PERK/CaN信号通路在糖尿病非阻塞性冠脉疾病微循环障碍中的作用机制及基因靶向沉默效果研究

Though without severe arterial stenosis, the MACE occurrence of non- obstructive coronary artery disease remains high. Microvascular dysfunction is not only the mechanism of non- obstructive coronary artery disease, but also one of the pathological characters of diabetes. Compared with non- diabetic patients, the prevalence of non- obstructive coronary artery disease is higher in patients with diabetes; the initial data of our clinical trial showed that the coronary microvascular dysfunction is more serious in patients with diabetes. Inhibition of coronary microvascular dysfunction becomes one of the treatment strategies of diabetic non- obstructive coronary artery disease. We propose that the activation of PERK/CaN/NFAT signaling pathway of endoplasmic reticulum stress in cardiac microvascular endothelial cells would induce coronary microvasuclar dysfunction by triggering apoptosis of endothelial cells, local inflammation and microthrombosis; while inhibition of activation of PERK signaling would improve microvascular dysfunction by protecting endothelial cells. In this study, a viral vector carrying small interference RNA against PERK would be administrated to animal model of diabetic non- obstructive coronary artery disease. Coronary angiography, index of microcirculatory resistance assessment, myocardial perfusion romographic imaging and methods of molecular biology would be applied. Results of this study would enrich our knowledge of mechanism of diabetic non- obstructive coronary artery disease. The effectiveness and feasibility of genetic therapy would be also discussed.

虽无严重血管狭窄，但非阻塞性冠脉疾病仍可导致不良心血管事件的发生。微循环障碍不仅是非阻塞性冠脉疾病的发病机制，也是糖尿病的主要病理特征。课题组的初步临床研究提示非阻塞性冠脉疾病微循环障碍在糖尿病患者中更为显著。抑制冠脉微循环障碍成为本病的一个治疗策略。结合前期工作，申请者提出冠脉微循环内皮细胞内质网应激PERK/CaN/NFAT信号通路激活可导致微循环内皮细胞凋亡、局部炎症反应以及微血栓形成，最终造成糖尿病非阻塞性冠脉疾病微循环障碍的发生；同时，对PERK信号通路的靶向抑制可能通过保护内皮细胞而改善微循环。本研究拟建立携带可沉默PERK表达的siRNA的病毒载体，对2型糖尿病非阻塞性冠脉疾病动物模型进行干预，利用冠状动脉造影术、冠脉微循环阻力指数、心肌核素显像以及分子生物学技术等手段，明确糖尿病非阻塞性冠脉疾病微循环的发病机制，探讨PERK为抑制靶点的基因治疗的疗效及可行性。

目的：在选择性冠状动脉造影术(CAG)证实的冠状动脉无明显狭窄的患者出现心脏缺血甚至心肌梗死的情况。这种状况被命名为非阻塞性冠脉疾病(NoCAD)，呈现出显著的微循环内皮功能障碍。糖尿病也表现出明显的内皮功能障碍。本研究旨在观察糖尿病是否会加剧NoCAD微循环障碍，并对发病机制进行探讨。..方法：对77例NoCAD患者进行了研究，采用压力导丝微循环阻力指数(IMR)对冠脉微循环进行评估。分析了糖尿病与NoCAD患者微循环障碍的关系。建立了NoCAD及糖尿病小鼠模型。使用重组腺相关病毒9(AAV9)将特异性siRNA导入小鼠体内及原代微循环内皮细胞(CMECs)对PERK基因进行敲减。使用HE染色评估小鼠前降支狭窄情况；使用心脏超声检测冠脉血流速度储备(CFVR)评估冠脉微循环；TUNEL法及流失细胞术评估细胞凋亡；免疫荧光染色评估NFAT核转位；WB及q-PCR信号通路关键分子的表达及磷酸化；ELISA法对炎症因子水平进行检测。..结果：糖尿病是导致NoCAD患者冠脉微循环障碍的危险因素。NoCAD患者冠脉IMR值与糖化血红蛋白成显著正相关。本研究成功建立了AAV9-perk-siRNA病毒载体，并证实该载体能够有效敲减CMECs内PERK并进而抑制其磷酸化。发现NoCAD糖尿病动物CFVR显著降低，CMECs凋亡增加，局部IL6、TNFα及TXB2分泌增多，心肌局部活化血小板聚集。AAV9-perk-siRNA病毒载体注射能够显著改善NoCAD糖尿病动物CFVR，抑制CMECs凋亡，降低局部IL6、TNFα及TXB2分泌并抑制活化血小板聚集。AAV9-perk-siRNA病毒载体转染能够显著抑制高糖诱导的原代CMECs凋亡及IL6、TNFα及TXB2分泌。体内及体外研究均证实，AAV9-perk-siRNA病毒载体是抑制CMECs内PERK通路激活，进而抑制NFAT核转位，最终使其下游凋亡相关靶基因FasL/Fas，炎症相关靶基因IL6，TNFα以及血栓形成靶基因COX2表达降低。..结论：糖尿病通过诱导微循环内皮细胞凋亡、局部炎症以及血栓形成等功能障碍加剧NoCAD。微循环内皮细胞内质网应激PERK/NFAT信号通路激活参与了该过程。AAV9-perk-siRNA病毒载体对该信号通路的特异性抑制能够显著改善糖尿病NoCAD冠脉微循环障碍。