【作者】 张英梅；

【导师】 王海昌；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 博士

【摘要】 研究背景:据国际糖尿病联合会提供的数据,目前全球糖尿病患者超过2.5亿,预计到2025年这个数字将变成3.8亿。由于人口基数大,中国将成为仅次于印度的糖尿病第二大国。因其起病隐匿且造成人体各个器官的血管损害,糖尿病绝对称得上是人体的定时炸弹。目前糖尿病患者死亡的第一位原因就是冠心病。糖尿病合并冠心病患者多为多支血管病变且病变弥漫,PCI术后的无复流发生率和严重程度均明显升高。糖尿病合并冠心病患者无复流现象的预防和治疗已经成为心血管界介入治疗和药物治疗面临的新挑战。心脏血运重建的过程中出现无复流现象,无复流现象的可能机制为:①微血管舒张功能障碍及中性粒细胞栓塞;②血小板及中心粒细胞形成微血栓堵塞微血管;③细胞肿胀挤压微血管;④血液粘滞性变化等。可见无复流现象与心肌微血管内皮密切相关,但其发生的具体机制目前尚不清楚。糖尿病以引起多器官的微血管、小血管的病变而著称,糖尿病患者无复流现象的发生很可能与心肌微血管的损伤密切相关。多项研究证实,糖尿病患者胰岛素受体后信号转导通路受损,引起eNOS表达减少,内皮细胞分泌NO的量及活性降低,可能与内皮依赖性舒张功能障碍、血小板聚集等导致无复流发生的因素有关。而胰岛素参与调节另一途径即分裂原活化蛋白激酶(MAPK)途径则保持完好,甚至加强,产生促进内皮细胞的凋亡效应,这就是近年来有些学者提出的“选择性”胰岛素抵抗。I/R也是引起MAPK信号通路激活的常见刺激因素。在I/R的刺激下,糖尿病患者的心肌微血管内皮细胞会发生怎样的变化,以及这一变化产生的可能机制目前尚不清楚。因此,本研究的实验目的:1.明确糖尿病大鼠缺血/再灌注后心肌微血管的损害是否比正常大鼠严重。2.如果是,糖尿病大鼠心肌缺血/再灌注后PI3K信号转导通路及MAPK通路的影响。这两条信号通路之间是否存在相互关系。3.如果两条信号通路间的相互关系存在,此种关系在糖尿病引起的心肌微血管损害中起到什么样的作用。4.阻断两通路间的相互关系是否可以改善糖尿病引起的心肌微血管损害。实验方法1.糖尿病大鼠模型的制备:高脂高糖饮食饲养雄性成年SD大鼠(体重200 g左右)3月后,给予低剂量STZ腹腔注射(50 mg/kg),检测造模前后血糖值,发现造模后大鼠血糖显著增高,达糖尿病诊断标准。大鼠血糖升高1周后用于实验。2.缺血/再灌注模型的制备:成年SD雄性大鼠,腹腔注射戊巴比妥麻醉,呼吸机辅助呼吸,于3～4肋间开胸1.5 cm,迅速暴露心脏,冠状动脉左室支下穿线活扣结扎,以标Ⅱ导联心电图改变为阻断血流指标,连续缺血30 min,松线再灌注开始,关胸,制备大鼠心肌I/R模型。I/R术中通过八道生理记录仪持续记录大鼠心电图、动脉压及左室内压等血流动力学指标。再灌注3 h后采用伊文蓝-硫磺素S双染法测定心肌微血管损伤范围,伊文蓝-三苯四唑氯盐(TTC)双染法确定心肌梗死范围。CD31免疫荧光染色确定各组心肌微血管内皮细胞的数量。再灌注后24 h,心脏B超测量各组大鼠心功能。3.建立心肌微血管内皮细胞(CMECs)H/R模型:无菌分离成年SD大鼠左心室组织,去除心内外膜及冠脉组织,取心尖部心肌组织剪为1 mm3组织块,经胰蛋白酶、胶原酶消化,条件培养及细胞纯化后获得CMECs。分离培养得到的CMECs置于缺氧细胞培养箱(95% N2 / 5% CO2 , 37℃)1 h后重新置于正常细胞培养箱3 h,硝酸还原法测定各组细胞NO的分泌量,流式细胞仪检测细胞凋亡,western blot方法检测Caspase-3、P-JNK,JNK,PI3K、MEKK1、IRS-1等蛋白的表达及磷酸化水平。实验结果1.糖尿病(DM)大鼠缺血/再灌注后微血管损伤范围显著大于N + I/R大鼠(1.08±0.07 % vs 3.03±0.11%,P < 0.01)梗死范围显著大于非糖尿病大鼠(57.49±1.25% vs 68.39±2.31%, P < 0.05)。DM大鼠缺血/再灌注后-dp/dtmax低于非糖尿病大鼠(-3613±88.67 vs. -4065.09±99.31,P < 0.05)。DM大鼠缺血/再灌注后左室射血分数较N + I/R组显著降低(32.5±10.0% vs 51.6±9.3%,P < 0.05),也显著低于D组(32.5±10.0% vs 57.3±8.5%,P < 0.05)DM大鼠缺血/再灌注后3 h心肌CD31阳性的细胞密度较N+I/R组显著降低(1600±82/mm vs 2653±41/mm,P < 0.05),较D组也显著降低(1600±82/mm vs 2590±52/mm,P < 0.05)。提示糖尿病大鼠缺血/再灌注后心肌微血管损伤及心功能损伤较非糖尿病大鼠加重。2.缺氧/复氧(H/R)损伤显著增加糖尿病及正常大鼠CMEC的凋亡率(2.23±0.07% vs. 31±2.32%, P < 0.01和2.33±0.16% vs. 38.17±1.97%, P < 0.01)。Caspase-3的表达也呈现出相同的趋势,另外,H/R处理后糖尿病大鼠CMECs表达Caspase-3的量显著高于非糖尿病大鼠(P < 0.05)。H/R处理后糖尿病及非糖尿病大鼠CMECs分泌NO的能力均显著下降(86.67±2.18% vs. 56.67±1.28%, P < 0.01 and 100% vs. 74.67±0.96%, P < 0.01),H/R后糖尿病大鼠CMECs分泌NO的量显著低于非糖尿病大鼠(74.67±0.96% vs. 56.67±1.28%, P < 0.01)。这些结果提示,H/R对糖尿病大鼠CMECs的分泌功能及凋亡影响更大。3.DM大鼠CMECs IRS-1的磷酸化水平显著降低(105.33±3.50% vs.56.27±4.60%, P < 0.05)。PI3K的表达在糖尿病大鼠CMECs中的表达也有明显下降(110.00±3.31% vs. 82.22±3.31%, P < 0.01),在糖尿病(82.22±3.31% vs. 35.67±1.78%, P < 0.01)和非糖尿病(110.00±3.31% vs. 89.67±2.91%,P < 0.05)大鼠中均可见到H/R引起的PI3K的表达下降。H/R的刺激正常大鼠的CMECs,MEKK1表达量(101.33±5.18% vs. 299±7.36%, P < 0.05 )JNK的磷酸化水平(97.36±0.76% vs. 162.33±4.49%,P < 0.05)均显著升高,H/R的刺激糖尿病大鼠的CMECs时,JNK的磷酸化水平增加更加显著(148.47±12.27% vs. 399.67±7.77%,P < 0.01)。