Bcl-2-associated athanogene 3在Streptozotocin誘導第一型糖尿病大鼠心肌缺血再灌流傷害角色探討

The Role of Bcl-2-associated athanogene 3 in Streptozotocin-Induced Type 1 Diabetic Rats Myocardial Ischemia/Reperfusion Injury

温庭睿(Wen Ting-Jui)

指導教授 ： 鄭劍廷

國立臺灣師範大學/理學院/生命科學系/碩士(2019年)

https://doi.org/10.6345/NTNU201900553

摘要

隨著糖尿病患人口持續增加，在21世紀的現代社會裡，糖尿病已經成為全球最重要的公共衛生議題之一。糖尿病是心血管疾病的危險因子，糖尿病患者發生心血管疾病的機率是非糖尿病患者的2到4倍。Bcl-2-associated athanogene 3 (BAG3) 是一種抗細胞凋亡的蛋白質。現在的研究已經了解BAG3對於心肌缺血再灌流傷害具有保護效果，但是在糖尿病的條件下，心肌缺血再灌流中BAG3的角色還不清楚。本篇研究主要的目的在於了解BAG3在糖尿病的條件下，與心肌缺血再灌流傷害所造成的氧化壓力和細胞凋亡的關聯。本篇研究使用streptozotocin (STZ) 誘發大鼠產生的第一型糖尿病，並使用冠狀動脈結紮，造成缺血再灌流傷害。在STZ誘發前與誘發後的第1, 2, 3, 4週，測量體重與空腹血糖值，並在STZ誘發後的第4週，進行代謝籠實驗，記錄24小時的飲水量、進食量、排尿量與糞便重量，確認第一型糖尿病誘發狀況。在STZ誘發後的第8週，測量腎臟的參數，包括腎臟重量，使用西方墨點法與免疫組織化學 (IHC) 分析腎臟4-hydroxynonenal (4-HNE) 的表現情形，與H＆E染色測定腎臟病理變化。為了了解糖尿病對血管的影響，使用腸繫膜阻力動脈的第二分支進行離體血管張力測定。在STZ誘發後的第8週，在冠狀動脈左前降支的位置進行結紮，造成心肌缺血，持續45分鐘。心肌缺血45分鐘後，進行4小時的再灌流。左心室心舒末期壓力 (LVEDP)、壓力上升最大速率 (+dp/dt) 與壓力下降最大速率 (-dp/dt) 會透過左心室壓進行分析，並使用雷射都普勒血流影像儀分析缺血再灌流過程中，心臟表面血流微循環的變化。在完成心肌缺血再灌流手術後，使用MDA assay分析心臟組織MDA的量，使用西方墨點法分析心臟組織BAG3、Bcl-2、caspase 3、PARP的表現情形，與心臟組織切片H&E染色、IHC染色和TUNEL染色，以了解病理與生理的作用機轉。STZ誘發會出現體重下降與高血糖的症狀，並產生喝多、尿多與吃多的症狀，確認第一型糖尿病誘發成功。STZ誘發第一型糖尿病會造成腎臟肥大與腎臟氧化壓力。在STZ誘發糖尿病第8週，糖尿病大鼠的血管舒張功能顯著低於正常大鼠，並且血管的收縮力顯著低於正常大鼠。STZ誘發第一型糖尿病會加劇心肌缺血再灌流所造成的左心室功能損害、增加心臟的氧化壓力、降低BAG3的表現。STZ誘發第一型糖尿病顯著降低Bcl-2的表現，並加劇心肌缺血再灌流所造成的caspase 3活性與細胞凋亡。本篇研究顯示在糖尿病的條件下，STZ誘發第一型糖尿病會加劇心肌缺血再灌流所造成的氧化壓力、BAG3表現量降低與促細胞凋亡機轉，進一步造成心臟的損傷。

