阿托伐他汀对急性心肌梗死大鼠心肌炎症和纤维化反应相关Notch1与TGF-β-Smad信号通路的作用以及对冠心病患者血浆

发布时间：2018-08-31 19:39

【摘要】：背景:急性心肌梗死(Acute myocardial infarction,AMI)的大多数病因是不稳定动脉粥样硬化斑块破裂形成血栓,从而中断冠状动脉血流。而AMI发病起始和发展过程中大量炎症因子相互作用的参与是重要因素。尽管现在患者使用抗栓药物或者采用冠状动脉介入治疗方法能早期使血管再通或使心肌再灌注,但是很多患者还会发生不同程度的左室重构,甚至心力衰竭。其中受损或梗死心肌细胞释放炎症因子,进而引发的炎症反应在心肌重构和心肌纤维化中起到了重要作用。为抑制或减少斑块发生和发展,今后防治冠心病的关键环节之一是抑制炎性反应等不利因素,然而,针对这些病理环节的治疗,目前尚未有公认的药物和其他有效的方法。曾有报道显示阿托伐他汀在治疗冠心病等心血管病过程中也发挥抗炎、抗氧化和抗纤维化等多样化效应,但阿托伐他汀对AMI大鼠心肌炎症及纤维化发展过程是否具有有益作用,其是否影响AMI大鼠心功能和血流动力学状态尚待验证,阿托伐他汀否通过调控TGF-β-smad和notch1信号通路起到上述有益作用尚还未知。此外,冠心病患者血浆炎性和纤维化因子半乳糖凝集素-3(Galectin-3)变化和他汀对Galectin-3的作用以及心房颤动(Atrial Fibrillation,AF)患者射频消融(radiofrequency ablationradiofrequency,RFCA)前后Galectin-3变化均尚未见报道。目的:1、观察大鼠AMI建模后第一天心功能变化及建模后第5天阿托伐他汀或氯沙坦钾治疗对大鼠血浆炎症因子TNF-α,IL-1β的作用;2、观察阿托伐他汀或氯沙坦钾治疗对大鼠AMI建模后第14天心功能和血流动力学影响,以及对心肌细胞外基质金属蛋白酶mmp2、mmp9及其抑制物TIMP2蛋白表达和血浆心功能标志物BNP的变化;3、观察大鼠AMI建模后第28天,阿托伐他汀或氯沙坦钾治疗对大鼠的心功能和血流动力学影响,观察心肌胶原蛋白CollagenⅠ、CollagenⅢ和notch1、TGF-β1、Smad2、Smad7、Galectin-3蛋白表达及血浆BNP变化;观察阿托伐他汀或氯沙坦钾是否通过抑制notch1-TGF-β-smad信号通路抑制AMI大鼠心肌炎症和纤维化反应;4、观察阿托伐他汀或氯沙坦钾对AMI大鼠建模后第14天、28天心肌细胞结构和心肌胶原纤维的影响;5、临床试验:观察冠心病患者包括稳定心绞痛组(Stable angina pectoris,SAP)、不稳定心绞痛组(Unstable angina pectoris,UAP)和AMI患者血浆炎性因子Galectin-3变化及与病情严重程度的相关关系,观察阿托伐他汀80mg对AMI患者短期治疗前后Galectin-3变化;观察心房颤动(Atrial Fibrillation,AF)患者射频消融(radiofrequency ablationradiofrequency,RFCA)前后Galectin-3变化。方法:1、大鼠分为四组:对照组(假手术组):单纯分离前降支,未行结扎;心肌梗死组(AMI建模组):单纯结扎冠状动脉前降支,建模后无药物治疗;他汀组:前降支结扎+阿托伐他汀(10mg/kq/d)治疗;氯沙坦组:前降支结扎+氯沙坦钾治疗(5mg/kq/d)。除对照组大鼠外,其余组入选大鼠EF均≤50%,评价建模后各组大鼠第14天、28天心功能和血流动力学变化,并观察AMI后大鼠心肌细胞炎症、纤维化发展变化过程。2、采用Elisa方法观察大鼠AMI建模第5天血浆炎症因子TNF-α、IL-1β变化和建模后第14天、28天血浆BNP变化。3、采用生物学Q-PCR、western blot技术和/或免疫组化方法检测大鼠AMI建模后心肌细胞炎症因子TNF-α、IL-1β、Galectin-3、心肌胶原蛋白CollagenⅠ、CollagenⅢ、金属基质蛋白酶mmp2、mmp9及其抑制物TIMP2和信号通路蛋白notch1、TGF-β1、Smad2、Smad7,观察其变化。4、采用HE染色和MASSON染色观察大鼠AMI建模后第14天、28天不同组大鼠心肌细胞结构变化和心肌胶原纤维变化。5、临床试验:采用Elisa方法测定不同冠心病类型患者和AF患者RFCA前后血浆炎性因子Galectin-3含量。AMI患者经皮冠状动脉介入(percutaneous coronary intervention,PCI)术前给予80mg阿托伐他汀治疗,用Elisa方法测定对AMI患者PCI前后Galectin-3变化;观察冠心病患者血浆Galectin-3水平与LVEF相关性。结果:1、大鼠AMI建模后24h心功能明显下降,EF、FS值低于正常对照组;大鼠AMI建模后第5天血浆炎症因子TNF-α、IL-1β升高,用阿托伐他汀或氯沙坦钾治疗后明显下降(p0.05);2、大鼠AMI建模后第14天,阿托伐他汀组或氯沙坦钾治疗组心功能均较AMI未治疗组提高,血流动力学指标dp/dt Max、dp/dtMin等明显好转(p0.05);应用Q-PCR和/或WesternBlot技术和/或免疫组化方法所检测结果显示,阿托伐他汀组或氯沙坦治疗组大鼠心肌mmp2、mmp9、TNF-α、IL-1β蛋白表达降低,TIMP2蛋白表达增加(p0.05);建模14天后血浆心功能标志物BNP升高,阿托伐他汀组或氯沙坦治疗组大鼠血浆BNP降低(p0.05)。3、大鼠AMI建模28天后,阿托伐他汀组或氯沙坦治疗组心功能均较AMI未治疗组提高,血流动力学指标dp/dt max、dp/dt min等明显好转(p0.05);应用Q-PCR和/或WesternBlot技术和/或免疫组化方法所检测结果显示,阿托伐他汀或氯沙坦抑制大鼠心肌胶原蛋白CollagenⅠ、CollagenⅢ表达(p0.05),并抑制notch1、TGF-β1、Smad2、Galectin-3蛋白表达,增加蛋白Smad7表达(p0.05);阿托伐他汀或氯沙坦通过抑制notch1-TGF-β-smad信号通路抑制AMI大鼠心肌炎症和纤维化反应;建模28天后血浆心功能标志物BNP升高,阿托伐他汀组和氯沙坦治疗组大鼠血浆BNP降低(p0.05);4、AMI大鼠建模14天和28天后,阿托伐他汀组或氯沙坦治疗组心肌细胞结构紊乱现象好转,炎症细胞减少,心肌胶原纤维组织范围减少;5、临床试验显示:AMI组患者血浆Galectin-3含量高于UAP组(p0.05),UAP组患者Galectin-3含量高于SAP组(p0.05);冠状动脉多支病变组患者Galectin-3水平高于单只病变组(p0.05);AF行RFCA术后转复为窦性心律患者Galectin-3水平较术前有所下降,但前后比较无差别(p0.05);AMI患者PCI术前给予80mg阿托伐他汀治疗可降低血浆Galectin-3水平,但前后比较无差别;Galectin-3水平与冠心病患者左室射血分数(Left ventricular ejection fraction,LVEF)值成负相关性(r=-0.405,p0.05)。结论:1、大鼠AMI建模后心肌发生炎症、纤维化反应、炎症因子TNF-α、IL-1β、Galectin-3升高和纤维化因子mmp2、mmp9、CollagenⅠ、CollagenⅢ升高,同时心功能受损,心室发生重构,而阿托伐他汀或氯沙坦可通过notch1-TGF-β-smads通路抑制心肌炎症及纤维化,减少炎症及纤维化指标表达,减轻心肌重构,在一定程度上对心功能和血流动力学起到改善作用。2、阿托伐他汀或氯沙坦可改善AMI大鼠建模后心肌细胞坏死和结构紊乱,并较少心肌胶原纤维范围;3、临床试验显示,冠心病患者血浆中炎症和纤维化因子Galectin-3含量与心肌缺血、损伤严重程度相关,随着心肌缺血、损伤加重,Galectin-3含量逐步升高;Galectin-3与冠心病患者LVEF成负性相关;4、Galectin-3是心肌炎症和纤维化因子,AF患者Galectin-3水平升高与心房纤维化有关,RFCA术后Galectin-3水平有所下降,但术前术后比较无差别,可能与样本量较少有关;阿托伐他汀降低AMI患者PCI术后Galectin-3水平,但前后比较无差别,可能与服药次数较少有关。
