IL-18导致心脏舒张功能不全机制研究

Diastolic heart failure (DHF), with increasing prevalence and incidence, is the most common type of heart failure. Evidence accumulated in recent years suggests that patients with DHF, as well as patients with systolic heart failure (SHF), have poor prognosis as there are few efficient therapies for treating them. Therefore, investigating the mechanisms involved in the development of diastolic dysfunction and finding novel ways to improve cardiac function and survival rate of patients with diastolic dysfunction are of significance..Clinical studies demonstrate that serum interleukin (IL)-18 is elevated in patients with heart failure. IL-18 experimentally has been shown to induce production of tumor necrosis factor-α , IL-1β , IL-6 and inducible nitric oxide synthase, which have all been associated with myocardial dysfunction. To date, however, the mechanism of IL-18 on diastolic dysfunction remains obscure..Recent studies have shown that daily administration of IL-18 in healthy mice induces LV dysfunction and blunts β-adrenergic responsiveness to isoproterenol. Moreover, induction of myocardial hypertrophy and cardiac fibrosis by IL-18 indicates a role for this cytokine in myocardial remodeling. Our group has also proved that daily administration of IL-18 in healthy mice induces cardiac fibrosis and myocardial hypertrophy. In this model, endothelial cells contribute to fibroblast accumulation through an endothelial-mesenchymal transition (EndMT) were found. In vitro, our previous data showed that IL-18 could induce cardiac endothelial cells gradual loss of endothelial markers such as CD31, VE cadherin; with the gradual appearance of fibroblastic markers such as Vimentin, alpha-smooth muscle actin (α-SMA). Our MicroRNA array data revealed that miR-21 levels were significantly increased during EndMT. The mRNA level of PTEN, a bonafide target of miR-21, was downregulated in EndMT-derived fibroblast-like cells. We also found that the mTOR activity increased significantly and autophagy was inhibited in endothelial cells and cardiac myocytes. However, whether IL-18 induced EndMT and myocardial hypertrophy via autophagy in diastolic dysfunction remain unclear? Thus, we propose a hypothesis that IL-18 induce EndMT and myocardial hypertrophy causing diastolic dysfunction via autophagy: the mechanism is that IL-18 suppress autophagy by miR-21—PTEN—mTOR signal pathway, which may inhibit the apoptosis of cardiac endothelial cells and promote EndMT and myocardial hypertrophy. In this project, we aim to provide reliable evidence to clarify detailed mechanisms involved in IL-18 induced diastolic dysfunction through the methods such as PCR, western blotting, siRNA, tracking in vivo of cardiac endothelial cells in Atg5+/- mice.

炎性细胞因子是导致心脏舒张功能不全重要原因之一。我们前期实验发现IL-18导致小鼠心肌细胞肥厚及心脏间质纤维化，导致心脏舒张功能不全，其中可见心脏内皮细胞向成纤维细胞转化（EndMT）。IL-18是否通过自噬引起心肌细胞肥大及EndMT，导致心脏舒张功能不全，仍不清楚？进一步体外实验发现IL-18能诱导心脏内皮细胞发生EndMT，通过microRNA芯片发现miR-21表达明显增加，同时能使miR-21靶基因PTEN下调，mTOR蛋白活性增加，自噬减弱。因此我们提出IL-18导致心脏舒张功能不全的新机制：IL-18通过激活miR-21转录调控，下调PTEN表达，抑制自噬，促使心肌细胞肥大，介导内皮细胞发生EndMT，引起心脏间质纤维化，导致心脏舒张功能不全。本项目将采用多种分子生物学技术，活体示踪及基因敲除等动物模型，从体内外证实上述机制，为临床预防/逆转舒张性心力衰竭治疗提供新的科学证据

心脏舒张功能不全导致的心力衰竭，又称左室射血分数尚保留的心力衰竭，占临床心力衰竭患者的一半以上。迄今为止，临床上针对心脏舒张功能不全的治疗进展极少。因此阐明心室舒张心功能不全的发生机制，以寻找新的治疗方法和手段，改善心功能，提高生存率有着重要意义，已经成为了心血管科研领域的热点。.炎性细胞因子是导致心脏舒张功能不全重要原因之一。本项目体外证明IL-18 通过miR-21抑制PTEN的表达，诱导心脏内皮细胞发生 EndMT。体外证明IL-18通过miR-21-PTEN信号通路参与内皮细胞EndMT的机制。但在动物实验中，给小鼠注射IL-18后，miR-21的表达变化并不稳定，预在体心肌细胞中特异性过表达或敲低miR-21，亦未能实现，因此未能从体内实验直接证明上述三者的调控关系。在体外证实自噬介导了EnMT及心肌细胞肥大，导致IL-18所致心脏舒张功能不全。在体外IL-18诱导的小鼠EndMT及心肌细胞肥大细胞模型中，可见内皮细胞及心肌细胞自噬减弱，p-ULK1水平明显增加（p-ULK1为mTOR蛋白作用底物，说明mTOR活性明显增强）。但由于在体实验中，给小鼠注射IL-18后，自噬水平的检测结果不稳定，因此未能从动物实验证明该机制。.因此本项目转而研究造成心脏功能不全的另一重要病因——急性心肌梗死，证明急性心肌梗死后能诱导心脏功能不全心肌纤维化的重要细胞组分——心脏成纤维细胞，产生内质网应激，通过增强自噬作用下调纤连蛋白。.冠状动脉粥样硬化斑块破裂是引起急性心肌梗死的主要病因，而平滑肌细胞的功能紊乱在其中起关键作用，本项目另外发现雷帕霉素诱导凋亡的大鼠主动脉平滑肌细胞释放亲环蛋白A，但不通过自噬小体影响胞吐作用。ROCK，肌动蛋白重塑和突触小泡不参与细胞凋亡相关的CyPA释放。肌球蛋白II的激活调节了血管平滑肌细胞的凋亡和雷帕霉素诱导的凋亡细胞死亡中CyPA的释放。为临床通过自噬抑制EndMT，从而抑制心脏纤维化，为临床预防/逆转舒张性心力衰竭治疗提供新的科学证据。且为将来从抑制ER应激和自噬的角度改善心梗后修复提供理论支持。

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