姜黄素在血管紧张素Ⅱ致大鼠心肌纤维化中的作用及机制研究

发布时间：2018-06-03 02:52

本文选题：心肌纤维化 + 血管紧张素II　；参考：《山西医科大学》2016年博士论文

【摘要】：研究背景:目前,各种心血管疾病如高血压、缺血性心脏病或心脏瓣膜病等导致的心肌纤维化是40%-50%的心脏衰竭患者左室舒张功能不全的主要原因。心肌纤维化的主要病理特点是纤维状胶原蛋白堆积,由血管周围向心肌组织间隙呈弥漫性分布。大量的胶原蛋白堆积,且得不到及时的降解,使心肌顺应性减弱,心室充盈受阻,最终导致心脏衰竭。大量的基础研究及临床试验表明,通过激活肾素-血管紧张素系统(renin-angiotensin system)产生的血管紧张素II(Angiotension II,Ang II),是高血压、心肌纤维化及心脏衰竭等发生的重要病理基础。随着对这一激素体液系统在心血管损伤方面的深入研究,以及针对Ang II在体内含量变化所采取的相应治疗手段的进展,人们对Ang II与各种心血管疾病的发生发展关系,有了深刻的理解和认识。众所周知,血管紧张素转换酶抑制剂(Angiotensin converting enzyme inhibitor,ACEI)与血管紧张素受体拮抗剂(Angiotensin receptor blocker,ARB)为临床治疗各类心血管病的常规药。通过ACEI减少Ang II的生成,以及利用ARB阻断Ang II的作用,从而达到治疗目的。但在实际临床应用中,这两类药物具有一定的副作用。ACEI可引起干咳,血管神经性水肿,具有高肾素敏感性,且肾动脉狭窄患者禁用;ARB可导致低血压,而且存在患者药物敏感性差异显著等,这些问题均限制了ACEI、ARB在一些患者中的使用。因此,寻找其它的具有减轻Ang II损伤作用,进而改善心血管患者预后的药物,并澄清其作用机制,具有重要的临床意义。姜黄素,是从植物姜黄的根茎中提取的黄色素,已被证实具有多种潜在的机体保护作用,如抗氧化,抗炎和抗纤维化等。因此,姜黄素受到人们极大的关注。本研究小组曾报道,在心梗引发心衰的大鼠模型中,姜黄素可以减小缺血后心脏梗死面积,进而改善心脏功能。然而,姜黄素在心衰发生发展过程中是否通过抗纤维化发挥保护作用,且是否与调制血管紧张素转换酶或血管紧张素II受体有关还不清楚。为此,本研究通过在大鼠缺血再灌注引起的心衰模型中,观察姜黄素对心肌纤维化的作用及机制。研究结果发现姜黄素可通过抑制心肌纤维化,进而改善心功能,且其发挥抗纤维化作用是通过双重调节血管紧张素转换酶与血管紧张素II受体实现的。为了进一步证实姜黄素对Ang II所致心肌纤维化的保护效应并研究其发挥保护作用的信号通路,我们利用微渗透泵人工直接输注Ang II引起高血压的大鼠模型。结果发现姜黄素的抗纤维化作用是通过下调AT1受体,上调AT2受体,进一步抑制TGFβ1/Smad信号通路,以及增强ACE2表达实现的。本实验研究包括以下两部分:第一部分姜黄素通过抑制血管紧张素转化酶及拮抗血管紧张素ⅡAT1受体发挥抗心肌纤维化作用研究目的:观察心梗后心肌纤维化时血管紧张素转化酶、血管紧张素II受体的变化特点,探讨姜黄素在血管紧张素II所致纤维化中的保护作用及可能机制。研究假设:已有的工作证实,Ang II是心肌纤维化、心衰发生的重要病理性基础。姜黄素具有抗纤维化作用。我们曾发现,姜黄素减小心肌缺血后的梗死范围,并能改善心衰患者的心功能。提示姜黄素减轻心梗后心肌纤维化可能与调节Ang II有关。这些结果将为我们进一步澄清姜黄素的心脏保护效应,提供工作基础。我们假设,姜黄素抑制纤维化过程改善心功能的保护效应是通过改变血管紧张素转化酶及血管紧张素Ⅱ受体的表达而实现的。方法:体内实验在缺血再灌注的大鼠模型进行,胃管给予姜黄素(150 mg/kg/day)。分别在再灌注后7天和42天,超声检测心脏功能及室壁厚度;提取心肌组织,利用Masson’s trichrome染色检测心肌纤维化;Western-blot进行蛋白定量分析,包括ACE、AT1R、AT2R;免疫组化染色观察各受体原位表达情况。体外实验在过氧化氢刺激下的H9c2细胞进行,检测AT1受体表达情况,细胞活性检测采用MTT及PI染色。结果:超声心动图结果表明,姜黄素组梗死前壁厚度明显厚于对照组。给予姜黄素的大鼠左室短轴缩短率和射血分数增加。Masson’s trichrome染色结果提示姜黄素不仅减少了纤维化心肌层的胶原沉积,也缩小了富含胶原的瘢痕范围,使具有收缩功能的心肌细胞增多。与对照组相比,姜黄素能显著降低ACE和AT1受体的蛋白表达水平,免疫组化染色显示姜黄素减少心肌组织和冠脉中ACE和AT1受体表达。同时,与对照组相比,姜黄素可显著增加AT2受体的表达。在培养的心肌细胞,姜黄素抑制由氧化剂引起的AT1受体的表达,并能增强细胞活性。结论:上述结果表明,再灌注过程中,口服姜黄素抑制ACE和AT1受体的表达;同时增强AT2受体表达。姜黄素的保护效应由AT1受体与AT2受体的比值减小可证明。与此发现相一致,缺血/再灌注的心室壁上胶原沉积、纤维化的组织减少,这也进一步证明姜黄素可以增加心室壁厚度。此外,左室短轴缩短率和射血分数增加,表明心脏收缩功能得到改善。体外实验表明,以氯沙坦处理的细胞作为对照,姜黄素抑制H2O2诱导的H9c2细胞AT1受体的表达,提高细胞的存活率,提示姜黄素能够通过调节AT1受体保护细胞免受氧化剂诱导的细胞损伤。第二部分姜黄素通过血管紧张素II受体/TGFβ1/Smad通路及血管紧张素转化酶2抑制心肌纤维化研究目的:探讨姜黄素抗心肌纤维化的作用机理。研究假设:实验研究的第一部分提示,Ang II具有致心肌纤维化作用,这些变化可被姜黄素所逆转。我们已发表的研究发现,姜黄素通过平衡胶原的合成与降解使心肌梗死后的心功能得到改善。在心肌纤维化的发生发展中,纤维化转化生长因子-β1(Transforming growth factor beta-1,TGFβ1)和Smads调定下游胶原为主要的信号转导通路。但姜黄素逆转组织纤维化是否有这一通路的参与目前仍不明确。另外,Ang II可激活其AT1和AT2两种受体;AT1具有缩血管,促进组织纤维化的作用;而活化AT2受体产生相反的保护效应。血管紧张素转化酶2(Angiotensin-converting enzyme 2,ACE2)与ACE同源,将Ang II转化成Ang(1-7)并发挥心肌保护效应。因此,我们假设,姜黄素具有调定AT1、AT2受体和ACE2的作用,进而抑制Ang II导致的心肌纤维化。方法:实验在背部皮下包埋植入微型渗透泵直接输注Ang II(500 ng/kg/min)的大鼠模型进行,胃管给予姜黄素(150 mg/kg/day)。无创监测输注Ang II后0,7,14,28天的血压变化。分别在2周和4周后,提取心肌组织,利用Masson染色检测心肌纤维化;Western-blot测定AT1、AT2、ACE2、TGFβ1、p-Smad2,3、I型胶原蛋白含量;免疫组化染色检测各受体和通道蛋白原位表达、巨噬细胞聚集及成纤维细胞增生情况。结果:Ang II输注7天后,与假手术(Sham)组相比,对照组(Control)血压明显升高,并且随着输注时间的延长逐渐升高,到21天时血压值达到高峰,且一直保持较高水平,直至实验结束。