因此PI3K和JNK很可能是联系糖尿病和缺血/复氧两个病理过程的重要中间分子。4.H/R处理前1 h给于PI3K的抑制剂LY294002(10μM)可进一步增加H/R导致的DM大鼠CMECs的凋亡(39.07±5.48% vs. 47.55±3.46%, P < 0.01)。LY294002也进一步增加H/R引起的DM大鼠CMECs Caspase-3蛋白的表达(338.00±29.53% vs. 450.00±15.51%, P < 0.05),减少H/R导致的大鼠CMECs分泌NO的量的减少(56.67±3.34% vs. 41.00±5.72%, P < 0.05)。LY294002进一步上调了H/R引起的DM大鼠CMECs JNK磷酸化水平的增加(260.75±8.76% vs. 373.5±10.74%,P < 0.01)。此部分结果说明,PI3K的抑制剂可通过激活JNK进一步恶化H/R导致的DM大鼠CMECs细胞凋亡和分泌功能下降。5.H/R使DM大鼠CMECs分泌NO的量显著减低(86.67±5.67% vs. 56.67±3.34%,P < 0.01),H/R处理前给于JNK的抑制剂SP600125(10μM)可部分改善DM大鼠CMECs NO分泌的减少(56.67±3.34% vs. 68.75±8.58%,P < 0.05)。SP600125降低H/R引起的糖尿病大鼠CMECs Caspase-3蛋白的表达增加(338.00±29.53% vs. 260.67±26.91%, P < 0.05)。H/R刺激下调DM大鼠PI3K的表达(82.00±9.93% vs. 35.00±5.35%,P < 0.01),JNK的抑制剂可部分上调因H/R导致的PI3K的表达下降(35.00±5.35% vs. 62.00±4.54%,P < 0.05)。此部分结果说明,JNK的抑制剂通过激活PI3K改善H/R导致的DM大鼠CMECs的分泌功能,减少凋亡。结论1.糖尿病大鼠缺血/再灌注后较非糖尿病大鼠心肌微血管损伤范围、梗死范围增加,心肌收缩、舒张能力均显著下降,心肌微血管内皮细胞数量减少。2.DM大鼠CMECs缺氧/复氧处理后凋亡增加,NO分泌量下降,同时伴有JNK磷酸化水平增加及PI3K的表达减少。3. H/R刺激通过下调PI3K的表达减少DM大鼠CMECs分泌NO的量,而JNK的抑制剂可通过上调PI3K的表达,改善DM大鼠CMECs分泌NO的能力。4.H/R也激活JNK,引起DM大鼠CMECs的凋亡,PI3K的抑制剂促使JNK进一步活化,增加H/R引起的DM大鼠CMECs的凋亡。上述结果提示MAPK信号通路与PI3K信号通路的相互关系参与缺血/再灌注引起的糖尿病大鼠心肌微血管的损伤。以JNK和PI3K为连接点的两条信号通路的相互关系的存在为I/R对糖尿病患者造成的心肌微血管损害提供可能的分子机制和治疗靶点。更多还原

【Abstract】 BackgroundAccording to the data provided by International Diabetes Faderation (IDF), the number of the diabetic patients is over 250 million, and the number will become 380 million by 2025. China will be the second biggest diabetic country in the world. The first cause of death in diabetic patients is cardiovascular disease. Multivessel coronary artery disease is always seen in diabetic coronary artery disease patients. The incidence of no-reflow after PCI treatment is much higher in diabetic patients compared with those patients without diabetes. The therapy and prevention of no-reflow penomenon have become a new challenge in the field of cardiovascular disease.The possible mechanism of no-reflow penomenon in PCI treatment are the following points:①Microvessel vasodialation dysfunction and neutrophilic granulocyte blocking in microvessles;②Thrombocyte occludes in microvessles;③Endothelial cells swelling;④Change of the stagnation of the blood viscosity. It seems that no-reflow phenomenon is associated with microvascular injury, but the accurate mechanism is not clear. Diabetes is famous of causing mulp-organ microvessle injury. The no-reflow phenomenon seen in diabetic patients may be associated with cardia microvessle injury.Many data reported that the insulin receptor pathway was hurt and the expression of eNOS and secretion of NO were decreased in diabetic patients. These changes were associated with endothelium-dependent vasodialation dysfuncrion and thrombocyte aggragation which were causes of no-reflow phenomenon. But another pathway associated with insulin named MAPK