ALDH2对1型糖尿病致心肌损伤的保护作用及其机制研究

发布时间：2019-06-21 05:13

【摘要】：研究背景糖尿病是一种慢性代谢性疾病,严重威胁人类的健康水平,其心血管并发症是糖尿病患者死亡的重要病因。糖尿病心肌病是以心脏结构和功能障碍,伴有心肌肥厚,心肌间质纤维化和细胞凋亡为特征的疾病,最终发展为心力衰竭,导致患者死亡。如何减轻糖尿病对心脏组织造成的损伤,降低糖尿病患者的死亡率,提高其生活质量,是急需解决的问题。乙醛脱氢酶2(ALDH2)是位于线粒体基质的氧化酶,能够氧化细胞内外的醛类物质,减少醛类物质对心肌造成的损害。研究表明,ALDH2参与细胞内多种信号通路的调节,减轻心肌损伤;Alda-1能选择性激活ALDH2,改善心肌缺血再灌注引起的损伤。自噬是细胞清除衰老或受损细胞器或蛋白质的重要方式,对维持细胞内稳态至关重要;且自噬与多种心脏疾病关系密切。但是,ALDH2对糖尿病状态下心肌细胞及细胞内自噬的影响及其具体机制尚不清楚。研究目的动物和细胞水平观察ALDH2对糖尿病状态下心脏功能及细胞凋亡指数、线粒体膜电位和细胞内自噬水平的影响,证实ALDH2是否通过调节细胞内自噬,实现对心肌细胞的保护作用,明确ALDH2对自噬调节的分子机制。研究方法通过STZ腹腔注射的方法建立糖尿病小鼠模型,测试心肌细胞的收缩及舒张功能,检测心肌细胞Ca2+调节能力的改变及心肌细胞横截面面积;以33mmol/L浓度葡萄糖处置H9C2心肌细胞72h,应用ALDH2激动剂Alda-1(20μM)、溶酶体抑制剂Bafilomycin A1(BAF,50 n M)、自噬抑制剂3-甲基化腺嘌呤(3-MA,10 m M)和自噬激活剂Rapamycin(Rap,100 n M)干预细胞,用TUNEL法检测细胞凋亡指数,JC-1荧光染色检测线粒体膜电位水平,Western blot法检测自噬标志蛋白LC3、p62及ULK1、p-ULK1的表达水平,GFP-LC3质粒转染检测细胞内自噬体数量。实验结果1.与野生型小鼠相比,过表达ALDH2小鼠能拮抗STZ引起的心肌细胞收缩及舒张功能障碍,能部分恢复心肌细胞的Ca2+调节能力,减轻心肌细胞肥大性改变2.与对照组相比,高糖导致心肌细胞凋亡增加,线粒体膜电位下降,自噬蛋白LC3II表达减少,p62表达增加,ULK1磷酸化程度下降;应用Alda-1激活ALDH2能够降低细胞凋亡,提升线粒体膜电位,增加细胞内LC3表达,p62表达减少,ULK1磷酸化程度升高(p0.05);3.与对照组相比,应用自噬抑制剂3-MA及BAF能部分拮抗ALDH2的保护作用,而应用自噬激活剂Rap可增强ALHD2的保护作用,说明ALDH2通过激活细胞内自噬实现对心肌细胞的保护作用;4.与对照组相比,应用si RNA干扰ULK1表达后,ULK1磷酸化程度下调,自噬水平下降,部分抵消ALDH2的心肌保护作用,说明ALDH2通过上调ULK1磷酸化,激活细胞内自噬而发挥心肌保护作用。结论ALDH2对高糖致心肌损伤具有保护作用,其保护作用是通过促进ULK1的磷酸化,上调自噬水平实现的。
[Abstract]:Background Diabetes is a chronic metabolic disease, which seriously threatens the health level of human beings, and its cardiovascular complications are an important cause of death in diabetic patients. Diabetic cardiomyopathy is a disease characterized by cardiac structural and functional disorders, accompanied by myocardial hypertrophy, myocardial interstitial fibrosis and apoptosis, which eventually develops into heart failure and leads to death. How to reduce the damage of heart tissue caused by diabetes mellitus, reduce the mortality of diabetic patients and improve their quality of life is an urgent problem to be solved. Acetaldehyde dehydrogenase 2 (ALDH2) is an enzyme located in the matrix of mitochondria, which can oxidize aldehydes inside and outside cells and reduce the damage to myocardium caused by aldehydes. Studies have shown that ALDH2 is involved in the regulation of various intracellular signaling pathways to alleviate myocardial injury, and Alda-1 can selectively activate ALDH2, to improve myocardial ischemia-reperfusion injury. Autophagy is an important way for cells to eliminate aging or damaged organelle or protein, which is very important to maintain intracellular homeostasis, and autophagy is closely related to a variety of heart diseases. However, the effect of ALDH2 on cardiomyocytes and intracellular autophagy in diabetic state and its specific mechanism are not clear. Objective to observe the effects of ALDH2 on cardiac function, apoptosis index, mitochondrial membrane potential and intracellular autophagy in diabetic rats, and to confirm whether ALDH2 can protect cardiomyocytes by regulating intracellular autophagy and clarify the molecular mechanism of ALDH2 on autophagy. Methods Diabetic mouse model was established by intraabdominal injection of STZ. The systolic and diastolic function of cardiomyocytes, the changes of Ca2 regulation ability and the cross section area of cardiomyocytes were measured. H9C2 cardiomyocytes were treated with 33mmol/L glucose for 72 h. ALDH2 agonist Alda-1 (20 渭 M), lysosome inhibitor Bafilomycin A1 (BAF,50 n M), autophagy inhibitor 3-methyladenine (3 鈮，

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