Ang II输注2周后,心肌纤维化开始发生变化,但不具有统计学差别;而在4周后,心肌纤维化出现了明显的组织病理学改变,表现为:心肌组织小动脉、小静脉以及毛细血管管周围纤维组织明显增生,结构紊乱;心肌组织中纤维化明显,呈条索或片状散在分布于心肌层。CD68标记的巨噬细胞和由a-平滑肌肌动蛋白(a-smooth muscle actin,a-SMA)标记的肌成纤维细胞,在组织中聚集明显增多,心肌组织Ⅰ型胶原含量增加。姜黄素对输注Ang II 2周时的组织变化无显著影响;但在4周时,与对照组相比,姜黄素显著减小了心肌纤维化的范围;巨噬细胞和活化的成纤维细胞聚集减少,I型胶原含量减少。与Sham组相比,对照组在AngⅡ慢性输注2周,AT1受体表达稍有增加,无统计学差别;AT2受体表达减少;但对照组在输注4周后,AT1受体表达明显增加,AT2受体表达减少;免疫组化染色检测也发现,心肌血管壁周围和心肌组织中的AT1受体表达增强和AT2受体表达减弱。与这些变化相一致,TGFβ1蛋白和p-Smad2、p-Smad3表达增加。此外,ACE2表达减少。姜黄素干预4周后,上述变化明显得到改善。表现为:AT1受体表达明显减少,AT2受体表达增加,免疫组化染色检测也证实,血管壁周围和心肌组织中的AT1受体减弱,AT2受体表达增强。与姜黄素抑制胶原蛋白减少纤维化作用一致,TGFβ1、p-Smad2、p-Smad3表达减少。此外,ACE2表达增加;免疫组化染色显示心肌组织中ACE2表达增强。结论:上述结果表明,Ang II具有显著的致心肌组织纤维化作用。Ang II可引起组织间隙巨噬细胞和成纤维细胞聚集增加,并伴有Ⅰ型胶原增生。这些现象在Ang II输注4周时最为明显。实验发现,姜黄素具有明显的抗心肌纤维化作用。探究其作用机制,参与Ang II致心肌纤维化的信号传导通路为血管紧张素II AT1/TGFβ1/Smad。Ang II通过改变AT1/AT2相对表达含量,进而使TGFβ1受体表达增加,并持续激活下游Smad通路,引起心肌细胞纤维化。姜黄素一方面平衡此通路中AT1、AT2的表达水平,另一方面还上调ACE2的表达,使大量Ang II降解为Ang1-7。同以往研究结果一致,姜黄素具有心血管保护作用。在本研究中,姜黄素可抑制Ang II直接刺激后的心脏损伤,为进一步研究姜黄素对心血管系统的保护作用提供直接、有力的证据。本研究展望我们的研究首次证明姜黄素抗纤维化作用与拮抗血管紧张素II受体和血管紧张素转化酶有关,与此同时,我们观察到姜黄素还可以降低血管紧张素II引起的高血压,这些结果为进一步明确姜黄素的心血管保护作用提供了直接的实验依据。姜黄素,作为一种植物提取物,在印度被人们作为调料每日食用,其药力动力学、安全性及疗效已被临床前研究及临床试验所证实。本实验结果还提示,药食两用的姜黄素有望与其它临床常规使用药物,如ACE抑制剂,AT1受体阻断剂或β受体阻断剂等联合用药,以减轻患者对其它药物的副作用,并为对此类药物禁忌的患者带来福音,进而提高药物在治疗高血压,缺血性心脏病和心衰等心血管疾病的疗效,并改善患者的预后。
[Abstract]:Background: myocardial fibrosis caused by various cardiovascular diseases such as hypertension, ischemic heart disease or heart valvular disease is the main cause of left ventricular diastolic dysfunction in patients with 40%-50% heart failure. The main pathological feature of myocardial fibrosis is fibrous collagen accumulation, from the perivascular to the interventricular space. Diffuse distribution. A large amount of collagen accumulation, and not timely degradation, makes myocardial compliance weakens, ventricular filling is blocked, and eventually leads to heart failure. A large number of basic research and clinical trials have shown that the angiotensin II (Angiotension II, Ang II) produced by the renin angiotensin system (renin-angiotensin system) is activated. It is an important pathological basis for hypertension, myocardial fibrosis and heart failure. With the deep research on the cardiovascular damage of this hormone body fluid system and the progress of the corresponding treatment for the changes in the content of Ang II in the body, people have a profound relationship with the development of Ang II and the development of various kinds of cardiovascular diseases. Understanding and understanding. It is known that the angiotensin converting enzyme inhibitor (Angiotensin converting enzyme inhibitor, ACEI) and angiotensin receptor antagonist (Angiotensin receptor blocker, ARB) are the conventional drugs for the clinical treatment of all kinds of cardiovascular diseases. In actual clinical applications, but in practical clinical applications, these two kinds of drugs have a certain side effect.ACEI can cause dry cough, angioedema, high renin sensitivity, and renal artery stenosis patients are prohibited; ARB can cause hypotension, and