DPP-4抑制剂阿格列汀减轻糖尿病兔心房重构及改善线粒体功能和生物合成

发布时间：2018-04-05 22:06

  本文选题：心房颤动　切入点：糖尿病　出处：《天津医科大学》2017年博士论文

【摘要】：目的:糖尿病是心房颤动(房颤)的独立的危险因素,但目前其发病机制尚不明确。最新证据显示,线粒体功能障碍参与了房颤发生的病理生理过程。二肽基肽酶-4(DPP-4)抑制剂被发现具有改善线粒体功能的作用。在本研究中,我们探讨DPP-4抑制剂阿格列汀对心房线粒体重构的影响,以及对与代谢应激相关房颤的预防作用。方法:健康日本大耳白兔用于建立家兔糖尿病模型,随机分为三组:对照组(Control,C组),经耳缘静脉注射生理盐水4ml;糖尿病组(Diabetes Mellitus,D组),经耳缘静脉注射四氧嘧啶120mg/kg;糖尿病+阿格列汀组(Diabetes+Alogliptin,DA组),口服阿格列汀12.5mg/Kg/d。每组30只,自由饮食,饲养8周。造模成功后,各组中随机抽取10只用于以下三部分实验:一、心脏超声、血流动力学、血液学检查、病理学和分子生物学实验:心脏超声测定各心腔大小、心室壁厚度及射血分数;血流动力学测定主动脉内压、心室内压及心室内压最大上升和下降速率(±dp/dtmax);血液学检查测定包括部分生化指标、胰岛素(INS)、胰高血糖素样肽-1(GLP-1),以及超氧化物歧化酶(SOD)、超敏C反应蛋白(hs-CRP)、丙二醛(MDA)、8-羟基脱氧鸟苷(8-OHDG)等氧化应激和炎症指标;病理学检查应用HE和Masson染色评价心肌细胞横截面积和胶原容积分数,透射电镜观察细胞核、肌丝及线粒体形态学;分子生物学应用Western blot方法测定TGF-β1、NF-κBp65蛋白表达水平,以及线粒体生物合成信号通路、动力学相关蛋白和ATP合酶亚基ATP5A的蛋白的表达,应用RT-PCR的方法测定电子传递链复合体Ⅰ-Ⅴ亚基的m RNA水平,以及PGC-1α、NRF-1、Tfam的m RNA表达水平。二、心脏电生理实验:利用Langendorff离体灌流模型测定心房各点的有效不应期(AERP)、有效不应期离散度(AERPD)、房间传导时间(IACT),计算房颤诱发率。三、线粒体功能实验,测定线粒体呼吸控制率(RCR)、膜电位(Δψ)及ROS生成速率。结果:1.心脏超声结果显示,与C组相比,D组和DA组左心房内径(LAD)、室间隔厚度(IVS)和左室后壁(PWLV)均增加(P0.01),而在DA组LAD、IVS及PWLV较D组降低(P0.01);三组间EF值及血流动力学参数无统计学差异。2.与C组相比,D组血清INS(8.01±1.66 mmol/L vs 17.36±3.35 mmol/L,P0.01)和GLP-1(0.35±0.07 pmol/L vs 1.21±0.38 pmol/L,P0.01)水平降低,而阿格列汀治疗后GLP-1的浓度(0.80±0.19 pmol/L vs 0.35±0.07 pmol/L,P0.01)较D组明显升高。3.D组与C组相比,MDA、8-OHDG和hs-CRP浓度升高(P0.01),而SOD活性降低(P0.05),阿格列汀治疗后降低了D组MDA、8-OHDG和hs-CRP的浓度(P0.01),但对SOD活性无明显影响。4.与C组相比,D组心肌横截面积、胶原容积分数增大(P0.05),而阿格列汀治疗后显著抑制了这种病理改变(P0.05)。5.电生理研究显示,D组AERPD较C组和DA组明显延长(P0.01);D组IACT在基础起搏周长150ms、200ms及250ms时较C组和DA组均明显延长(P0.01)。与C组相比,D组房颤的诱发率从1.33%上升至5.56%,而DA组房颤的诱发率为2.44%,升高幅度明显低于D组(P0.05)。6.线粒体功能实验显示,D组RCR值及ΔΨm较C组明显降低(P0.05),而阿格列汀治疗后逆转了这种病例生理改变(P0.05或0.01);线粒体ROS生成速率在D组明显高于C组(P0.05),阿格列汀显著抑制了糖尿病线粒体ROS的生成速率(P0.05)。7.Western blot结果显示,D组TGF-β1和NF-κBp65蛋白表达水平明显高于C组,阿格列汀治疗后显著抑制了这两种蛋白的表达;D组线粒体生物合成相关蛋白APN、p AMPK、PGC-1α、NRF1、Tfam蛋白表达水平较C组下调;而与D组比较,DA组中这些蛋白表达水平均上调;D组线粒体分裂蛋白Drp1、融合蛋白Mfn1及OPA1表达下调,而DA组中蛋白表达水平较D组均上调;三组间AMPK和ATP5A的蛋白表达水平无显著差异。8.RT-PCR实验,D组PGC-1α和Tfam的m RNA表达水平低于C组和DA组,而NRF1则在三组间无差异;D组复合体Ⅰ、Ⅱ、Ⅳ亚基NDUFB8、SDHB、MTCO1的m RNA表达水平明显低于C组和DA组,而D组复合体Ⅲ亚基UQCRC2的m RNA表达水平高于C组和DA组;三组间ATP5A的m RNA表达水平无统计学差异。结论:DPP-4抑制剂改善糖尿病心房基质,对房颤具有潜在的预防作用,其机制涉及减轻线粒体氧化应激,改善线粒体呼吸功能和促进线粒体生物合成。