[Abstract]:BACKGROUND: Most of the causes of acute myocardial infarction (AMI) are unstable atherosclerotic plaque rupture, resulting in thrombosis, which interrupts coronary flow. The involvement of a large number of inflammatory factors in the initiation and development of AMI is an important factor, although patients now use antithrombotic drugs or take them. Coronary artery interventional therapy can early revascularize or reperfusion myocardium, but many patients also have different degrees of left ventricular remodeling, and even heart failure. Injured or infarcted myocardial cells release inflammatory factors, which in turn trigger inflammatory reactions in myocardial remodeling and myocardial fibrosis play an important role. To prevent or reduce the occurrence and development of plaque, one of the key links in the prevention and treatment of coronary heart disease in the future is to inhibit inflammatory reaction and other adverse factors. However, there are no recognized drugs and other effective methods for the treatment of these pathological links. It has been reported that atorvastatin also plays an anti-inflammatory role in the treatment of cardiovascular diseases such as coronary heart disease. However, whether atorvastatin has beneficial effects on the development of myocardial inflammation and fibrosis in AMI rats, and whether it affects cardiac function and hemodynamic status in AMI rats remains to be verified. Whether atorvastatin plays the above beneficial role by regulating TGF-beta-smad and Notch1 Signaling pathways remains to be determined. In addition, the changes of plasma inflammatory and fibrotic factors galectin-3 (Galectin-3) and the effect of statin on Galectin-3 in patients with coronary heart disease and the changes of Galectin-3 in patients with atrial fibrillation (AF) before and after radiofrequency ablation (RFCA) have not been reported. Changes of cardiac function on the first day after modeling and effects of atorvastatin or losartan potassium on plasma inflammatory factors TNF-a and IL-1 beta in rats on the fifth day after modeling; 2. Effects of atorvastatin or losartan potassium on cardiac function and hemodynamics and extracellular matrix metalloproteinase mmp2, MMP9 in rats with AMI on the fourteenth day after modeling were observed. The expression of TIMP2 protein and the changes of plasma cardiac function marker BNP were observed. 