there is a difference in drug sensitivity in patients. These problems are all limited to ACEI, ARB in some It has important clinical significance to find other drugs that can reduce the Ang II damage and improve the prognosis of cardiovascular patients and clarify its mechanism. Curcumin, a yellow pigment extracted from the rhizomes of the plant Curcuma, has been proved to have a variety of potential protective effects, such as antioxidant and anti-inflammatory. Therefore, curcumin has been greatly concerned. This research team has reported that curcumin can reduce the infarct size and improve cardiac function in rats with heart failure caused by myocardial infarction. However, curcumin plays a protective role in the development of heart failure through anti fibrosis, and it is a protective effect of curcumin on the development of heart failure. It is not clear that the modulation of angiotensin converting enzyme or angiotensin II receptor is not clear. To this end, this study observed the effect and mechanism of curcumin on myocardial fibrosis in the model of heart failure induced by ischemia-reperfusion in rats. The results showed that curcumin could inhibit cardiac fibrosis and then improve cardiac function, and it exerts resistance to the heart. The effect of fibrosis is realized by double regulation of angiotensin converting enzyme and angiotensin II receptor. In order to further confirm the protective effect of curcumin on Ang II induced myocardial fibrosis and to study the signaling pathway of its protective effect, the rat model of hypertension induced by Ang II induced by artificial infusion of microosmotic pump was used. The anti fibrosis effect of curcumin was found by down regulating the AT1 receptor, up regulating the AT2 receptor, further inhibiting the TGF beta 1/Smad signaling pathway and enhancing the expression of ACE2. This experimental study includes the following two parts: Part 1: Curcumin exerts anti myocardial fiber by inhibiting angiotensin converting enzyme and antagonizing angiotensin II AT1 receptor Objective: To observe the changes of angiotensin converting enzyme (angiotensin converting enzyme) and angiotensin II receptor (angiotensin) receptor (angiotensin) receptor (angiotensin) in myocardial fibrosis after myocardial infarction (MI) and explore the protective effect and mechanism of curcumin in angiotensin II induced fibrosis. The research hypothesis: Ang II is an important pathological basis for myocardial fibrosis and heart failure. Curcumin has an anti fibrosis effect. We have found that curcumin reduces the infarct range after myocardial ischemia and improves cardiac function in patients with heart failure. It is suggested that curcumin may be related to the regulation of Ang II after myocardial infarction. These results will provide a basis for further clarification of the protective effect of curcumin on the heart. We hypothesized that the protective effect of curcumin on the improvement of cardiac function by inhibiting the process of fibrosis is achieved by changing the expression of angiotensin