【作者】 李永胜；

【导师】 郑智；

【作者基本信息】 华中科技大学 ， 急诊医学， 2007， 博士

【摘要】 心肌肥厚是心脏对多种疾病包括高血压、机械压力负荷、心肌梗塞、心律失常、内分泌机能紊乱以及心肌收缩蛋白基因突变而产生的适应性病理反应。在疾病早期,因心脏作功增加可引起代偿性心肌肥厚反应。持续性的心肌肥厚能够引起扩张性心肌病、心律失常、心力衰竭甚至猝死。心肌肥厚是引起心血管疾病发病率和病死率的独立危险因素。研究发现在心肌肥厚时左心室重量普遍增高,心血管事件发生率是无心肌肥厚者的2-4倍。心肌肥厚不仅是一个病理生理过程,而更是一个影响其短期和长期预后的失代偿性变化。它的不可逆性进展可以直接导致心力衰竭。目前临床上用于治疗心肌肥厚的治疗药物主要有血管紧张素转化酶抑制剂、血管紧张素受体拮抗剂和钙离子通道阻滞剂,但皆因存在不同程度的副作用而使其临床应用受到一定限制。而心肌肥厚的治疗是一个长期的过程,因此寻找一些疗效好而毒副作用低的药物显得十分必要。丹参酮ⅡA是中药丹参的主要脂溶性成分。目前已发现丹参酮ⅡA可以减轻、逆转高血压左心室肥厚,但其具体作用机制尚未阐明。本研究拟在腹主动脉缩窄的高血压大鼠上构建心肌肥厚模型,将丹参酮ⅡA与经典的抗心肌肥厚药物血管紧张素1型受体拮抗剂――缬沙坦比较,以探讨丹参酮ⅡA逆转心肌肥厚的分子生物学机制。第一部分丹参酮ⅡA对腹主动脉缩窄高血压大鼠心肌肥厚超声影像学及病理形态学的影响目的:研究丹参酮ⅡA对腹主动脉缩窄术后大鼠左室心肌肥厚的作用。方法:40只健康SD大鼠,行腹主动脉缩窄术建立高血压左室心肌肥厚模型,另取8只大鼠行假手术。术后4周将手术大鼠随机分为心肌肥厚组、丹参酮低剂量组(10mg/Kg/d腹腔注射)、丹参酮高剂量组(20mg/Kg/d腹腔注射)及缬沙坦组(10mg/Kg/d灌胃)。用药8周后通过超声心动图测定左室后壁、室间隔的厚度及左室射血分数(EF);取左心室组织检测左心室质量指数(left ventricular mass index,LVMI)、病理切片HE染色测量心肌纤维直径(myocardial fiber dimension,MFD)。结果:1.心肌肥厚组、丹参酮组(高、低剂量)的血压显著高于假手术组及缬沙坦组(p﹤0.01),丹参酮高、低剂量组及心肌肥厚组之间无统计学差异(p﹥0.05),缬沙坦组的血压值降至正常、但仍明显高于假手术组(p﹤0.05)。2.丹参酮高、低剂量组和缬沙坦组的室间隔厚度均高于假手术组(p﹤0.05),显著低于心肌肥厚组(p﹤0.01);丹参酮两组的室间隔厚度无差异(p﹥0.05),但高于缬沙坦组(p﹤0.05)。3.丹参酮低剂量、高剂量组及缬沙坦组之间左室后壁值数据没有显著性差异(P>0.05),此三组左室后壁值均明显低于心肌肥厚组(p﹤0.01),但尚未恢复至正常水平(p﹤0.05)。4.所有各组的EF值均在正常值范围(>60%),尚未表现出心功能衰竭。但心肌肥厚组的EF值小于其他各组(p﹤0.05),丹参酮组的EF也小于缬沙坦组(p﹤0.05)。5.丹参酮低剂量和高剂量组间LVMI和MFD没有显著性差异(P>0.05),缬沙坦组和丹参酮各组的LVMI、MFD均高于假手术组(p﹤0.05),显著低于心肌肥厚组(p﹤0.01);丹参酮两组的LVMI、MFD均高于缬沙坦组(p﹤0.05)。结论:丹参酮Ⅱ-A对心肌肥厚的逆转作用是非血压依从性的。丹参酮Ⅱ-A与血管紧张素Ⅱ1型受体拮抗剂缬沙坦均可阻止、逆转高血压心肌肥厚的发展,但其作用弱于缬沙坦。第二部分丹参酮ⅡA对腹主动脉缩窄大鼠肥厚心肌血管紧张素受体的影响目的:通过研究丹参酮ⅡA对腹主动脉大鼠肥厚心肌血管紧张素受体及细胞内游离钙离子的影响,探讨丹参酮ⅡA逆转高血压左心室肥厚的分子生物学机制。方法:取各组大鼠的左心室组织,采用逆转录-聚合酶链式反应(RT-PCR)、免疫印记法(Western blotting)分别检测AT1、AT2受体mRNA和蛋白的表达水平。利用激光共聚焦显微镜测定心肌细胞内Ca2+浓度的变化。结果:1.心肌肥厚组的AT1 mRNA和蛋白的表达显著高于其他各组(p﹤0.01);丹参酮ⅡA高、低剂量组间无差异(p﹥0.05),丹参酮ⅡA组高于缬沙坦组(P﹤0.05);丹参酮ⅡA组和缬沙坦组的AT1基因表达均未降至假手术组水平(p﹤0.05)。2.缬沙坦组的AT2 mRNA和蛋白表达水平较其他各组升高(p﹤0.05),其他各组之间无统计学差异(p﹥0.05)。3.心肌肥厚组细胞内[Ca2+]i明显高于其他各组(p﹤0.01);丹参酮高剂量组与假手术组之间无统计学差异(p﹥0.05);缬沙坦组和丹参酮低剂量组仍高于假手术组和丹参酮高剂量组(p﹤0.05);丹参酮低剂量组细胞内钙浓度与缬沙坦组之间无差异(p﹥0.05)。结论:丹参酮ⅡA和缬沙坦均可通过下调AT1基因表达、阻止心肌细胞的钙离子内流发挥逆转心肌肥厚的作用,AT2有可能参与了缬沙坦降低血压、逆转心肌肥厚的作用。第三部分丹参酮IIA对腹主动脉缩窄高血压大鼠肥厚心肌一氧化氮合酶的影响目的:通过研究丹参酮ⅡA对腹主动脉缩窄大鼠肥厚心肌一氧化氮合酶的影响,探讨丹参酮ⅡA逆转高血压左心室肥厚的分子生物学机制。方法:取各组大鼠的左心室组织,利用硝酸还原酶法测定心肌组织的NO含量,采用逆转录-聚合酶链式反应(RT-PCR)、免疫印记法(Western blotting)分别检测eNOS的基因表达水平和蛋白激酶C(PKC)的活性。结果:1.假手术组的心肌组织NO含量明显高于其他各组(p﹤0.01);丹参酮高、低剂量组与缬沙坦组之间无统计学差异(p﹥0.05),但此三组的NO含量明显高于心肌肥厚组(p﹤0.01)。2.心肌肥厚组的eNOS mRNA和蛋白表达水平明显低于其他各组(p﹤0.01),缬沙坦组低于假手术组和丹参酮组(p﹤0.05),丹参酮高、低剂量组与假手术组之间无差异(p﹥0.05)。3.心肌肥厚组的PKC蛋白水平明显高于其他各组(p﹤0.01),缬沙坦组低于假手术组和丹参酮组(p﹤0.05),丹参酮高、低剂量组与假手术组之间无差异(p﹥0.05)。结论:心肌局部的NO/NOS系统与心肌肥厚的病理过程密切相关。丹参酮ⅡA可能通过抑制PKC的蛋白活性、促进心肌局部eNOS的基因表达和内源性NO的产生,发挥其逆转心肌肥厚的药理作用。更多还原

【Abstract】 Cardiac hypertrophy is an adaptive response of the heart to virtually all forms of cardiac diseases, including those arising from hypertension, mechanical load, myocardial infarction ,cardiac arrhythmias, endocrine disorders, and genetic mutations in cardiac contractile protein genes. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, hypertrophy can lead to dilated cardiomyopathy, heart failure and sudden death. cardiac hypertrophy was an absolute and dangerous factor that affected mortality and morbidity for cardiovascular diseases.Left ventricular weight rise in patients with cardiac hypertrophy. Cardiac hypertrophy was not only a pathophysiological process, but also a discompensatory change that affected short-term and long-term prognosis. Nowdays drugs for curing cardiac hypertrophy mostly have angiotenesin converting