3. The effects of atorvastatin or losartan potassium on cardiac function and hemodynamics were observed on 28 days after AMI in rats, and the expressions of collagen I, Collagen III and notch1, TGF-beta 1, Smad2, Smad7, Galectin-3 protein and plasma BN were observed. To observe whether atorvastatin or losartan potassium can inhibit myocardial inflammation and fibrosis in rats with AMI by inhibiting notch1-TGF-beta-smad signaling pathway; 4. To observe the effects of atorvastatin or losartan potassium on myocardial cell structure and myocardial collagen fibers in rats with AMI on the 14th and 28th days after modeling; 5. Clinical trial: To observe the patients with coronary heart disease. The changes of plasma inflammatory factor Galectin-3 in patients with stable angina pectoris (SAP), unstable angina pectoris (UAP) and AMI and their correlation with the severity of the disease were observed. The changes of Galectin-3 in patients with AMI before and after treatment with atorvastatin 80 mg were observed. Methods: 1. Rats were divided into four groups: control group (sham operation group): isolated anterior descending branch without ligation; myocardial infarction group (AMI modeling group): ligation of anterior descending branch of coronary artery without drug therapy; statin group: ligation of anterior descending branch + atrovir Statin (10mg/kq/d) treatment; Losartan group: anterior descending branch ligation + losartan potassium treatment (5mg/kq/d). Except for the control group rats, the EF of the other groups were all less than 50%. The changes of cardiac function and hemodynamics were evaluated on the 14th and 28th day after modeling, and the changes of myocardial inflammation and fibrosis were observed after AMI. 2. Elisa method was used. The changes of plasma inflammatory factors TNF-a, IL-1 beta and BNP were observed on the fifth day after AMI modeling and on the fourteenth and twenty-eighth days after AMI modeling. 3. Biological Q-PCR, Western blot and/or immunohistochemical methods were used to detect the levels of inflammatory factors TNF-a, IL-1 beta, Galectin-3, Collagen I, Collagen III and metal matrix in myocardial cells of rats after AMI modeling. Proteinase MMP 2, MMP 9 and their inhibitors TIMP 2 and signal pathway proteins notch 1, TGF - beta 1, Smad 2, Smad 7 were observed. 4. HE staining and MASSON staining were used to observe the changes of myocardial cell structure and myocardial collagen fibers in different groups of rats on the 14th and 28th days after AMI modeling. 5. Clinical trial: Elisa method was used to determine different types of coronary heart disease. Levels of plasma inflammatory factor Galectin-3 in patients with type I and AF before and after RFCA were measured by Elisa method. Results: 1, 24 hours after AMI, the cardiac function of rats decreased significantly, EF, FS values were lower than the normal control group; 5 days after AMI, the plasma inflammatory factors TNF-a, IL-1 beta increased, and significantly decreased after treatment with atorvastatin or losartan potassium (p0.05); 2, 14 days after AMI, the cardiac function of atorvastatin group or losartan potassium treatment group were significantly lower than that of AMI untreated rats. The results of Q-PCR and/or Western Blot and/or immunohistochemistry showed that the expression of mmp-2, mmp-9, TNF-alpha, IL-1 beta protein in myocardium of rats in atorvastatin group or losartan treatment group decreased, and the expression of TIMP-2 protein