【作者】 王东娟；

【导师】 王海昌； 曹丰；

【作者基本信息】 第四军医大学 ， 内科学， 2013， 博士

【摘要】 实验背景：糖尿病是危害人类健康的重大疾病，具有高发病率，高致残率和高致死率的特点。糖尿病心血管并发症是引发糖尿病患者死亡的首要原因。2007年ESC/EASD（欧洲心脏病学会/欧洲糖尿病研究学会）指南中指出与非糖尿病人群相比，男性糖尿病患者心血管疾病的发生风险增加了2-3倍，而女性糖尿病患者心血管疾病的发生风险增加了3-5倍。另有研究发现，糖尿病患者急性心肌梗死后给予血管再通治疗时无复流现象的发生率是非糖尿病患者的4倍，而急性心肌梗死后充血性心力衰竭的发生率是非糖尿病患者的3倍。因此，寻找有效的干预靶点早期防治糖尿病心血管并发症对于糖尿病患者的预后具有十分重要的意义。糖尿病心肌微血管损伤在糖尿病心血管并发症的发生发展中起着非常重要的作用。心肌微血管位于循环的末梢部分，决定心肌灌注水平，在一定程度上影响冠脉储备。在糖尿病状态下，心肌微血管损伤早于大血管及心肌细胞。目前研究已发现糖尿病患者心肌微血管内皮细胞连接不完整，屏障功能降低，同时心肌微血管密度降低，微血管数/心肌纤维数比率降低，这些变化导致了心肌微循环障碍。心肌微循环是心肌细胞与血液进行物质交换的场所，微循环障碍会影响微循环灌注，造成心肌局部或整体氧供与血流量失衡，从而引起心脏的结构变化和功能障碍。即使无明显冠脉大血管狭窄，糖尿病心肌微循环障碍仍可导致临床不良事件的发生。因此，减轻糖尿病心肌微血管损伤对于糖尿病心血管并发症的防治非常重要，但是，目前仍无有效的措施干预并延缓糖尿病心肌微血管损伤的发生发展。胰高血糖素样肽-1（glucogan like peptide-1，GLP-1）是由空、回肠上皮L细胞分泌的一种小分子多肽。近年研究发现，GLP-1除了降血糖作用外，对心血管系统也有直接的保护作用，如减轻心肌缺血/再灌注损伤、舒张血管、促进病理状态下心室功能的恢复等，而抗氧化应激损伤是其发挥心血管保护作用的重要机制之一。已有研究指出，氧化应激是引起糖尿病和心血管疾病的“共同土壤”，内皮细胞氧化应激可导致内皮功能失调，从而引发心血管疾病的发生。但是，GLP-1对于糖尿病心肌微血管是否具有保护作用尚未见报道，其发挥保护作用的机制是否是通过抗氧化应激损伤仍需进一步的研究。因此，本课题拟分别从整体动物、组织和细胞水平观察GLP-1对糖尿病心肌微血管损伤的保护作用，并进一步研究其发挥保护作用的下游分子机制。研究目的：1.建立糖尿病大鼠模型，明确vidgliptin和exenatide对糖尿病大鼠心肌微血管损伤的保护作用，并进一步明确心肌微血管功能的改善对心脏葡萄糖摄取能力及心脏功能的影响。2.分离培养心肌微血管内皮细胞，明确心肌微血管内皮细胞是否表达GLP-1受体，并进一步观察GLP-1对高糖作用下心肌微血管内皮细胞的保护作用。3.明确GLP-1发挥心肌微血管内皮细胞保护作用的机制，致力于阐明cAMP/PKA/Rho信号通路在其中的作用。研究方法：1.选取体重为200-220g雄性SD大鼠，腹腔注射35mg/kg链脲佐菌素（streptozocin，STZ），连续3天，以2次随机血糖≥16.7mmol/L视为糖尿病造模成功。2.将糖尿病大鼠随机分为6组，分别为：糖尿病+vildagliptin（1mg/kg），糖尿病+exenatide（1nmol/kg），糖尿病+insulin（0.5U），糖尿病+insulin（1U），糖尿病+insulin（1.5U），糖尿病+insulin（2U），测各组血糖，连续监测14天，选择与vildagliptin和exenatide降糖能力相当的insulin剂量。3.将糖尿病大鼠随机分为4组，分别给予vehicle，vildagliptin（1mg/kg），exenatide（1nmol/kg）或insulin（1.5U）治疗12周，以正常大鼠作为对照组，检测各组大鼠体重、血糖及胰岛素水平的变化。4.以树脂（Mercox，SPI）灌注各组大鼠心脏，扫描电镜下观察心肌微血管内皮细胞连接完整性。5.取各组大鼠心脏悬挂于Langendorff装置，硝酸镧灌流液以生理压力灌流20分钟，透射电镜下检测各组大鼠心肌微血管的屏障功能变化。6.采用小动物超声检测各组大鼠心脏功能。7.采用小动物PET/CT检测各组大鼠心脏葡萄糖摄取能力。8.分离培养心肌微血管内皮细胞，分为以下4组：对照组（5.5mmol/L），高糖组（25mmol/L）+vehicle，GLP-1预处理组（10nmol/L），高糖+GLP-1预处理组。9.采用免疫荧光及Western blot法检测大鼠心肌微血管内皮细胞GLP-1受体的表达。10.采用超氧化物试剂盒及超氧化物阴离子探针检测各组心肌微血管内皮细胞ROS的生成。11.通过TUNEL法及caspase3表达测定心肌微血管内皮细胞凋亡。12.采用Western blot法检测心肌微血管内皮细胞p47^phox，gp91^phox，p22^phox，p40^phox，Rho及ROCK的表达。研究结果：1.每日给予长效insulin1.5U时，其降糖作用与vildagliptin（1mg/kg/d）及exenatide（1nmol/kg/d）的降糖作用相当。2.糖尿病大鼠心肌微血管内皮细胞连接不完整，屏障功能降低，给予小剂量insulin治疗其改善不明显，给予vildagliptin或exenatide治疗12周后，其心肌微血管内皮细胞连接完整性及心肌微血管屏障功能明显改善。3.糖尿病大鼠心脏葡萄糖摄取能力明显降低，给予小剂量insulin治疗其葡萄糖摄取能力无明显改善，而给予vildagliptin或exenatide治疗12周以后，其心脏葡萄糖明显改善。4.糖尿病大鼠心脏舒张功能明显降低，给予小剂量insulin治疗其心脏舒张功能改善不明显，而给予vildagliptin或exenatide治疗12周后，其心脏舒张功能明显改善，但是其收缩功能无显著变化。5.心肌微血管内皮细胞呈“铺路石样”生长，乙酰低密度脂蛋白吞噬实验阳性。6.免疫荧光及Western blot法检测均发现心肌微血管内皮细胞表达GLP-1受体。7.高糖可诱导心肌微血管内皮细胞ROS生成增加，给予GLP-1预处理后，ROS生成减少。8.高糖作用下心肌微血管内皮细胞NADPH氧化酶活性升高，p47^phox，gp91^phox，p22^phox及p40^phox表达增加，给予GLP-1预处理后，NADPH氧化酶活性下降，p47^phox，gp91^phox，p22^phox及p40^phox表达降低。9.高糖可诱导心肌微血管内皮细胞凋亡增加，给予GLP-1预处理后，心肌微血管内皮细胞凋亡减少。10.高糖作用下，心肌微血管内皮细胞Rho及ROCK的表达升高，给予GLP-1预处理后Rho及ROCK表达下降，同时给予PKA抑制剂H89后，Rho及ROCK表达再次升高。11.分别给予GLP-1或Rho抑制剂fasudil后均可抑制高糖诱导的心肌微血管内皮细胞ROS的生成，但同时给予GLP-1和fasudil并不能进一步抑制ROS的生成，H89可逆转GLP-1抑制ROS生成的作用。12.分别给予GLP-1或Rho抑制剂fasudil后均可降低高糖诱导的心肌微血管内皮细胞p47^phox及gp91^phox的表达，但同时给予GLP-1和fasudil并不能进一步降低p47^phox及gp91^phox的表达，H89作用下p47^phox及gp91^phox的表达再次升高。研究结论：1. Vidgliptin和exenatide可保护糖尿病心肌微血管的屏障功能及内皮细胞连接完整性，从而进一步改善了糖尿病心脏葡萄糖摄取能力及心脏舒张功能。2. GLP-1可抑制高糖诱导下心肌微血管内皮细胞的氧化应激并减少细胞凋亡。3. GLP-1的心肌微血管保护作用可能是通过cAMP/PKA/Rho通路实现的。更多还原

