【作者】 李素红；

【导师】 韩德五； 许瑞龄；

【作者基本信息】 山西医科大学 ， 生理学， 2012， 博士

【摘要】 第一部分高糖高脂饮食诱导IETM在代谢综合征相关AS发生发展中的作用及机制探讨目的：探讨高糖高脂饮食诱发肠源性内毒素血症(IETM)在代谢综合征相关的AS发生、发展中的作用及机制。研究血浆中持续高表达的LPS触发代谢综合征相关的AS发生、发展的作用机制,为防治代谢综合征相关的AS寻求新思路及新途径。方法：1、64只SD大鼠随机分为对照组(normal group; NC)和模型组(model group; MG),每组各32只；每组再随机分3月、6月、9月、12月4个时段进行观察,每时段8只。对照组喂饲基础饲料,模型组喂饲高糖高脂饲料。2、放免法及酶联免疫吸附实验测定大鼠外周血内毒素(LPS)、肿瘤坏死因子(TNFα)、C反应蛋白(CRP)、巨噬细胞趋化蛋白-1(MCP-1)及一氧化氮(NO)、内皮素-1(ET-1)、PAI-1的水平。3、酶联免疫吸附试验测定外周血丙氨酸氨基转移酶(ALT)、游离脂肪酸(FFA)、甘油三酯(TG)、总胆固醇(Tch)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、血糖(FPG)、空腹胰岛素(FINS)的水平,并计算胰岛素抵抗指数(HOMA-IR)。4、摘取主动脉及肝左叶,病理组织学观察主动脉壁粥样斑块的形成及肝脏组织炎症、脂变、纤维化病变。5、免疫组化法检测CD68阳性的单核／巨噬细胞在动脉壁的浸润情况；检测动脉壁iNOS和NF一κB的表达；检测Tunnel在动脉壁的表达分析细胞凋亡情况。6、苏月IV(Sudan IV)特殊染色法观察动脉壁及肝细胞的脂质异位沉积。结果：1、模型组各时段大鼠外周血LPS及炎症相关因子TNF-a、CRP、MCP-1的表达水平明显高于对照组,差异有统计学意义(P<0.05)；血管活性因子ET-1、PAI-1的表达水平明显高于对照组,而NO的表达水平低于对照组,差异有显著性(P<0.05)。2、模型组各时段大鼠外周血中ALT、FFA、TG、Tch、LDL的表达水平明显高于对照组,而HDL的水平明显低于对照组,差异有统计学意义(P<0.05)；模型组FPG、FINS的表达水平明显高于对照组,胰岛素抵抗指数HOMA-IR的数值显著高于对照组,差异有显著性(P<0.05)。3、主动脉病理组织学：对照组各时段主动脉壁未见明显异常病变；模型组第3月主动脉内皮增生；6月内皮增生进一步加重,内膜明显增厚,内皮下脂质呈条带状沉积；中膜平滑肌细胞增生及弹力纤维紊乱；内膜／中膜比值增大；9月组动脉壁内膜进一步增厚,脂质沉积进一步增多,而中膜开始出现萎缩,内膜／中膜比值进一步增大；粥样斑块形成,其内出现了小灶钙化；12月组内膜/中膜比值保持高水平,差异有统计学意义(P<0.05),显示动脉壁钙化明显加重。4、主动脉单核/巨噬细胞的浸润：对照组动脉壁未见明显单核/巨噬细胞粘附及浸润；模型组第3月主动脉内皮表面粘附个别单核/巨噬细胞,6月组内皮下可见单核/巨噬细胞浸润；9月及12月单核/巨噬细胞在内皮下浸润进一步增多,且主要集中在粥样斑块中,差异有统计学意义(P<0.05)。5、主动脉iNOS的表达：对照组动脉壁内皮细胞及平滑肌细胞iNOS散在少许细胞弱阳性表达。模型组大鼠动脉壁内皮细胞及平滑肌细胞表达iNOS随观察时间的延长阳性表达细胞逐渐增多,强度逐渐增强,各时段间的表达差异有统计学意义(P<0.05)。6、主动脉NF-κB的表达：对照组动脉壁内皮细胞和平滑肌细胞NF-κB的阳性表达呈均匀一致的胞浆弱阳性表达,核无阳性表达；模型组动脉壁内皮细胞和平滑肌细胞出现了核阳性表达,随着观察时间的延长,核阳性表达逐渐增多,胞浆阳性变得不均匀,阳性部位多聚集于核附近,模型组各时段核阳性表达有统计学差异(P<0.05)。7、主动脉Tunel的表达：Tunel检测显示对照组动脉壁内皮细胞和平滑肌细胞仅个别细胞核阳性表达；模型组3月组即可看到动脉壁内皮细胞和平滑肌细胞明显阳性表达；6月组核阳性表达增多；9月组和12月组动脉壁内皮细胞和平滑肌细胞呈弥漫核阳性表达,差异有显著性(P<0.05)。8、肝脏的病理组织学观察：对照组肝细胞未出现明显病变,仅在汇管区见散在个别炎症细胞浸润；模型组从第3个月始大鼠先后发生了脂肪肝、脂肪性肝炎,第6个月出现纤维化,第12个月发展为早期肝硬化。结论：1、高糖高脂饮食诱导大鼠发生了IETM,血浆中LPS持续高表达。2、肝脏单核/巨噬细胞被LPS激活,使肝脏发生IR及NAFLD,糖代谢、脂代谢发生紊乱,出现代谢综合征症候群。3、代谢综合征时,增多的脂质异位沉积到动脉壁,使动脉发生IR；4、持续慢性炎症、IR及脂质异位沉积损伤了内皮细胞、平滑肌细胞功能,最终导致AS的发生发展。5、高糖高脂饮食促使IETM发生,血浆高表达的LPS触发了代谢综合征相关的AS的发生及发展。第二部分高糖高脂饮食诱导IETM在代谢综合征相关的心肌损伤中的作用和机制研究目的：探讨高糖高脂饮食诱发IETM在代谢综合征相关的心肌损伤发生、发展中的作用和机制,分析血浆中持续高表达的LPS损伤心肌功能的作用机制,为预防代谢综合征相关的心肌损伤提供新思路。方法：1、动物分组：同第一部分。2、血清学检测：同第一部分。3、摘取心肌组织进行病理组织学观察,观察心肌细胞变性、凋亡及纤维化的发生情况。4、免疫组化法检测CD68阳性的单核／巨噬细胞在心肌中的浸润；检测心肌细胞iNOS和NF-kB的表达；Tunnel检测心肌细胞的凋亡情况。5、苏丹Ⅳ (Sudan Ⅳ)特殊染色观察心肌中的脂质异位沉积情况。结果：1、模型组大鼠血清学检测结果：同第一部分。2、心肌病理组织学观察：对照组大鼠心肌细胞排列整齐、规则,心肌细胞大小、直径正常,未见变性发生,无明显炎症细胞浸润；模型组大鼠3月时段心肌细胞排列尚整齐规则,部分心肌细胞体积增大,心肌纤维间可见散在炎症细胞浸润；6月时段大部分心肌细胞直径增大,炎症细胞浸润明显；9月心肌出现了小灶状坏死,12月组坏死呈多灶状,纤维组织增生,炎症细胞浸润。3、心肌内脂质异位沉积：心肌内及心肌肌束间于模型组第3月组即可见小点灶状脂质沉积,第6个月脂质沉积增多,第9-12月明显加重,呈斑片状,差异有统计学意义(P<0.05)。4、心肌细胞凋亡：对照组心肌细胞很少发生凋亡；模型组第3月时段散在心肌细胞发生凋亡,6月时段凋亡较明显,9-12月组大部分心肌细胞显示凋亡,差异有显著性(P<0.05)。结论：1、高糖高脂饮食下大鼠发生了IETM及代谢综合征相关的心肌损伤.2、血浆中LPS持续高表达,激活体内单核／巨噬细胞系统,诱导心肌发生炎症、IR及脂质异位沉积,最终导致心肌细胞凋亡,损伤心肌功能。3、高糖高脂饮食促使IETM发生,进而诱导了代谢综合征相关的心肌损伤。第三部分内质网应激在心血管疾病发生发展中的机制研究目的：探讨内质网应激在代谢相关的AS及心肌损伤发生发展中的机制方法：1、动物分组：同第一部分2、血清学检测：同第一部分3、摘取主动脉及心脏,原位杂交检测CHOP mRNA及Grp78mRNA在动脉壁和心肌中的表达结果：1、对照组动脉壁内皮细胞、平滑肌细胞及心肌细胞Grp78mRNA为阴性表达或仅少部分弱阳性表达,模型组动脉壁及心肌细胞浆Grp78mRNA阳性表达,且随着观察时间的延长,表达逐渐增强,差异有显著性意义(P<0.05)。2、对照组动脉壁内皮细胞和平滑肌细胞及心肌细胞CHOP mRNA阴性表达,模型组动脉壁及心肌细胞浆CHOP mRNA表达阳性,随鼠龄增大及观察时段延长,CHOP mRNA表达逐渐增强,差异有显著性意义(P<0.05)。结论：1、模型组动脉壁和心肌中CHOP mRNA及Grp78mRNA的表达显著增强,且与病变程度相关。2、内质网应激在代谢相关的AS及心肌损伤发生发展机制中具有重要作用。更多还原

