【作者】 钱令波；

【导师】 夏强； 王会平；

【作者基本信息】 浙江大学 ， 生理学， 2006， 硕士

【摘要】 背景 糖尿病是一组由遗传和环境因素相互作用而引起的代谢疾病,由于胰岛素分泌不足以及靶组织细胞对胰岛素敏感性降低,引起糖、蛋白质、脂肪、水和电解质等一系列代谢紊乱,而以高血糖为主要共同标志。 糖尿病发病率在近十年中显著增长,据世界卫生组织预测,到2025年全世界大约有3亿糖尿病患者(占世界人口的5.4%)。目前我国糖尿病患者有大约2000万人,预计到了2010年糖尿病患者有可能达到6300万。糖尿病患病率不断增高,给人类带来的危害也越来越大,它是继心血管疾病、癌症之后,致残率、致死率最高的第三大疾病,诸如冠心病、心肌梗塞、动脉粥样硬化之类的心血管并发症是造成糖尿病患者死亡的主要原因。 糖尿病心血管并发症发病机理复杂,牵涉到一系列的细胞信号转导通路,细胞因子作为细胞间的交流信号,在诱导舒血管活性物质(如一氧化氮、花生四烯酸衍生物等)的合成及调控心血管系统的正常功能中起着重要作用。研究发现血管内皮功能障碍在糖尿病早期就出现,可能是糖尿病心血管病变的关键起始因子,并且不同糖尿病模型的各种血管中均存在内皮依赖性血管舒张功能的减弱,高血糖被认为是内皮功能障碍的促进因素。动物实验证明高浓度葡萄糖水平导致氧化应激增加以及血管舒张因子一氧化氮的生物合成及活性减少是糖尿病血管内皮依赖性舒张功能障碍的共同特点。据报道,糖尿病患者或动物血更多还原

