基于内质网应激-自噬稳态失衡研究糖尿病心肌病“毒损心络”病理机制及滋阴益气活血解毒法的干预作用

Myocardial apoptosis in diabetic cardiomyopathy (DCM) phase highlights important early pathological changes, but the exact mechanism has not yet been elucidated. Recent studies have shown that endoplasmic reticulum stress (ERS) is a key mechanism of occurrence and development of DCM has the important status in the myocardium, and autophagy may as an important protection mechanism against ERS for tissue cells is of great significance to maintain stable internal environment and function, but the imbalance ERS - Autophagy homeostasis mediated myocardial apoptosis in DCM research, so far unreported at home and abroad. In the previous study, we found that the damaged myocardial cells appear mRNA expression markers of endoplasmic reticulum stress protein GRP78 increased, while the autophagic membrane marker protein LC3-Ⅱ protein downregulated , speculated that imbalance between endoplasmic reticulum stress and autophagy homeostasis may be a new mechanism of myocardial cell injury in DCM. To provide that hypothesis,we will study the molecular mechanism of ERS-Autophagy homeostasis in injured myocardial cells from the models of DCM and in vitro using the examination technology of molecular biology, Laser scanning ConfocalMicroscopy and so on. At the same time, all will be verified by the traditional Chinese medical prescription based the method of tonifying yin and qi, promoting blood flow and detoxifcating. Our aim is to enrich the scientific connotation of the pathogenesis of “accumulation of poison and blood stasis” and “damage of heart by poison” in DCM, and provide to scientfic basis for clinical diagnosis and look forward to new therapeutic targets for DCM.

心肌细胞凋亡在糖尿病心肌病(DCM)早期病变阶段凸显重要，但确切机制尚未阐明。新近研究表明，内质网应激(ERS)是心肌细胞凋亡及DCM发生发展的关键，而自噬可能作为对抗ERS的重要保护机制，对组织细胞的内环境稳定与功能维持具有重要意义。但ERS—自噬稳态失衡介导DCM心肌细胞凋亡的研究，迄今国内外尚无报道。我们前期研究发现，受损心肌细胞出现ERS分子伴侣GRP78 mRNA及蛋白表达增高，而自噬体膜标记蛋白LC3-Ⅱ蛋白表达下调，推测ERS—自噬稳态失衡可能是DCM心肌细胞损伤的新机制。因此，我们拟以DCM小鼠(转基因MKR鼠)和体外诱导受损心肌细胞为模型，采用分子生物学、激光扫描共聚焦显微检测等技术，探讨ERS—自噬稳态失衡在DCM心肌细胞凋亡中的分子机制，并以滋阴益气活血解毒法中药复方干预进行反证。旨在丰富DCM“因毒（瘀）致虚，毒损心络”病机的科学内涵，为其治疗提供新理论与新方法。

糖尿病心肌病是糖尿病的主要心血管并发症之一，为糖尿病患者高心力衰竭发生率和高死亡率的主要原因。心肌细胞凋亡及心肌间质纤维化是DCM发生发展的重要病理机制。.已有研究证实，内质网应激（ERS）在糖尿病及其并发症脏器损害过程中存在并发挥着重要作用，糖尿病及其心血管并发症中存在诸如高血糖、氧化应激、AGEs、肾素-血管紧张素系统激活、脂质代谢障碍等多种可诱发心肌细胞ERS的因素，并进而诱导心肌细胞凋亡。提示过度ERS在糖尿病心肌细胞损伤以及心肌重构进程中扮演重要角色。而自噬对于维持胰岛β细胞、心肌细胞、血管内皮细胞以及足细胞等结构和功能亦至关重要。自噬可能是对抗ERS的一种重要保护机制，自噬对于内质网应激状态下组织细胞的内环境稳定和功能维持具有重要意义。.本课题结合前期研究工作，以MKR鼠2型糖尿病心肌病转基因动物模型 、以及体外高糖诱导的心肌细胞为研究对象，分别从在体、体外实验两方面，旨在探讨探讨ERS—自噬稳态失衡在糖尿病心肌病心肌细胞凋亡中的分子机制及滋阴益气活血解毒立法的左归降糖舒心方的干预作用与干预机制。研究结果表明：左归降糖舒心方具有明显的降血糖作用，改善心肌病理形态学损害、降低心肌损害相关指标以及胶原蛋白Ⅰ、Ⅲ的表达水平，并可通过调节Bax/Bcl-2比值以改善心肌细胞凋亡。同时，MKR糖尿病心肌病模型小鼠心肌及高糖损伤心肌细胞均具有内质网应激与基础自噬水平的异常，给予内质网应激抑制剂4苯基丁酸、自噬激动剂雷帕霉素、自噬抑制剂3-甲基腺嘌呤后，内质网应激与自噬标志物及其相关信号分子明显表达上调或下调。而左归降糖舒心方具有明显增强模型小鼠心肌及高糖损伤心肌细胞的双层膜囊泡结构自噬小体及自噬体膜标志蛋白Beclin-1、LC3-Ⅱ表达，增强了自噬水平；同时，该方具有调控内质网应激标志蛋白GRP78及其内质网应激-自噬相关信号分子PI3K、Akt、mTOR、p-AMPK、p-TSC2、p-mTOR的mRNA与蛋白质表达。通过对上述内质网应激-自噬相关信号分子的调控，从而延缓并改善糖尿病心肌损伤。