【作者】 马永刚；

【导师】 胡卓伟；

【作者基本信息】 北京协和医学院 ， 药理学， 2010， 博士

【摘要】 充血性或慢性心力衰竭,简称心衰,是各种原因所引起的心脏疾病的终末阶段,心脏收缩及(或)舒张功能明显降低,以致心脏排血量不能满足机体代谢所需。它的病因很多,主要包括冠心病、高血压、心肌病、心脏毒素、瓣膜疾病和先心病等。随着我国进入老年化社会,慢性心衰的发病率仍在增加。尽管抗心衰的药物、介入疗法和外科手术已取得了长足的进展,但这些治疗方法只能改善慢性心衰患者的症状和体征、提高其生活质量及延缓病情的进展,对重度心衰除了心脏移植外尚没有更有效的办法。以心肌细胞丢失、心肌肥厚和组织纤维化为特征的心脏组织异常重构是各种慢性心脏病的核心病理改变。近些年的研究发现免疫炎症反应在心脏组织重构过程中发挥了重要作用。细胞损伤释放的内源性损伤相关分子模式(DAMPs)激活模式识别受体(如Toll样受体)、活化免疫炎症反应并引起组织损伤,参与心衰的发生发展。文献报道Toll样受体-2(TLR2)缺失可以减轻阿霉素诱导的心功能障碍、降低局部炎症反应、抑制心肌细胞凋亡及提高动物生存率,表明TLR2介导了阿霉素诱导的心功能失调。本实验想观察在小鼠形成慢性心衰以后再阻断TLR2能否有治疗作用。我们的研究发现,用TLR2中和性抗体(TLR2Ab)治疗性阻断TLR2活性能明显减轻阿霉素引起的心功能障碍和心肌异常重构,抑制心肌局部炎症反应。接下来,我们检测了几种与TLR2密切相关的DAMPs的表达,发现阿霉素可以明显上调高迁移率族蛋白-1(HMGB1), Hsp70, S100A2和S100A8的表达,而阻断TLR2能抑制HMGB1和Hsp70的表达,尤其对HMGB1的抑制作用更为明显。而且,免疫共沉淀和激光共聚焦显微镜结果显示TLR2Ab明显抑制了HMGB1与TLR2的相互作用。因此,我们推测阿霉素诱导组织细胞死亡释放大量HMGB1,后者通过激活TLR2参与心衰的发生发展。为了验证这个假说,我们通过使用甘草甜素抑制细胞外HMGB1的功能,观察能否发挥改善作用。结果显示,预防性给予甘草甜素能明显改善阿霉素诱导的心功能障碍和心肌重构,并抑制HMGB1与TLR2的相互作用。因此,我们的结果证实TLR2Ab明显抑制HMGB1的表达及HMGB1与TLR2的相互作用,从而改善阿霉素引发的心衰。为了进一步证实阻断TLR2活性改善心衰的普遍作用,我们使用了冠脉结扎诱导的小鼠心肌缺血模型。同样发现,阻断TLR2活性明显改善缺血诱导的心功能障碍、减轻炎症、氧化应激、心肌纤维化及心肌细胞凋亡,其机制可能与上调AMPK通路活性密切相关。我们的研究结果不仅有助于深入理解免疫系统尤其是TLR2在心肌损伤和心脏重塑过程中的作用,同时也为开发抗慢性心衰和心梗药物提供了研究线索和潜在靶点。更多还原

【Abstract】 Congestive or chronic heart failure (HF) is a common terminal phase of heart diseases induced by a variety of injury. Cardiac output of failing hearts fails to meet the need of body metabolism because of impairment of myocardial systolic and (or) diastolic function. Etiologies of HF include coronary heart diseases, hypertension, cardiomyopathy, cardiotoxins, valvular diseases, congenital heart diseases, and so on. As China enters the aging society, the incidence rate of HF is gradually increasing. Though significant progress in therapy of HF including drugs, intervention as well as surgery, these treatments only ameliorate symptoms and signs of patient with HF, improve their quality of life and delay the disease progression. Except for heart transplantation, there is no effective therapy for advanced HF.Cardiac remodeling, including loss of cardiomyocytes, myocardial hypertrophy and fibrosis, is a major pathological characteristic of multiple chronic heat diseases. Accumulating evidence demonstrated that immune-inflammatory responses played vital roles in adverse cardiac remodeling. Endogenous damage associated molecular patterns (DAMPs) released by damaged cells activated pattern recognition receptors (for example, toll like receptor) leading to inflammation and tissue injury, and were involved in the development and progression of HF. Research results showed that TLR2deficiency could mitigate doxorubicin (Dox) induced cardiac dysfunction, inflammation, cardiomyocytes apoptosis as well as improve survival rate, suggesting that TLR2mediated Dox triggered cardiac malfunction. However, we wonder whether inhibition of TLR2activity by anti-TLR2neutralizing antibody (TLR2Ab) would exert therapeutic efficiency against established cardiac remodeling and dysfunction triggered by chronic Dox administration. The findings indicated that therapeutic blocking TLR2activity markedly attenuated Dox induced cardiac dysfunction and remodeling as well as inflammatory responses in the heart. Then through detecting expressions of several important DAMPs strongly related to TLR2, we found Dox significantly upregulated levels of high mobility group box-1(HMGB1), Hsp70, S100A2and S00A8. However, blocking TLR2inhibited the upregulation of HMGB1and Hsp70, with more obvious suppression on HMGB1. Furthermore, TLR2Ab inhibited the interaction of HMGB1and TLR2as assessed by co-immunoprecipitation and confocal microscopy. Therefore, we speculated that Dox resulted in cardiac myocytes death and large mounts of HMGB1were released from these injured cells, and HMGB1participated in the development and progression of HF via engagement of TLR2. To validate the hypothesis, we wonder if administration of glycyrrhizin, functional antagonist of extracellular HMGB1, would exert protective action against injury induced by Dox, as TLR2Ab did. Our results demonstrated that prophylactic usage of glycyrrhizin significantly improved Dox induced myocardial dysfunction and remodeling, as well as repressed the interaction of HMGB1with TLR2. In conclusion, our findings validated that therapeutic blocking TLR2markedly ameliorated HF triggered by doxorubicin by suppressing the of HMGB1expression and the interaction between HMGB1and TLR2.To further confirm the universal role of targeting TLR2in improvement of HF, we utilized myocardial infarction model in mice prepared by surgical occlusion of left coronary artery. Similarly, blockage of TLR2mitigated functional impairment, cardiac inflammation, oxidative stress, cardiac fibrosis and cardiomyocytes apoptosis induced by ischemia, which were associated with activation of AMP-activated protein kinase (AMPK). Collectively, the results of our study not only contribute to understanding the roles of immune system (especially TLR2) in myocardial injury and remodeling, but also provide the research clues and potential targets for the development of new anti-HF and anti-myocardial infarction drugs.更多还原