内质网应激转录因子XBP1对糖尿病心肌病的保护作用及其分子机制的研究

The diabetic cardiomyopathy is one of the diabetic mellitus-independent complications, is characterized by myocardial hypertrophy and fibrosis independent of hypertension，coronary artery disease or other idiopathic heart diseases．But the exact pathogenesis is still not completely clear. The unfolded protein response transcription factor X-box–binding protein-1 (XBP1) was reported to improve glucose homeostasis and regulate hepatic lipogenesis, highlighting its critical role in obesity and associated metabolic syndrome. Our previous studies showed that cardiac expression of ER stress marker XBP-1 were decreased in the maladaptive phase in hypertrophic and failing heart and may play a protective role in cardiac hypertrophy and heart failure. We further found that cardiac expression of XBP1 was decreased in the maladaptive phase in High fat diet(HFD)-induced diabetic cardiomyopathy. Therefore, the nature of XBP1 in the models of diabetic cardiomyopathy needs to be further investigated. In this project, classic animal models of diabetic cardiomyopathy were used in in vivo study of the overall effects of XBP1. HFD feeding was used to induce obesity and diabetes in wild type mice or mice lacking XBP1,decreases the expression of XBP1 in mouse hearts. Secondly, we measured the effects and molecular mechanism of XBP1 on cell energy metabolism, mitochondrial morphology and dynamics in cardiac myocytes. This work will provide important theoretical and experimental basis for XBP1 targeted therapy for prevention and treatment of diabetic cardiomyopathy.

糖尿病心肌病是糖尿病患者的主要心脏并发症之一，可引起心脏收缩功能不全等严重后果，但其发病机制尚不完全明确。XBP1是由内质网应激反应诱导产生，并在糖脂代谢和胰岛素抵抗等方面中有重要调控作用的转录因子。我们前期研究发现XBP1对心肌肥厚和心力衰竭有保护性作用，而且在2型糖尿病模型心脏中XBP1表达水平是下降的。本项目将在此基础上采用rAAV9介导XBP1差异性表达动物模型、心肌细胞特异性XBP1敲除小鼠，建立高脂饲养诱导2型糖尿病，在体内和体外研究XBP1调节糖尿病心脏糖脂代谢、改善心肌组织线粒体功能障碍的作用和分子机制，明确XBP1在糖尿病心肌病心肌细胞能量代谢和线粒体功能调控中的作用，并寻找XBP1上下游调控机制。这将帮助阐明XBP1在糖尿病心肌病中的作用，从而完善对糖尿病心肌病发病机制的认识，为糖尿病心肌病的防治提供新的思路。

【研究目的】明确XBP1在高脂饮食诱导的糖尿病心肌病模型中的保护作用，并探究其下游机制.【研究方法】在高脂饲养诱导的2型糖尿病模型中，采用western blot和real-time PCR检测心脏组织内质网应激激活和XBP1的表达和小鼠心功能的变化，观察XBP1的动态改变与糖尿病心肌病进展之间的相关性；采用rAAV9-cTNT-GFP载体，包装rAAV9-cTNT-XBP1s病毒，在高脂饲养小鼠心脏中特异性过表达XBP1s，检测XBP1s对心功能的影响；同时通过对小鼠心脏组织进行mRNA测序，筛选XBP1s靶基因，进行western blot、real-time PCR、荧光报告基因实验和染色质免疫共沉淀PCR实验验证XBP1s对靶基因的转录调控作用。.【研究结果】高脂饲养C57BL小鼠后，4个月时超声心动图检测显示E/A下降，6个月时超声检测显示E/A和EF (Ejection Fraction, 射血分数)下降，同时millar导管检测显示dP/dt max下降和dP/dt min升高，心脏切片HE染色显示心肌细胞肥大，Masson染色显示心肌组织纤维化增加，油红染色显示心肌细胞内脂滴聚集增多；Western blot显示高脂饲养后心脏组织中IRE1α磷酸化和XBP1s下调，XBP1u上调；在心脏灌流得到的成纤维细胞和心肌细胞中，XBP1剪切检测和real-time PCR显示高脂饲养后XBP1s转录本表达下调；心脏切片免疫荧光染色和western显示高脂喂养导致XBP1s入核减少；同时在心脏特异性过表达XBP1s后，超声心动图和millar导管检测显示E/A升高，dP/dt max升高和dP/dt min下降；转录组测序显示心脏组织中的ACOT1在XBP1s过表达后有显著改变；双荧光素酶报告基因实验提示XBP1s在ACOT1启动子上-1040bp到-560bp区间内有结合区；染色质免疫共沉淀(Chromatin Immunoprecipitation assay, ChIP) PCR实验显示XBP1s在ACOT1启动子区上有富集。.【研究结论】我们的研究明确了高脂饲养可以抑制心脏组织中的IRE1α-XBP1轴，导致XBP1s转录本和蛋白表达下调，入核减少，进而影响XBP1s转录调控下游基因。同时，我们发现ACOT1是XBP1s保护糖尿病心肌病的潜在靶基因。