【作者】 周寿红；

【导师】 刘立英；

【作者基本信息】 中南大学 ， 药理学， 2010， 博士

【摘要】 目的：探讨毒死蜱对高脂饮食致动脉粥样硬化形成的影响及相关机制。方法：1.32只健康雄性新西兰兔随机分为如下四组(n=8)：对照组、毒死蜱组、高脂饮食组、高脂饮食+毒死蜱组。高脂饮食配方为通用配方,毒死蜱的剂量为(20 mg/kg/d,毒死蜱兔的口服LD50为1000-2000 mg/kg),连续观察6个月。2.动物处死前从耳缘静脉取血并分离出血清储存,用于检测血脂水平和胆碱酯酶(AChE)、对氧磷酶1(PON1)和超氧化物歧化酶(SOD)的活性以及丙二醛(MDA)的含量,及相关肝、肾功能指标。3.收集并培养腹腔巨噬细胞,测定其胆固醇流出率。4.切取一段胸主动脉采用离体血管环灌流法检测血管舒缩功能。5.采用苏丹Ⅳ染色法观察胸主动脉粥样硬化斑块,计算斑块与血管内皮表面积的百分比。颈总动脉、主动脉弓作石蜡切片,用以观察血管的形态学变化。6.采用实时定量PCR和蛋白印迹法分别检测肝、血管组织和腹腔巨噬细胞中三磷酸腺苷结合盒转运体A1 (ABCA1)的表达以及肝脏PON1的表达。结果：1.高脂饮食6个月,其血浆总胆固醇和脂蛋白水平明显升高,SOD活性降低,而MDA水平升高,其肝脏、主动脉和腹腔巨噬细胞中ABCA1的表达升高,腹腔巨噬细胞中胆固醇流出增加,肝脏中PON1的表达降低,与对照组相比,均有显著性差异；其主动脉和颈总动脉有明显的动脉粥样硬化斑块形成。但血清AChE有轻度升高,但无显著性差异。2.毒死蜱组兔血清AChE和PON1、和SOD活性和HDL-C水平降低,MDA水平升高,与对照组比较有显著性差异；肝肾功能指标与对照组比,无显著性差异。主动脉和腹腔巨噬细胞ABCA1的表达、肝脏PON1的表达均有降低,腹腔巨噬细胞中胆固醇流出减少,与对照组比较有显著性差异。3.高脂饮食+毒死蜱组PON1、和SOD活性和HDL-C水平降低,MDA水平升高,主动脉和腹腔巨噬细胞ABCA1的表达、肝脏PON1的表达均有降低,腹腔巨噬细胞中胆固醇流出减少,与单高脂饮食组或毒死蜱组相比,上述指标的改变更明显,统计学处理有显著性差异。主动脉和颈总动脉粥样硬化斑块更加明显。但血清AChE无明显改变。结论：亚毒性剂量的毒死蜱可加快高脂饮食致动脉粥样硬化形成,加重血清中总胆固醇、脂蛋白、及其它生化指标的改变,其机制可能与毒死蜱消耗有抗氧化作用的PON1和降低其活性,从而间接或直接触发氧化应激,损伤内皮功能,降低体内ABCAl的表达,继而减少胆固醇流出有关。目的：探讨对氧磷对Ox-LDL诱导的鼠源巨噬细胞性泡沫细胞形成及相关机制方法：1.将RAW264.7巨噬细胞,分为下例5组：(1)对照组,(2)Ox-LDL泡沫细胞模型组,(3-5)组分别为Ox-LDL+对氧磷(1、10和100μmol／1)组。各组细胞置5%CO2培养箱,37℃，常规培养72 h。2.油红染色进行形态学观察,高效液相色谱法测定细胞内总胆固醇(TC)、游离胆固醇(FC)水平,及胆固醇流出率。3.采用实时定量PCR和蛋白印迹法分别检测巨噬细胞中CD36、ABCA1、和酰基辅酶A胆固醇酰基转移酶1(ACAT1)的表达。结果：1. Ox-LDL处理48 h后,与正常巨噬细胞相比,油红0染色阳性细胞显著增加,细胞体积明显增大,形态多呈圆形或不规则形；对氧磷(10, 100μmol/l)组细胞油红0染色阳性细胞比Ox-LDL模型组形态学改变更明显,细胞体积明显增大,而对氧磷(1μmol/l)组细胞与模型组相比没有显著性差异。2.泡沫细胞模型组细胞内TC、FC、CE水平显著高于对照组,且CE/TC有显著性差异；对氧磷(10,100μmol/l)组细胞内TC、FC、CE水平及CE/TC均有显著性增加,与泡沫细胞模型组相比,有显著性差异,对氧磷(1μmol/l)组TC、FC、CE与泡沫细胞模型组差异无显著性。3. Ox-LDL泡沫细胞模型组细胞胆固醇流出率显著性增加,与对照组相比,有显著性差异；对氧磷(10和100μmol/l)组细胞胆固醇流出率显著性降低,与泡沫细胞模型组相比,有显著性差异；对氧磷(1μmol/l)处理组与泡沫细胞模型组相之间无显著性差异。4. Ox-LDL刺激的泡沫细胞组与对照组比CD36 mRNA表达显著增加,Ox-LDL刺激的泡沫细胞加对氧磷(10和100μmol/l)组与泡沫细胞组相比CD36 mRNA表达显著增加。对氧磷(1μmol/l)处理组与泡沫细胞模型组相比没有显著性差异。5.泡沫细胞模型组ABCA1表达较对照组显著增加；与模型组相比,对氧磷(10和100μmol/l)显著地下调了泡沫细胞中ABCA1的表达,而对氧磷(1μmol/l)没有影响泡沫细胞中ABCA1的表达。6.泡沫细胞模型组ACAT1mRNA表达较对照组显著增加；与模型组相比,对氧磷(10和100μmol/l)组显著地增加了泡沫细胞中ACAT1的表达,而对氧磷(1μmol/l)处理组没有明显增加泡沫细胞的ACAT1 mRNA表达。结论：对氧磷能加速Ox-LDL诱导的鼠源巨噬细胞性泡沫细胞形成。其机制可能与对氧磷上调泡沫细胞中CD36和ACAT1表达而降低ABCA1的表达有关。目的：探讨对氧磷下调鼠源巨噬细胞性泡沫细胞ABCA1表达和胆固醇流出的机制方法：1.将Ox-LDL刺激形成的鼠源巨噬细胞性泡沫细胞分为以下组：(1)对照组,(2)对氧磷处理组：对氧磷(1,10和100μmol/1)孵育泡沫细胞24h；对氧磷(100μmol/1)孵育泡沫细胞(6,12,24)h。2.高效液相色谱法测定测定细胞内TC及FC水平,计算CE,测定胆固醇流出率。3.采用ELISA法检测泡沫细胞内cAMP水平、腺苷酸环化酶和磷酸二脂酶的活性。4.采用实时定量PCR和蛋白印迹分别检测泡沫细胞中ABCA1的mRNA和蛋白水平的表达。结果：1.对氧磷以时间和剂量依赖的方式增加RAW264.7巨噬细胞源性泡沫细胞中TC、FC和CE水平,降低ABCA1表达和胆固醇流出。2.对氧磷降低了细胞中cAMP的水平及腺苷酸环化酶(AC)的活性和增加磷酸二酯酶(PDE)的活性。3. cAMP的类似物双丁酰环腺苷酸(dBcAMP)能抑制对氧磷降低ABCA1表达和部分阻断对氧磷降低胆固醇流出的作用。