【作者】 张峰；

【导师】 曾秋棠；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 第一部分:大鼠糖尿病性心肌病模型的建立及PPARα-FFA信号传导通路在糖尿病性心肌病中的作用目的建立2型糖尿病及糖尿病性心肌病大鼠模型,研究过氧化物酶体激活物受体α-游离脂肪酸(PPARα-FFA)信号传导通路以及心肌重塑在糖尿病性心肌病病程中的作用。材料和方法:65只雌性Wistar大鼠,随机选出8只作为对照组(CON)给予普通饮食;其余57只用以诱导2型糖尿病,饲以高脂高糖饮食,4周后检测空腹血糖、胰岛素。以小剂量链脲佐菌素腹腔注射,48小时后,空腹血糖≥16.7mmol/L为2型糖尿病模型。随机选取8只糖尿病鼠(其余留作他用)为糖尿病性心肌病组(DCM),继续饲养16周后用超声心动图评价心功能,取血检测血糖、胰岛素、FFA水平,心脏称重,免疫组化检测心肌组织胶原分数,观察Ⅰ、Ⅲ型胶原和PPARα的表达,并用电镜观察心肌超微结构。结果:(1)有40只大鼠符合2型糖尿病标准,与CON(n=8)组相比,糖尿病大鼠表现为高血糖、高胰岛素血症。血游离脂肪酸水平增加(208±18.3μmol/L vs 128±11.3μmol/L,P<0.01)。(2)糖尿病性心肌病(DCM,n=6)组2只死亡,心功能减退,以舒张功能减退为主;与CON组比较,心脏重量指数增加(DCM组4.42±0.46 vs CON组3.54±0.34,P<0.01);胶原分数增加(DCM组2.21±0.56 vs CON组1.61±0.36,P<0.01);Ⅰ、Ⅲ型胶原比率显著增加。心肌超微结构显示心肌纤维变性坏死,间质胶原纤维增多,有大量糖原颗粒沉积,微血管基底膜轻度增厚等。(3)PPARα的表达DCM组显著高于CON组(DCM组65.34±3.7vs CON组48.69±4.9, P<0.01)。结论:实验成功地构建以胰岛素抵抗为特征的2型糖尿病及糖尿病心肌病模型,心肌组织中PPARα表达上调、血FFA水平增加与心肌重塑,心肌病进展密切相关。第二部分:过氧化物酶体激活物受体激动剂非诺贝特抑制STZ诱导的大鼠糖尿病心肌病的心肌炎症和心肌重塑目的:通过构建的糖尿病性心肌病大鼠模型,观察过氧化物酶体激活物受体α(PPARα)激动剂非诺贝特对糖尿病心肌病心肌炎症因子的作用以及对心肌重塑的影响。方法:对照组(CON)大鼠普通饲料喂养,由STZ诱导的2型糖尿病鼠继续给予高脂高糖饲料并随机分为三组:糖尿病心肌病组(DCM),非诺贝特低剂量干预组(LFIB, 4 mg·kg-1·day-1)和高剂量干预组(HFIB, 40 mg·kg-1·day-1),每组8只,干预16周。超声心动图评价心功能,测定血糖、胰岛素、胆固醇、甘油三脂(TG)以及游离脂肪酸(FFA)含量,评价心脏重量指数,胶原容积分数(CVF),免疫组化以及RT-PCR检测肿瘤坏死因子(TNF-α)、白介素10(IL-10)和PPARα的表达,观察心肌超微结构。结果:(1)超声心动图检查显示糖尿病大鼠心功能减退,DCM组尤为显著,两组不同剂量的非诺贝特干预后与DCM组相比心功能明显改善(P<0.01),但两干预组之间无显著差异(P>0.05)。(2)与CON组比较,糖尿病大鼠血糖、血胰岛素水平显著升高,血胆固醇、TG以及FFA水平增加。与DCM组比较,非诺贝特对血糖、胰岛素无显著影响,但可显著降低血胆固醇、TG以及FFA水平;血FFA浓度在HFIB组显著低于LFIB组(187±13.6 vs 168±17.3μmol/L,P<0.05)。(3)DCM组TNF-α、IL-10以及PPARα的表达明显增加,病理检查及超微结构可见心肌损伤以及心肌重塑,药物干预可降低TNF-α、IL-10以及PPARα的表达,减轻心肌重塑;RT-PCR分析进一步显示TNF-α的mRNA表达在DCM组上调(0.83±0.08),在LFIB、HFIB干预组下调,分别为(0.58±0.07、0.55±0.07),与DCM组相比差异有非常显著意义(P<0.01),而与CON组相比,仍有显著差异(P<0.01)。与CON组相比,IL-10mRNA表达在DCM组显著升高,干预组表达上调更加明显(P<0.01)。结论:心肌能量代谢和炎症因子表达的变化、心肌重塑与糖尿病性心肌病病程密切相关,激活PPARα通路可能通过平衡炎症因子表达改善糖尿病大鼠的心肌重塑和心脏功能。第三部分曲美他嗪对糖尿病性心肌病大鼠心肌重塑以及转化生长因子β1的影响目的:研究曲美他嗪对糖尿病性心肌病心肌重塑及对转化生长因子β1表达的影响。方法:采用高脂高糖饲养并且以小剂量STZ诱导出2型糖尿病动物模型,随机分为未治疗的糖尿病心肌病组(DCM)、曲美他嗪干预组(TMZ, 10 mg·kg-1·day-1)、卡托普利干预组(CAP, 50 mg·kg-1·day-1),另设正常对照组(CON),每组8只。16周后,采用超声心动图等评价各组的心功能改变,评价心脏重量指数、胶原容积分数(CVF),免疫组织化学以及逆转录-聚合酶链反应(RT-PCR)检测心肌组织中TGF-β1含量以及mRNA表达。结果:(1)DCM组出现了心肌病的特征,表现为现舒张功能减退,E/A比值下降,IVRT缩短,-dp/dt max下降, CVF增加为(0.38±0.09 vsCON组0.16±0.04),TGF-β1的免疫组化平均积分光密度值(IDP)显著增加(1.03±0.12vsCON组0.22±0.06), mRNA水平显著增加(1.01±0.1 vsCON组0.42±0.07),均为P<0.01。(2)与DCM组相比,TMZ和CAP干预组心脏重量指数下降(P<0.05),舒张功能明显改善,CVF减少,两组分别为(0.27±0.05)、(0.24±0.06),与DCM组(0.38±0.09)相比都有显著差异(均P<0.01);TGF-β1的IDP在两干预组分别为(0.75±0.08)、(0.74±0.08),与CON组(1.03±0.12)相比均显著降低(均P<0.01);TGF-β1mRNA表达在两干预组明显下调,TMZ和CAP干预组分别为(0.71±0.09)、(0.73±0.22),与CON组相比均有显著差异(均P<0.01)。结论:曲美他嗪可以减轻糖尿病性心肌病中心肌胶原沉积,减轻心室重塑,其作用可能与抑制心肌组织中TGF-β1的表达有关。更多还原

