【作者】 贾俊海；

【导师】 陈克平；

【作者基本信息】 江苏大学 ， 食品科学， 2008， 博士

【摘要】 目的:本实验旨在探讨糖尿病心肌缺血再灌注损伤的发生机理,观察药物预处理和缺血预处理对糖尿病性心脏的保护作用并探讨临床价值。方法:（1）制作糖尿病模型和在体心肌缺血再灌注模型。将大鼠随机分为四组:非糖尿病假手术(C_sham)组,非糖尿病心肌缺血再灌注(C_IR)组,糖尿病假手术(D_sham)组和糖尿病心肌缺血再灌注(D_IR)组。进行血糖、血脂、血流动力学、心肌梗死范围和心室/体重指数测定,HE染色观察各组形态学变化。（2）检测C_sham组、C_IR组、D_sham组和D_IR组血清、心肌丙二醛（MDA）含量、超氧化物酶（SOD）和谷胱甘肽-过氧化物酶（GSH-P_X）活性;检测血清、心肌一氧化氮（NO）含量、一氧化氮合酶（NOS）活性;检测心肌线粒体Na⁺,K⁺-ATP酶、Mg²⁺-ATP酶、Ca²⁺-ATP酶活性;放射免疫法检测血浆血管紧张素Ⅱ（AngⅡ）、醛固酮（ALD）和血清胰岛素样生长因子-1（IGF-1）水平;免疫组织化学法检测心肌细胞间粘附分子-1（ICAM-1）和基质金属蛋白酶-2（MMP-2）蛋白表达;Western blot检测心肌RhoA和ROCK蛋白表达。（3）用维生素E预处理,检测C_IR组、D_IR组、非糖尿病维生素E(C_VE)组、糖尿病维生素E(D_VE)组血清、心肌MDA含量、SOD和GSH-P_X活性;检测心肌线粒体ATP酶活性;检测心肌ICAM-1蛋白表达;测定心肌梗死范围。（4）用灯盏花素预处理,检测C_IR组、D_IR组、非糖尿病灯盏花素(C_Bre)组、糖尿病灯盏花素(D_Bre)组血浆AngⅡ、ALD和血清IGF-1水平;检测血清、心肌MDA含量、SOD和GSH-P_X活性;检测心肌线粒体ATP酶活性;检测心肌ICAM-1蛋白表达;测定心肌梗死范围。（5）在缺血再灌注前进行缺血预处理,检测C_IR组、D_IR组、非糖尿病缺血预处理(C_IP)组、糖尿病缺血预处理(D_IP)组血清、心肌MDA含量、SOD和GSH-P_X活性;检测血清、心肌NO含量及血清、心肌NOS活性;检测心肌线粒体ATP酶活性;检测心肌MMP-2、RhoA和ROCK蛋白表达;进行心肌梗死范围测定;HE染色观察各组形态学变化。结果:（1）糖尿病大鼠血糖水平显著高于非糖尿病对应组（p<0.001）;而体重显著轻于非糖尿病对应组（p<0.01）;缺血期和再灌注期,各组间的心率、左心室收缩压、左心室舒张末压不存在显著差异;C_IR组较C_sham组心肌梗死面积增加（P<0.01）;D_IR组较D_sham组心肌梗死面积增加（P<0.05）;与C_IR组比较,D_IR组心肌梗死面积和心室重/体重比增加（P<0.001）;与C_sham组比较,D_sham组心肌梗死面积和心室重/体重比增加（P<0.001）,HE染色显示:糖尿病经历心肌缺血再灌注后,心肌损害加重。（2）与C_IR组相比,D_IR组血清和心肌MDA含量明显升高（P<0.05）;血清IGF-1含量明显降低（P<0.05）;心肌线粒体Na⁺,K⁺-ATP酶活性明显降低（P<0.05）;血清NO明显升高（P<0.01）,心肌NO明显升高（P<0.05）;心肌NOS明显升高（P<0.01）;心肌ICAM-1表达明显升高（P<0.001）;心肌MMP-2、RhoA和ROCK蛋白表达明显升高（P<0.05）。（3）糖尿病大鼠用维生素E预处理后,与D_IR组相比,D_VE组血清、心肌MDA含量明显降低（P<0.05）,血清SOD活性明显升高（P<0.01）,心肌SOD活性明显升高（P<0.05）,血清和心肌GSH-P_X活性明显升高（P<0.05）;心肌线粒体Na⁺,K⁺-ATP酶、Mg²⁺-ATP酶、Ca²⁺-ATP酶活性明显升高（P<0.05）;心肌ICAM-1蛋白表达显著降低（P<0.05）;心肌梗死面积有所降低,但无统计学意义（P>0.05）。（4）糖尿病大鼠用灯盏花素预处理后,与D_IR组相比,D_Bre组血浆AngⅡ浓度明显降低（P<0.05）,血浆ALD浓度明显降低（P<0.01）,血清IGF-1含量明显升高（P<0.05）;血清、心肌MDA含量明显降低（P<0.05）,血清SOD活性明显升高（P<0.001）,心肌SOD活性明显升高（P<0.05）,血清和心肌GSH-PX活性明显升高（P<0.05）;心肌线粒体Na⁺,K⁺-ATP酶、Ca²⁺-ATP酶活性明显升高（P<0.05）,Mg²⁺-ATP酶活性明显升高（P<0.01）;心肌ICAM-1蛋白表达显著降低（P<0.05）;心肌梗死面积有所降低,但无统计学意义（P>0.05）。（5）对糖尿病大鼠进行心肌缺血预处理后,与D_IR组相比,D_IP组血清、心肌MDA含量明显降低（P<0.05）,血清SOD活性明显升高（P<0.001）,心肌SOD活性明显升高（P<0.05）,血清、心肌GSH-P_X活性明显升高（P<0.05）;心肌线粒体Na⁺,K⁺-ATP酶活性明显升高（P<0.05）,Mg²⁺-ATP酶活性明显升高（P<0.01）;血清和心肌NO含量明显降低（P<0.05）,血清NOS活性明显降低（P<0.01）,心肌NOS活性明显降低（P<0.05）;心肌MMP-2、RhoA和ROCK蛋白表达显著降低（P<0.05）;心肌梗死范围降低（P<0.05）。结论（1）早期糖尿病可加重心肌缺血再灌注损伤。（2）糖尿病参与心肌缺血再灌注损伤可能与增加MDA和NO含量、增加NOS活性、增加心肌ICAM-1、MMP-2、RhoA和ROCK蛋白表达、降低Na⁺,K⁺-ATP酶活性、下调IGF-1浓度有关。（3）维生素可通过减轻脂质过氧化和自由基损伤,下调ICAM-1蛋白表达,拮抗糖尿病大鼠心肌缺血再灌注损伤。（4）灯盏花素可通过减轻脂质过氧化和自由基损伤,减低血浆AngⅡ、ALD浓度,增加血清IGF-1浓度,下调ICAM-1蛋白表达,减轻糖尿病大鼠心肌缺血再灌注损伤。（5）缺血预处理可减轻早期糖尿病大鼠心肌缺血再灌注损伤,可能与减轻脂质过氧化和自由基损伤,增加Na⁺,K⁺-ATP酶、Mg²⁺-ATP酶活性、下调MMP-2、RhoA和ROCK蛋白表达、降低心肌梗死范围等有关。更多还原