【Abstract】 Objiective:Investigate the mechanism of intestinal endotoxemia(IETM) induced high-sucrose and high-fat diet in the development of metabolic syndrome-related AS. Analysi the molecular mechanism of sustained high expression of LPS in plasma trigger the occurrence and development of metabolic-related AS.Seek the new idea and new way of prevention and treatment of metabolic syndrome-related AS.Methods:1.64SD rats were randomly divied into nomal control group (NC group)(n=32) and model group(MG)(n=32);then randomly divided the two group into3months,6months,9months,12months.8rats per month.The NC group was fed basal diet,MG was fed high-sucrose and high-fat diet.2.The levels of endotoxin(LPS),tumor necrosis factor-α(TNF-α),C reactive protein(CRP), monocyte chemoattractant protein-1(MCP-1), nitrogen monoxidum(NO), endothelin-1(ET-1), PAI-1in serum by radio immunoassay and ELASA.3.The levels of alanine transaminase(ALT), free fatty acids(FFA), triglyceride(TG), total cholesterol(Tch), high-density lipoprotein(HDL), low-density lipoprotein(LDL), fasting plasm glucose(FPG), fasting plasm insulin(FINS) in serum were measured by ELISA. and the insulin resistance index(HOME-IR) was calculated.4.The aorta and liver left lobe were prepared for HE to observe the formation of aortic atherosclerosis and inflammation, steatosis, fiborosis of liver.5.To detect the CD68+monocyte/macrophage infiltration in the liver and artery; To detect the expression of iNOS and NF-kB in the artery; To anslysis the expression of Tunnel in artery.6.The artery and liver specimens were prepared for Sudan Ⅳ staining, and the lipid ectopic deposition were observed.Results:1.Compared with normal control groups,the levels of LPS,CRP,TNF-a,MCP-1in the serum of model group of per month were significantiy higher,and the differences had statisitic significance(P<0.05); The levels of Vasoactive factors ET-1,PAI-1also significantiy higher,but the level of NO reduced,and the differences had statisitic significance(P<0.05);2. Compared with normal control groups,the levels of FFA, Tch, TG, LDL, ALT in the serum of model group of per month were remarkably higher,but the level of HDL reduced, and the differences had statisitic significance(P<0.05); the levels of FBG,FIN were remarkably higher,HOMA-IR also significantiy higher, and the differences had statisitic significance(P<0.05);3. Histopathology of the aortic wall:the aortic wall of normal control groups of per month show no abnormal lesions.The model group of3months show aortic endothelial hyperplasia,but lipid deposition is not obvious;The aortic endothelial hyperplasia aggravated in6months,the lipid was banded deposition in subendothelial, atherosclerotic plaque was visible; Theendometrial obvious thickening, The ratio of the intima and the membrane was increased,the Smooth muscle cells proliferation and elastic fibers were disorded; The lipid deposition inarterial wall was aggravated in9months, The endometrial further thickening, The membrane began to shrink, The ratio of the intima and the membrane was further increased; Small focal calcification was appeared within the plaque; The ratio of the intima and the membrane was keeping in a high level in12months, The calcification was significantly increased, and the differences had statisitic significance(P<0.05);4. Aortic monocyte/macrophage infiltration:the aortic wall of normal