【作者】 张茜；

【导师】 肖新华；

【作者基本信息】 北京协和医学院 ， 内分泌， 2014， 博士

【摘要】 目的糖尿病严重危害人们的健康及生存质量。天麦消渴片和阿卡波糖在临床上表现出很好的降糖疗效。本研究旨在探讨天麦消渴片和阿卡波糖对糖尿病大鼠体重、血糖、血脂、胰岛素和胰高血糖素样肽-1(GLP-1)等相关代谢指标的影响,并且利用全基因组表达谱芯片、miRNA表达谱芯片和实时定量RT-PCR探讨天麦消渴片和阿卡波糖降血糖的机制。方法(1)SD大鼠通过高脂饮食/注射STZ法构建糖尿病大鼠模型。72小时后,空腹血糖高于11.1mmol/L的SD大鼠判定为糖尿病模型建立成功。(2)将SD大鼠分为小剂量天麦消渴片组(8只,给予50mg/kg/d的天麦消渴片粉末悬浊液)、大剂量天麦消渴片组(8只,给予100mg/kg/d的天麦消渴片粉末悬浊液)、小剂量阿卡波糖组(30mg/kg/d)、大剂量阿卡波糖组(60mg/kg/d)、糖尿病模型组(8只,给予等体积生理盐水)和正常对照组(8只,给予等体积生理盐水),均连续灌胃8周。(3)每2周测定SD大鼠空腹血糖(FBG)和体重。7周末进行口服糖耐量实验(OGTT),测空腹和葡萄糖负荷后血糖和血浆GLP-1水平。8周末测定大鼠空腹血糖、血清胰岛素和血脂水平,观察天麦消渴片和阿卡波糖对糖尿病大鼠血糖和血脂的改善作用。(4)取大鼠骨骼肌组织进行全基因组表达谱芯片实验,取胰腺组织和小肠组织进行miRNA表达谱芯片实验,并运用实时定量RT-PCR验证芯片结果,以期探讨天麦消渴片和阿卡波糖对糖尿病大鼠降血糖的机制。结果(1)血糖结果：干预后,大剂量天麦消渴片组大鼠较糖尿病模型组空腹血糖(15.4±5.3mmol/L vs24.1±2.5Inmol/L第2周,14.5±3.5vs25.3±3.1mmol/L第4周,16.3±4.3vs24.8±4.8mmol/L第6周,15.3±5.3vs23.6±4.3mmol/L第8周)和OGTT曲线下面积(AUC)(35.7±4.6vs53.7±3.4mmol/L,P<0.05)显著下降。干预后,大剂量阿卡波糖组较糖尿病模型组空腹血糖(14.6±4.3mmol/L vs23.8±4.9mmol/L第2周,15.4±2.5vs23.0±4.6mmol/L第4周,14.7±4.6vs24.6±5.4mmol/L第6周,15.3±6.2vs23.7±4.9mmol/L第8周)和OGTT曲线下面积(AUC)(31.7±4.7vs52.6±6.3mmol/L,P<O.05)显著下降。(2)血清胰岛素结果：干预8周后,大剂量天麦消渴片组空腹血清胰岛素(FINS)(15.79±3.75vs31.90±4.68μIUU/mL,P<O.01)和胰岛素抵抗指数(HOMA-IR,15.83±4.87vs33.46±8.30)较糖尿病模型组显著降低(P<0.01)。大剂量天麦消渴片组胰岛β细胞分泌指数(HOMA-β,31.85±3.65vs21.92±2.46)较糖尿病模型组显著升高(P<0.01)。(3)糖负荷后血浆GLP-1结果：干预7周后,大剂量天麦消渴片组糖负荷后15分钟的血浆GLP-1(6.7±0.6vs5.7±0.3pmol/L)较糖尿病模型组显著升高(P<0.05)。(4)血脂结果：干预8周后,大剂量天麦消渴片组血总胆固醇(TC)(1.27±0.01vs1.44±0.01mmol/L)和甘油三酯(TG)(0.49±0.04vs0.83±0.05mmol/L)较糖尿病模型组显著降低(P<0.05)。(5)全基因组表达谱芯片结果：大剂量天麦消渴片组较糖尿病模型组有1752个基因表达上调,471个基因表达下调。基因分类分析(GO)和基因生物学功能通路分析(Kegg)等生物信息学分析显示,大剂量天麦消渴片组骨骼肌差异表达基因主要涉及三个通路：胰岛素信号通路、糖酵解/糖异生通路和三羧酸循环通路。实时定量RT-PCR结果显示,大剂量天麦消渴片组骨骼肌Akt1(胸腺病毒原癌基因1,fold-change=5.83±0.15)和Irs2(胰岛素受体底物2,3.62±0.021)基因较糖尿病模型组显著上调。大剂量天麦消渴片组骨骼肌Fox03(叉头框蛋白3,0.24±0.009)、Pck2(磷酸羧化酶,0.21±0.017)和Ptpnl(又称Ptplb,蛋白酪氨酸磷酸酶1B,0.34±0.013)基因较糖尿病模型组显著下调。(6)miRNA表达谱芯片结果：大剂量天麦消渴片组胰腺较糖尿病模型组有18个miRNA上调,3个miRNA下调。实时定量RT-PCR结果显示证实的这一结果。miRNA靶基因预测和通路分析结果揭示,天麦消渴片能上调胰腺miR-375和miR-30d,从而改善胰腺功能；通过miRNA,抑制胰腺炎症因子表达。大剂量阿卡波糖组小肠较糖尿病模型组有6个miRNA上调,2个miRNA下调。实时定量RT-PCR结果显示证实的这一结果。miRNA靶基因预测和通路分析结果揭示,阿卡波糖能通过激活miR-10a-5p和miR-664改善小肠MAPK通路和抑制炎症因子改善糖尿病大鼠糖代谢。结论(1)天麦消渴片不仅能有效降低糖尿病大鼠FBG,改善胰岛素敏感性,增加糖负荷后血浆GLP-1分泌,还能调节脂代谢。阿卡波糖能改善糖尿病大鼠血糖。(2)天麦消渴片可能是通过改善骨骼肌胰岛素信号通路、降低PCK2、Fox03和PTP-1B水平;上调胰腺miR-375和miR-30d水平,刺激胰岛β细胞增殖,抑制胰岛α细胞增殖,增加胰岛素基因表达；上调胰腺let-7b、let-7e、miR-142-5p和miR-375,抑制细胞因子及受体相互作用通路和MAPK通路的功能,从而改善糖尿病大鼠血糖和胰岛素抵抗状态。(3)阿卡波糖可能通过激活miR-10a-5p和miR-664改善小肠MAPK通路和抑制炎症因子改善糖尿病大鼠糖代谢。更多还原