【Abstract】 ObjectiveThe purpose of this study was to explore the pathophysiological mechanisms underlying ischemia reperfusion injury in diabetic and non diabetic rat heart.To investigate the protective effects of pharmacological preconditioning （PPC） and ischemia preconditioning（IPC） on myocardial ischemia reperfusion injury in diabetic and non diabetic rats and to search the clinical value.Methods（1）The diabetic rat model was established by streptozotocin injection to the abdominal cavities of Spraque-Dawlay rats.Four weeks later, the myocardial ischemia reperfused models were established in anesthetic SD rats. The rats were randomly divided into two groups（diabetic group and non diabetic group）,and each group divided into two subgroups including sham group and ischemia reperfusion（IR） group. In sham group,there were made no procedures after surgery. In IR group, the injury was induced by occlusion of the left anterior descending coronary after for 30 min followed by reperfusion for 120 min. A micro-catheter was inserted into left ventricular through right carotid artery to measure the left ventricular pressure.Hemodynamic data were continuously monitored and simultaneously digitized.Blood samples were drawn from caudal vein to measure blood glucose. At the end of the experiment, ventricular blood was sampled to measure triglyceride and cholesterol.The myocardial infarct size and ventricle weight index were determined,After embedded in paraffin, heart was formed into sections, then which were stained with hematoxylin and eosin.（2）The model and protocol were performed as previously described.The changes of plasma angiotensinⅡ（AngⅡ） and aldosterone（ALD） and serum insulin-like growth factor-1 （IGF-1）were examined with RIA.The contents of malonaldehyde（MDA） and nitric oxide（NO） in serum and myocardial tissues were detected. The activities of nitric oxide synthase（NOS） and superoxide dismutase（SOD） and glutathione peroxidase（GSH-Px） in serum and myocardial tissues were measured.The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.The intercellular adhesion molecule-1（ICAM-1）,matrix metalloproteinase-2（MMP-2） protein expressions in myocardium were evaluated by immunohistochemical method. The RhoA and Rho correlation kinase（ROCK） protein expressions were assessed by western blot assay. （3）The effect of Vitamin E administrating orally for 4 weeks against myocardial ischemia reperfusion injury in diabetic and non diabetic rats was investigated.The contents of MDA in serum and myocardial tissues were detected. The activities of SOD and GSH-Px in serum and myocardial tissues were measured. The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.The ICAM-1 protein expressions were evaluated. The myocardial infarct size was determined.（4）The diabetic and non diabetic rats were given Breviscapine by stomach perfusion to investigate the protective effect of in vivo pharmacological preconditioning of Breviscapine.The changes of plasma AngⅡ,ADD and serum IGF-1 were examined by RIA. The contents of MDA in serum and myocardium were detected. The activities of SOD and GSH-P_X in serum and myocardium were measured. The activities of Na⁺,K⁺-ATPase, Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.The ICAM-1 protein expressions were evaluated. The myocardial infarct size was determined.