【作者】 王鑫；

【导师】 黄从新；

【作者基本信息】 武汉大学 ， 内科学， 2013， 博士

【摘要】 背景：心肌梗死(myocardial infarction,MI)是心血管疾病致死及致残的主要原因。心肌梗死后,神经体液调节机制激活、炎症、细胞因子引起心肌细胞及细胞间质形态结构的改变,使心脏结构功能按照一定的模式进行重塑,导致心脏形态结构和血流动力学异常,最终发展为心力衰竭(heart failure, HF)。因此,如何延缓、逆转心肌梗死后心室重构,是心肌梗死后临床治疗的关键。番茄红素是一种来源于番茄的类胡萝卜素。大量研究报道,番茄红素具有心血管保护作用。血清高浓度的番茄红素与心肌梗死、猝死的发病风险呈负相关。但对于番茄红素是否能改善心室重构较少有报道。本研究通过结扎大鼠冠状动脉前降支制备心肌梗死模型,观察番茄红素对心肌梗死后心功能及心室重构的影响,并探讨其机制。第一部分：番茄红素对大鼠心肌梗死后心功能及心室重构的影响目的：探讨番茄红素对心肌梗死后大鼠心功能及心室重构的影响。方法：通过结扎冠状动脉前降支制备心肌梗死模型,术后第一天开始给予番茄红素(番茄红素组)或生理盐水(心梗对照组)灌胃。假手术组冠状动脉前降支只穿线,不结扎。28天后,观察各组大鼠心率、体重、相对心脏体重比及相对肺脏体重比；心脏超声及血流动力学检测心脏结构功能改变；HE染色观察各组心肌组织结构变化。结果：(1)各组大鼠心率、平均动脉压、体重差异无统计学意义(P>0.05),(2)心梗对照组大鼠相对肺脏体重比较假手术组升高(P<0.05),番茄红素组较心梗对照组降低(P<0.05)；(3)与假手术组相比,心肌梗死后大鼠心功能及血流动力学受损(P<0.05),番茄红素组心脏超声及血流动力学指标显著优于心梗对照组(P<0.05)；(4)HE染色切片发现,与假手术组相比较,心肌梗死与番茄红素组心肌梗死区变薄,心肌结构紊乱,纤维化增加,非梗死区残存心肌梗细胞代偿性肥大(P<0.05),番茄红素组心肌组织非梗死区结构明显改善(P<0.05),梗死面积减小(P<0.05)。结论：番茄红素干预改善大鼠心肌梗死后心功能及心室重构。第二部分：番茄红素对大鼠心肌梗死后炎症反应、心肌细胞凋亡的影响及机制研究目的：炎症反应是心肌梗死后心室重构的重要环节,是慢性心力衰竭发展的基本机制。心肌细胞凋亡在心室重构中起着重要作用。NF-κB信号通路活化可以促进炎症反应及心肌细胞凋亡。有研究报道番茄红素可抑制肺纤维化模型中NF-κB信号通路的激活,所以我们推测番茄红素可能通过NF-κB通路逆转心肌梗死后心室重构。本实验研究目的观察番茄红素对大鼠心脏梗死周边区巨噬细胞的浸润、TNF-α表达、心肌细胞凋亡及NF-κB信号通道的影响。方法：通过结扎冠状动脉前降支制备心肌梗死模型,术后第一天开始给予番茄红素(番茄红素组)或生理盐水(心梗对照组)灌胃。假手术组冠状动脉前降支只穿线,不结扎。行心肌脏超声及血流动力学检测后,处死大鼠,留取心脏标本。(1)免疫组织化学法检测梗死周边巨噬细胞抗原(ED-1)的表达；(2) Tunnel染色法检测梗死周边心肌细胞凋亡指数,western blot法检测梗死周边心肌组织caspase-3的蛋白表达；(3) real-time PCR及western blot法检测梗死区周边心肌TNF-α的表达；(4) western blot法检测梗死周边心肌组织NF-κB/p65、IκBα、IKKα/β的蛋白表达。结果：(1)梗死周边区ED-1阳性巨噬细胞浸润增多(P<0.05),番茄红素组ED-1阳性巨噬细胞浸润较心梗对照组减少(P<0.05);(2)Tunnel染色发现,心梗对照组梗死周边区心肌细胞凋亡指数及caspase-3蛋白表达增高(P<0.05),番茄红素组改善心肌细胞凋亡(P<0.05)；(3)心肌梗死后梗死周边区心肌组织TNF-a的mRNA及蛋白水平升高(P<0.05),番茄红素组低于心梗对照组(P<0.05)。(4)与假手术组相比,心梗对照组NF-κB/p65蛋白表达、IκBa及IKKα/β的磷酸化水平增加,番茄红素组抑制IKBα、IKKα/β磷酸化水平,从而抑制NF-κB/p65活化(P<0.05)。结论：番茄红素降低梗死周边区心肌细胞凋亡及caspase-3表达,减轻梗死周边区巨噬细胞的浸润及TNF-α的表达,可能与其抑制NF-κB信号通路的激活有关,这可能是番茄红素改善心肌梗死后心室重构的部分机制。第三部分：番茄红素对大鼠心肌梗死后心肌纤维化的影响及机制研究目的：有研究证实番茄红素能够减轻博来霉素介导的大鼠实验性肺纤维化。我们推测番茄红素可抑制心肌梗死后心肌间质纤维化改善心室重构。本实验研究目的是观察番茄红素对大鼠心肌梗死后心肌间质纤维化,并探讨可能涉及的机制。方法：通过结扎冠状动脉前降支制备心肌梗死模型,术后第一天开始给予番茄红素(番茄红素组)或生理盐水(心梗对照组)灌胃。假手术组冠状动脉前降支只穿线,不结扎。行心肌脏超声及血流动力学检测后,处死大鼠,留取心脏标本。(1)Masson染色检测心肌纤维化,计算出梗死周边区胶原容积分数；(2) Western blot法检测梗死周边区Ⅰ胶原的蛋白表达；(3) Western blot法检测梗死周边区MMP-9及p38MAPK信号通路的改变。结果：与假手术组比较,心梗对照组梗死周边区心肌纤维化增加(P<0.05),MMP-9蛋白表达增高(P<0.05), p38MAPK磷酸化水平升高(P<0.05),番茄红素改善纤维化,降低Ⅰ胶原蛋白的增生,同时抑制MMP-9的表达及p38MAPK信号通路的激活(P<0.05)。结论：番茄红素可能通过抑制p38MAPK信号通路及MMP-9来改善心肌梗死后心室重构。更多还原