【Abstract】 PartⅠModel construction and effect of PPARα-FFA signal pathway in STZ–induced diabetic cardiomyopathy ratsObjective To construct ideal diabetic cardiomyopathy model in streptozotocin (STZ) induced type 2 diabetic rats,and investigate the effect of peroxisome proliferator -activated receptor alpha(PPARα)and free fatty acids (FFA) signals transduction pathway in the development of diabetic cardiomyopathy through detecting cardiac expression changes of PPARαand metabolic alteration of FFA.Methods 65 female Wistar rats were randomized into control group (CON, n=8), feeding with general diet all the time, and experimental group ( n=57), which were fed with high glucose and hyperlipid diet for 4 weeks, to induce insulin resistance. Then diabetes mellitus was induced by a single injection of STZ (35 mg/kg; i.p.), Hyperglycemia (≥16.7mmol/L) was confirmed 48h later.The diabetic rats were fed with high glucose and hyperlipid diet for 16 weeks continually and 8 of them were randomized into diabetic cardiomyopathy group(DCM), Hemodynamic characterization was determined by echocardiography,the myocardial pathologic and ultrastructure changes was observed by microscope and electron microscope. The collagen volume fraction(CVF, VG staining)and expression of types I, III collagen and PPARαwere quantified by digital image analysis. Levels of blood glucose, insulin, total cholesterol, triacylglycerol and FFA were determined using biochemical or RIA methods.Results (1) Hyperglycemia was displayed in 40 STZ-induced rats followed with hyperinsulinemia, 2 rats of DCM group were died at the end of experiment. Diastolic function deteriorated in DCM-group(n=6) exhibited low E wave to A wave ratio (E/A) (DCM, 0.83±0.21 versus CON, 1.28±0.18, P<0.01)and shortened isovolumic relaxation time (IVRT)(DCM, 29.13±7.10 versus CON, 45.06±5.03, P<0.01). Histopathology revealed ventricular remodeling in STZ–DCM heart. The HW/BW ratio, ColⅠ/ ColⅢratio in DCM group (4.42±0.46, 15.49±0.87, respectively) is higher than that in CON group(3.54±0.34, 6.51±0.96, P<0.01), the CVF of LV was significantly higher in DCM (2.21±0.56) than CON (1.61±0.36, P<0.01) rats. The ultrastructure of DCM heart presented focal degeneration and disarranged order of myofilament, increased interstitial collagen and glycogen granule deposite, etc.(2) Systemic and cardiac metabolism changes: Compared with CON group, there were elevated plasma concentrations of glucose, insulin, Chol, Tg and FFA in DCM group. This was paralleled by abundant PPARαexpression in DCM-group (65.34±3.67) versus CON-group (48.69±4.88, P<0.01).Conclusion STZ-induced insulin-resistant type 2 diabetic and diabetic cardiomyopathy rats model were established successfully. The expression alterations of PPARαand metabolism changes of FFA involve in the abnormality of myocardial metabolism and myocardial remodeling, and possibly play an important role in the development of diabetic cardiomyopathy.PartⅡPPARαagonist fenofibrate inhibits myocardial inflammation and remodeling in STZ-induced diabetic cardiomyopathy