艾塞那肽通过脂联素途径改善糖尿病性心肌病的作用及其机制研究

发布时间：2018-08-28 10:33

【摘要】：研究背景糖尿病心肌病(Diabetic Cardiomyopathy,DC)最初由Rubler于1972年首先描述,定义为无冠状动脉疾病、高血压或其他潜在病因的由糖尿病引起的一种特异性心肌病。常表现为心脏顺应性降低,舒张期充盈受阻或收缩功能不全。研究发现高血糖及相关的代谢紊乱可直接损伤心肌,从而导致DC,因此糖尿病心血管并发症的患者均可合并DC。临床上DC拥有很大的发病人群,对患者造成很大的危害。早期的观点认为在DC病理生理改变中,心肌破坏的主要形式为心肌细胞坏死,而随后大量的研究发现,心肌坏死更多发生在心肌梗塞的急性期等急性病变中,而糖尿病的心肌病理改变是慢性长期的过程,主要表现为心肌间质纤维化、心肌细胞肥大和心肌细胞凋亡,其中心肌细胞凋亡在DC的发生发展中占据主要地位[1,2],故抑制心肌细胞凋亡应是临床防治糖尿病性心肌病的关键。在既往的研究中已发现上调脂联素途径关键蛋白脂联素受体连接蛋白1(APPL1)可改善糖尿病性心肌病[3],进一步的研究发现脂联素途径保护心血管系统作用环节在于激活脂联素信号途径中的APPL1-AMPK轴,抑制核因子NF-?B的表达[4,5]。此外,其他实验室和我们均已发现脂联素信号途径中的PPARα能抑制NF-?B,直接参与防止糖尿病心肌细胞凋亡[6],而PPARα为AMPK的重要信号下游分子。据此,我们推测在脂联素途径中可能存在“APPL1-AMPK-PPARα轴”,其在防止糖尿病心肌细胞凋亡方面可能占有重要地位。又有研究发现胰高血糖素样肽-1(GLP-1)对心血管系统有一定保护作用,可以减少包括糖尿病心肌细胞的凋亡,并可促进脂联素的分泌,且该作用独立于降糖效应之外[7-9]。因此,现在广泛认可的观点是GLP-1具有对糖尿病患者心血管保护作用,其机制可能与激活脂联素途径有关,我们进一步推测GLP-1可能具有减少糖尿病心肌细胞凋亡的作用,其作用机制在于激活心肌细胞脂联素信号通路,活化APPL1-AMPK-PPARα轴。为证明我们的设想,本实验拟从临床观察、动物模型和细胞水平进行深入研究。目前,临床上尚无明确的糖尿病性心肌病的诊断标准,主要以心功能参数作为参考指标。我们拟首先收集临床2型糖尿病病例,并与正常体检人群对照,探索2型糖尿病患者血清脂联素水平变化与心功能参数改变的相关关系。其次拟通过小剂量链脲佐菌素(stz)+高脂饮食诱导2型糖尿病大鼠模型,予以glp-1受体激动剂艾塞那肽为干预措施,观察艾塞那肽对糖尿病心肌细胞凋亡的影响,并观察艾塞那肽通过调节脂联素途径关键连接蛋白appl1水平影响心肌细胞凋亡的作用。最后,在培养大鼠原代心肌细胞中检测艾塞那肽对心肌细胞脂联素信号途径的激活作用及对细胞凋亡的影响。目的观察2型糖尿病血清脂联素水平与心脏功能之间的相关关系以及艾塞那肽对心肌细胞凋亡的影响,阐明艾塞那肽通过激活脂联素途径改善糖尿病性心肌病的作用及其机制。方法1.收集2型糖尿病患者及健康体检者的临床基本资料,采集患者空腹静脉血,elisa法检测血清高分子量脂联素水平,增强磁共振测定患者心功能指标;2.构建2型糖尿病sd大鼠模型,高脂饮食喂养,分为正常对照组(n组),糖尿病组(d组),糖尿病加胰岛素治疗组(di组),糖尿病加艾塞那肽治疗组(de组),成模后干预12周,取大鼠髂静脉血,elisa法测定血清胰岛素、脂联素,多媒体生物信号记录仪测定血流动力学,tuenl法检测心肌细胞凋亡变化,取心脏组织行免疫组化及western-blotting检测信号分子appl1、ampk、pparα、nf-?b表达水平;3.培养原代鼠心肌细胞,分为正常对照组(c组),高糖高脂组(d组),高糖高脂+艾塞那肽组(de组),高糖高脂+艾塞那肽+appl1过表达腺病毒组(oe组),高糖高脂+艾塞那肽+appl1干扰腺病毒组(bl组),elisa法测定细胞培养液中高分子量脂联素水平,tuenl法检测原代心肌细胞凋亡变化,取心肌细胞行western-blotting检测信号分子appl1、ampk、pparα、nf-?b表达水平。结果本实验共收集到30例2型糖尿病患者(t2dm组)及30例健康体检患者(n组),两组患者一般资料无明显差异,t2dm组患者糖化血糖水平(hba1c)、空腹血糖水平、homa-ir指数明显高于n组患者,c肽水平低于n组患者,而两组间胰岛素水平无明显统计学差异。t2dm组患者血清hdl水平(1.23±0.40mmol/l)明显低于n组患者(1.61±0.45mmol/l),tg水平(3.73±3.44mmol/l)明显高于n组患者(1.56±0.86mmol/l),差异具有统计学意义(p0.05)。二组患者的tc及ldl水平无差别(p0.05)。t2dm组患者血清高分子量脂联素水平(10.83±2.81mg/l)明显低于n组(14.90±3.26mg/l)(p0.05)。经心脏增强磁共振检测,t2dm组患者心脏左室舒张末期容积(edv)(59.70±7.26ml/m2)及每搏输出量(sv)(34.77±6.48ml/m2)较n组edv(65.84±9.67ml/m2)及sv(38.40±4.75ml/m2)降低,而收缩末期容积(esv)(29.00±6.60ml/m2)较n组(24.25±5.51ml/m2)明显增加,差异均有统计学意义(p0.05)。相关分析显示高分子量脂联素水平与糖尿病患者每搏输出量之间存在一定的正相关关系[r=0.376,p=0.041]。在动物实验中,di组及de组血糖较d组明显下降,血清胰岛素上升,差异均有统计学意义(p0.05),而di与de两组间血糖水平无明显差异。d组及di组血清高分子量脂联素水平较n组明显下降,de组血清高分子量脂联素水平较di组明显上升,差异均有统计学意义(p0.05)。d组心肌细胞凋亡率(55.71%±3.84%)明显增加,de组大鼠心肌细胞凋亡率(27.43%±3.63%)明显下降,而与de组比较,di组大鼠心肌细胞凋亡率(43.91%±4.23%)明显升高。心功能检测显示相较于c组,d组大鼠心脏lvsp(105.87±4.08mmhg)及di组大鼠心脏lvsp(107.19±4.09mmhg)明显降低,而d组大鼠心脏lvedp(17.62±1.74mmhg)及di组大鼠心脏lvedp(17.48±1.49mmhg)明显增加,差异均具有统计学意义(p0.05)。相较于di组,de组大鼠心脏lvsp(119.11±5.11mmhg)明显增加,而lvedp(13.64±1.25mmhg)明显降低,差异具有统计学意义(p0.05)。对心肌细胞信号分子的检测显示相较于c组,d组及di组心肌appl1、p-ampk、pparα表达量均明显降低,但nf-?b表达量较c组明显升高。de组appl1、p-ampk、pparα表达量分别为0.65±0.02、0.78±0.04、1.72±0.05,较d组及di组明显增加,de组大鼠心肌nf-?b表达量较d组和di组心肌明显升高,差异有统计学意义(p0.05)。细胞实验中,我们发现心肌细胞具有自分泌脂联素的能力,并观察到de组、oe组、bl组大鼠细胞上清高分子量脂联素水平分别为9.40±0.16mg/l、8.70±0.34mg/l、和9.30±0.30mg/l,较d组明显上升(p0.05),de、oe、bl组细胞上清脂联素水平比较差异没有统计学意义(p0.05)。但细胞凋亡检测结果提示de组细胞凋亡(20%~29%)较d组(34%~42%)有降低趋势。oe组中这种趋势更加明显(18%~24%),但相反的bl组细胞凋亡率较de组明显上升(43%~52%),差异均有统计学意义(p0.05)。另外,各组心肌细胞appl1、p-ampk、pparα、nf-?b检测结果与动物实验结果具有相同变化趋势。结论2型糖尿病患者心脏收缩及舒张功能受损与循环中高分子量脂联素水平降低密切相关,运用glp-1受体激动剂艾塞那肽干预糖尿病后通过上调血清和心肌细胞自分泌脂联素水平及激活APPL1-AMPK-PPARα轴信号通路,抑制了心肌细胞内NF-?B活化和心肌细胞凋亡,改善糖尿病心肌功能。