converting enzyme and angiotensin II receptor. Methods: in the rat model of ischemia reperfusion in vivo, the gastric tube was given curcumin (150 mg/kg/day), 7 days and 42 days after reperfusion, respectively. The cardiac function and wall thickness were detected; myocardial tissue was extracted and Masson 's trichrome staining was used to detect myocardial fibrosis; Western-blot was used for quantitative analysis of protein, including ACE, AT1R, AT2R; immunohistochemical staining was used to observe the expression of each receptor in situ. In vitro experiments were carried out in H9c2 cells stimulated by hydrogen peroxide to detect the expression of AT1 receptor. MTT and PI staining were used to detect the cell activity. Results: the results of echocardiography showed that the thickness of the anterior wall of the curcumin group was significantly thicker than that of the control group. The short axis shortening rate and the ejection fraction of curcumin increased by.Masson 's trichrome staining results suggested that curcumin not only reduced the collagen deposition of the fibrotic myocardium, but also reduced the collagen deposition. Cicatricial areas rich in collagen made the myocardial cells with contractile function increased. Compared with the control group, curcumin could significantly reduce the protein expression level of ACE and AT1 receptors. The immunohistochemical staining showed that curcumin reduced the expression of ACE and AT1 receptors in the myocardium and coronary artery. Meanwhile, curcumin could significantly increase the AT2 receptor compared with the control group. In cultured cardiac myocytes, curcumin inhibited the expression of AT1 receptor induced by oxidant and enhanced cell activity. Conclusion: the above results show that the oral curcumin inhibits the expression of ACE and AT1 receptors and increases the expression of AT2 receptor in the process of reperfusion. The protective effect of curcumin decreases from the ratio of AT1 receptor to AT2 receptor. It was proved that, in accordance with this discovery, the collagen deposition on the ventricular wall of ischemia / reperfusion and the fibrosis tissue decreased, which further demonstrated that curcumin could increase the thickness of the ventricular wall. In addition, the short axis shortening rate and the ejection fraction of the left ventricle increased, indicating that the systolic function of the heart was improved. In vitro experiments showed that the cells treated with losartan were used in vitro. Curcumin inhibits the expression of AT1 receptor in H9c2 cells induced by H2O2 and improves cell survival, suggesting that curcumin can protect cells from AT1 receptors to protect cells from oxidative induced cell damage. Second part curcumin can inhibit myocardial fibrosis through angiotensin II receptor /TGF beta 1/Smad pathway and angiotensin converting enzyme 2 Objective: To explore the mechanism of curcumin against myocardial fibrosis. Research hypothesis: the first part of the experimental study suggests that Ang II can induce myocardial fibrosis, and