【Abstract】 BackgroundDiabetes is a metabolic disease induced by interaction of gene and environment. Either lack of insulin secretion or hyposensitive to insulin of target tissues and cells can lead to a serious of metabolic disorder including sugars, proteins, fats, water and electrolytes metaboly, which is characterized by hyperglycemia.The incidence and prevalence of diabetes has significantly increased in recent decades. The World Health Organization estimates that, by 2025, there will be 300 million diabetic patients (5.4% of the world population). At present there are about 20 million diabetic patients in China and it is forecasted that, by 2010, this population will be 63 million. The endangerment of diabetes to human being’s health becomes more and more great as its morbidity is increased constantly. Diabetes has been the third principal disease of deformity and mortality after cardiovascular disease and cancer, and cardiovascular complications including coronary heartdisease, myocardial infarction, and atherosclerosis are the principal causes of mortality in patients with diabetes.The pathogenesis of diabetic cardiovascular complications is very complex, which involves a serious of cell signal transduction pathways. Cytokines acting as communication signals between cells modulate cardiovascular functions via inducing synthesis of vasoactive substances such as nitric oxide (NO) and arachidonate derivatives in vessel walls. It has been reported that vascular endothelium dysfunction appears in early duration of diabetes, and loss of the modulatory role of the endothelium may be a critical initiating factor of diabetic cardiovascular complications. The endothelium controls the tone of underlying vascular smooth muscle by producing vasodilator mediators, and impaired endothelium-dependent vasodilatation has been demonstrated in various vascular beds of different models of diabetes. Among the many metabolic disturbances in diabetes, hyperglycemia appears to be the main cause of endothelial dysfunction. High glucose in vitro or in vivo inhibits acetylcholine (ACh)-induced endothelium-dependent vasodilatation via impairing the synthesis of NO, and generating reactive oxygen species. In diabetic patients and animals the level of plasma interleukin-2 (IL-2), a proinflammatory cytokine, is significantly changed, and we recently found that IL-2 can lead to endothelium-dependent vasodilatation via the NO-guanylyl cyclase pathway and the cyclooxygenase-dependent pathway, and cardioprotection against injury induced by ischemia and reperfusion via the K-opioid receptor. Therefore we presume that IL-2 may have an important effect on cardiovascular function changes in diabetes or high glucose. However, the cardiovascular effect of IL-2 in high glucose concentration is still not clear.ObjectivesOne experimental diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ) and IL-2 is subcutaneously injected once per day for 5 weeks, the other model was that aortas and hearts isolated from non-diabetic rats perfused with high glucoseconcentration to simulate the cardiovascular injury induced by diabetes. The study was carried out on the isolated rat aortic rings and isolated rat hearts to determine the effect of IL-2 on the vascular response and cardiac function in diabetes (or high glucose concentration) and explore the related underlying mechanisms. The main point of this study is to make clear the exact role of IL-2 in endothelium-dependent vasodilatation in hyperglycemia.Methods1. Establishment of diabetic rat modelMale Sprague-Dawley rats were injected with a single dose of STZ (60 mg/kg, i.p.) to induced diabetes. Rats with glucose levels >15 mM were considered diabetic about 72 h after STZ treatment. After the onset of diabetes rats were subcutaneously injected with a low (5000 U/kg/d) or a high dose (50000 U/kg/d) of IL-2 for 5 weeks according to experiment protocols. Another model of cardiovascular injuries induced by diabetes was established by perfusion aortas and hearts with high glucose concentration, and IL-2 was added into perfusion solution according to Protocols of experiments.2. Organ bath experimentAortic rings were mounted between two stainless steel hooks and suspended in an organ bath containing Krebs-Henseleit solution (K-H solution), and the tension of the aortic rings was recorded in a data acquisition system (MedLab, Nanjing Medease Co. Ltd., China)3. Measurement of NO level, SOD and GSH-PX activities in serumUsing kits purchased from Jiancheng Bioengineering Institute (Nanjing, China), NO level and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in serum were assayed by the Griess method, xanthine-xanthine oxidase method and DTNB colorimetry, respectively.4. Organ bath experiment in high glucose concentrationAortic rings isolated from normal rats were incubated in an organ bath containing K-H solution of high glucose concentration (44 mM) for 4 h, then their tensions were recorded in a data acquisition system (MedLab, Nanjing Medease Co. Ltd., China).5. Measurement of NOS and SOD activities in aortasThe activities of nitric oxide synthase (NOS) and SOD in aortas isolated from normal rats, which were incubated in K-H solution of high glucose concentration (44 mM) for 4 h, were assayed by using kits of Jiancheng Bioengineering Institute (Nanjing, China).6. Isolated rat heart constant flow perfusionImmediately after stunning and cervical dislocation, the normal male Sprague-Dawley rat hearts were rapidly excised, cannulated via the aorta and perfused retrogradely for 2 h under constant flow (10 ml/min) using a calibrated roller pump with K-H solution containing a high (33.3 mM) or normal (11 mM) concentration of glucose. Hemodynamic parameters including coronary perfusion pressure (CPP), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP) and maximal rate of rise/fall of left ventricular pressure (±dP/dtmax) were monitored continually.Results1. Chronic IL-2 treatment (5000 or 50000 U/kg/d, s.c.) for 5 weeks had no effect on blood glucose level in diabetic rats, but lessened the decrease of body weight induced by diabetes.2. ACh-induced endothelium-dependent vasodilatation (EDR) was significantly impaired in diabetic rats compared with normal controls, which was attenuated by chronic treatment withIL-2 (5000 or 50000 U/kg/d, s.c). Sodium nitroprusside (SNP)-induced endothelium-independent vasodilatation (EIR) was no difference among all groups.3. Chronic treatment with IL-2 (5000 or 50000 U/kg/d, s.c.) significantly attenuated the reduction of serum NO level compared with untreated diabetic rats, but did not inhibit the decrease of both serum SOD and GSH-PX activities induced by diabetes.4. Incubation of aortic rings with high glucose (44 mM) for 4 h resulted in a significant inhibition of EDR, but had no effects on EIR. Co-incubation with IL-2 (100 or 1000 U/ml) for 40 min prevented the inhibition of EDR caused by high glucose in a concentration-dependent manner, which can be reversed by pretreatment with L-NAME (0.1 mM). SNP induced EIR was not significantly different among the groups and high mannitol concentration (11 mM glucose+33 mM mannitol) had no effect on both EDR and EIR.5. Incubation of isolated aortic segments in high glucose for 4 h resulted in a decrease of total NOS, constitutive NOS (cNOS) and SOD activity in aortic tissue. Treatment with IL-2 (1000 U/ml) in the high glucose group significantly attenuated the decrease of these parameters. High mannitol concentration had no effect on NOS and SOD activity.6. In the hearts perfusion with high glucose (33.3mM) for 2 h, there was a marked increase in CPP. Pretreatment with IL-2 (200U/ml) for 10 min significantly attenuated the increase of CPP induced by high glucose, which effect was not abolished by pretreatment with norbinaltorphimine (5 uM), a selective K-opioid receptor antagonist, for 10 min. High mannitol concentration (11 mM glucose+22.3 mM mannitol) had no effect on CPP.7. In the hearts perfusion with high glucose (33.3mM) for 2 h, there was no difference in LVDP, but a marked decrease in ±dP/dtmax, and a marked increase in LVEDP. Pretreatment withIL-2 (200U/ml) for 10 min aggravated this left ventricular dysfunction, and norbinaltorphimine pretreatment for 10 min partly canceled this effect of IL-2. High mannitol concentration (11 mM glucose+22.3 mM mannitol) had no effect on left ventricular function.ConclusionsHyperglycemia significantly inhibits endothelium-dependent vasodilatation and left ventricular systolic and diastolic function. IL-2 can attenuate the inhibition of endothelium-dependent vasodilatation induced by high glucose or diabetes, in which activation of the NO pathway and decrease of oxidative stress injury are involved. Activation of the K-opioid receptor system may involve in the effect of IL-2 on left ventricular function in high glucose concentration.更多还原