enzyme inhibitors ,angiotensin receptor blocker and calcium channel antagonist. But these drugs have their limit for some adverse reaction. So it is important to look for several drugs which have good therapeutic effect and little side effect in the long-term cure for myocardial hypertrophy.TanshinoneⅡA is the main lipid soluble component of danshen, a kind of traditional Chinese drug. Now it has been found that tanshinoneⅡA could alleviate and reverse left ventricular hypertrophy causing by hypertension, but its mechanism is still unknown.In this study, we would construct the model of myocardial hypertrophy in hypertensive rats caused by abdominal aorta constriction and compare tanshinoneⅡA with an angiotensin receptor inhibitor-valsartan, a classical drug for resisting cardiac hypertrophy to explore the molecular biological mechanism for tanshinoneⅡA reversing myocardial hypertrophy. Part one : the ultrasound imageological and pathomorphological effect of tanshinoneⅡA on hypertrophic myocardium due to abdominal aorta stenosis in ratsObjective: To study the effect of tanshinoneⅡA on hypertrophic myocardium caused by abdominal aorta stenosis in rats.Methods: Taking 40 rats who were suffered abdominal aorta constriction and 8 rats who were suffered artificial surgery. 4 weeks later, these rats would be divided into 4 groups: myocardial hypertrophy, low dose tanshinoneⅡA treatment (10mg/kg/d peritoneal injection), high dose tanshinoneⅡA treatment (20mg/kg/d peritoneal injection) and valsartan treatment (10mg/kg/d intragastric administration). After accepting therapy for 8 weeks, to measure the thickness of posterior ventricular wall and interventricular septum and left ventricular ejection fraction(EF) by echocardiogram ; then to take the tissue of left ventricle to detect left ventricular mass index ( LVMI) and myocardial fiber dimension (MFD) by pathological section and HE stain.Results :1. The blood pressure in the group of myocardial hypertrophy and tanshinoneⅡA treatment (low and high dose) was obviously higher than that in the group of artificial surgery and valsartan treatment (p<0.01). But it was not different between myocardial hypertrophy group and tanshinoneⅡA treatment (low and high dose) groups (p>0.05).2. The thickness of interventricular septum in the groups of tanshinoneⅡA and valsartan was more than that in artificial surgery (p<0.05), but less than that in myocardial hypertrophy. And two tanshinoneⅡA treatment groups had similar result(p>0.05), but they both were thicker than valsartan treatment group.3. There were not significant difference about thickness of posterior left ventricle among two tanshinoneⅡA treatment groups and valsartan treatment group(p<0.05). They were all obviously less than that in myocardial hypertrophy group(p<0.01), but did not get to normal lever(p<0.05).4. The EF value in all groups were in normal range(>60%). Though it was not found heart failure in myocardial hypertrophy group, the EF value in this group were less than that in other groups(p<0.05). And EF value in tanshinoneⅡA groups was less than that in valsartan group(p<0.05).5. There were not significantly different in LVMI and MFD between two tanshinoneⅡA treatment groups(p>0.05). MFD in valsartan group and two tanshinoneⅡA groups were obviously less than that in myocardial hypertrophy group(p<0.01) and more than that in artificial surgery group(p<0.05); and two tanshinoneⅡA groups more than valsartan group(p<0.05).Conclusion: The effect of tanshinoneⅡA on