【Abstract】 Myocardial infarction (MI) is the leading cause of mortality and disability for cardiovascular disease. Ventricular remodeling, a key role in development of heart failure, is a therapeutic target in myocardial infarction. Lycopene is one of the major carotenoids. A large number of studies have reported that lycopene has a protective effect on cardiovascular. High serum concentration is associated with markedly reduced risk of cardiovascular disease, neurodegenerative disorders, serum lipid oxidation and cancers. However, it is unknown whether lycopene improves ventricular remodeling after myocardial infarction. In this study, we attempt to investigate the cardioprotection of lycopene after myocardial infarction. The study was divided into three parts as bellow.Part I:Effects of lycopene on cardiac function in the rat model of myocardial infarctionObjective:To explore the effects of lycopene on cardiac function and ventricular remodeling after myocardial infarction.Methods:Rat MI model was established by Left anterior descending coronary artery ligation. After operation, rats received lycopene or saline. After28days, rats underwent echocardiography and hemodynamic detection, and were sacrificed. Cardiac pathology change was analyzed by HE staining.Results:Compared with the MI group, lycopene significantly improved histomorphology and cardiac function, and reduced infarct size after myocardial infarction.Conclusion:Lycopene improved the cardiac function and ventricular remodeling in MI rats. Part II:Effects of lycopene on inflammatory response and myocardial apoptosis in the rat model of myocardial infarctionObjective:Inflammatory response is the basic mechanism of the development of chronic heart failure. Cardiomyocyte apoptosis plays an important role in ventricular remodeling after MI. NF-κB pathway could promote inflammation and myocardial apoptosis. It had reported that Lycopene inhibited cigarette smoke extract-mediated NF-kB activation. Therefore, we hypothesized that lycopene may improve ventricular remodeling by NF-κB pathway. The purpose of this part study was to observe the effects of lycopene on macrophage infiltration, TNF-a expression, cardiomyocyte apoptosis and NF-κB signaling pathway.Methods:Rat MI model was established by Left anterior descending coronary artery ligation. After operation, rats received lycopene or saline. After28days, rats underwent echocardiography and hemodynamic detection, and were sacrificed. Immunohistochemistry analysis was observed to macrophage infiltration. The expression of TNF-a and caspase-3, and NF-κB pathway activation in ischemic zone surrounding MI were detected PCR and western blot analysis.Results:Lycopene reduced myocardial apoptosis and caspase-3expression, inhibited macrophage infiltration and TNF-a expression, and attenuated NF-κB signaling pathway activation in ischemic zone surrounding MI.Conclusion:Lycopene inhibited NF-κB signaling pathway to reduce cardiomyocyte apoptosis and inflammatory response after MI, which could be mechanism of cardioprotective effects of lycopene. Part Ⅲ:Effect of lycopene on myocardial fibrosis in the rat model of myocardial infarctionObjective:It had reported that lycopene inhibited cigarette smoke extract-mediated matrix metalloproteinase-9(MMP-9) activation, and alleviated bleomycin-mediated pulmonary fibrosis. We speculated that lycopene could inhibit myocardial fibrosis and improve ventricular remodeling after MI. The purpose of this part study is to observe the effect of lycopene on myocardial fibrosis and to explore the possible mechanisms involved.Methods:Rat MI model was established by Left anterior descending coronary artery ligation. After operation, rats received lycopene or saline. After28days lycopene or saline, rats underwent echocardiography and hemodynamic detection, and were sacrificed. Myocardial fibrosis was observed by Masson staining. I type collagen MMP-9and MAPK protein expression was detected in ischemic zone surrounding MI by western blot.Results:Compared with MI group, lycopene improved myocardial fibrosis, inhibited the expression of p38MAPK, MMP-9and I type collagen.Conclusion:Lycopene improved ventricular remodeling by inhibiting p38MAPK activation and MMP-9expression.更多还原