【摘要】：研究背景糖尿病是一種慢性代謝性疾病,嚴(yán)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(jié)構(gòu)和功能障礙,伴有心肌肥厚,心肌間質(zhì)纖維化和細(xì)胞凋亡為特征的疾病,最終發(fā)展為心力衰竭,導(dǎo)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(zhì)量,是急需解決的問題。乙醛脫氫酶2(ALDH2)是位于線粒體基質(zhì)的氧化酶,能夠氧化細(xì)胞內(nèi)外的醛類物質(zhì),減少醛類物質(zhì)對心肌造成的損害。研究表明,ALDH2參與細(xì)胞內(nèi)多種信號(hào)通路的調(diào)節(jié),減輕心肌損傷;Alda-1能選擇性激活A(yù)LDH2,改善心肌缺血再灌注引起的損傷。自噬是細(xì)胞清除衰老或受損細(xì)胞器或蛋白質(zhì)的重要方式,對維持細(xì)胞內(nèi)穩(wěn)態(tài)至關(guān)重要;且自噬與多種心臟疾病關(guān)系密切。但是,ALDH2對糖尿病狀態(tài)下心肌細(xì)胞及細(xì)胞內(nèi)自噬的影響及其具體機(jī)制尚不清楚。研究目的動(dòng)物和細(xì)胞水平觀察ALDH2對糖尿病狀態(tài)下心臟功能及細(xì)胞凋亡指數(shù)、線粒體膜電位和細(xì)胞內(nèi)自噬水平的影響,證實(shí)ALDH2是否通過調(diào)節(jié)細(xì)胞內(nèi)自噬,實(shí)現(xiàn)對心肌細(xì)胞的保護(hù)作用,明確ALDH2對自噬調(diào)節(jié)的分子機(jī)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(xì)胞的收縮及舒張功能,檢測心肌細(xì)胞Ca2+調(diào)節(jié)能力的改變及心肌細(xì)胞橫截面面積;以33mmol/L濃度葡萄糖處置H9C2心肌細(xì)胞72h,應(yīng)用ALDH2激動(dòng)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(yù)細(xì)胞,用TUNEL法檢測細(xì)胞凋亡指數(shù),JC-1熒光染色檢測線粒體膜電位水平,Western blot法檢測自噬標(biāo)志蛋白LC3、p62及ULK1、p-ULK1的表達(dá)水平,GFP-LC3質(zhì)粒轉(zhuǎn)染檢測細(xì)胞內(nèi)自噬體數(shù)量。實(shí)驗(yàn)結(jié)果1.與野生型小鼠相比,過表達(dá)ALDH2小鼠能拮抗STZ引起的心肌細(xì)胞收縮及舒張功能障礙,能部分恢復(fù)心肌細(xì)胞的Ca2+調(diào)節(jié)能力,減輕心肌細(xì)胞肥大性改變2.與對照組相比,高糖導(dǎo)致心肌細(xì)胞凋亡增加,線粒體膜電位下降,自噬蛋白LC3II表達(dá)減少,p62表達(dá)增加,ULK1磷酸化程度下降;應(yīng)用Alda-1激活A(yù)LDH2能夠降低細(xì)胞凋亡,提升線粒體膜電位,增加細(xì)胞內(nèi)LC3表達(dá),p62表達(dá)減少,ULK1磷酸化程度升高(p0.05);3.與對照組相比,應(yīng)用自噬抑制劑3-MA及BAF能部分拮抗ALDH2的保護(hù)作用,而應(yīng)用自噬激活劑Rap可增強(qiáng)ALHD2的保護(hù)作用,說明ALDH2通過激活細(xì)胞內(nèi)自噬實(shí)現(xiàn)對心肌細(xì)胞的保護(hù)作用;4.與對照組相比,應(yīng)用si RNA干擾ULK1表達(dá)后,ULK1磷酸化程度下調(diào),自噬水平下降,部分抵消ALDH2的心肌保護(hù)作用,說明ALDH2通過上調(diào)ULK1磷酸化,激活細(xì)胞內(nèi)自噬而發(fā)揮心肌保護(hù)作用。結(jié)論ALDH2對高糖致心肌損傷具有保護(hù)作用,其保護(hù)作用是通過促進(jìn)ULK1的磷酸化,上調(diào)自噬水平實(shí)現(xiàn)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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