【作者】 王敏；

【导师】 傅国胜；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 糖尿病是由于胰岛素分泌绝对或者相对不足导致慢性持续性高血糖状态引起的一组代谢异常综合症,可伴有或者不伴有胰岛素抵抗。流行病学调查表明,到2030年,糖尿病的患病率将从2000年的2.8%上升到4.4%。糖尿病心肌病(Diabetic Cardiomyopathy, DC)是糖尿病最常见的心血管并发症之一,是独立于冠状动脉病变、微血管病变和心脏自主神经病变之外的心脏结构改变和功能失调。大量临床以及实验研究表明,慢性高血糖状态是糖尿病微血管病变及心血管并发症最重要的始动因子,高血糖可以通过多种信号转导系统,影响心肌细胞的结构和功能,最终导致心功能不全。糖尿病患者心力衰竭进展快,病死率高,但是在病程早期以心脏舒张功能不全为主要表现,具有隐蔽性,给诊断带来困难,因此最理想的治疗应该是在糖尿病心肌病早期进行干预,阻断其向心力衰竭发展的路径。糖尿病心肌损害早期就已经存在神经内分泌系统过度激活、氧化应激反应增加、蛋白激酶C(Protein kinase C, PKC)表达水平以及活性异常等改变,所有这些因素综合作用共同导致心肌重构以及功能障碍。近年来研究认为,高血糖可以通过激活PKC相关信号转导系统,调节细胞的生长,影响细胞的收缩性和通透性,使细胞外基质成分和基因表达发生改变,引起心肌细胞肥大,心脏微血管病变和间质纤维化,影响心功能。因此,PKC在糖尿病心肌病的发病机制中起重要作用,抑制心肌细胞PKC的表达对糖尿病心肌病的防治具有重要意义。灯盏花素(breviscapine)是从菊科属植物短亭飞蓬全草灯盏细辛中提取的一类黄酮类成分,具有扩张血管,降低血管阻力,增加血流量,改善微循环以及抗血小板聚集的作用,目前已广泛应用于心脑血管疾病的治疗,具有良好的耐受性和安全性。近年来,随着对灯盏花素研究的不断深入,发现其具有较强的抑制PKC活性的作用,并可抑制高糖所诱导的c-fos,c-jun蛋白表达与Ⅳ型胶原合成增加。已有研究表明,灯盏花素对糖尿病肾病具有保护性作用,其机理可能与其抑制PKC的表达和活性有关。但是,关于灯盏花素对糖尿病心肌病是否同样具有保护作用及其相关机制,目前尚未明确。鉴于糖尿病心肌病中存在PKC的高表达以及灯盏花素具有抑制PKC的作用,我们从理论上推测灯盏花素可以改善糖尿病导致的心脏结构和功能的损害,对糖尿病心肌病具有保护作用,其机理可能与其抑制心肌细胞PKC的表达有关。因此我们通过建立糖尿病大鼠模型,观察灯盏花素治疗是否能改善糖尿病大鼠的心功能,并且逆转糖尿病导致的心肌肥大和结构损害,同时检测PKC以及相关蛋白的表达水平,试图从PKC角度探讨其对糖尿病心肌病的保护作用。1灯盏花素对糖尿病大鼠心功能的改善作用及钙离子调控蛋白表达的影响目的：探讨灯盏花素对链脲佐菌素诱导的糖尿病大鼠心功能和PKC(PKC-α、PKC-β2)以及相关钙离子调控蛋白表达水平的影响。方法：雄性Sprague-Dawley大鼠一次性腹腔注射STZ诱导糖尿病模型,对照组注射枸橼酸盐缓冲液。建模成功4周后分成4组：(1)正常对照组；(2)糖尿病大鼠组；(3)糖尿病大鼠小剂量灯盏花素处理组(10mg／kg／d)；(4)糖尿病大鼠大剂量灯盏花素处理组(25mg／kg／d)。继续分组处理6周后,用心脏超声以及血流动力学方法测定相关心功能指标,采用Western blot方法检测PKC-α、p-PKC-α、PKC-β2、p-PKC-β2以及相关钙离子调控蛋白即蛋白磷酸酶抑制子-1(protein phosphatase inhibitor-1,PPI-1),磷酸受纳蛋白(phospholamban, PLB)以及肌浆网钙泵(sarco/endoplasmic reticulum Ca2+-ATPase-2, SERCA-2)表达水平的变化,采用Ca2+-ATPase酶测定试剂盒检测心肌组织Ca2+-ATPase酶的活性。结果：与对照组相比,糖尿病大鼠表现出明显的心功能不全,同时心肌组织PKC-α、p-PKC-α、PKC-β2、p-PKC-β2以及PLB的表达水平明显增加,而PPI-1、p-PLB、SERCA-2的表达水平以及Ca2+-ATPase酶的活性明显下降,灯盏花素治疗后糖尿病大鼠的心功能得到改善,并且心肌组织PKC-α、p-PKC-α、PKC-β2、p-PKC-β2以及PLB的表达水平明显降低,同时PPI-1、p-PLB、SERCA-2的表达水平以及Ca2+-ATPase酶的活性增加。结论：灯盏花素治疗可以改善糖尿病大鼠的心功能,对糖尿病心肌病具有保护作用,其机制可能与其抑制PKC的表达,并且调节相关钙离子调控蛋白的表达水平和活性有关。2灯盏花素通过抑制PKC的表达逆转糖尿病大鼠心肌肥大以及相关机制目的：探讨灯盏花素对链脲佐菌素诱导糖尿病大鼠心肌肥大的影响及其相关机制。方法：雄性Sprague-Dawley大鼠一次性腹腔注射STZ诱导糖尿病模型,对照组注射枸橼酸盐缓冲液。建模成功4周后分成4组：(1)正常对照组；(2)糖尿病大鼠组；(3)糖尿病大鼠小剂量灯盏花素处理组(10mg／kg／d)；(4)糖尿病大鼠大剂量灯盏花素处理组(25mg／kg／d)。继续分组处理6周后,通过HE染色以及透射电镜观察心肌细胞结构和形态变化,采用Western blot亏法检测PKC-α、p-PKC-α、PKC-β2、p-PKC-β2以及NF-κB、p-NF-κB、c-fos等蛋白表达水平的变化,采用RT-PCR方法检测TNF-α的mRNA表达水平变化。结果：与对照组相比,糖尿病大鼠表现出明显的心肌细胞肥大以及超微结构异常,同时心肌组织PKC-α、p-PKC-α、PKC-β2、p-PKC-β2以及NF-κB、p-NF-κB、c-fos的蛋白表达水平和TNF-α的mRNA表达水平明显增加,灯盏花素治疗后糖尿病大鼠心肌肥大以及心肌细胞超微结构损害得到改善,并且PKC-α、p-PKC-α、PKC-β2、p-PKC-β2以及NF-κB、p-NF-κB、c-fos的蛋白表达水平和TNF-α的mRNA表达水平均明显降低。结论：灯盏花素治疗可以逆转糖尿病大鼠心肌肥大以及心肌细胞超微结构损害,对糖尿病心肌病具有保护作用,其机制可能与其抑制PKC的表达,并进一步调节NF-κB、TNF-α和c-fos的表达水平有关。更多还原

【Abstract】 Diabetes mellitus is a state of chronic hyperglycemia due to an absolute or relative deficiency of insulin secretion that may or may not be associated with insulin resistance. The world wide prevalence of diabetes was estimated to be 2.8% in 2000 and is projected to reach 4.4% by 2030. Diabetic cardiomyopathy, characterized by structure changes and function impairment of cardiomyocytes, is one of the most prevalent cardiovascular complications of diabetes mellitus that occurs independently of coronary artery disease, microangiopathy and autonomic neuropathy of the heart. Many epidemiological and clinical studies have shown that chronic hyperglycemia is a major initiator