these changes can be reversed by curcumin. Our published studies have found that the cardiac function of curcumin can be modified by the synthesis and degradation of the balance collagen. Good. In the development of myocardial fibrosis, fibrotic transforming growth factor - beta 1 (Transforming growth factor beta-1, TGF beta 1) and Smads regulating downstream collagen are the main signal transduction pathways. However, the involvement of curcumin in tissue fibrosis is still unclear. In addition, Ang II activates two kinds of receptors of AT1 and AT2. AT1 has a vasoconstrictor that promotes tissue fibrosis, and activates the AT2 receptor to produce an opposite protective effect. Angiotensin converting enzyme 2 (Angiotensin-converting enzyme 2, ACE2) is homologous to ACE, transforming Ang II into Ang (1-7) and exerting myocardial protective effect. Therefore, we hypothesize that curcumin has the role of regulating AT1, AT2 receptors and ACE2. And then inhibit the myocardial fibrosis caused by Ang II. Methods: the rat model was implanted subcutaneously into the back subcutaneously implanted into the rat model of Ang II (500 ng/kg/min), and the gastric tube was given curcumin (150 mg/kg/day). The blood pressure changed after Ang II without invasive monitoring. The myocardial tissue was extracted after 2 weeks and 4 weeks, and Masso was extracted with Masso respectively. N staining was used to detect myocardial fibrosis; Western-blot was used to determine the content of AT1, AT2, ACE2, TGF beta 1, p-Smad2,3, I type collagen; immunohistochemical staining was used to detect the expression of each receptor and channel protein in situ, macrophage aggregation and fibroblast proliferation. Results: Ang II infusion 7 days later, compared with the sham group (Sham), the control group (Control) blood pressure was obviously increased High, and as the time of infusion increased gradually, the blood pressure reached a peak at 21 days and kept a high level until the end of the.Ang II infusion. After 2 weeks, the myocardial fibrosis began to change, but no statistical difference was found; and after 4 weeks, the myocardial fibrosis appeared obvious histopathological changes, manifested as: myocardium Tissue arterioles, small veins and capillary tubes were obviously proliferated and disorganized around the capillary tube, and the fibrosis was obvious in the myocardium. The myofibroblast and myofibroblast marked by.CD68 and a- muscle actin (a-Smooth muscle actin, a-SMA) were marked in the myocardium. In addition, the content of type I collagen in myocardial tissue increased. Curcumin had no significant effect on the tissue changes at the time of infusion of Ang II at the 2 week, but at the 4 week, curcumin significantly reduced the range of myocardial fibrosis, the accumulation of macrophages and activated fibroblasts decreased, and the content of type I collagen decreased. Compared with the Sham group, the control group was Ang II. After 2 weeks of chronic infusion, the expression of AT1 receptor was slightly increased, and the expression of AT2 receptor