【作者】 尚福军；

【导师】 赵连友；

【作者基本信息】 第四军医大学 ， 内科学， 2007， 博士

【摘要】 研究背景及目的高血压是心血管疾病最重要的危险因素,而心脏重构是导致高血压患者心血管事件发生率显著增加的独立危险因素,是心脏功能由代偿向失代偿转化的重要病理表现。因此,心脏重构的发病机制和防治研究已成为国内外热点研究课题。现已明确,神经体液因素的异常激活是心脏重构发生的重要机制,但在多种神经体液因素阻断治疗已成为高血压常规治疗的今天,仍然无法阻遏心脏功能向失代偿发展的最终趋势,心力衰竭发病率仍然居高不下。目前,随着对炎症反应在多种心血管疾病发病机制中作用的认识,炎性细胞因子作为炎症反应的重要介质,已经成为与心血管疾病相关的另一类重要的神经体液因素。大量研究表明,炎性细胞因子可通过诱导心肌细胞肥大、细胞凋亡、调节细胞外基质的合成和降解等多种途径参与心脏重构的形成。此外,心肌内多种组成细胞可以与炎症细胞一样合成炎性细胞因子,通过自分泌和旁分泌方式影响心肌组织的损伤、修复和结构的改变。但这些研究以体外研究为主,体内有关研究也主要集中在心肌梗死和心肌炎等有明显炎症基础的疾病导致的心脏重构。虽然临床资料显示高血压患者血清促炎性细胞因子表达上调,但是由于临床研究的限制,心肌组织内炎性细胞因子的表达情况尚不清楚,而后者在心脏重构研究中的意义则更为重要。高血压是否能够引起心肌组织表达炎性细胞因子以及表达机制如何,目前尚不明确。新近研究显示,活性氧(ROS)与高血压的发生和发展密切相关。ROS不仅可以直接导致组织和细胞损伤,而且ROS本身是重要的信号分子,可激活多种氧敏感性激酶和转录因子,从而调节多种蛋白基因的表达。文献报道,ROS可以调控血管内皮细胞和巨噬细胞表达炎性细胞因子,而且在高血压患者和高血压动物模型心肌组织内均发现ROS水平的升高。但高血压是否通过增加心肌组织内ROS水平调控炎性细胞因子的表达,从而推动心脏重构的发生和发展,目前尚未见报道。他汀类药物是目前调脂治疗的首选药物。大量研究表明,他汀具有诸多非调脂作用。本课题组以往研究显示,他汀类药物可抑制心肌细胞肥大以及心脏成纤维细胞增殖和胶原合成;在自发性高血压大鼠模型中也证实,辛伐他汀(Sim)可延缓心肌肥厚的发展。这些研究提示他汀类药物可调控心脏重构过程,但其分子机制仍不十分清楚。本课题动态观察高血压大鼠心肌组织炎性细胞因子表达特点及其与心脏重构和心功能变化的关系;研究ROS在心肌组织和心肌细胞炎性细胞因子表达机制中的作用;初步探讨Sim对高血压心脏重构的防治作用及其分子机制。旨在炎症介质角度阐明高血压心脏重构的发病机制和他汀类药物防治心脏重构的分子机制。为高血压心脏重构的防治提供新思路和理论依据。研究方法本研究先后以压力负荷高血压大鼠和体外培养的新生SD大鼠心肌细胞为实验模型,采用组织病理学分析、心脏超声检查、血流动力学分析、比色法、荧光探针技术、细胞色素C还原试验、ELISA、RT-PCR、Western blot等方法和技术,动态观察:(1)压力负荷大鼠心肌组织内促炎因子TNF-α、IL-6和抗炎因子IL-10表达变化及其与心脏结构和功能的关系;(2)压力负荷大鼠对心肌组织ROS水平及NADPH氧化酶活性的影响;(3)脂多糖(LPS)对心肌细胞内ROS水平、NADPH氧化酶活性以及p38 MAPK活性的影响;(4)抗氧化剂氮乙酰半胱氨酸(NAC)干预对心脏重构以及心肌组织和心肌细胞炎性细胞因子表达的影响;(5) Sim干预对心脏重构以及心肌组织和心肌细胞ROS水平和炎性细胞因子表达的影响。研究结果(1)腹主动脉缩窄(AC)组大鼠血压在1周时就显著高于假手术(Sham)组(P<0.01),2周后左室重量指数(LVWI)、室壁厚度、心肌细胞内径、心肌组织胶原含量与Sham组比较明显升高(P<0.01),4周后左室舒张功能降低(P<0.05),随着观察时间的延长,这些改变进行性发展。在8周的观察时间内未发现左室收缩功能障碍。(2) AC组大鼠心肌组织内促炎因子TNF-α、IL-6以及抗炎因子IL-10 mRNA表达水平在2周后开始明显升高(P<0.05),在4周时达到最高水平,分别比Sham组升高116%(P<0.01)、183%(P<0.01)、83%(P<0.05)。(3) AC组大鼠心肌组织内TNF-α、IL-6和IL-10蛋白表达水平在4周时明显升高,分别比Sham组升高179%(P<0.01)、114%(P<0.01)和38%(P<0.05)。而AC组和Sham组之间血清内这些炎性细胞因子的蛋白表达水平无显著性差异(P>0.05)。(4)相关分析结果显示,心肌组织TNF-α、IL-6蛋白表达水平与心肌胶原蛋白含量、左室舒张末期压力(LVEDP)均呈显著正相关(P<0.01),而与心肌细胞内径无显著相关性(P>0.05)。心肌组织IL-10蛋白表达水平与胶原蛋白含量、LVEDP以及心肌细胞内径均无明显相关关系(P>0.05)。(5) AC组大鼠心肌组织ROS水平和NADPH氧化酶活性在1周时即显著升高,与Sham组比较有非常显著性差异(P<0.01)。2周时达到最高水平,分别比Sham组升高140%和114% ,4周和8周组ROS水平和NADPH氧化酶活性有所下降,但与对照组比较仍有非常显著性差异(P<0.01)。(6) NAC治疗后,压力负荷大鼠LVWI、室壁厚度、胶原含量以及左室舒张功能均明显改善,(P<0.05或P<0.01),而血压和心肌细胞内径无显著变化(P>0.05)。心肌组织TNF-α、IL-6 mRNA和蛋白表达水平均明显下降,与AC组比较有非常显著性差异(P<0.01)。而两组之间IL-10 mRNA和蛋白表达水平无显著性差异(P>0.05)。(7)早期或晚期给予Sim治疗后,压力负荷大鼠血压虽然无明显变化,但LVWI、室壁厚度、胶原含量、心肌细胞内径以及左室舒张功能均明显改善(P<0.05或P<0.01)。而且Sim早期治疗组对各指标的干预效果明显优于Sim晚期治疗组(P<0.05)。(8)早期或晚期给予Sim治疗后,压力负荷大鼠心肌组织TNF-α、IL-6 mRNA和蛋白表达水平明显下降,与AC组比较有显著性差异(P<0.05或P<0.01),而且Sim早期治疗组与Sim晚期治疗组之间亦有显著性差异,早期治疗组TNF-α、IL-6表达水平下降得更为明显(P<0.05或P<0.01)。Sim治疗对IL-10 mRNA和蛋白表达水平无显著性影响(P>0.05)。(9) Sim治疗后,压力负荷大鼠心肌组织ROS水平和NADPH氧化酶活性明显降低(P<0.05或P<0.01),而且与Sim晚期治疗组比较,Sim早期治疗组ROS水平和NADPH氧化酶活性下降得更为显著(P<0.05)。(10)在1μg/ml LPS作用下,体外培养的新生SD大鼠心肌细胞内TNF-α、IL-6和IL-10 mRNA和蛋白表达水平明显增加,并具有时间依赖性,与对照组比较有显著性差异(P<0.05或P<0.01)。(11)在1μg/ml LPS作用下,心肌细胞ROS水平和NADPH氧化酶活性明显升高,与对照组比较有非常显著性差异(P<0.01)。心肌细胞ROS水平和NADPH氧化酶活性的升高在时间上早于炎性细胞因子的表达。给予NAC或NADPH氧化酶抑制剂二亚苯基碘鎓(DPI)预处理后,心肌细胞TNF-α、IL-6蛋白表达水平较LPS组明显降低(P<0.01),而IL-10蛋白含量与LPS组无显著差异。