中药苦瓜、地骨皮、翻白草提取物干预2型糖尿病胰岛素抵抗的研究

【作者】 严哲琳；

【导师】 刘铜华；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的观察中药苦瓜、地骨皮、翻白草提取物对2型糖尿病胰岛素抵抗动物模型糖脂代谢的影响,并研究肝脏和骨骼肌组织中胰岛素PI3K/Akt信号通路和AMPK信号通路的相关蛋白表达,探讨三种中药提取物干预2型糖尿病胰岛素抵抗的分子机制。方法1.采用雄性3周龄Wistar大鼠80只,按照随机数字表法随机选取10只作为对照组,予标准饲料喂养,其余70只高脂喂养16周后,腹腔注射小剂量STZ (35mg·kg-1·bw-1)诱发2型糖尿病胰岛素抵抗Wistar大鼠模型,将造模成功的大鼠(共63只),随机选出56只分为8组,即苦瓜提取物高、低剂量组、翻白草翻白草提取物高、低剂量组、地骨皮提取物高、低剂量组、西药对照组,每组各7只,余下造模成功大鼠7只设为模型组。另取从正常饮食组随机取7只作为对照。以噻唑烷二酮类胰岛素增敏剂吡格列酮(4.05mg·kg-1·bw-1)作为西药对照、苦瓜、翻白草、地骨皮提取物高剂量组(400mg·kg-1·bw-1)和低剂量组(200mg·kg-1·bw-1),灌胃给药,正常组及模型组按10ml·kg-1·bw-1灌服无菌水。药物干预4周后,进行高胰岛素—正糖钳夹实验(Hyperinsulinemic-Euglycemic Clamp)评价三种中药对IR的干预效果,检测各组动物空腹血糖(Fasting Blood Glucose, FBG)、口服糖耐量(Oral Glucose Tolerance Test, OGTT)、空腹血清胰岛素(Fasting Serum Insulin, FINS)及胰岛素敏感指数(Insulin Sensitive Index, ISI)、血清甘油三酯(TG)、血清总胆固醇(TC)、血清游离脂肪酸(FFA)、血清低密度脂蛋白(LDL-C)和血清高密度脂蛋白(HDL-C);采用Western Blot检测各组大鼠肝脏组织中AMPK、Akt蛋白及其磷酸化水平；采用RT-PCR检测肝脏组织AMPKα1-mRNA、AMPKα2-mRNA基因转录水平。2.采用雄性6周龄BKS.Cg-m +/+ Leprdb/J (db/db)小鼠48只,按照随机数字表法分组,分为苦瓜、翻白草、地骨皮提取物高、低剂量组、西药对照组、每组6只,模型组6只。C57BL/KsJ(+/-)小鼠6只为正常对照组。以噻唑烷二酮类胰岛素增敏剂吡格列酮(1.2g.kg-1.bw-1)作为西药对照、苦瓜、翻白草、地骨皮提取物高剂量组(4g.kg-1.bw-1)和低剂量组(2g.kg-1.bw-1),灌胃给药,正常组及模型组按1ml·kg-1·bw-1灌服无菌水。药物干预4周后,检测各组动物空腹血糖(FBG)、口服糖耐量(OGTT)、空腹血清胰岛素(FINS)及胰岛素敏感指数(ISI)、血清甘油三酯(TG)、血清总胆固醇(TC)、血清游离脂肪酸(FFA)、血清低密度脂蛋白(LDL-C)和血清高密度脂蛋白(HDL-C);采用Western Blot检测各组小鼠骨骼肌组织中AMPK、Akt、AS160蛋白及其磷酸化水平；采用RT-PCR检测骨骼肌组织中GLUT4-mRNA基因转录水平。结果1苦瓜、地骨皮、翻白草提取物对2型糖尿病IR大鼠的干预作用1.1大鼠糖钳实验的结果显示：治疗4周后,与模型组相比,吡格列酮组、苦瓜、翻白草、地骨皮提取物高剂量组的葡萄糖输注率明显升高(P<0.05),低剂量组有升高趋势,说明各组大鼠治疗后胰岛素敏感性增加。1.2大鼠血生化指标显示：与模型组比较,中药苦瓜、翻白草、地骨皮提取物干预4周后,显著改善大鼠OGTT、降低FBG、升高FINS,显著降低TC、TG、LDL-C并升高HDL-C水平(P<0.05)。1.3大鼠肝脏HE染色结果显示：与正常组相比,模型组可见轻度的肝细胞脂肪变性,光镜下脂肪变性的肝细胞沿中央静脉呈向心性分布,胞浆内周边可见许多小的脂滴空泡,细胞核位置无明显改变,肝小叶结构完整。苦瓜、地骨皮、翻白草提取物干预4周后肝细胞脂肪变形明显减轻,个别可见到局灶性的肝细胞胞浆周边细小的脂滴空泡,细胞核位置无明显改变,肝小叶结构完整。1.4大鼠肝脏的Western Blotting结果显示：与模型组相比,中药苦瓜、地骨皮、翻白草提取物高、低剂量组都能上调大鼠的肝脏组织中P-AMPK、P-Akt (Ser 473)、P-AS160蛋白的表达(P<0.05),三种中药的上述功效与噻唑烷二酮类胰岛素增敏剂吡格列酮组相比,无显著差异。1.5大鼠肝脏RT-PCR结果显示：与模型组相比,中药苦瓜、地骨皮、翻白草提取物高、低剂量组都能上调肝脏组织中AMPKα1-mRNA和AMPKα2-mRNA的表达含量(P<0.05)。2苦瓜、地骨皮、翻白草提取物对BKS.Cg-m +/+ Leprdb/J (db/db)小鼠的干预作用2.1小鼠血生化指标显示：与模型组比较,中药苦瓜、翻白草、地骨皮提取物干预4周后,都能显著改善大鼠OGTT、降低FBG、升高FINS,显著降低TC、TG、LDL-C并升高HDL-C水平(P<0.05)。2.2小鼠骨骼肌的Western Blotting结果显示：与模型组相比,中药苦瓜、地骨皮、翻白草提取物高、低剂量组都能上调小鼠骨骼肌组织中P-AMPK、P-Akt (Ser 473)、P-AS160蛋白的表达(P<0.05),三种中药的上述功效与噻唑烷二酮类胰岛素增敏剂吡格列酮组相比,无显著差异。2.3小鼠骨骼肌RT-PCR结果显示：与模型组相比,中药苦瓜、地骨皮、翻白草提取物高、低剂量组都能上调骨骼肌组织中GLUT 4-mRNA的表达含量(P<0.05)。结论中药苦瓜、地骨皮、翻白草提取物可以改善长期高脂喂养加小剂量STz诱导的IR大鼠模型以及IR小鼠模型BKS.Cg-m +/+ Leprdb/J(db/db)小鼠的胰岛素抵抗。苦瓜、地骨皮、翻白草提取物改善IR的机制是激活AMPK,活化的AMPK促进GLUT4转位,加快葡萄糖利用。同时,三味中药激活PI3k/Akt途径,活化的Akt进一步激活AS160,加强GLUT4转运,进而改善胰岛素抵抗。更多还原

【Abstract】 ObjectiveTo investigate the effects of three Chinese herb-extracts:Ku Gua (Momordica Charantia L., MC) extract, Di Gu Pi (Lycium Chinense Mill.,LCM) extract, and Fan Bai Cao(Potentilla Discolor bunge,PDB) extract on glucose and lipid metabolism in insulin resistance and type 2 diabetic animal models. There are also further studies on liver and skeletal muscle of insulin signaling pathway:the PI3K/Akt signaling pathway and insulin independent signaling pathway:the AMPK signaling pathway by evaluating several related protein expressions to explore the molecular mechanism of the three Chinese herb extracts on the improvement of insulin resistance and type 2 diabetes.Methods1. Eighty male Wistar rats,3 weeks old, are divided randomly into two groups according to random data