【作者】 王春艳；

【导师】 高长斌；

【作者基本信息】 吉林大学 ， 内科学， 2009， 博士

【摘要】 近年来,糖尿病已经成为严重危害人类生活质量的重大疾病之一。研究证实,心绞痛,心肌梗死是糖尿病常见的心血管并发症,心肌梗死在糖尿病患者中的病死率较无糖尿病的患者高3倍～4倍。此外,心力衰竭和再梗死的发生率在合并糖尿病的心肌梗死患者中也明显增加。因此,糖尿病的治疗不仅要立足于降低血糖,更重要的是改善糖尿病造成的心肌代谢紊乱,降低心脑血管并发症。罗格列酮(Rosiglitazone, RSG)是噻唑烷二酮类降糖药,具有提高机体对内源性胰岛素的敏感性,减轻2型糖尿病患者的胰岛素抵抗,纠正多种代谢紊乱和降低血糖作用,近年来在临床上广为应用。由于2型糖尿病患者本身是心血管疾病的高危人群,心血管疾病又是2型糖尿病的常见并发症,因此寻求具有改善糖尿病心血管并发症的降血糖药物并对其心血管保护机制进行研究具有重要意义。本课题拟观察罗格列酮对正常及糖尿病大鼠急性心肌缺血再灌注损伤心肌梗死面积,心律失常发生率和心肌病理学改变的影响,并深入探讨罗格列酮对2型糖尿病大鼠心肌缺血保护作用的机制。本研究首先以结扎正常大鼠左冠状动脉前降支复制急性心肌缺血模型,观察缺血再灌注损伤后心肌梗死面积,心律失常发生次数和类型以及血压的变化,判断罗格列酮对正常大鼠心肌缺血的保护作用,结果发现罗格列酮组心肌梗死面积减小,心肌梗死面积与左心室面积百分率以及与危险区面积比值减少,心律失常评分降低,并且可以改善心肌病理组织学和心肌细胞超微结构损伤。说明罗格列酮对急性心肌损伤有良好的保护作用。实验第二部分,我们继续观察了罗格列酮对糖尿病大鼠缺血再灌注损伤的保护作用。以高脂饮食联合小剂量多次腹腔注射链脲霉素(streptozotocin,STZ)建立大鼠2型糖尿病模型,结扎大鼠左冠状动脉前降支复制急性心肌缺血模型,同样,观察缺血再灌注损伤后心肌梗死面积,心律失常的发生及血压的变化。结果显示,罗格列酮给药组糖尿病大鼠心肌各项指标亦有明显改善。提示罗格列酮对糖尿病心肌病亦有显著的疗效。糖尿病心肌病的发病机制目前仍然不是十分清楚,目前研究显示,能量代谢障碍是糖尿病心肌病发生的重要因素。心脏是机体对能量需求最高的器官之一。在糖尿病心肌细胞存在严重的糖脂代谢紊乱,由于胰岛素缺乏和(或)胰岛素抵抗使心肌细胞利用葡萄糖氧化来提供的能量减少,从利用葡萄糖和脂肪酸供能转变为几乎全部通过脂肪酸β-氧化提供能量。可以说,糖代谢异常是糖尿病心肌病发生的始动因素,脂质代谢障碍是糖尿病心肌病的促进因素。过氧化物酶体增殖物激活型受体(peroxisome proliferator-activated receptor,PPAR),是由Issemann等于1990年发现的能被过氧化酶体增殖物激活的一类核转录因子。现证实其存在三种异构体,PPARα、PPAR-β/δ、PPARγ。其被配体激活后,通过结合目标基因启动子内反应元件,在转录水平调节相关基因表达。PPARγ对于调节全身组织代谢有极其重要的意义,尤其是脂肪酸及葡萄糖代谢。PPARγ参与脂质代谢、促进脂肪细胞分化的基因转录,增加脂肪酸转运蛋白和脂肪酸转运酶表达,刺激细胞对脂肪酸的摄入及向脂酞转化。由于罗格列酮是PPARγ的特异性激动剂,因此,我们猜测罗格列酮对糖尿病心肌病的治疗作用可能是通过上调心肌组织中PPARγ的表达,PPARγ与配体结合后,通过调整下游基因表达,进而增强胰岛素的反应性及组织摄取糖,进而明显改善心肌能量代谢的作用。因此,我们检测了与心肌能量代谢相关的蛋白PPARγ,PPARγ辅助因子(PPARγcoactivator 1α, PGC-1α),解耦联蛋白2(UCP2),解耦联蛋白3(UCP3)表达的改变。结果显示,2型糖尿病大鼠心肌组织中PPARγ、PGC1、UCP2、UCP3蛋白表达增高。而与2型糖尿病大鼠相比,糖尿病伴缺血再灌注损伤组大鼠PPARγ、PGC1蛋白表达下调,UCP2蛋白急剧增高, UCP3蛋白表达亦明显上调;与缺血再灌注损伤组相比,罗格列酮组PPARγ、PGC1表达增高、UCP2及UCP3蛋白表达下调。实验结果表明,糖尿病心肌缺血大鼠PPARγ及PGC-1α表达下调,脂肪酸氧化障碍,心肌能量供给不足;同时PPARγ降低可上调心脏线粒体解耦联蛋白UCP2,UCP3过度表达,使物质氧化与磷酸化解耦联,从而阻止ATP合成,使能量以热能形式散失,表现为心肌能量耗竭。而罗格列酮组PPARγ及PGC-1α蛋白表达上调,UCP2及UCP3蛋白下调,提示罗格列酮可能是通过该途径改善心肌能量代谢,从而发挥其心肌保护作用。更多还原