(12)在20~500μmol/l外源性H2O2作用下,心肌细胞培养上清TNF-α和IL-6蛋白含量与对照组比较均明显升高,有显著性差异(P<0.05)。20~500μmol/l H2O2对IL-10蛋白含量无明显影响(P>0.05)。(13)给予0.1~10μmol/l Sim预处理后再给予LPS刺激,TNF-α、IL-6 mRNA和蛋白表达水平随着Sim浓度的增加逐步下降,其中1μmol/l和10μmol/l Sim干预组表达水平与LPS组比较有非常显著性差异(P<0.01)。而在1μmol/l Sim的基础上同时给予0.1 mmol/l甲羟戊酸(MVA)预处理,TNF-α、IL-6 mRNA和蛋白表达水平与1μmol/l Sim+LPS组比较显著升高(P<0.01),而与LPS组比较无显著性差异(P>0.05)。0.1~10μmol/l Sim对LPS诱导的IL-10 mRNA和蛋白表达水平无明显影响(P>0.05)。(14)在1μmol/l Sim预处理后再给予100μmol/l H2O2刺激,心肌细胞培养上清TNF-α和IL-6蛋白含量与H2O2组比较无显著差异(P>0.05)。(15)在1μg/ml LPS作用下,心肌细胞p38 MAPK活性显著增强(P<0.01),并且在时间上早于炎性细胞因子的表达。NAC、DPI以及Sim预处理可明显抑制p38 MAPK活性(P<0.01),MVA可逆转Sim对p38 MAPK活性的抑制作用。(16)在LPS作用的基础上给予p38 MAPK抑制剂SB203580预处理后,心肌细胞培养上清TNF-α、IL-6蛋白含量明显降低,与LPS组比较有非常显著性差异(P<0.01),而IL-10蛋白含量与LPS组比较无显著性差异(P>0.05)。研究结论(1)高血压可上调心肌组织促炎性细胞因子表达,虽然与心脏重构的启动无关,但参与了心脏重构的进一步发展,与间质重构和舒张性心脏功能衰竭相关。(2)高血压可上调心肌组织ROS水平,ROS介导的信号通路是心肌组织和心肌细胞内促炎性细胞因子表达的重要机制之一,p38 MAPK可能是该信号通路的一个重要下游分子。抗氧化剂可以通过下调促炎性细胞因子表达延缓高血压心脏重构的进程。(3) Sim可在一定程度上延缓和逆转高血压心脏重构的发生,早期治疗效果可能优于晚期治疗。(4) Sim抑制心肌组织和心肌细胞NADPH氧化酶活性,降低ROS水平,部分阻断ROS信号通路,从而抑制促炎性细胞因子表达,可能是Sim延缓和逆转心脏重构的重要机制之一。综上所述,不难看出高血压可上调促炎性细胞因子表达,促炎性细胞因子的表达可促进心脏重构的形成和发展;活性氧介导的信号通路至少部分参与了心肌组织和心肌细胞炎性细胞因子表达的信号传导机制;他汀类药物可通过抑制氧化应激和炎性细胞因子表达防治高血压心脏重构。由此可见,本项研究的结果有望为高血压心脏重构的防治提供新思路和理论依据。更多还原

【Abstract】 Background and objective Hypertension is the most important risk factor for cardiovascular disease. Cardiac remodeling in patients with hypertension is an independent risk factor for cardiovascular events, and is the key pathological manifestations during the transition of heart function from compensate to decompensate. Therefore, the study on the pathogenesis and control of cardiac remodeling has become a worldwide hot topic. In the past decades, many strides have been made in the field, but the pathogenesis mechanism of cardiac remodeling still remains unclear. It is wellknown that abnormal activation of neurohumoral factors is an important mechanism for cardiac remodeling. Nowadays, blockages of a variety of neurohumoral factors have become routine medication, however, the tendency of heart failure in patients is still unable to prevent and the incidence of heart failure remains high. Proinflammatory cytokines, the important mediators of inflammatory response, have become another important neurohumoral factors associated with cardiovascular system. A growing body of studies has shown that inflammatory cytokines play an important role in cardiac remodeling. Inflammatory cytokines promote the progression of cardiac remodeling in various ways, including cardiomyocytes hypertrophy, cell apoptosis and extracellular matrix synthesis and degradation. Furthermore, just like inflammatory cells, many kinds of cells in myocardium may synthesis and secrete inflammatory cytokines, which affect the injury, repairing and structural change of heart in autocrine and paracrine manner. But most of the studies are in vitro research-based, and the studies in vivo were mainly confined in the cardiovascular diseases with obvious inflammation response such as myocardial infarction and myocarditis. Clinical data show the serum levels of proinflammatory factors in patients with hypertension increase in