定向干预脂肪酸代谢的关键酶对糖尿病心脏重构的影响及其机制研究

A common disorder presented in various heart diseases is the impaired fatty acid (FA) catabolism, which always occurs ahead of cardiac remodeling. Lipid overaccumulation and reactive oxygen species (ROS) overproduction induced by increased FA metabolism during diabetic status may lead to endoplasmic reticulum stress (ER stress) which promotes diabetic cardiac remodeling by inducing cardiomyocytes apoptosis and influencing intracellular calcium handling. Cardiospecific downregulation of fatty acid translocase (CD36) and carnitine palmitoyltransferase-1b (CPT-1b), which are two important enzymes in the FA catabolism pathway, may improve the abnormality of FA catabolism in diabetic heart. .We hypothesize that ER stress and calcium mishandling are important mechanisms underlying the diabetic cardiac remodeling, which is characterized by increased FA catabolism. Suppression of FA uptake and utilization may protect diabetic heart from remodeling by meliorating ER stress and calcium mishandling..Db/db mice which have high FA catabolism at 4 weeks of age and obvious cardiac remodeling at 12 weeks of age are used as diabetic models in this project. Lentivirus vector containing short hairpin RNAs targeting CD36 and CPT-1b are constructed and injected into the hearts directly to selectively downregulation the expression of CD36 and CPT-1b in the heart. Biochemical, functional, morphological, histological, and molecular profiles will be evaluated. We try to illuminate the role of ER stress and calcium mishandling in the developing of diabetic cardiac remodeling, and try to find out a strategy based on metabolism interference for cardiac remodeling control.

心肌细胞脂肪酸（FA）代谢异常是多种心脏病共有的表现，常早于心脏重构发生。其所致的脂质蓄积和有害代谢副产物活性氧簇的增加可能引起内质网应激，进而介导心肌细胞凋亡并影响细胞钙调控，促进糖尿病心脏重构的发生。下调FA代谢的2个重要酶—脂肪酸转位酶（CD36）和肉毒碱脂酰转移酶1b（CPT-1b）的表达可能有助于改善糖尿病心肌细胞FA代谢异常。我们假设：内质网应激和钙调控异常是心肌细胞FA高代谢率导致糖尿病心脏重构的重要机制，逆转FA高代谢率可通过改善上述异常而抑制心脏重构。本项目以心肌细胞FA代谢率增高且12周龄后出现心脏重构的db/db小鼠为研究对象，构建LshCD36和LshCPT-1b慢病毒载体选择性下调其心肌细胞中CD36和CPT-1b的表达。观察血清生化参数、心脏形态学、功能学、组织学及分子生物学的改变。力图阐明FA高代谢率致糖尿病心脏重构的机制，从代谢角度为抗心脏重构治疗提供思路。

本研究旨在通过RNA干扰下调糖尿病小鼠心肌组织中脂肪酸代谢的关键酶CD36或CPT-1b的表达，观察此干预对糖尿病心脏重构的影响，并探索其潜在机制。.本课题组成功构建了靶向CD36和CPT-1b慢病毒，并采用高脂饮食+链脲佐菌素多次经腹腔注射的方法构建了小鼠糖尿病模型，通过心肌内注射慢病毒的方式成功下调了小鼠心肌组织中目标基因的表达。.研究结果提示：1、心肌内注射慢病毒未导致生化指标的不良效应，其安全性良好。2、糖尿病导致小鼠心脏质量减轻，左室收缩功能下降（EF%及FS%均减低）；下调两种目标基因的表达均可改善上述异常。3、油红O染色证实，下调CD36或CPT-1b的表达可分别导致糖尿病小鼠心肌内中性脂质含量的减少及增加。4、高效液相色谱-质谱串联分析证实，下调CD36或CPT-1b的表达均可降低糖尿病小鼠心肌内毒性脂质神经酰胺的含量。5、采用DCFH-DA和DHE两种染色方法均证实，糖尿病引起心肌细胞内活性氧簇的含量显著增加，下调两种目标基因的表达均可显著减少活性氧簇生成。6、电镜检查发现，糖尿病小鼠心肌细胞中线粒体的数量减少、排列紊乱，部分出现花斑及嵴溶解，线粒体占细胞总面积的比例增加；下调两种目标基因的表达均改善了线粒体超微结构的异常，减少了线粒体相对面积。7、组织学染色证实，糖尿病引起心肌细胞凋亡增多，并有导致心肌纤维化的趋势，下调两种目标基因的表达均显著减少心肌细胞的凋亡，抑制心肌纤维化。8、糖尿病导致小鼠心肌细胞内钙超载、肌浆网钙调控紊乱，通过RNA干扰可改善甚至逆转糖尿病小鼠心肌细胞钙调节障碍。9、分子生物学结果证实，糖尿病引起心肌细胞发生内质网应激，后者介导了凋亡相关基因CHOP、caspase12等表达增加，下调目标基因的表达可改善内质网应激，抑制凋亡相关基因的表达；此外，糖尿病还导致IRS-PI3K-mTOR信号通路激活，下调CD36的表达可抑制该效应，可能是其抗心脏重构的分子生物学机制。.上述结果表明，干预脂肪酸代谢的关键酶可能成为治疗代谢相关心脏重构的有效手段。.此外，本实验研究对象还包括肥胖小鼠模型。