control groups of per month show no monocyte/macrophage infiltration; The model group of3months show sporadic monocyte/macrophage in aortic,The infiltration further increased in6months,and the infiltration was more increased in9and12months, the differences also had statisitic significance(P<0.05);5. The expression of iNOS in aortic:the endothelial cells in aortic wall and smooth muscle iNOS of normal control groups were scattered expression. The expression of aortic iNOS in model group gradually increased with the extension of the observed time,intensity gradually increased,and the expression of per month had statisitic significance(P<0.05);6. The expression of NF-kB in aortic:The expression of NF-kB in control group show the cytoplasm of arterial wall endothelial cells and smooth muscle cells were uniform positive expression, nuclear non-positive; The arterial wall cell nuclear were positive expressed in model group,and the expression gradually increased with the extension of the observed time,the cytoplasm positive becoming uneven, The positive parts are clustered near the nuclear,the expression of Nuclear positive in model group has statisitic significance(P<0.05);7. The expression of Tunel in aortic:The Tunel detection show:The arterial wall endothelial cells and smooth muscle cells only sporadic positive expression in control group;The3months model group show the positive expression of arterial wall endothelial cells and smooth muscle cells is increased.and more evident in6months. The positive expression of arterial wall endothelial cells and smooth muscle cells is diffused in9and12months.8. Liver histopathology:The changed of liver cells is not obvious in the control group,only a little inflammatory cell in the portal area was found;The model group of3months show the rats have fatty liver, steatohepatitis, the fibrosis was appeared in6months,the early cirrhosis was formed in12months.Conclusion:1IETM can be induced by high-sucrose and high-fat diet, and there is the high level of LPS in plasma..2. The monocyte/macrophage in liver and fat were activated, resulting in IR and NAFLD of liver, disorder of glucose metabolism and lipid metabolism, Metabolism syndrome occurred.3. Lipid ectopic deposition into the artery wall in the the during of MS, resulting in the occurrence of arterial wall IR.4. The sustaining inflammation, IR and lipid ectopic deposition to promote and exacerbate the injury of endothelial cell and smooth muscle cell, and ultimately lead to the occurrence of AS.5. The high-sucrose and high-fat diet impel the occurrence of IETM, the high expression of LPS in plasma triggers the occurrence and development of the metabolic syndrome-related AS. Objiective:Investigate the mechanism of intestinal endotoxemia(IETM) induced high-sucrose and high-fat diet in the development of metabolic syndrome-related heart disease; analysis the mechanism of sustained high expression of LPS in plasma triggers the injury of myocardial cell; and provide new ideas of prevention and treatment of metabolic syndrome-related heart disease.Methodes:L.rats grouping:With the first part.2. Serological tests:With the first part.3.Collect heart tissue for histopathology, the change of myocardial cell degeneration, necrosis and fibrosis.4. Detect the CD68+monocyte/macrophage infiltration in the myocardial; detect the expression of