of diabetic cardiovascular complications, as high glucose may regulate the growth of cardiomyocytes via activating several signal transduction pathways. The characteristic of diabetic heart failure, manifested by early diastolic dysfunction, followed by late systolic dysfunction, is faster deteriorating with high mortality and is difficult to diagnose in the early stage. Thus, we tried to seek for an ideal treatment to inhibit the pathway towards heart failure in the earlier period of diabetic cardiomyopathy.There was a consensus that there were excessive activity of sympathetic nerve system, oxidative stress as well as higher expression and activation of protein kinase C isoforms in the early stage of diabetic cardiomyopathy, all of which accelerated cardiac remodeling and dysfunction. Studies have revealed that hyperglycemia could increase the formation and activity of PKC isoforms, and PKC signal pathway is now considered to be one of the most important intracellular transduction pathways that functions as a core effect in the onset and progression of diabetic cardiomyopathy. Increased PKC activity in diabetic cardiomyopathy could regulate the growth, contractility and permeability of cardiomyocytes, and is associated with remodeling of extracellular matrix, thickening in basement membrane, hypertrophy of cardiomyocytes, as well as microangiopathy and autonomic neuropathy of the heart, which eventually lead to the structure and function impairment of cardiomyocytes in diabetic cardiomyopathy. So inhibiting the expression of PKC and blockade of PKC signal transduction pathway may have preventative and therapeutical effect on the diabetic cardiomyopathy.Breviscapine is a flavonoid extracted from Erigeron breviscapus and prepared into a Chinese patent medicine, its essential active ingredient is flavones. Its pharmacologic action is dilating blood vessel, reducing blood viscosity and improving microcirculation. It also possesses an anti-platelet action and can decrease plasma fibrin content and promote fibrinolytic activity. Recent studies have also shown that breviscapine could inhibit the expression of PKC, and decrease the protein expression of c-jun, c-fos and synthesis of type IV collagen induced by hyperglycaemia. There were also researches indicated that breviscapine had a protective effect on diabetic nephropathy through inhibiting the increased expression and activity of PKC-P2. But little is known about the influence of breviscapine on diabetic cardiomyopathy and its underlying mechanisms.On the basis of above considerations that PKC is activated in diabetic mellitus, and breviscapine could inhibit the activation and expression of PKC, we hypothesized that breviscapine may have protective effect on the structure and function injury of cardiomyocytes in diabetic cardiomyopathy via inhibiting the cardiac overexpression of PKC. In this study, we use diabetic rats as an animal model to investigate the influence of breviscapine treatment on the cardiac structure and function in diabetic cardiomyopathy, as well as the myocardial expression of PKC and associated proteins and its underlying mechanisms. Part 1 Breviscapine ameliorates cardiac dysfunction and regulates the myocardial Ca2+-cycling proteins in diabetic ratsObjective:To investigate the influence of breviscapine on the cardiac function in diabetic rats as well