table:control group (n=10 with rat standard diet) and high-fat diet group (HFD) (n=70 rats). At the end 16 a week feeding period, in order to induce insulin resistance and type 2 diabetes, the 70 HFD group of Wistar rats are given an intraperitoneal-injection of small doses of STZ (35mg·kg-1·bw-1). Then,56 rats are randomly selected from the successful induced insulin resistance and type 2 diabetic model (the sum of successful induced-model is a total of 63) and are divided into 8 groups which are the three Chinese herb-extract high and low dose groups, respectively, as well as western medicine compared group. There are 7 rats in each group with the remaining 7 successfully induced model rats set as a model group. Another 7 rats are randomly selected from the normal diet group. The Thiazolidine (TZDs) insulin sensitizer Pioglitazone is used as western medicine control group (4.05mg/kg). The three Chinese herb extracts are obtained by high-dose group (400mg·kg-1·bw-1) and low dose group (100mg·kg-1·bw-1) with intragastric administration, respectively. The normal control group and model group fed with distilled water at 10ml·kg-1·bw-1. After 4 weeks of drug intervention, to evaluate the effect of insulin resistance improvement by three Chinese herb extracts, the Hyperinsulinemic-Euglycemic Clamp (HEC) experiments are examined. The fasting blood glucose (FBG), oral glucose tolerance (OGTT), fasting serum insulin (FINS), insulin sensitivity index (ISI), serum triglyceride (TG), serum total cholesterol (TC), serum free fatty acid (FFA), serum low-density lipoprotein (LDL-C) and serum high density lipoprotein (HDL-C) are also examined. The liver AMPK, Akt protein, and phosphorylation levels are detected by Western Blot. The liver AMPKα1-mRNA/AMPKa2-mRNA gene transcription are detected by RT-PCR.2. Forty-eight male BKS.Cg-m +/+ Leprdb/J db/db mice,6 weeks old, are divided randomly into 8 groups according to random data table:the three Chinese herb-extract high and low dose group, respectively, as well as western medicine compared group. There are 6 mice in each group, and the remaining 6 mice are set as model group. Another 6 mice were randomly selected from the normal C57BL/KsJ (+/-) group. The Thiazolidine (TZDs) insulin sensitizer Pioglitazone is used as western medicine control group (1.2g·kg-1·bw-1), three Chinese herb-extracts are obtained by high-dose group (4g·kg-1·bw-1) and low dose group (1g·kg-1·bw-1) with intragastric administration, respectively. The normal control group and model group are fed with distilled water at 1ml·kg-1·bw-1. After 4 weeks of drug intervention; fasting blood glucose (FBG), fasting serum insulin (FINS), insulin sensitivity index (ISI), serum triglyceride (TG), serum total cholesterol (TC), serum free fatty acid (FFA), serum low-density lipoprotein (LDL-C) and serum high density lipoprotein (HDL-C) are examined. The skeletal muscle AMPK, Akt, AS 160 protein, and phosphorylation levels are detected by Western Blot. The skeletal muscle GLUT4-mRNA gene transcription is detected by RT-PCR.Results1 The effects on IR by the three Chinese herb-extracts in type 2 diabetic IR rats1.1 Hyperinsulinemic-Euglycemic Clamp (HEC) experiment shows that:after 4 week treatments (compared with the MO group, the KG group, the DGP group, and the FBC group with high dose significantly increase the glucose infusion rate [GIR] (P<0.05) the low-dose group with the three Chinese