（5）Following 4 weeks of streptozotocin-induced diabetic rats,all hearts were divided randomly into four groups:non-diabetic ischemia reperfusion group(C_IR group),non-diabetic ischemia preconditioning group(C_IP group),diabetic IR group(D_IR group),diabetic ischemia preconditioning group(D_IP group).In IR group ,the injury was induced by occlusion of the left anterior descending coronary after for 30 min followed by reperfusion for 120 min.In IP group,preconditioning consisted of three cycles of 5 min ischemia and 5 min reperfusion,prior to ischemia.At the end of the experiment,checked infarct size.The protein expressions of MMP-2 were evaluated by immunohistochemical method. The RhoA and ROCK protein expressions were assessed by western blot assay.The contents of MDA and NO in serum and myocardium were detected. The activities of SOD, GSH-Px and NOS in serum and myocardium were measured. The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.Results（1） The effects of diabetes on myocardial ischemia reperfusionAfter four weeks of diabetic duration, blood glucose levels significantly increased （P<0.001）.while body weight significantly decreased in diabetic group compared with non diabetic group （P<0.01）. There were no significant differences in Hemodynamic data（such as HR,LVSP,LVEDP）among all groups.Compared with that of C_IR group, the myocardial infarct size was increased significantly（P<0.001）,ventricular weight index was increased at 4 weeks of D_IR group as compared to C_IR group （P<0.001）.By HE staining:in the myocardial ischemia reperfusion of diabetic rats we observed disorganized cardiac muscle.（2）The effects of diabetes on myocardial ischemia reperfusionCompared with that of C_IR group, the contents of MDA and NO in myocardium were increased significantly in D_IR group（P<0.05）,the activities of NOS in myocardium were increased significantly in D_IR group（P<0.01）.The levels of IGF-1 were decreased significantly in D_IR group（P<0.05）. And the activities of Na⁺-K⁺-ATPase, Mg²⁺-ATPase, Ca²⁺-ATPase were decreased significantly in D_IR group （P<0.05）. Compared with C_IR group, the ICAM-1 and MMP-2 and RhoA and ROCK protein expressions were increased significantly in D_IR group.（3） The effects of Vitamin E on myocardial ischemia reperfusion in diabetic ratsCompared with that of D_IR group, the activities of Na⁺-K⁺-ATPase, Mg²⁺-ATPase in myocardial mitochondria were increased significantly in D_VE group（P<0.05）.The levels of MDA in serum and myocardium were decreased significantly （P<0.05）.whereas the activities of SOD and GSH-P_X increased. Compared with that of C_IR group, the levels of ICAM-1 in myocardium were decreased significantly in Dve group （P<0.05）. The myocardial infarct size were not reduced significantly（P>0.05）.