[Abstract]:Background Diabetic cardiomyopathy (DC), originally described by Rubler in 1972, is defined as a specific cardiomyopathy caused by diabetes without coronary artery disease, hypertension or other underlying causes. It is often characterized by decreased cardiac compliance, diastolic filling impairment or systolic dysfunction. Blood glucose and related metabolic disorders can directly damage the myocardium and lead to DC, so patients with diabetic cardiovascular complications can be combined with DC. Clinically, DC has a large number of patients, causing great harm to patients. A large number of studies have found that myocardial necrosis occurs more in the acute phase of myocardial infarction and other acute lesions, and diabetic myocardial pathological changes are a chronic long-term process, mainly manifested as myocardial interstitial fibrosis, cardiomyocyte hypertrophy and cardiomyocyte apoptosis, in which cardiomyocyte apoptosis plays a major role in the occurrence and development of DC [1,2]. It has been found that up-regulation of adiponectin pathway key protein adiponectin receptor connexin 1 (APPL1) can improve diabetic cardiomyopathy [3]. Further studies have found that adiponectin pathway protects cardiovascular system by activating lipids. The APPL1-AMPK axis in the adiponectin signaling pathway inhibits the expression of nuclear factor NF-? B [4,5]. In addition, other laboratories and we have found that PPARa in the adiponectin signaling pathway inhibits NF-? B and directly participates in the prevention of apoptosis of diabetic cardiomyocytes [6]. PPARa is an important downstream molecule of AMPK signal. There may be an "APPL1-AMPK-PPAR alpha axis" which may play an important role in preventing apoptosis of diabetic cardiomyocytes. Some studies have found that glucagon-like peptide-1 (GLP-1) has a protective effect on cardiovascular system, which can reduce apoptosis of cardiomyocytes including diabetes mellitus and promote adiponectin secretion, and this effect is independent of Therefore, it is widely accepted that GLP-1 has cardiovascular protective effects in diabetic patients. The mechanism may be related to the activation of adiponectin pathway. We further speculate that GLP-1 may reduce the apoptosis of diabetic cardiomyocytes by activating adiponectin signaling pathway. To prove our hypothesis, we intend to conduct in-depth study from clinical observation, animal model and cell level. At present, there is no definite clinical diagnostic criteria for diabetic cardiomyopathy, mainly with cardiac function parameters as