【Abstract】 BackgroundDiabetes mellitus （DM） is recognized as a major risk factor for cardiovascular disease,the leading etiology of morbidity and mortality in the diabetic population. Diabeticcardiovascular disease results from many causes such as microangiopathy, myocardialmetabolic abnormalities and fibrosis. Under microangiopathy, the vessel wall ofmicrovessels become thicker and vulnerable for bleeding, protein leakage, and slow bloodflow. Accumulating evidence has demonstrated that microvascular injury plays a veryimportant role in the diabetic cardiovascular dysfunction. However, there are still feweffective strategies to prevent the progress of the microvascular dysfunction in diabetesmellitus. Glucagon-like peptide-1（GLP-1） is a hormone predominately synthesized andsecreted by intestinal L-cells. Pharmacological modulation of the GLP-1system hasemerged as a treatment option for diabetes mellitus. In addition to its glucose loweringproperties, GLP-1was found to have multiple cardioprotective effects. Impaired cardiacmicrovascular function is thought to contribute greatly to the diabetes cardiovasculardisease. Yet the effects of GLP-1on cardiac microvessels remained unclear, this studywas to assess if GLP-1could protect the cardiac microvessels and subsequently improvecardiac function and glucose metabolism and to investigate the underlying regulatorymechanism in diabetes.Methods1. Male Sprague-Dawley （SD） rats （weight,200-220g） were made diabetic usingstreptozotocin （STZ） injection. Blood glucose levels were tested1week after STZinjection. Animals with glucose levels≥16.7mmol/L were considered diabetic rats.2. To determine the dosage of insulin to control blood glucose in DM group at the similarlevel as vildagliptin-and exenatide-treated groups, diabetic rats were randomized intothe following groups:（1） Vildagliptin group that received daily treatment ofvildagliptin at1mg/kg of body weight;（2） Exenatide group that received dailytreatment of exenatide at1nmol/kg of body weight;（3） Insulin group that receiveddaily treatment of insulin at0.5U,1U,1.5U, and2U. Blood glucose was measuredonce a day for2weeks.3. STZ-induced diabetic rats were randomized to12weeks of treatment with vehicle,vildagliptin （DPP-4inhibitor,1mg/kg/d）, exenatide （GLP-1analogue,1nmol/kg/d）,or insulin （1.5U）. Before and after treatment, blood glucose levels, weight, and plasmainsulin levels were assessed.4. Cardiac microvascular barrier function was detected by scanning electron microscopyand transmission electron microscopy.5. Cardiac function was examined by echocardiographic measurements.6. Cardiac glucose metabolism was examined by18F-FDG PET/CT. 7. Adult rat cardiac microvascular endothelial cells （CMECs） were isolated and replacedto different conditions after confluence: normal glucose medium （5.5mmol/L）, normalglucose plus GLP-1（10nmol/L）, high glucose medium （25mmol/L） plus vehicle（DMSO）, high glucose plus GLP-1（10nmol/L）.8. Glucagon-like peptide-1receptor （GLP-1R） expression was detected byimmunofluorescence stainning and Western blot.9. Superoxide assay kit and dihydroethidine （DHE） staining were used to assessoxidative stress.10. TUNEL staining and caspase3expression were used to assess the apoptosis ofCMECs.11. H89was used to inhibit cAMP/PKA pathway; fasudil was used to inhibit Rho/ROCKpathway. The protein expression of Rho, ROCK, p47^phox, gp91^phox, p22^phox, and p40^phoxwere examined by