【作者】 司延斌；

【导师】 龚培力；

【作者基本信息】 华中科技大学 ， 药理学， 2006， 硕士

【摘要】 心肌肥厚作为心力衰竭的前奏,可增加心律失常、心肌梗死及猝死的发生率,被认为是心血管疾病的独立危险因素之一。尼可地尔(Nicorandil,Nic)是近年来研制开发的一种兼有钙拮抗作用的KATP通道开放剂。研究已证实,Nic除了能增加冠脉血流,减少心肌氧耗外,还可同时降低心脏的前、后负荷,且不引起反射性心动过速等作用。本研究通过建立不同动物心肌肥厚模型,运用免疫学、电生理学、分子生物学等实验方法,从氧自由基损伤、能量代谢障碍、细胞动作电位、细胞凋亡、细胞内钙超载及相关基因表达诸方面探讨Nic对实验性心肌肥厚的作用及其机制,为Nic的临床应用提供实验依据。第一部分尼可地尔抗心肌肥厚的药效学研究一、尼可地尔对异丙肾上腺素诱发小鼠心肌肥厚的保护作用采用连续多次皮下注射小剂量异丙肾上腺素(isoproterenol,Iso) 2 mg?kg-1的方法建立小鼠心肌肥厚模型,观察:Nic对小鼠心重指数和组织病理改变的影响; Nic对常压密闭缺氧条件下小鼠存活时间的影响;Nic对小鼠心肌组织超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量的影响。结果显示:(1) Nic明显减轻小鼠心重指数和纤维化病理改变。(2) Nic延长心肌肥厚小鼠缺氧后存活时间(P<0.05或P<0.01),增强整体动物在缺氧条件下的生存能力。(3) Nic提高肥厚心肌组织中SOD和GSH-Px活性,减少MDA含量,其中尤以中、高剂量组效果明显(P<0.05或P<0.01)。二、尼可地尔对甲状腺素诱发大鼠心肌肥厚的保护作用采用连续腹腔注射左旋甲状腺素(L-thyroxine,L-thy)建立大鼠心肌肥厚模型,检测Nic对大鼠心肌线粒体组织超氧化物歧化酶(SOD)、丙二醛(MDA)、Na+,K+-ATPase和Ca2+-ATPase活性的影响,同时使用KATP通道阻滞剂格列苯脲(glibenclamide,Glib),观察其对Nic作用的影响。研究发现:Nic能提高心肌线粒体组织中SOD活性,减少MDA含量(P<0.05或P<0.01);Nic能增加心肌线粒体组织中Na+,K+-ATPase和Ca2+-ATPase活性(P<0.01)。Glib可不同程度地减弱Nic对心肌线粒体组织的上述作用。第二部分尼可地尔抗心肌肥厚的机制研究一、尼可地尔对豚鼠心室乳头状肌动作电位的影响本节以豚鼠心室乳头状肌为研究对象,采用标准微电极技术记录心肌动作电位,研究Nic对心肌细胞动作电位的作用。本实验检测了在不同浓度条件下,Nic对乳头状肌动作电位幅值(action potential amplitude,APA)、动作电位时程(action potential duration,APD)APD50及APD90的影响;冲洗换液后给予一定量的KATP通道阻滞剂Glib后,同样在上述不同Nic浓度条件下,观察Nic对AP作用的变化。结果发现:Nic可浓度依赖性的缩短动作电位的APD50及APD90,其中对APD90的影响尤为显著(P<0.01),对APA无显著作用(P>0.05);当加入Glib后,心室乳头状肌细胞动作电位APD50和APD90的缩短率均显著减小(P<0.01),这表明Glib可明显减弱Nic对APD50及APD90的作用。二、尼可地尔对心肌肥厚大鼠心肌细胞凋亡的影响采用腹腔注射L-thy建立大鼠心肌肥厚模型。用免疫组织化学染色法观察Nic对Fas和Bcl-2蛋白表达的影响。结果表明:与模型组比较,Nic可明显抑制肥厚心肌组织Fas蛋白的表达(P<0.01),促进Bcl-2蛋白的表达(P<0.05或P<0.01)。与单用Nic组相比,合用Glib后Fas蛋白表达明显上调(P<0.01),而Bcl-2蛋白表达明显下调(P<0.01)。三、尼可地尔对心肌肥厚大鼠c-fos、c-jun和TGF-β1表达的影响采用定磷法测定大鼠心肌组织CaN活力,观察Nic对其活力的影响;用半定量RT-PCR法检测Nic对心肌组织增殖相关基因c-fos、c-jun和转化生长因子β(1TGF-β1)mRNA表达的影响;用ELISA法检测Nic对心肌组织TGF-β1蛋白表达的影响。结果显示:Nic可显著降低心肌肥厚大鼠心肌组织CaN活力(P<0.01),减少c-fos、c-jun及TGF-β1mRNA含量(P<0.05或P<0.01),下调TGF-β1蛋白的表达(P<0.01);Glib可减弱Nic的上述作用(P<0.05或P<0.01)。结论:1. Nic能显著抑制Iso引起的小鼠心肌肥厚,此作用可能与Nic增强心肌组织清除氧自由基能力、减少脂质过氧化物生成等有关。2. Nic能显著抑制L-thy诱发的大鼠心肌肥厚,此作用可能与Nic抑制心肌细胞线粒体脂质过氧化,显著改善能量代谢等有关。3. Nic可浓度依赖性的缩短心肌细胞动作电位的APD50及APD90,其中对APD90的作用更为显著,Glib可减弱Nic对APD的作用,提示Nic缩短APD50及APD90的作用与其开放钾通道关系密切。4. Nic可抑制Fas蛋白的表达而增加Bcl-2蛋白的表达,表明Nic具有抑制心肌肥厚时心肌细胞凋亡的作用。5. Nic可降低心肌肥厚大鼠心肌组织CaN活力,减少c-fos、c-jun及TGF-β1 mRNA含量,下调TGF-β1蛋白表达,Glib可明显减弱Nic的上述作用,提示Nic可通过开放KATP通道继而抑制相关增殖基因表达而发挥心肌保护作用。更多还原

【Abstract】 Cardiac hypertrophy, the prelude of heart failure, could increase the incidence rates of cardiac arrhythmia, myocardial infarction and sudden death, which was generally accepted as an independent risk factor of cardiovascular disease. Nicorandil was an ATP-sensitive potassium channel opener with a calcium blocking effect. It was confirmed that nicorandil could not only increase coronary blood, decrease oxygen consumption, but also reduce preload and