comparison with normotensive, nevertheless the expression of inflammatory cytokine in local myocardium is not yet clear, which is more important in the formation of cardiac remodeling. Whether hypertension can cause expression of inflammatory cytokines in myocardium and the expression mechanism, is not yet clear. Latest studies show that ROS is associated with the genesis and development of hypertension. Increasing evidence is emerging that ROS is an important signaling molecule that can activate oxygen sensitivity kinases and transcription factors and modulate a variety of genes expression. Documents have reported that ROS is involved in the expression of inflammatory cytokines in vascular endothelial cells and macrophages. And up-regulation of ROS was detected in myocardial tissue of both patients with hypertension and hypertensive animals. But whether hypertension can up-regulate the expression of inflammatory cytokines by increasing ROS level of myocardium and subsequently promote the development of cardiac remodeling has not yet been reported. Our previous studies demonsrated that HMG-CoA reductase inhibitors, named as statins, inhibited the hypertrophy of cardiomyocytes and the proliferation and collagen synthesis of cardiac fibroblasts. Statins attenuated the cardiac hypertrophy in SHR model. These studies suggest the inhibitory effects of statins on cardiac remodeling, but the mechanism remains unclear. This study was therefore designed to observe the temporal profile of the expression of inflammatory cytokines and analyze its relationship with hypertensive cardiac remodeling; to study the role of ROS in the expression of cytokines in myocardium and cardiomyocytes; to investing the effects and the mechanism of simvastatin (Sim) in the prevention and treatment of cardiac remodeling. The purpose of the study is to elucidate the cellular and molecular mechanisms of cardiac remodeling associated with hypertension and the interventional effects of Sim on it, and provide novel theoretical evidences and strategy for prevention and treatment of hypertensive cardiac remodeling.Methods In this study, pressure-overload rats and cultured cardiac myocytes of neonatal SD rats were used as experiment models. Histopathologic assay, echocardiography, hemodynamic measurement, colorimetric method, fluorescent probe technique, cytochrome C reduction assay, ELISA, RT-PCR and Western blot were applied to identify: (1) the dynamic changes of expression of TNF-α, IL-6 and IL-10 in pressure-overload rats and its relationship with structure and function of heart; (2) the dynamic changes of ROS level and NADPH oxidase activity in pressure-overload rats; (3) the effects of LPS on the ROS level, NADPH oxidase activity and p38 MAPK activation in cultured cardiomyocytes. (4) the effects of N-acetyl cysteine (NAC) on the expression of inflammatory cytokines in myocardium of pressure-overload rats and cultured cardiomyocytes. (5) the effects of Sim on the ROS level and the expression of cytokines in myocardium of pressure-overload rats and cultured cardiomyocytes.Results (1) Compared with sham operation (Sham) rats, blood