【Abstract】 OBJECTIVEThe incidence of type2diabetes mellitus is increasing rapidly worldwide, reaching8.3percent in developed countries. It is the fourth prior considerable disease which is the fifth main causes of death after carcinoma, AIDS, cardiovascular disease, imposing a major burden on the health. Chromium is an essential mineral that is thought to be necessary for normal glucose homeostasis. Numerous researches give evidence that acarbose and chromium picolinate which is a formulation designed to improve absorption moderate glucose and insulin resistance. The main ingredient of Tianmai Xiaokepian is chromium picolinate. The research is to explore the effects of Tianmai Xiaokepian and acarbose on the weight, blood glucose, lipid metabolism, serum insulin and glucagon-like peptide-1(GLP-1) in diabetic rats and to investigate its possible glucose-lowing mechanism.METHODSSD rats were randomly divided into four groups:low dosage of Tianmai Xiaokepian group (TML, treated with50mg/kg/d Tianmai Xiaokepian, n=8), high dosage of Tianmai Xiaokepian group (TMH, treated with100mg/kg/d Tianmai Xiaokepian, n=8), low dosage of acarbose group (AcarL, treated with30mg/kg/d acarbose, n=8), high dosage of acarbose group (AcarH, treated with60mg/kg/d, n=8), diabetic model group (n=8) and control group(n=8). The fasting blood glucose and weight were tested on week0,2,4,6and8. The OGTT test was done on week7to measure glucose and plasma GLP-1. Serum insulin, TC, TG, HDL-c and LDL-c were detected on week8. Roche NimbleGen gene array experiment was done using skeletal muscle of rats. miRcury LNATM miRNA array experiment was performed using pancrease and ileum of rats. Real time RT-PCR was done to verify the results of array.RESULTSWe found high dosage of Tianmai Xiaokepian could significantly decrease the level of fasting blood glucose (15.4±5.3vs24.1±2.5mmol/L week2,14.5±3.5vs25.3±3.1mmol/L week4,16.3±4.3vs24.8±4.8mmol/L week6,15.3±5.3vs23.6±4.3mmol/L week8), area under curve of blood glucose in oral glucose tolerance test (AUC,35.7±4.6mmol/L vs53.7±3.4mmol/L), serum fasting insulin (15.79±3.75vs31.90±4.68μIU/mL), HOMA-IR index (15.83±4.87vs33.46±8.30), TC (1.27±0.01vs1.44±0.01mmol/L) and TG (0.49±0.04vs0.83±0.05mmol/L), increase HOMA-β (31.85±3.65vs21.92±2.46) and plasma GLP-1at15min after glucose loading (6.7±0.6vs5.7±0.3pmol/L) compared with those of diabetic group (P<0.05or P<0.01). High dosage of acarbose could significantly decrease the level of fasting blood glucose (14.6±4.3mmol/L vs23.8±4.9mmol/L week2,15.4±2.5vs23.0±4.6mmol/L week4,14.7±4.6vs24.6±5.4mmol/L week6,15.3±6.2vs23.7±4.9mmol/L week8). Gene array showed that Tianmai Xiaokepian up-regulated1752gene expression, down-regulated471gene expression. Based on KEGG pathway analysis, we found that the most three significant pathways were "insulin signaling pathway","glycolysis/gluconeogenesis" and "citrate cycle (TCA)". Real time RT-PCR showed the expression of Akt1(fold-change=5.83±0.15) and Irs2(3.62±0.021) increased in high dosage of Tianmai Xiaokepian group; while Fox03(0.24±0.009)、Pck2(0.21±0.017) and Ptpnl (0.34±0.013) reduced. miRNA array showed18miRNAs increased and3miRNAs decreased in high dasage of Tianmai Xiaokepian group. miR-448, let-7b, miR-540, miR-296, miR-880, miR-200a, miR-500, miR-10b, miR-336, miR-30d, miR-208, let-7e, miR-142-5p, miR-874, miR-375, miR-879, miR-501and miR-188were up-regulated, while miR-301b, miR-134and miR-652were down-regulated in TMH group. Real time RT-PCR showed miR-375and miR-30d, which can stimulate insulin secretion in islet significantly increased. We found that miR-151*, miR-10a-5p, miR-205, miR-17-5p, miR-145and miR-664were up-regulated in the AcarH group, while miR-541and miR-135b were down-regulated. Real time PCR verified these results. Our data suggest that acarbose can improve blood glucose in diabetic rats through the MAPK pathway and can down-regulate pro inflammatory factors by activating miR-10a-5p and miR-664in the ileum.CONCLUSIONTianmai Xiaokepian acts the functions of regulating the levels of fasting blood glucose and lipid metabolism, moderating insulin resistance in diabetic rats. Acarbose can reduce the blood glucose in diabetic rats. The glucose-lowing mechanisms may be correlated with insulin signaling pathway, reducing PCK2, Fox03and PTP-1B in skeletal muscle; incresing miR-375and miR-30d to moderate insulin secretion and islet β cell function, increasing let-7b, let7e, miR-375and miR-142-5p to moderate cytokine-cytokine receptor interaction and MAPK signaling pathway in pancrease. Acarbose can improve blood glucose in diabetic rats through the MAPK pathway and can down-regulate proinflammatory factors by activating miR-10a-5p and miR-664in the ileum.更多还原