姜黄素对2型糖尿病模型大鼠及3T3-L1前脂肪细胞PTP1B通路的影响研究

【作者】 秦培洁；

【导师】 高思华；

【作者基本信息】 北京中医药大学 ， 中医基础理论， 2014， 博士

【摘要】 目的：糖尿病(DM)是一种严重危害人类健康的慢性、代谢性疾病,已成为当今中国乃至世界公共卫生的重大问题之一,糖尿病的研究已经越来越受重视。立足肝脾肾,三脏同治辨证论治糖尿病是最能够切合糖尿病病机的中医治疗方法,临床和实验研究均证实其安全有效。郁金是治疗糖尿病调肝治法的常用中药,既往的研究证实其发挥作用的主要物质是其所含的姜黄素。本课题旨在通过观察姜黄素对2型糖尿病模型大鼠、3T3L-1脂肪细胞的降糖、改善胰岛素抵抗的疗效,以及对PTP1B通路各因子的影响,以期明确姜黄素降糖及改善胰岛素抵抗的作用靶点,探讨调肝治法在2型糖尿病治疗中的作用机制,为临床治疗运用调肝法治疗2型糖尿病提供实验依据。方法：一、将3T3-L1前脂肪细胞诱导分化后,应用XTT法观察姜黄素对其分化及增值的影响,用生化法观察姜黄素对葡萄糖摄取量的影响,用real-time PCR法检测姜黄素对脂肪细胞PPARγ、C/EBPαmRNA、SREBP-1表达、内脏脂肪内脂素、抵抗素及脂联素mRNA表达的影响。二、将sd大鼠应用高脂饮食联合小剂量stz造模成功后,按体重、血糖分层随机分为3组,分别为姜黄素、吡格列酮、模型组,8周后处死动物,观察如下指标：(1)糖代谢基本生化指标：体重、葡萄糖耐量、胰岛素耐量、空腹血糖、空腹胰岛素、HOMA-IR,ISI指数、血清总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白、血清FFA、TNF-α及肝肾功变化；(2)用real-time PCR法和western-blot法检测大鼠肝脏组织PTP1B、 IRS-2、IRS-1mRNA表达及PTP1B、IRS-2蛋白表达；(3)用real-time PCR法和western-blot法大鼠肌肉组织PTP1B、IRS-1、GLUT4mRNA及PTP1B、GLUT4蛋白表达。结果：一、姜黄素能够显著降低高脂饲料联合小剂量STZ诱导的2型糖尿病大鼠血糖、血脂水平、体重；显著降低2型糖尿病大鼠FFA、TNF-a,从而增加2型糖尿病大鼠对胰岛素的敏感性,改善糖尿病大鼠的胰岛素抵抗。姜黄素可以明显降低2型糖尿病大鼠的肝重及肝脏指数,缓解其脂肪肝,降低大鼠肝脏PTP1B、IRS-1mRNA的表达及蛋白表达,增强IRS-2mRNA表达。姜黄素可以明显降低2型糖尿病大鼠肌肉组织的PTP1B、IRS-1mRNA的表达及PTP1B的蛋白表达,增强GLUT4mRNA表达。二、姜黄素能够增加脂肪细胞葡萄糖摄取和利用、促进脂肪细胞分化关键因子PPARγ和C/EBP a mRNA的表达其效应与PPARγ激动剂吡格列酮相似。同时,姜黄素可能通过抑制内脂素,抵抗素mRNA的表达,增强脂联素mRNA的表达而低浓度时促进脂肪细胞增殖,高浓度时则抑制其增殖、同时促进前脂肪细胞的分化。结论：一、体内实验：姜黄素能够通过作用于PTP1B通路而起到降糖及改善胰岛素抵抗的作用。二、体外实验：姜黄素能够促进脂肪细胞分化抑制脂肪细胞增殖,促进脂肪细胞葡萄糖摄取和利用而改善脂肪细胞的胰岛素抵抗。本实验以姜黄素作为调肝法治疗2型糖尿病研究靶点,初步证实了姜黄素是通过作用于PTP1B通路而起到了改善糖脂代谢的作用,为调肝法治疗2型糖尿病提供了实验依据。更多还原

【Abstract】 Objective:Diabetes mellitus(DM) is a chronic, metabolic diseases, which cause serious harm to human health. The prevalence of obesity has increased dramatically, it has been a problem about the healthy of world people, so it has become a focus of medicine scientific researcher to find an effect drug to prevent and control obesity. The purpose of our research is to observe the effect on the treatment of curcumin to T2DM.,And to find the mechanism of curcumin.Method1.After induction of differentiation on3T3-L1preadipocytes, we observeed the effect of curcumin on differentiation and glucose uptake, and detect the effect of curcumin on adipocyte’s PPARy, C/EBPamRNA, SREBP-1expression by Real-time PCR..2. After induction of differentiation on3T3-L1preadipocytes, we observeed the effect of curcumin on differentiation and glucose uptake, and detected the effect of curcumin on adipocyte’s visfatin, resistin and adiponectinexpression by Real-time PCR.3. The T2DM rats were divided into3groups. The changes of weight, blood glucose, blood lipid,insulin, HOMA-IR,ISI TNF-α、FFA were detected, respectively.4. The T2DM rats were divided into3groups. The changes of Liver weight, liver index, liver pathological, PTP1B. IRS-2、IRS-1mRNA expression and PTP1B, IRS-2protein expression were detected, respectively.5. The T2DM rats were divided into3groups. The changes of muscle PTP1B, GLUT4, IRS-1mRNA expression and GLUT4and PTP1B protein expression were detected, respectively.RESLUTS1. Curcumin can increase glucose uptake and utilization of fat cells, and promote the key factor of adipocyte differentiation---PPARγ and C/EBPamRNA’s expression.Its effect is similar as PPARC agonist pioglitazone.2. Curcumin can inhibit visfatin, resistin mRNA expression, and enhance expression of adiponectin mRNA. promote adipocyte proliferation at low concentrations, inhibit their proliferation at high concentrations, while promoting the differentiation of preadipocytes.3. Curcumin can significantly reduce the glucose and lipid levels, body weight of fat diet combined with low doses of STZ-induced type2diabetic rats. Its also can increase sensitivity to insulin in type2diabetic rats, and improve insulin resistance,.4. Curcumin can significantly reduce type2diabetic rats’FFA TNF-a, thereby increasing sensitivity to insulin in type2diabetic rats, and improveing insulin resistance.5. Curcumin can significantly reduce liver weight and liver in type2diabetic rats index to ease their fatty liver. Its also can significantly reduce PTP1B expression and protein expression in type2diabetic rat liver, and can enhance IRS-1mRNA IRS-2mRNA expression,thereby improving hepatic insulin resistance in type2diabetic rats.6. Curcumin can significantly reduce PTP1B and IRS-1mRNA expression enhance GLUT4mRNA expression and protein expression, thereby improving muscle insulin resistance in type2diabetic rats.Conclusion:The treatment of T2DM with curcumin have a good effect. And the mechanism of that may be the herbs can inhibit the expression of PTP1B pathway.更多还原