的重要疾病之一,全球的发病率呈上升趋势。2型糖尿病的病因及病理机制极为复杂,目前尚未完全清楚,但其发病的中心环节是胰岛素抵抗和胰岛素的分泌缺陷,胰岛素分泌缺陷在从NGT到IGT和IGT到糖尿病的过程,起了决定性的作用。长期高血糖、高血脂是胰岛素分泌缺陷发生的始动因素。因而如何保护胰腺,保护β细胞,阻止其功能衰退,增加胰岛素的分泌是2型糖尿病治疗中的关键问题。槟榔碱是从天然植物槟榔中的提取的生物碱。研究发现槟榔碱体外给药能抑制口腔角质细胞内白介素和肿瘤坏死因子的表达,下调氧化低密度脂蛋白和高糖致血管内皮细胞或巨噬细胞中炎症因子的表达,具有抗动脉粥样硬化的作用。我们前期的研究工作已经证明,槟榔碱具有降血糖的作用,由此推测槟榔碱可能通过某种机制保护胰腺,保护β细胞,促进胰岛素分泌,降低血糖浓度。本研究在高果糖诱发大鼠2型糖尿病模型上,给予不同浓度槟榔碱腹腔注射,寻找槟榔碱在大鼠整体水平有效的降糖剂量,通过检测血生化指标,观察胰腺形态特征,检测胰腺中重要的2型糖尿病相关因子的表达,探讨槟榔碱对2型糖尿病大鼠胰腺细胞的保护作用及其机制。【方法】1.建立高果糖诱导的2型糖尿病大鼠模型2.采用HI-TACH717全自动生化分析仪测定空腹血糖、血脂水平,放射免疫法测定空腹血清胰岛素及胰高血糖素含量。3.用HE染色检测不同浓度槟榔碱对糖尿病大鼠胰腺细胞形态的影响。4.TUNEL法检测不同浓度槟榔碱对糖尿病大鼠胰腺细胞凋亡的影响。5.逆转录多聚酶链反应( reverse transcription polymerase chain reaction, RT-PCR )检测槟榔碱对胰腺细胞内Bcl2,Bax,PDX-1,insulin,GLUT2,GK,IRS1,IRS2 mRNA表达的影响。6.Western blot检测不同浓度槟榔碱对胰腺细胞内FoxO1,AKT, p-AKT蛋白的影响。【结果】1.槟榔碱对高果糖诱导的2型糖尿病大鼠行为,体重,血生化及胰腺毒性的影响1)糖尿病模型大鼠分别以0.5, 1, 5, 10, 20, 50mg/kg槟榔碱腹腔注射4周,除0.5mg/kg组外,各处理组大鼠血糖、血脂、体重、胰岛素水平均明显下降,统计学差异具有显著性(p<0.01)。2) HE染色法检测胰岛形态学变化由胰腺HE染色图片可以观察到,糖尿病模型组及0.5mg/kg槟榔碱处理组大鼠胰岛边缘β细胞出现聚集,细胞核形状发生变化; 5,10mg/kg槟榔碱处理组,胰岛形态相对规则,边缘平整,β细胞分布均匀; 20, 50mg/kg槟榔碱处理组,胰岛界限模糊。3) TUNEL法检测胰腺细胞凋亡观察结果显示模型组及0.5mg/kg槟榔碱处理组凋亡细胞数量较对照组显著增加, 1,5mg/kg槟榔碱处理组胰腺细胞凋亡率下降, 10,20,50mg/kg槟榔碱处理组细胞凋亡率明显升高。以上结果显示, 1,5mg/kg槟榔碱为本实验有效降糖剂量。2.槟榔碱对2型糖尿病大鼠胰腺细胞凋亡相关因子Bcl2/Bax的影响1)与对照组相比,糖尿病模型组大鼠胰腺细胞内Bcl2/Bax比值降低。2) 1,5,10mg/kg槟榔碱处理对糖尿病模型组大鼠胰腺细胞内Bcl2/Bax比值的降低有逆转作用。3) 1,5mg/kg槟榔碱不影响正常大鼠Bcl2/Bax比值。3.槟榔碱对2型糖尿病大鼠胰腺胰岛素分泌及糖代谢相关因子基因表达的影响1)糖尿病模型组大鼠胰腺细胞内PDX-1,insulin,GLUT2,GK基因表达较对照组显著降低。2) 1,5mg/kg槟榔碱可以显著逆转PDX-1,insulin基因表达的降低, 5mg/kg槟榔碱处理明显逆转高糖引起的GLUT2,GK基因表达的降低。3) 1,5mg/kg槟榔碱不影响正常大鼠PDX-1,insulin, GLUT2,GK基因表达。?4.槟榔碱对2型糖尿病大鼠胰腺胰岛素信号通路的影响。1)与正常组相比,糖尿病模型组大鼠胰腺细胞中IRS1、IRS2 mRNA及p-AKT蛋白表达显著降低, FoxO1蛋白表达显著提高。2) 1,5mg/kg槟榔碱处理明显增强糖尿病模型组大鼠胰腺IRS1、IRS2 mRNA及AKT蛋白表达,降低FoxO1的表达。3) 1,5mg/kg槟榔碱处理不引起正常大鼠胰腺IRS1、IRS2 mRNA及AKT、FoxO1蛋白表达变化。【结论】1. 1,5mg/kg槟榔碱能以剂量依赖性方式降低2型糖尿病大鼠血糖、血脂,胰岛素, 0.5mg/kg槟榔碱为无效剂量; 10,20,50mg/kg槟榔碱具有毒性作用。2.槟榔碱降糖的机制可能是通过胰腺胰岛素信号转导通路中胰岛素受体底物,经PI3K/AKT通路,上调抑凋亡基因表达,保护胰腺,保护β细胞,抑制FoxO1的表达,上调胰岛素分泌及糖代谢相关的Insulin,GLUT2,GK基因的转录,促进胰岛素分泌,增强糖代谢。更多还原

【Abstract】 [BACKGROUD AND OBJECTIVE]Although the development of type 2 diabetes is more complex and incompletely clear in etiology,it is usually associated with a combination of pancreaticβ-cell dysfunction and insulin resistance.Moreover,β-cell dysfunction has played a decisive role in the progress that diabetes come into being. Chronically elevated levels of glucose and lipids are known as the initiated factors and also the hallmark of diabetes.Arecoline is extracted from natural plant alkaloid. In vitro study, found that oral administration of arecoline in keratinocyte cells inhibits interleukin, and tumor necrosis factor expression, reduced ox- LDL and high glucose in vascular endothelial cells and macrophages in the expression of inflammatory factors, with anti-atherosclerosis The role of atherosclerosis;. Speculated that arecoline can protectβcells, thus improving diabetes.With regards to this, we will investigate the effect of different concentrations of arecoline on the levels of fasting blood glucose and the expression of insulin secretion factors, transcription