as the expression of PKC-α、PKC-β2 and Ca2+-cycling proteins.Methods:Diabetes were induced in male Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin and the control rats were injected with citrate buffer solution. After the induction of diabetes for 4 weeks, the animals were divided into four groups:(1)normal rats as control; (2)diabetic rats; (3)diabetic rats treated with low dose of breviscapine(10mg/kg/day); (4)diabetic rats treated with high dose of breviscapine(25mg/kg/day). After treatment with breviscapine for 6 weeks, the echocardiographic parameters and invasive cardiac function were measured. The expression of PKC-α、p-PKC-α、PKC-β2、p-PKC-β2 and calcium handling regulators, such as protein phosphatase inhibitor-1(PPI-1), phospholamban(PLB) and sarco/endoplasmic reticulum Ca2+-ATPase-2(SERCA-2) were detected by western blot. The activity of Ca2+-ATPase was measured using Ca2+-ATPase kit.Result:Diabetic rats showed impaired cardiac function compared with control rats. The expression of PKC-α、p-PKC-α、PKC-β2、p-PKC-β2 and PLB in diabetic rats increased significantly compared with control rats, while the expression of PPI-1, p-PLB, SERCA-2 and activity of Ca2+-ATPase decreased. Treatment with breviscapine could reverse the cardiac dysfunction in diabetic cardiomyopathy rats, and decrease the expression of PKC-α、p-PKC-α、PKC-β2、p-PKC-β2 and PLB, as well as increase the expression of PPI-1, p-PLB, SERCA-2 and the activity of Ca2+-ATPase.Conclusion:This study showed that breviscapine could ameliorate cardiac dysfunction in diabetic rats, and have protective effect on diabetic cardiomyopathy via regulating the expression of PKC and Ca2+-cycling proteins. Part 2 Breviscapine ameliorates cardiac hypertrophy of diabetic rats via inhibiting the expression of PKC and its mechanismsObjective:To investigate the influence and mechanism of breviscapine on the hypertrophy of cardiomyocytes in diabetic cardiomyopathy rats.Methods:Diabetes were induced in male Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin and the control rats were injected with citrate buffer solution. After the induction of diabetes for 4 weeks, the animals were divided into four groups:(1)normal rats as control; (2)diabetic rats; (3)diabetic rats treated with low dose of breviscapine(10mg/kg/day); (4)diabetic rats treated with high dose of breviscapine(25mg/kg/day). After treatment with breviscapine for 6 weeks, heart tissues were obtained for HE stain and electron microscopestudy. The expression of PKC-α、p-PKC-α、PKC-β2、p-PKC-β2 and NF-kB、p-NF-KB、TNF-α、c-fos were detected by western blot or RT-PCR.Result:Diabetic rats showed cardiac hypertrophy and impaired cardiac structure compared with control rats, the protein expression of PKC-α、p-PKC-α、PKC-β2、p-PKC-β2、NF-kB、p-NF-KB、c-fos and the mRNA expression of TNF-αall increased significantly. Treatment with breviscapine could reverse the cardiac hypertrophy and structure impairment in diabetic cardiomyopathy rats, as well as decrease the protein expression of PKC-α、p-PKC-α、PKC-β2、p-PKC-β2、NF-kB、p-NF-kB、c-fos and the mRNA expression of TNF-a.Conclusion:This study showed that breviscapine could ameliorate cardiac hypertrophy and structure impairment in diabetic rats, and have protective effect on diabetic cardiomyopathy via inhibiting the activity of PKC and then the subsequent expression of NF-kB、TNF-αand c-fos.更多还原