pathway was actived which resulted in endothelial cells apoptosis. This phenomenon was called selective insulin resistance. I/R is another important stimulus for MAPK pathway. What will happen to endothelial cells when stimulated by I/R in diabetic patients? It is not clear now.Objectives1. To determine whether cardia microvessle injury in diabetic rats was more severe than in normal rats following the treatment of I/R.2. If so, to investigate the effect of I/R on PI3K pathway and MAPK pathway, and the possible relationship between the two pathways.3. If the relationship between the two pathways was exsited, to see the role of it in the cardia microvessle injury caused by I/R in diabetic rats.4. To elucidate whether inhibition of the relationship between the two pathways was helpful to improve cardia microvessle injury caused by I/R in diabetic rats.Methods1. Male Sprague-Dawley rat, 200 g, dieted with high glucose and high cholesterol for 12 weeks, diabetes was induced with a single intraperitoneal injection of streptozotocin (50 mg/kg, Sigma). Random serum glucose greater than 16.7 mmol/L were considered success.2. Rats were anesthetized and myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk and making a slipknot around the left anterior descending coronary artery. After 30 minutes of ischemia, the slipknot was released and the myocardium was reperfused for 3 hours. Hemodynamic data were continuously monitored on a polygraph (RM-6200C) and simultaneously digitized by using a computer interfaced with an analog-to-digital converter. Even’s blue and TTC staining was used to determine the infarction area, and Even’s blue and Thioflavin-S staining was used to determine the no-reflow area after 30 min ischemia and 3 hours after reperfusion. Immunofluorescence assay of CD31 was used to determine the number of endothelial cells. Echocardiography was performed at 24 hours after reperfusion.3. Followed by removal of the endocardial endothelium and the epicardial coronaries, the left ventricles were cut into small pieces and incubated in 2 ml 0.2% collagens, isolated and purified cardiac microvascular endothelium cells(CMECs). The isolated primary CMECs were put into hypoxia incubator (95% N2 / 5% CO2 , 37℃) for 1 h and then returned to normal incubator for 3 h. Nitrate reduction kit was used to analyse the secretion of NO. Flow cytometry assay was used to detect the apoptosis of cells, and the expression of PI3K, P-JNK, JNK, MEKK1、IRS-1 were determined by western blot at the end of 3 h reoxygenation.Results1. The No-reflow size in diabetic rats significantly larger than that in normal rats after I/R (1.08±0.07 % vs 3.03±0.11%,P < 0.01). The infarct size (IS)/AAR ratio in diabetic rats was significantly larger than in normal rats (57.49±1.25% vs 68.39±2.31%, P < 0.05)too. I/R decreased the -dp/dtmax in both diabetic rats