ratsObjective To construct diabetic cardiomyopathy(DCM)model in streptozotocin (STZ) induced type 2 diabetic rats,and investigate the effect of peroxisome proliferator-activated receptor alpha(PPARα)agonist fenofibrate on myocardial inflammatory factors and myocardial remodeling.Methods Control rats were fed with common diet. Type 2 diabetes rats was induced by high energy diet, then treated with lower dose of STZ and continuous high glucose and hyperlipid diet. The model rats were randomized into three groups : diabetic cardiomyopathy (DCM) group, intervention group treated with low dose fenofibrate(LFIB, 4 mg·kg-1·day-1) and intervention group treated with high dose fenofibrate(HFIB, 40 mg·kg-1·day-1)for 16 weeks, respectively, with 8 rats in each group. Hemodynamic characterization was determined by echocardiography, the myocardial ultrastructure changes was observed by electron microscope, and the plasma biochemical indicators,heart weight index,typeⅠandⅢcollagens, collagen volume fraction, myocardial cytokine gene and protein expression (TNF-α, IL-10, PPARα) were detected respectively by biochemical methods, semi-quantity RT-PCR and immunohistochemistry at the end of experiment.Results (1) The decreased left ventricular systolic and diastolic function, myocardial remodeling were manifested in all diabetic rats, and these abnormalities were more significant in DCM group. Fenofibrate intervention groups with two different doses significantly improved the cardiac function compared with DCM group(P<0.01). But the two intervention groups had no significant difference(P>0.05). (2) Compared with CON group, there were significant elevated plasma concentrations of glucose, insulin, Chol, Tg and FFA in DCM group. Fenofiberate treatment had no effect on the level of blood glucose and insulin, but significantly decreased the concentration of blood Chol, Tg and FFA. The level of FFA in HFIB group was significantly lower than that in LFIB group(187±13.6 vs 168±17.3μmol/L,P<0.05). (3) The expression of TNF-α, IL-10 and PPARαwas increased significantly,the myocardial impairment and remodeling was displayed.in DCM . In contrast , The expression of TNF-α, IL-10 and PPARαwas decreased and the myocardial remodeling was attenuated by fenofiberate treatment, moreover, RT-PCR analysis further suggested that mRNA expression of TNF-αwas upregulated in DCM group(0.83±0.08), which was down regulated in LFIB and HFIB intervention groups. The values in these two groups were 0.58±0.07 and 0.55±0.07, respectively, both of which had significant difference compared with that of DCM group(P<0.01), while which still had significant difference compared with that of CON group. On the contrary, mRNA expression of IL-10 increased significantly in DCM group, while the expression was obviously upregulated in two intervention groups(P<0.01).Conclusion The alteration of myocardial energy