factors, and key factors of insulin signal pathway in type 2 diabetes rats induced by high-glucose diet, and explore the underlying mechanisms. [METHODS]1) A type 2 diabetic rat model was established by fed with high fructose diet;2) The blood glucose, lipid level ,fasting plasma glucagon and fasting insulin were measured;3) Pathogenic changes of rats pancreatic tissue by Hematoxylin-eosin staining ;4) Apoptosis of rats pancreatic tissue by tunel staining ;5) The mRNA expression of pancreatic Bcl2 , Bax, PDX-1 , Insulin, GLUT2 , GK , IRS-1 , IRS-2 were detected by Reverse transcription polymerase chain reaction (RT-PCR);6) The protein expression of p-AKT and FoxO1 was detected by western blotting.[RESULTS]1. arecoline can safely decrease the level of fasting blood glucose and TG in type 2 diabetes rats.1) Type 2 diabetes rats were treated with 0.5, 1, 5, 10, 20, 50 mg/kg arecoline with abdominal injection for 4 weeks. The level of body weight, fasting blood glucose, lipid level, fasting plasma glucagon and fasting insulin were decreased, but not 0.5 mg/kg arecoline (p<0.05) .2) HE staining of pancreatic shows that 1, 5 mg/kg arecoline can improve pancreatic injury in type 2 diabetes rats induced by high-glucose diet, such as decreased the number of impairment cells. But 10, 20, 50 mg/kg arecoline have toxicants in pancreatic. 3) The apoptotic index measured by TUNEL increased gradually with adding the concentrations of 10, 20, 50 mg/kg arecoline.2. arecoline can augmentation the rate of Bcl2/Bax in type 2 diabetes rats.1) The results showed that the mRNA levels of Bcl2 reduced in type 2 diabetes rat .2) 1, 5 mg/kg arecoline can increased the mRNA levels of Bcl2 in type 2 diabetes rat paccreatic.3) Control+arecoline groups was not significantly different compared to control group.3. arecoline can increase the mRNA PDX-1、Insulin、GLUT2 and GK level of in type 2 diabetes rats.1) The results showed that the mRNA levels of PDX-1、Insulin、GLUT2 and GK were reduced in type 2 diabetes rat .2) 1, 5 mg/kg arecoline can increased the mRNA levels of PDX-1、Insulin、GLUT2 and GK in type 2 diabetes rat pancreatic.3) Control+arecoline groups was not significantly different compared to control group.4. arecoline can improve the insulin sensitivity of pancreatic in type 2 diabetes rats .1) IRS-2, IRS-2 mRNA and the p-AKT protein were down-regulated in type 2 diabetes rats liver. 2) 1, 5 mg/kg arecoline can up-regulated IRS-2,IRS-2 mRNA and the p-AKT protein in type 2 diabetes rats pancreatic.3) Control+arecoline groups was not significantly different compared to control group.[CONCLUSION]1. can be with adding the concentrations arecoline (1, 5 mg/kg) in type 2 diabetes rats2. The mechanism of improve about glucose metabolism may be through the insulin signal pathway in pancreas ,up-regulate the insulin receptor substrate, by the PI3K/AKT pathway, to influence antiapoptotic gene expression, protectβcells, or inhibit the expression of FoxO1, increases insulin secretion and glucose metabolism gene transcription, promote insulin secretion, enhance glucose metabolism更多还原