【Abstract】 In recent years, diabetes has become a seriously diseases endangered the human life. It is confirmed that angina pectoris and myocardial infarction are frequent cardiovascular complications in diabetic patients. The mortality of myocardial infarction in patients with diabetes was 3-4 times higher than those without it. In addition, heart failure and re-infarction are increased in the myocardial infarction patients combined with diabetes significantly. Therefore, the treatment of diabetes should not only be based on lowering blood glucose and, more importantly, to improve the diabetic metabolic disorder and reduce cardiovascular and cerebrovascular complications.Rosiglitazone, a hypoglycemic agent of thiazolidinediones (TZDs), has attracted remarkable scientific interest on novel and potent improveing insulin- sensitizing agents, and to lighten insulin resistance, more over, to correct metabolic disturbance. Patients with type 2 diabetes are at a high risk for cardio- vascular disease,resulting in increased mortality rates. So there is an urgent need to develop a hypoglycemic agent with protective effects on the cardiova- scular system. In this topic, we will research the protective effects of Rosiglitazone on acute myocardial I/R injure in normal and Type 2 diabetic (T2DM) rats.Rosiglitazone is a specific agonist of PPARγ. PPARγis an important factor for regulating body metabolism, in particular metabolism of fatty acid and glucose. It can promote adipocyte differentiation; increase the expression of fatty acid transport protein and fatty acid transporter enzyme. It also could stimulate fatty acids be intake and transformation, and enhance insulin-responsive and glucose uptake by adjusting the expression of downstream gene, thereby improve the energy metabolism of myocardial significantly. Therefore, we proposed to investigate the mechanism of rosiglitazone on the perspective of energy metabolism of myocardial.Methods:1.The effects of rosiglitazone on myocardial ischemia-reperfusion ( I/R) in normal rats: I/R injury rat model was induced via ligating left anterior descending branch of coronary artery for 30 min and reperfusing for 120 min; Rats were randomly divided into rosiglitazone, I/R and shame- operated group. Myocardial infarct size (IS) and the ratio of IS and area at risk (AAR) were determined, arrhythmias score was evaluated according to Lambeth conventions, and blood pressure (BP) was recorded 1 w after intragastric administration and I/R injury operation. The ultrastr- ucture of cadiocyte was observed by electron microscope.2.The effects of rosiglitazone on myocardial ischemia-reperfusion (I-R) in T2DM rats: T2DM ratmodelwas established by high lipid diet combined multiple low dose strep tozocin (STZ); T2DM rats were randomly divided into rosiglitazone, I/R and shame-operated groups.3. Myocardial protein was extracted in each group. The protein level of PPARγ、PGC1、UCP2 and UCP3 in cardiac muscle was measured using western blot. Actin,a conservative protein,was used as an interner control to adjust the concentration of each sample.Results:1. The effects of rosiglitazone on I/R in normal rats: Compared with IR group, rosiglitazone decreased the ration of IS/LV (29.3±4.9vs 37.6±3.2) and the rate of AAR /LV (76.1±9.6 vs 93.5±7.4), reduced scores of arrhythmias (2.6±0.4 vs 4.2±0.6) and ameliorated the restore of BP in reperfusion period (P < 0.05).2. The effects of rosiglitazone on I/R in T2DM rats: Compared with IR group, rosiglitazone decreased the ration of IS/LV (30.3±3.6 vs 38.7±3.0) and the rate of AAR /LV (76.0±2.6 vs 93.2±3.0) , reduced scores of arrhythmias (2.6±0.4 vs 4.4±0.8) and ameliorated the restore of BP in reperfusion period (P < 0.05).3. Compared with control group, PPARγ、PGC1、UCP2 and UCP3 are increased in the cardiac musculature. It is suggested that in diabetes, due to elevated blood sugar and abnormal glucose metabolism, there have been some changes on energy metabolism of myocardial and myocardial fatty acid oxidation may be enhance compensatory. However, the protein level of PPARγand PGC-1 decreased in DM-I-R group, with UCP2 sharply raised up. The UCP3 was also up-regulated in I-R group, compared with DM group. It shows that when myocardial ischemia was developed in diabetic rats, fatty acid dysoxidation was happen, combined with a serious shortage of oxygen supply, myocardial energy supply was insufficient; At the same time UCP2, UCP3 were over-expression, which made the oxidative phosphorylation uncoupling. Thereby it prevented ATP synthesis, and enhanced energy deficiency, manifested as myocardial energy depletion; And RSG can noteably swtch these changes, compared with I-R group.Conclusions:Rosiglitazone has the protective effects on acute myocardial I/R injure in normal and T2DM rats and it may be associated with the improvment of energy metabolism of cadiocyte.更多还原