（4）The effects of Breviscapine on myocardial ischemia reperfusion in diabetic ratsCompared with that of D_IR group, the levels of AngⅡand ALD in plasma were decreased significantly and the levels of IGF-1 were increased significantly in D_Bre group.The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase,Ca²⁺-ATPase in myocardial mitochondria were increased significantly in D_Bre group compared with D_IR group.The levels of MDA in serum and myocardium were decreased significantly in D_Bre group, whereas activities of SOD and GSH-Px in serum and myocardium increased significantly.The expression levels of ICAM-1 protein were decreased significantly in D_Bre group compared with D_IR group.And the myocardial infarct size were not reduced significantly（P>0.05）.（5）The effects of ischemia preconditioning on myocardial ischemia reperfusion in diabetic ratsCompared with that of D_IR group, the levels of MDA in serum and myocardium were decreased significantly, the activities of SOD and GSH-Px in serum and myocardium were increased significantly in D_IP group. Compared with that of Dm groups.the expression of MMP-2 and RhoA and ROCK in D_IP group were decreased.Compared with that of D_IR group, the contents of NO in serum and myocardium were decreased significantly,the activities of NOS in serum and myocardium were decreased significantly in D_IP group. The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase in myocardial mitochondria were increased significantly in D_IP group compared with D_IR group.And the myocardial infarct size were reduced significantly （P<0.05）. Conclusion（1） Early diabetic during ischemia significantly exacerbates myocardial ischemia reperfusion injury, as evidenced by significantly enlarged infarct size, and worse myocardial structure following myocardial IR.（2） Early diabetic during ischemia significantly exacerbates myocardial ischemia reperfusion injury by increasing MDA,AngⅡ,ALD,NO,NOS,MMP-2,ICAM-1,RhoA and ROCK,decreasing Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase,SOD,GSH-P_X and IGF-l.（3） Vitamin E could relieve myocardial ischemia reperfusion injury and the damage of lipid peroxidation and free radical induced by MIR in diabetic rats, and this effect was mediated by reduction of the expression of ICAM-1 protein.（4） Breviscapine had obvious protective effects on myocardial ischemic and reperfusion injury in diabetic rats probably by scavenging hydroxyl free radicals, decreasing the level of plasma AngⅡand ALD, the level of MDA in serum and myocardium, the ICAM-1protein expression in myocardium and increasing the level of serum IGF-1,the level of SOD and GSH-Px in myocardium, the level of Na⁺,K⁺-ATPase,Mg²⁺-ATPase,Ca²⁺-ATPase inmyocardial mitochondria.（5） IPC could reduce the severity of reperfusion induced myocardial infarct size,Protects myocardial structure in non-diabetic rats.In 4 weeks diabetes afford the same myocardium protection as in non-diabetic rats. IPC had obvious cardioprotective effects on myocardial ischemic and reperfusion injury in diabetic rats probably by scavenging hydroxyl free radicals, increasing the level of Na⁺,K⁺-ATPase,Mg²⁺-ATPase,Ca²⁺-ATPase in myocardial mitochondria and decreasing the protein expression of MMP-2 and RhoA and ROCK in myocardium.更多还原