reference indicators. We intend to collect clinical type 2 diabetes mellitus cases and with normal. To explore the relationship between serum adiponectin level and cardiac function parameters in type 2 diabetes mellitus (T2DM) patients. Secondly, the model of type 2 diabetes mellitus (DM) rats was induced by low-dose streptozotocin (stz) + high-fat diet, and the intervention of GLP-1 receptor agonist exenatide was given to observe the effect of exenatide on diabetic cardiomyocyte apoptosis. Aim To observe the effect of exenatide on apoptosis of cardiomyocytes by regulating the level of key connexin appl 1 in adiponectin pathway. The relationship between diabetic cardiomyopathy and adiponectin level and cardiac function, and the effect of exenatide on cardiomyocyte apoptosis were studied to elucidate the effect and mechanism of exenatide on diabetic cardiomyopathy by activating adiponectin pathway. Methods Serum high-molecular-weight adiponectin levels were detected and cardiac function indexes were measured by enhanced magnetic resonance imaging (MRI). 2. The model of type 2 diabetes mellitus (DM) rats were established and fed with high-fat diet. The rats were divided into normal control group (n group), diabetic group (d group), diabetic plus insulin group (di group), diabetic plus exenatide group (de group). The levels of serum insulin and adiponectin were measured by elisa, the hemodynamics was measured by multimedia bio-signal recorder, the changes of cardiomyocyte apoptosis were detected by tuenl, and the expressions of appl-1, ampk, PPAR-a and NF-B were detected by immunohistochemistry and western-blotting. 3. Group c, group d, group de, group oe, group bl, group d, group d, group d, group d, group de, group oe, group b, group b, group d, group d, group d, group d, group d, group d, group d, group c, group d, group d, group d, group d, group d, group c, group d, group c, group d, group d, group d, group d, group d, group c, group d, group d, group d, group d, group d, group c, group d, group d, group c, group c, group d, group c, group c, group Western-blotting was used to detect the expression of appl-1, ampk, PPAR-a and nf-b. Results 30 patients with type 2 diabetes mellitus (t2dm group) and 30 healthy people (n group) were collected. There was no significant difference in the general data between the two groups. The levels of hba1c, fasting blood glucose and HOMA-IR index in T2DM group were significantly higher than those in n group. The level of serum HDL (1.23 + 0.40 mmol / l) in T2DM group was significantly lower than that in N group (1.61 + 0.45 mmol / l), and the level of TG (3.73 + 3.44 mmol / l) was significantly higher than that in N group (1.56 + 0.86 mmol / l), the difference was statistically significant (p0.05). Serum high-molecular-weight adiponectin (10.83 (+ 2.81 mg / l)) in T2DM group was significantly lower than that in N group (14.90 (+ 3.26 mg / l) (p0.05). left ventricular end-diastolic volume (edv) (59.70 (+ 7.26 ml / m2) and stroke output (sv) (34.77 (+ 6.48 ml / m2)) in T2DM group were significantly lower than that in N group (65.84 (+ 9.67 ml / m2) and SV (38.4) by cardiac contrast-enhanced magnetic resonance imaging. The end systolic volume (esv) (29.00 + 6.60ml / m2) was significantly higher than that of N group (24.25 + 5.51ml / m2). correlation analysis showed that there was a positive correlation between high molecular weight adiponectin level and stroke output of diabetic patients [r = 0.376, P = 0.041]. The levels of high molecular weight adiponectin in group D and di were significantly lower than those in group n, and the levels of high molecular weight adiponectin in group de were significantly higher than those in group di (p0.05). Myocyte apoptosis rate (55.71% + 3.84%) was significantly increased, myocardial apoptosis rate (27.43% + 3.63%) was significantly decreased in de group, and myocardial apoptosis rate (43.91% + 4.23%) was significantly increased in di group compared with de group. cardiac function test showed that compared with C group, LVSP (105.87 + 4.08mmhg) in D group and LVSP (107.19 + 4.09mmhg) in Di Group rats. LVEDP (17.62 + 1.74 mmhg) in group D and LVEDP (17.48 + 1.49 mmhg) in group Di were significantly increased (p0.05). LVSP (119.11 + 5.11 mmhg) in group de was significantly increased, while LVEDP (13.64 + 1.25 mmhg) in group Di was significantly decreased (p0.05). Compared with group c, the expression of appl-1, p-ampk and PPAR-a in myocardium of group D and di decreased significantly, but the expression of NF-B increased significantly. the expression of appl-1, p-ampk and PPAR-a in myocardium of group de were 0.65 (+) 0.02, 0.78 (+) 0.04, 1.72 (+) 0.05) respectively, which were significantly higher than that of group D and di. the expression of NF-B in myocardium of group de was significantly higher than that of group D and di. The level of high molecular weight adiponectin in the supernatant of de group, OE group and BL group was 9.40 (+ 0.16 mg / l), 8.70 (+ 0.34 mg / l) and 9.30 (+ 0.30 mg / l) respectively, which was significantly higher than that of D group (p0.05), de, OE and BL group (p0.05). There was no significant difference in serum adiponectin levels (p0.05). however, the results of apoptosis detection showed that apoptosis in de group (20% ~ 29%) was lower than that in D group (34% ~ 42%). The results of appl-1, p-ampk, PPAR-alpha and NF-B in cardiomyocytes of group 2 showed the same trend as those of animal experiment. Conclusion The impairment of cardiac systolic and diastolic function in type 2 diabetes mellitus is closely related to the decrease of circulating high molecular weight adiponectin levels. Cell autocrine adiponectin level and activation of APPL1-AMPK-PPARalpha axis signaling pathway inhibited the activation of NF-? B and myocardial cell apoptosis, and improved diabetic myocardial function.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2

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6 郁U，

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