afterload of heart, in the meanwhile, without accompanying tachycardia reflexly. With the techniques of immunology, electro- physiology and molecular biology, we studied the effects and its mechanism of nicorandil on cardiac hypertrophy through mice and rats in vivo and guinea pig papillary muscles in vitro. To clarify the mechanism of its action, we investigated the degree of reactive oxidative damages, energy metabolism, action potential, cell apoptosis,intracellular calcium overload and related gene expression in myocardial tissue of cardiac hypertrophy when given nicorandil.Part I The effects of nicorandil on cardiac hypertrophy1. Protective effects of nicorandil on cardiac hypertrophy induced by isoproterenol in miceIn this study, cardiac hypertrophy of mice was induced by continuous subcutaneous administration of isoproterenol (2mg·kg-1). The cardiac indexes, morphological changes, the survival time by close normobaric hypoxia, the activities of superoxide dismutase(SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined. The results showed that nicorandil remarkably reduced the cardiac indexes, obviously prolonged the survival time of mice in the close normobaric hypoxia (P<0.05 or P<0.01)and significantly increased the activities of SOD,GSH-Px and decreased the content of MDA in mice, especially in middle and high dose(P<0.05 or P<0.01).2. Protective effects of nicorandil on cardiac hypertrophy induced by L-thyroxine in rats.Cardiac hypertrophy of rats was established by administration of ip L-thyroxine. The activities of superoxide dismutase (SOD), the content of malondialdehyde (MDA), Na+,K+-ATPase and Ca2+-ATPase in the mitochondria of cardiac tissue were determined. Glibenclamide, a blocker of the ATP-sensitive potassium channel, was used to examine the changed effects of nicorandil on cardiac hypertrophy. The results showed that nicorandil significantly increased the activities of SOD,Na+,K+-ATPase, Ca2+-ATPase (P<0.01) and decreased the content of MDA (P<0.05 or P<0.01)in the mitochondria of cardiac tissue.PartⅡThe action mechanism of nicorandil on preventing cardiac hypertrophy1. Effects of nicorandil on the action potential of cardiac tissue in guinea pig papillary muscles.The action potential of isolated guinea pig papillary muscles was determined by the standard microelectrode technique. Effects of nicorandil on the action potential and the action of glibenclamide, a blocker of the ATP-sensitive potassium channel, on nicorandil were observed. Under the different concentration (0.3、1、3、10、30μmol?L-1)of nicorandil, the effects of nicorandil on APA、APD50 and APD90 were examined. The whole procedure was duplicated after adding glibenclamide. The results showed that nicorandil could shorten APD50 and APD90 in a concentration dependent manner, especially on