herb extracts have an increased GIR intendancy which indicates that the insulin sensitivity is improved after treatment.1.2 Blood biochemical results show that after 4 week treatments (compared with the model group) the three Chinese herb extracts significantly improve OGTT, increased serum FINS and HDL-C levels level, and decreased serum FBG, TC, TG, LDL -C level (P<0.05).1.3 HE staining shows under a light microscope that (compared with the NO group) the MO group shows mild fatty degeneration of liver cells, fatty degeneration of liver cells along the central vein showed concentric distribution, many small visible lipid droplet vacuoles around the cytoplasm, no significant change in nuclear location, and the structure of hepatic lobule is integrated. After 4 week treatments, the three Chinese herb extracts significantly reduce fat distortion of the rat liver cells with occasional small lipid droplets cavity around the cytoplasm in focal liver cell, no significant change in nuclear location, and the structure of hepatic lobule is integrated.1.4 Western Blotting results of rat liver shows that (compared with the Model group) all of the three Chinese herb-extracts increase P-AMPK, P-Akt (Ser 473) protein expression (P<0.05) in liver. There is no significant difference among Chinese herb extracts and Pioglitazone group.1.5 RT-PCR results of liver and skeletal muscle shows that (compared with the MO group) all of the three Chinese herb extracts increase liver AMPKα1-mRNA and AMPKa2-mRNA’s expression level (P<0.05).2. The effects on insulin resistance by the three Chinese herb-extracts in BKS.Cg-m +/+ Leprdb/J db/db mice2.1 Blood biochemical results show that after 4 week treatments (compared to with the model group), the Chinese herb extracts significantly increase serum FINS and HDL-C levels, and decrease serum FBG,TC,TG, LDL -C level (P<0.05).2.2 Western Blotting results of skeletal muscle shows that (compared with the model group) all of the three Chinese herb-extracts increase P-AMPK, P-Akt (Ser 473), P-AS160 protein expression (P<0.05) in skeletal muscle tissue. There is no significant difference among Chinese herb extracts and Pioglitazone group.2.3 RT-PCR results of skeletal muscle shows that (compared with the model group) all of the three Chinese herb extracts increase skeletal muscle GLUT 4-mRNA expression levels (P<0.05).ConclusionThe three Chinese herb-extracts:Ku Gua (Momordica Charantia L.,MC) extract, Di Gu Pi (Lycium Chinense Mill.,LCM) extract, and Fan Bai Cao (Potentilla Discolor Bunge,PDB) extract can improve insulin resistance in both long-term high-fat-diet with the low-dose STZ-induced Wistar IR rat model, and the leptin-receptor-deficient BKS.Cg-m+/+Leprdb/J db/db IR mice model. The improvement mechanism of insulin resistance by the three Chinese herb extracts is through the activation of AMPK which can increase GLUT4 translocation, so that glucose utilization is accelerated. Meanwhile, the three Chinese herb-extracts also active PI3k/Akt signaling pathway by the activation of Akt which leads to further activation of AS 160, so as to strengthen GLUT4 translocation. The three Chinese herb extracts regulate the AMPK signaling pathway through increasing the genetic transcription of AMPKal-mRNA and AMPKa2-mRNA levels in the liver and promoting the GLUT4-mRNA level in the skeletal muscle tissue, thereby the overall insulin resistance can be ameliorated.更多还原