increased 14 days after modeling (p0.05). Plasma BNP was elevated, plasma BNP was decreased in atorvastatin group or losartan treatment group (p0.05). After 28 days of AMI modeling, cardiac function in atorvastatin group or losartan treatment group was improved, and hemodynamic indexes such as dp/dt max, dp/dt min were improved significantly (p0.05). And/or immunohistochemical staining showed that atorvastatin or losartan inhibited the expression of collagen Collagen I and Collagen III in rat myocardium (p0.05), inhibited the expression of notch 1, TGF-beta 1, Smad2, and Galectin-3, and increased the expression of Smad7 (p0.05); atorvastatin or losartan inhibited the notch 1-TGF-beta-smad signaling pathway by inhibiting the expression of Smad7 (p0.05). Myocardial inflammation and fibrosis in AMI rats were induced; plasma BNP increased after 28 days of modeling, plasma BNP decreased in atorvastatin group and losartan group (p0.05); 4. After 14 and 28 days of modeling, the structural disorder of myocardial cells in atorvastatin group or losartan group improved, inflammatory cells decreased, and myocardial glue decreased. 5, clinical trials showed that the level of Galectin-3 in plasma of AMI patients was higher than that of UAP patients (p0.05), and the level of Galectin-3 in UAP patients was higher than that of SAP patients (p0.05); the level of Galectin-3 in coronary artery disease group was higher than that of single lesion group (p0.05); the level of Galectin-3 in AF patients converted to sinus rhythm after RFCA was higher than that before operation. The level of Galectin-3 was negatively correlated with the left ventricular ejection fraction (LVEF) in patients with coronary artery disease (r = - 0.405, P 0.05). Conclusion: 1. Rat AMI was established. Inflammation, fibrosis, elevation of inflammatory factors TNF-alpha, IL-1beta, Galectin-3 and fibrosis factors mmp2, mmp9, Collagen I and Collagen III occur in the myocardium after cardiac infarction. Atorvastatin or losartan can inhibit myocardial inflammation and fibrosis through notch1-TGF-beta-smads pathway and reduce inflammation and fibrosis. Atorvastatin or losartan can improve myocardial cell necrosis and structural disorder in AMI rats, and less myocardial collagen fibers range; 3. Clinical trials showed that inflammation and fibrosis factor Gale in plasma of patients with coronary heart disease. The content of ctin-3 was correlated with the severity of myocardial ischemia and injury, with the increase of myocardial ischemia, the content of Galectin-3 increased gradually; Galectin-3 was negatively correlated with LVEF in patients with coronary heart disease; 4, Galectin-3 was a factor of myocardial inflammation and fibrosis; the elevation of Galectin-3 in AF patients was related to atrial fibrosis, and the level of Galectin-3 decreased after RFCA. Atorvastatin decreased the level of Galectin-3 in patients with AMI after PCI, but there was no difference before and after PCI, which may be related to the fewer times of taking drugs.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R542.22

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