decreased, but in the control group, the expression of AT1 receptor was significantly increased and the expression of AT2 receptor decreased after 4 weeks of infusion. Immunohistochemical staining also found that the expression of AT1 receptor in the surrounding and myocardial tissue of the myocardium was enhanced and the expression of AT2 receptor weakened. In addition, the expression of TGF beta 1 protein and p-Smad2 and p-Smad3 was increased. In addition, the expression of ACE2 decreased. After 4 weeks of curcumin intervention, the above changes were obviously improved. The expression of AT1 receptor expression was significantly reduced, the expression of AT2 receptor was increased, and immunohistochemical staining showed that the AT1 receptor in the surrounding and myocardial tissues of the vascular wall weakened, and the expression of AT2 receptor increased. In addition, the expression of TGF beta 1, p-Smad2 and p-Smad3 decreased. In addition, the expression of ACE2 was increased, and the expression of ACE2 in the myocardium was enhanced by immunohistochemical staining. Conclusion: These results suggest that Ang II has a significant role in myocardial fibrosis and.Ang II can cause interstitial macrophages and interstitial macrophages. The aggregation of fibroblasts increased and was accompanied by type I collagen proliferation. These phenomena were most obvious at the 4 week of Ang II infusion. It was found that curcumin had obvious anti fibrosis effect. The mechanism of action was explored and the signal transduction pathway involved in Ang II induced myocardial fibrosis was angiotensin II AT1/TGF beta 1/Smad.Ang II by changing AT1/AT 2 relative expression levels, and then increase the expression of TGF beta 1 receptor, and continue to activate the downstream Smad pathway to cause myocardial fibrosis. Curcumin balanced the expression of AT1, AT2 in this pathway, on the other hand, up regulation of ACE2 expression, so that a large number of Ang II degrades to Ang1-7., and curcumin has cardiovascular protection. In this study, curcumin can inhibit the heart damage of direct stimulation of Ang II and provide direct and powerful evidence for the protective effect of curcumin on the cardiovascular system. This study looks forward to our study for the first time to demonstrate the anti fibrosis effect of curcumin and the antagonism of the blood angiotensin II receptor and angiotensin converting enzyme. At the same time, we observed that curcumin can also reduce the hypertension caused by angiotensin II. These results provide a direct experimental basis for further clarification of the protective effect of curcumin on cardiovascular protection. Curcumin, used as a plant extract, is eaten daily in India as a seasoning, and its pharmacological dynamics, safety and The results have been confirmed by preclinical studies and clinical trials. The results also suggest that curcumin is a promising alternative to other clinical practice.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.23

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