[Abstract]:Objective: diabetes is atrial fibrillation (AF) independent risk factors, but its pathogenesis is still not clear. Recent evidence suggests that mitochondrial dysfunction is involved in the pathophysiology of atrial fibrillation. Two dipeptidyl peptidase -4 inhibitors (DPP-4) were found to have improved mitochondrial function. In this study, we investigate the effects of DPP-4 inhibitors on atrial remodeling in A Glenn Dean mitochondria associated with metabolic stress, as well as for the prevention of atrial fibrillation. Methods: the rabbits were used to establish the rabbit model of diabetes, were randomly divided into three groups: control group (Control, group C), intravenous injection of saline 4ml the diabetic group (Diabetes; Mellitus, D group), intravenous injection of alloxan diabetes + four 120mg/kg; A Glenn Dean group (Diabetes+Alogliptin, group DA), oral A Glenn Dean 12.5mg/Kg/d. each group had 30 rats, free diet, feeding 8 weeks made. After the success of the model in each group, 10 rats were randomly selected for the following three parts: an experiment, echocardiography, hemodynamics, blood examination, pathology and molecular biology experiment: cardiac ultrasound determination of size of the cardiac chambers, ventricular wall thickness and ejection fraction; hemodynamic measurements of aortic pressure, intraventricular pressure and ventricular the maximum rate of pressure rise and fall (+ dp/dtmax); blood tests including determination of some biochemical indexes (INS), insulin, glucagon like peptide -1 (GLP-1), and superoxide dismutase (SOD), high sensitive C reactive protein (hs-CRP), malondialdehyde (MDA), 8- hydroxy deoxyguanosine glucoside (8-OHDG), oxidative stress and inflammation index; pathological examination by HE and Masson staining were used to evaluate the myocardial cell cross-sectional area and collagen volume fraction, TEM observation of nuclear, myofilament and mitochondrial morphology; molecular biology application of Western blot method for the determination of TGF- beta 1, NF- 魏Bp65铔嬬櫧琛ㄨ揪姘村钩,浠ュ強绾跨矑浣撶敓鐗╁悎鎴愪俊鍙烽，

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