metabolism and expression of inflammatory factors, myocardial remodeling are close related with the development of DCM. The activation of PPARαpathway could balance the expression of inflammatory factors and then improve myocardial remodeling and cardiac function.PartⅢInfluence of trimetazidine on myocardial remodeling and transforming growth factor-beta 1 expression in STZ-induced diabetic cardiomyopathy ratsObjective:To investigate the influence of trimetazidine on myocardial remodeling in diabetic cardiomyopathy in STZ-induced diabetic cardiomyopathy rats and the alteration of transforming growth factor-beta 1( TGF-β1).Method:Control rats were fed with common diet. Type 2 diabetes rats was established by high energy diet, then treated with lower dose of STZ and continuous high glucose and hyperlipid diet. The diabetic model animals were randomized into three groups: untreated DCM group, TMZ group and CAP group. A normal control group was set up in advance(n=8/group)After STZ injection and drug administration for sixteen weeks, LV function was evaluated by echocardiogram and heart weight index, collagen volume fraction (CVF )were calculated. myocardial cytokine gene and protein expression ( TNF-α, IL-10, PPARα) were detected. Myocardial TGF-β1 content and mRNA expression was quantified by immunohistochemistry and RT-PCR .Result:(1)DCM hearts demonstrated diabetic cardiomyopathy phenotype, including a significant reduction of LV diastolic function , reflected by lowered E/A ratio, shortened isovolumic relaxation time (IVRT) and decreased -dp/dt max .The CVF was increased to 0.38±0.09. The immunohistochemistry IDP and the level of mRNA were increased, which were (1.03±0.12)and (1.01±0.1), respectively. Compared with CON group, both groups had significant difference (P<0.01 in both groups). (2) In contrast, the ratio of heart weight/body weight was significantly decreased in TMZ and CAP rat groups compared with DCM rats(P<0.05); The diastolic function in TMZ and CAP groups was improved, the CVF was decreased(P<0.01)the values in both groups were (0.27±0.05) and(0.24±0.06), respectively. Compared with DCM group, both groups had significant difference (P<0.01 in both groups). IDP in both intervention groups were (0.75±0.08) and (0.74±0.08), respectively, which were significantly decreased compared with that in CON groups (P<0.01 in both groups). The mRNA expression of TGF-β1 was down regulated significantly, the values of TMZ and CAP groups were (0.71±0.09) and (0.73±0.22), respectively. Compared with CON group, both groups had significant difference (P<0.01 in both groups).Conclusion: Trimetazidine improved myocardial collagen deposition and myocardial remodeling in diabetic rats,associated with the decrease in expression of TGFβ1. Optimization of myocardial energy metabolism may be useful in prevention and treatment of diabetic myocardial disease.更多还原