發(fā)布時間：2019-06-21 05:13

【摘要】：研究背景糖尿病是一種慢性代謝性疾病,嚴重威脅人類的健康水平,其心血管并發(fā)癥是糖尿病患者死亡的重要病因。糖尿病心肌病是以心臟結構和功能障礙,伴有心肌肥厚,心肌間質纖維化和細胞凋亡為特征的疾病,最終發(fā)展為心力衰竭,導致患者死亡。如何減輕糖尿病對心臟組織造成的損傷,降低糖尿病患者的死亡率,提高其生活質量,是急需解決的問題。乙醛脫氫酶2(ALDH2)是位于線粒體基質的氧化酶,能夠氧化細胞內外的醛類物質,減少醛類物質對心肌造成的損害。研究表明,ALDH2參與細胞內多種信號通路的調節(jié),減輕心肌損傷;Alda-1能選擇性激活ALDH2,改善心肌缺血再灌注引起的損傷。自噬是細胞清除衰老或受損細胞器或蛋白質的重要方式,對維持細胞內穩(wěn)態(tài)至關重要;且自噬與多種心臟疾病關系密切。但是,ALDH2對糖尿病狀態(tài)下心肌細胞及細胞內自噬的影響及其具體機制尚不清楚。研究目的動物和細胞水平觀察ALDH2對糖尿病狀態(tài)下心臟功能及細胞凋亡指數、線粒體膜電位和細胞內自噬水平的影響,證實ALDH2是否通過調節(jié)細胞內自噬,實現對心肌細胞的保護作用,明確ALDH2對自噬調節(jié)的分子機制。研究方法通過STZ腹腔注射的方法建立糖尿病小鼠模型,測試心肌細胞的收縮及舒張功能,檢測心肌細胞Ca2+調節(jié)能力的改變及心肌細胞橫截面面積;以33mmol/L濃度葡萄糖處置H9C2心肌細胞72h,應用ALDH2激動劑Alda-1(20μM)、溶酶體抑制劑Bafilomycin A1(BAF,50 n M)、自噬抑制劑3-甲基化腺嘌呤(3-MA,10 m M)和自噬激活劑Rapamycin(Rap,100 n M)干預細胞,用TUNEL法檢測細胞凋亡指數,JC-1熒光染色檢測線粒體膜電位水平,Western blot法檢測自噬標志蛋白LC3、p62及ULK1、p-ULK1的表達水平,GFP-LC3質粒轉染檢測細胞內自噬體數量。實驗結果1.與野生型小鼠相比,過表達ALDH2小鼠能拮抗STZ引起的心肌細胞收縮及舒張功能障礙,能部分恢復心肌細胞的Ca2+調節(jié)能力,減輕心肌細胞肥大性改變2.與對照組相比,高糖導致心肌細胞凋亡增加,線粒體膜電位下降,自噬蛋白LC3II表達減少,p62表達增加,ULK1磷酸化程度下降;應用Alda-1激活ALDH2能夠降低細胞凋亡,提升線粒體膜電位,增加細胞內LC3表達,p62表達減少,ULK1磷酸化程度升高(p0.05);3.與對照組相比,應用自噬抑制劑3-MA及BAF能部分拮抗ALDH2的保護作用,而應用自噬激活劑Rap可增強ALHD2的保護作用,說明ALDH2通過激活細胞內自噬實現對心肌細胞的保護作用;4.與對照組相比,應用si RNA干擾ULK1表達后,ULK1磷酸化程度下調,自噬水平下降,部分抵消ALDH2的心肌保護作用,說明ALDH2通過上調ULK1磷酸化,激活細胞內自噬而發(fā)揮心肌保護作用。結論ALDH2對高糖致心肌損傷具有保護作用,其保護作用是通過促進ULK1的磷酸化,上調自噬水平實現的。
[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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