關鍵字

糖尿病 ； 缺血再灌流 ； Bcl-2-associated athanogene 3 ； 細胞凋亡 ； 氧化壓力

並列摘要

The number of diabetic patients is steadily increasing. Diabetes has become one of the most important public health issue in the 21st century for being an important risk factor for induction of cardiovascular disease. The morbidity of cardiovascular disease in diabetic patients was two to four times higher than non-diabetic patients. Bcl-2-associated athanogene 3 (BAG3) is an anti-apoptotic protein. Recent studies have shown that BAG3 can confer cardiac protection against ischemia/reperfusion injury. However, under diabetic condition, the role of BAG3 in ischemia/reperfusion injury is still not clear. The aim of this study was to investigate the relation between BAG3 and ischemia/reperfusion injury-induced oxidative stress and apoptosis under diabetic condition. In this study, we used streptozotocin (STZ) to induce type 1 diabetes mellitus in rats, following by coronary artery ligation to induce ischemia/reperfusion injury. Before and after STZ induction, we first measured body weight and fasted blood glucose for four weeks. In the 4th week of STZ induction, we used metabolic cage to measure 24-hr water intake, food intake, urine and feces. In the 8th week of STZ induction, we measured the renal parameters including weight of kidneys, renal expression of 4-hydroxynonenal (4-HNE) by western blotting and immunohistochemistry (IHC) and renal pathology determination by hematoxylin and eosin (H&E). To examine the diabetic effect on vascular dynamics, the second-order branches of mesenteric arteries were used to measure isometric myograph. In the 8th week of STZ induction, we used coronary artery ligation to induce myocardial ischemia for 45 min followed by 4-hr reperfusion. Left ventricular end-diastolic pressure (LVEDP), peak rate of pressure increase (+dp/dt) and peak rate of pressure decrease (-dp/dt) were analyzed by left ventricular blood pressure. Cardiac microcirculation was determined by a laser Doppler blood flow monitor during ischemia/reperfusion injury. The levels of MDA in heart tissues were measured by MDA assay. Myocardial BAG3, Bcl-2, caspase 3 and PARP were determined by western blotting. Heart sections were stained with H&E, IHC and TUNEL to investigate pathological and physiological mechanism. The induction of STZ caused body weight loss and hyperglycemia, and STZ-induced diabetic rats developed polyphagia, polydipsia and polyuria. STZ-induced type 1 diabetes caused renal hypertrophy and oxidative stress. In the 8th week of STZ induction, the degree of vasorelaxation and vasoconstriction was significantly decreased in STZ-induced diabetic rats. STZ-induced type 1 diabetes exacerbated ischemia/reperfusion-induced left ventricular dysfunction, oxidative stress and BAG3 downregulation. STZ-induced type 1 diabetes significantly decreased Bcl-2 expression and exacerbated ischemia/reperfusion-induced caspase 3 activity and apoptosis formation. In this study, we demonstrated that, under diabetic condition, STZ-induced type 1 diabetes exacerbated ischemia/reperfusion-induced oxidative stress, BAG3 downregulation and proapoptotic mechanism leading to further aggravated cardiac injury.

並列關鍵字

Diabetes mellitus ； ischemia/reperfusion ； Bcl-2-associated athanogene 3 ； apoptosis ； oxidative stress

參考文獻

1. Organization WH. Global report on diabetes. 2016.

2. Association AD. Economic costs of diabetes in the US in 2017. Diabetes care. 2018;41:917-928.

3. Care D. Standards of Medical Care in Diabetes 2019. Diabetes Care. 2019;42:S81.

4. Selvin E, Marinopoulos S, Berkenblit G, Rami T, Brancati FL, Powe NR and Golden SH. Meta-analysis: glycosylated hemoglobin and cardiovascular disease in diabetes mellitus. Ann Intern Med. 2004;141:421-31.

5. Gerstein HC, Islam S, Anand S, Almahmeed W, Damasceno A, Dans A, Lang CC, Luna MA, McQueen M, Rangarajan S, Rosengren A, Wang X and Yusuf S. Dysglycaemia and the risk of acute myocardial infarction in multiple ethnic groups: an analysis of 15,780 patients from the INTERHEART study. Diabetologia. 2010;53:2509-17.