pressure in abdominal aorta coarctation (AC) rats significantly increased at 1 week postoperation (P<0.01). Left ventricular weight index (LVWI), ventricular wall thickness, myocyte diameter and myocardial collagen content were elevated at 2 week (P<0.01). Left ventricle diastolic dysfunction was found at 4week (P<0.05). All the changes further progressed then. (2) In AC rats, the mRNA expression of TNF-α, IL-6 and IL-10 in myocardium increased at 2 week, and peaked at 4 week. Compared with Sham rats, the mRNA expression of TNF-α, IL-6 and IL-10 were increased by 126%, 183% and 83% respectively (P<0.05 or P<0.01). (3) In AC rats, the protein content of TNF-α, IL-6 and IL-10 in myocardium increased at 4 week. Compared with Sham rats, the protein content of TNF-α, IL-6 and IL-10 were increased by 179%, 114% and 38% respectively (P<0.05 or P<0.01).There was no significant difference of content of TNF-α, IL-6 and IL-10 in serum between AC and Sham groups (P>0.05). (4) Correlation analysis indicated the protein levels of TNF-αand IL-6 in myocardium were positively correlated with myocardial collagen content, left ventricular end-diastolic pressure (LVEDP) but not myocyte diameter. There was no significant correlation between protein levels of IL-10 in myocardium with myocardial collagen content, LVEDP and myocyte diameter. (5) In AC rats, ROS level and NADPH oxidase activity increased at 1 week, and peaked at 2week, preceding the increase of inflammatory cytokines expression. The ROS level and NADPH oxidase activity at 2 week increased by 140% and 114% (P<0.01). (6) In AC rats treated with NAC (0.2g/kg/d, in drinking water), LVWI, ventricular wall thickness, myocardial collagen content and left ventricular diastolic function were greatly improved (P<0.05 or P<0.01). The myocyte diameter had no change after NAC treatment. Compared with AC group, the mRNA and protein level of TNF-αand IL-6 in myocardium of AC rats treated with NAC markedly decreased (P<0.01). There was no significant change of IL-10 level (P>0.05). (7) In AC rats treated with Sim at both early and late stage, LVWI, ventricular wall thickness, myocyte diameter, myocardial collagen content and left ventricular diastolic function were all significantly improved (P<0.05 or P<0.01), but there was no change of blood pressure. The effects of Sim treatment at early stage was better than that at late stage (P<0.05). (8) In AC rats treated with Sim at both early and late stage, the mRNA and protein level of TNF-αand IL-6 in myocardium significantly decreased in comparison with AC rats (P<0.05 or P<0.01), and the effects of Sim treatment at early stage was better than that at late stage (P<0.05 or P<0.01). The level of IL-10 expression didn’t change after Sim treatment. (9) After Sim treatment, the ROS level and NADPH oxidase activity in myocardium significantly decreased (P<0.05 or P<0.01), and the effects of Sim treatment at early stage was better than that at late stage (P<0.05). (10) In cultured neonatal rats cardiomyocytes stimulated with 1μg/ml lipopolysaccharide (LPS), The mRNA and protein expression levels of TNF-α, IL-6 and IL-10 