APD90(P<0.01), with little effect on APA. The shortening rates of APD50 and APD90 significantly decreased ( P<0.01 ) after using glibenclamide, which indicated that glibenclamide could remarkably attenuate the above effects of nicorandil on APD50 and APD90.2. Effects of nicorandil on cell apoptosis in myocardial tissue of cardiac hypertrophy Cardiac hypertrophy was established by administration of ip L-thyroxine. Expression of Fas and Bcl-2 protein was measured by the technique of immuno- histochemistry. The results indicated that nicorandil could obviously reduce the expression of Fas protein(P<0.01)and enhance the expression of Bcl-2 protein(P<0.05 or P<0.01). Furthermore, when glibenclamide, a blocker of the ATP- sensitive potassium channel, was also used in the meanwhile, compared with using corresponding nicorandil, the expression of Fas protein was remarkably increased (P<0.01)while the expression of Bcl-2 significantly decreased(P<0.01).3. Effects of nicorandil on the expression of c-fos、c-jun and TGF-β1 in myocardial tissue of cardiac hypertrophy.Effect of nicorandil on the activity of calcineurin(CaN) in myocardial tissue of cardiac hypertrophy was determined. Expression of c-fos, c-jun and TGF-β1 was examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR); TGF-β1 protein levels were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that nicorandil significantly decreased the activity of CaN(P<0.01), nicorandil could induce mRNA expression of c-fos、c-jun and TGF-β1 (P<0.05orP<0.01) and decrease protein expression of TGF-β1 (P<0.01). Glibenclamide could reverse the above effects of nicorandil(P<0.05 or P<0.01). In conclusion:1. Nicorandil relieves cardiac hypertrophy induced by isoproterenol in mice. Nicorandil can protect cardiac tissue, which may be involved in inhibiting lipid peroxidation and eliminating oxygen free radical.2. Nicorandil can prevent cardiac hypertrophy induced by L-thy, which may be related to inhibiting lipid peroxidation in the mitochondria of myocytes and ameliorating energy metabolism.3. Nicorandil can shorten APD50 and APD90 in a concentration dependent manner. Glibenclamide can remarkably reverse the above effects of nicorandil, which indicate that its mechanism may be in correlation with opening potassium channel.4. Nicorandil can remarkably reduce the expression of Fas protein and enhance the expression of Bcl-2 protein, which indicate that nicorandil can inhibit cell apoptosis of hypertrophic myocardial.5. Nicorandil can decrease the activity of CaN, depress mRNA expression of c-fos、c-jun and TGF-β1 and reduce protein expression of TGF-β1 in myocardial tissue of cardiac hypertrophy. Glibenclamide can remarkably attenuate the above effects of nicorandil, which indicate that nicorandil can depress related gene expression by opening KATP channel.更多还原