and normal rats(-4326.75±108.90 vs. -3613±88.67, P < 0.01 and -4342.57±87.49 vs. -4065.09±99.31, P < 0.01), and -dp/dtmax was signaificantly lower in diabetic rats compared with normal rats after I/R treatment. The EF and FS value was attenuated in diabetic rats after I/R treatment (32.5±10.0% vs 57.3±8.5%,P < 0.05). The density of CD31 postive cells was lower in in diabetic rats after I/R campared with that in normal rats (1600±82/mm vs 2653±41/mm, P < 0.05).2. H/R resulted in a significant increased CMECs apoptosis index in both normal and diabetic rats (2.33±0.16% vs. 38.17±1.97%, P < 0.01 and 2.23±0.07% vs. 31±2.32%, P < 0.01). The expression of Caspase-3 showed the same trend. Further more, the expression of Caspase-3 in diabetic CMECs was significantly higher than in normal CMECs (P < 0.05). H/R reduced the secretion of NO in diabetic CMECs compared with normal CMECs after 3 h reoxygenation (74.67±0.96% vs. 56.67±1.28%, P < 0.01).3. The phosphorylation of IRS-1 and the expression of PI3K were significantly attenuated in diabetic CMECs compared with normal CMECs (P < 0.05). Following 3 h reoxygenation, the expression of PI3K was diminished in both diabetic CMECs and normal CMECs (P < 0.05). H/R increased the expression of MEKK1 and phosphorylation of JNK in both diabetic CMECs and normal CMECs (P < 0.01). The phosphorylation of JNK seems increased more in diabetic CMECs following H/R stimulus compared with in normal CMECs (148.47±12.27% vs. 399.67±7.77%, P < 0.01).4. PI3K inhibitor LY294002 (10μM) increased the H/R induced apoptosis in diabetic CMECs (39.07±5.48% vs. 47.55±3.46%, P < 0.01) and increased expression of Caspase-3 (338.00±29.53% vs. 450.00±15.51%, P < 0.05). LY294002 attenuated the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 41.00±5.72%, P < 0.05). The phosphorylation of JNK was increased by LY294002 followed by the treatment of H/R in diabetic CMECs (260.75±8.76% vs. 373.5±10.74%, P < 0.01).5. JNK inhibitor SP600125 (10μM) increased the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 68.75±8.58%, P < 0.05). SP600125 reduced the H/R increased expression of Caspase-3 (338.00±29.53% vs. 260.67±26.91%, P < 0.05). The reduced expression of PI3K induced by H/R was increased partly by SP600125 in diabetic CMECs (35.00±5.35% vs. 62.00±4.54%,P < 0.05).Conclusions1. Exaggerated no-reflow areas and infarction areas accompanied by severe impairment of cardiac function and endothelial cell density presented in diabetic rat model after I/R.2. H/R induced increased apoptosis and decreased NO secretion, accompanied by increased phosphorylation of JNK and attenuated expression of PI3K.3. JNK inhibitor improved the NO secretion ability of CMECs through increasing the expression of PI3K in diabetic CMECs.4. PI3K inhibitor increased the H/R induced apoptosis through activating JNK in diabetic CMECs.更多还原