Western blot analysis.Results1. Insulin treatment at1.5U dosage per day had the similar effect in blood glucose tothose elicited by vildagliptin and exenatide in diabetic rats.2. Significant deficit in barrier function of cardiac microvessels was found in diabeticrats. After12weeks of treatment with vildagliptin or exetinade, the cardiacmicrovascular barrier function was improved, in a manner more pronounced than theinsulin.3. Diabetes led to a defective18F-FDG uptake in the heart, the effect of which wassignificantly improved by vildagliptin or exenatide treatment. Insulin treatmentexhibited less18F-FDG uptake in the heart compared with administration ofvildagliptin or exenatide.4. Diabetic rats exhibited significantly dampened diastolic function, as manifested byincreased E/A ratio and LVEDD, the effects of which were mitigated by vildagliptin orexenatide treatment. Compared with insulin treated group, vildagliptin or exenatide further improved cardiac diastolic function in diabetic rats. However, there was littledifference in FS among the four groups studied.5. GLP-1R was expressed on CMECs.6. High glucose increased ROS production and NADPH activity after24hoursincubation in CMECs, while GLP-1decreased high glucose-induced ROS production.The protein expression of p47^phox, gp91^phox, p22^phox, and p40^phoxsubunits of NADPHoxidase were significantly increased in high glucose-induced CMECs compared withcontrol group. After treated with GLP-1, the expression of p47^phox, gp91^phox, p22^phox,and p40^phoxwere significantly decreased compared with high glucose-inducedCMECs.7. GLP-1exerted an anti-apoptotic effect on high glucose-induced CMECs.8. Incubation of CMECs with high glucose significantly upregulated Rho expression andelicited a significant increase in ROCK expression. Treatment of CMECs with GLP-1significantly alleviated high glucose-induced increase in Rho and ROCK expressionwithout affecting these parameters under normal glucose condition. Incubation of cellswith the PKA selective inhibitor H89abrogated GLP-1-induced effect on Rhosuppression.9. Chronic exposure of CMECs to high glucose promoted intracellular ROS levels, theeffect of which was obliterated by GLP-1. Incubation of CMECs with fasudil inhibitedROS production. However, combination of GLP-1and fasudil failed to furtherattenuate ROS production.10. The inhibitory effect of GLP-1on high glucose-induced ROS accumulation wassignificantly attenuated by treatment with H89.11. In line with the changes in intracellular ROS accumulation, the inhibitory effect ofGLP-1on high glucose-induced activation of p47^phoxand gp91^phoxwas reduced byH89. The combined treatment with GLP-1and fasudil failed to produce any additiveeffects on high glucose-induced p47^phoxand gp91^phoxexpression and oxidative stress. ConclusionsGLP-1could protect the cardiac microvessels against oxidative stress, apoptosis andthe resultant microvascular barrier dysfunction in diabetes rats, en route to improvedcardiac diastolic function and cardiac glucose metabolism. The protective effects ofGLP-1are dependent on downstream inhibition of Rho through a cAMP/PKA-dependentmanner, resulting in a subsequent decrease in the expression of NADPH oxidase. Thesefindings should provide important implications for diabetes patients with cardiovasculardisease, where GLP-1may hold promise for prevention and treatment.更多还原