increased in a time-dependent manner (P<0.05 or P<0.01). (11) The ROS level and NADPH oxidase activity in cardiomyocytes increased markedly in LPS group in comparison with that of control group (P<0.01), preceding the increase of expression of inflammatory cytokines. Pretreatment with NAC and diphenyleneiodonium (DPI), the NADPH oxidase inhibitor, inhibited the LPS-induced increase of protein content of TNF-αand IL-6 (P<0.01), but the IL-10 protein levels was not affected by NAC and DPI pretreatment. (12) Exogenous hydrogen peroxide (H2O2) also induced the increase of TNF-αand IL-6 protein level (P< 0.05), but not IL-10 protein level. (13) Pretreatment with Sim at concentrations of 0.1~10μmol/l attenuated the LPS-induced TNF-αand IL-6 mRNA and protein expression in a dose-dependent manner. The IL-10 level induced by LPS was not affected by treatment with Sim. Furthermore, mevalonate (MVA) pretreatment in combination with 1μmol/l Sim reversed the inhibitory effects of Sim on the expression of TNF-αand IL-6. Pretreatment with Sim or MVA alone didn’t significantly affect the basal levels of these cytokines expression. (14) Pretreatment with 1μmol/l Sim had no effects on the H2O2-induced TNF-αand IL-6 protein expression (P>0.05). (15) Western blot indicated that stimulation with LPS for 1h significantly increased p38 MAPK activity (P<0.01), preceding the increase of cytokines expression. Pretreatment with NAC, DPI and Sim all attenuated p38 MAPK activation induced by LPS (P<0.01). The effect of Sim on p38 MAPK activation was reversed by concomitant pretreatment with MVA (P<0.01). (16) 5μmol/l SB203580, a p38 MAPK inhibitor, attenuated LPS-induced increase of TNF-αand IL-6 protein level (P<0.01), but not that of IL-10.Conclusion Hypertension can up-regulate the expression of proinflammatory cytokines such as TNF-αand IL-6. Although the expression of proinflammatory cytokines has no effect on the initiation of cardiac remodeling, it is involved in the formation and progression of cardiac remodeling and is significant correlation with matrix remodeling and diastolic dysfunction. The ROS signal pathway, at least in part, mediates the expression of proinflammatory cytokines in myocardium and cardiomyocytes. The p38 MAPK probably is the important downstream molecular in the ROS signal pathway. Sim can prevent and reverse the progress of cardiac remodeling, and the interventional effects of Sim treatment at early stage is better that those at late stage. The inhibition of NADPH oxidase activity and the subsequent inhibition of ROS signal pathway and the expression of proinflammatory cytokines is one of important mechanism of the effects of Sim on cardiac remodeling. In short, hypertension up-regulates the expression of proinflammatory cytokines, the latter promote the progression of cardiac remodeling. ROS signal pathway is involved in the mechanism of cytokines expression. Sim can inhibit cardiac remodeling by the effects on oxidative stress and expression of proinflammatory cytokines. These will provide novel strategy and theoretic evidence to prevention and treatment of hypertensive cardiac remodeling.更多还原