iNOS and NF-κB in the myocardial cells; anslysis the expression of tunel in myocardial cells.5. Observe the ectopic deposition of lipid in the myocardium by Sudan IV staining.Results:1. The results of serological in model group:With the first part.2. Myocardial histopathology:The myocardial cells of control group are orderly and rules, the size of myocardial cells is normal, and no obvious degeneration, no significant infiltration; the myocardial cells of3months arranged in a basic orderly rules, a few myocardial cells increase in size, inflammatory cells can be seen scattered in Myocardial fibers; The cardiomyocyte diameter increased in6months, accompanied by inflammatory cell infiltration, lipid deposition can be seen in cardiac muscle bundle; the Myocardial show small focal myocardial necrosis in9months; the Necrotic area increased in12months, accompanied by lipid deposition, fibrosis, inflammatory cell infiltration.3. Lipid ectopic deposition of myocardial:Spotty lipid deposition of Myocardial and cardiac muscle bundle can be seen from3months, the lipid deposition aggravated in6months, and the lesions was significantly increased, becoming patchy in9and12months.4. Cardiomyocyte apoptosis:There is almost no cardiac myocyte apoptosis in control group; cardiomyocyte apoptosis is scattered in3months model group, and more obvious in6months, apoptosis becoming widely in9and12months.Conclusion:1IETM and metabolic syndrome-related myocardial injury can be induced by high-sucrose and high-fat diet.2The high expression of LPS in plasma activated the monocyte/macrophage system, resulting in Inflammation, IR and lipid ectopic deposition in myocardial, and ultimately lead to cardiomyocyte apoptosis, damaging the myocardial function.3IETM triggers the metabolic syndrome-related myocardial injury, pointed that the sustained high expression of LPS promotes the myocardial injury. Objiective:Discuss the effect about endoplasmic reticulum stress on the metabolic syndrome-related AS and myocardial injury.Methodes:1. Rats grouping:With the first part.2. Serological tests:With the first part.3. Collect the aorta and heart for detecting the expression of CHOP mRNA and Grp78mRNA in the arterial wall and myocardial through In situ hybridization.Results:1. The expression of Grp78mRNA in aortic endothelial cells, smooth muscle cells and myocardial cells in control group are negative or only weakly positive; The expression of Grp78mRNA in aortic endothelial cells and myocardial cells in model group are positive, and gradually increased with the extension of the observed time, the expression of per month had statisitic significance(P<0.05).2. The expression of CHOP mRNA in aortic endothelial cells, smooth muscle cells and myocardial cells in control group are negative; The expression of CHOP mRNA in aortic endothelial cells and myocardial cells in model group are positive, and gradually increased with the addition of the mouse age, the expression of per month had statisitic significance(P<0.05);Results:1. The expression level of Grp78mRNA and CHOP mRNA in aortic endothelial cells, smooth muscle cells and myocardial cells is coincidence with the seriousness of AS and injury of myocardium.2. Endoplasmic reticulum stress playing an important role in the development metabolism of metabolic syndrome-related AS and myocardial injury.更多还原