myocardial hypertrophy was not dependent on blood pressure. As angiotensinⅡreceptor antagonist-valsartan did , tanshinoneⅡA could block and reverse the development of myocardial hypertrophy, but its was a little weaker . Part two: The effect of tanshinoneⅡA on angiotensin receptor and free calcium ion in hypertrophic myocardium of rats due to abdominal aorta constriction.Objective: To explore the molecular biological mechanism about tanshinoneⅡA reversing left ventricular hypertrophy, it would be studied how tanshinoneⅡA gave an impact to angiotensin receptor and free calcium ion in hypertrophic myocardium of rats due to abdominal aorta constriction.Methods: To detect mRNA and protein’s expression of AT1 and AT2 receptors in left ventricular tissues of all groups by RT-PCR and Western blotting.Results:1. The mRNA and protein’s expression of AT1 receptor in myocardial hypertrophy group were obviously more than that in other groups(p<0.01); there were no difference within two tanshinoneⅡA groups(p>0.05), but tanshinoneⅡA groups was higher than valsartan group(p<0.05). The genic expression in these three groups did not recover to normal lever( artificial surgery group)(p<0.05).2. The mRNA and protein’s expression of AT2 in valsartan group was more than that in other groups(p<0.05), and there were no difference among the other groups(p>0.05).3. The concentration of calcium ion in myocardial hypertrophy group was highest( VS other groups, p<0.01); and that in the two tanshinoneⅡA groups and artificial surgery group had not statistically different(p>0.05) , but they were lower than that in valsartan group(p<0.05).Conclusion: TanshinoneⅡA and Valsartan both could resist the myocardial hypertrophy by downregulating the genic expression of AT1 receptor and blocking the inflow of calcium ion in cadiocyte. Moreover, AT2 receptor may participate the effect of lowering blood pressure and reversing hypertrophy as for valsartan. Part three: the effect of tashinone on the nitric oxide synthase in the hypertrophic cadiocyte of rats suffered abdominal aorta constriction.Objective: To explore the molecular biological mechanism for tanshinoneⅡA reversing left ventricular hypertrophy, it would be studying the effect of tashinone on the endothelial nitric oxide synthase( eNOS) in the hypertrophic cadiocyte of rats suffered abdominal aorta constriction.Methods: To detect the nitric oxide content by nitrate reductase, to detect the genic expression of eNOS by RT-PCR and to detect the activity of protein kinase C( PKC) by Western blotting.Results:1. The expressive lever of eNOSmRNA and protein in myocardial hypertrophy group was less than that in other groups(p<0.01). And valsartan group was less than tanshinoneⅡA groups and artificial surgery group(p<0.05), but there were no difference among the two tanshinoneⅡA groups and artificial surgery group(p>0.05).2. The expression of PKC in myocardial hypertrophy group was much more than that in other groups(p<0.01). valsartan group was less than artificial surgery group and tanshinoneⅡA groups(p<0.05), but there were no difference among two tanshinoneⅡA groups and artificial surgery group(p>0.05).Conclusion: NO/NOS system in local myocardium had close relationship with the pathological process for myocardial hypertrophy. TanshinoneⅡA could produce the pharmacological action to reverse myocardial hypertrophy by inhibiting the activity of PKC and promoting the genic expression of eNOS in local myocardium and the production of endogenous NO.更多还原