4.AC激动剂福斯高林(Forskolin)和PDE抑制剂3-异丁基-1-甲基黄嘌呤(IBMX)能拮抗对氧磷降低cAMP的作用。结论：对氧磷下调鼠源巨噬细胞性泡沫细胞ABCA1的表达,降低细胞内胆固醇流出和增加细胞内胆固醇堆积可能与干预cAMP信号通路有关目的：观察外源性PON1基因转染对对氧磷促鼠源巨噬细胞性泡沫细胞形成的影响方法：1.实验分为以下各组：(1)RAW巨噬细胞组、(2)Ox-LDL+RAW巨噬细胞组、(3)对氧磷+Ox-LDL+RAW巨噬细胞组、(4)对氧磷+Ox-LDL+PON1(+)RAW巨噬细胞组。将外源性的人的PON1基因转染RAW264.7细胞,用Ox-LDL(50μg／ml)诱导泡沫细胞形成,对氧磷处理组加对氧磷(100μmol/1)共孵育48 h。2.油红染色进行形态学观察,高效液相色谱法测定细胞内TC及FC水平,计算CE水平,测定胆固醇流出率。结果：1.Ox-LDL+RAW细胞组细胞内TC、FC、CE水平和CE／T值与RAW细胞组相比显著性增高；对氧磷(100μmol/1)+Ox-LDL+RAW细胞组组细胞内TC.FC.CE水平及CE/TC较Ox-LDL+RAW细胞组均有显著性增加,而PON1转染拮抗了对氧磷的作用。2.Ox-LDL+RAW组细胞胆固醇流出率与RAW细胞相比显著性增加；与Ox-LDL+RAW细胞组相比,对氧磷(100μmol/l)处理组细胞胆固醇流出率显著性降低,而PON1转染拮抗了对氧磷的作用。结论：外源性对氧磷酶1抑制了对氧磷促进鼠源巨噬细胞性泡沫细胞形成的作用更多还原

【Abstract】 Objective:To explore the effect of chlorpyrifos on the formation of atherosclerosis induced by the high fat diet in New Zealand rabbits and analyze the possible mechanisms.Methods:1.Thirty two healthy male New Zealand rabbits were divided randomly into four groups with eight rabbits in each group:control group, high-fat diet group, chlorpyrifos group and high-fat diet+chlorpyrifos group. Chlorpyrifos (20 mg/kg/d, the LD50 of chlorpyrifos by mouth in rabbit is 1000-2000mg/kg) was administered by lavage every day for six months.2.The levels of serum fat and activities of cholinesterase (CHE), paraoxonase 1(PON1) and alanine aminotransferase were measured respectively. Serum creatinine and blood urea nitrogen were measured respectively..The levels of serum superoxide dismutase (SOD) and malondialdehyde (MDA) were measured respectively.3.The vascular functions test was performed by using isolated blood vessel method.4.The peritoneal macrophages were assembled.and cellular cholesterol efflux was analyzed.5.Area of atherosclerosis plaque of thoracic aorta was measured by SudanⅣ. Common carotid artery and thoracic aorta were fixed in formalin, sliced and HE dyed and pathology analysis system was used.6.The expressions of ABCA1 and PON1 were detected by Real-time PCR and Western blot.Results:1.Serum activities of CHE and PON1 compared with control group were singificantly decreased, but there were not symptom of intoxation and injury of function of liver and kidney, and the level of HDL and SOD were markedly decreased, the level of MDA was increased, PON1 expression of liver was decreased, endothelium-depent and non-dependent relaxation of abdominal aorta was decreased, and expression of ABCA1 in liver, aorta and peritoneal macrophages and cholesterol efflux from peritoneal macrophages were markedly decreased in chlorpyrifos group compared with control group.2.Compared with control group, serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and triglyceride (TG) were singificantly increased, endothelium-depent and non-dependent relaxation of abdominal aorta was decreased, serum activity of PON1 and expression of ABCA1 in liver were singificantly, expression of ABCA1 in liver, aorta and peritoneal macrophages was markedly increased and cholesterol efflux was significantly increased in peritoneal macrophages in high-fat diet group. There was obvious atherosclerosis lesion in thoracic aorta and common carotid artery in high-fat diet group.3.Compared with high-fat diet group, serum activities of CHE and PON1 were singificantly decreased, but there were not symptom of intoxation and injury of function of liver and kidney, the level of SOD was markedly decreased, the level of MDA was increased, endothelium-depent and non-dependent relaxation of abdominal aorta was decreased, PON1 expression of liver was decreased and expression of ABCA1 and cholesterol efflux were singificantly decreased, the atherosclerosis lesion area in thoracic aorta and common carotid artery was increased in high-fat diet+chlorpyrifos group.Conclusion:Subtoxic dose of chlorpyrifos may accelerate formation of atherosclerosis induced by the high fat diet in New Zealand rabbits, which the mechanism may be related to the injury of endothelium, the decrease of ABCA1 expression and cholesterol efflux, the decrease of ABCA1 expression and increase of oxidative stress induced by chlorpyrifos.Objective:To explore the effect of paraoxon (PXN) on the formation of foam cells induced by the oxidized low density lipoprotein (Ox-LDL) and analyze the possible mechanisms.Methods:1.RAW 264.7 macrophages were divided into five groups:control group, foam cells model induced by Ox-LDL group, Ox-LDL+paraoxon (1μmol/l) group, Ox-LDL+paraoxon (10μmol/l) group, Ox-LDL+ paraoxon (100μmol/l) group. Cells were maintained at 37℃in 50% CO2-95% air in an incubator for 72 h.2.The cellular lipid accumulation was examined by oil red staining. The cellular contents of total cholesterol (TC) and free cholesterol (FC) were deteeted by high performance liquid chromatography assays. Cholesterol efflux from macrophages was examined.3.The expressions of CD36, ATP-binding Cassette Transporter A1 (ABCA1) and cholesterol acyltransferases-1 (ACAT-1) were detected by Real-time PCR and Western blot.Results:1.Oil red-staining positive cells were found and macrophages were filled with lipid droplet in foam cells model group. Compared with the foam cells, paraoxon (10μmol/l,100μmol/l) significantly increased the amount of oil red-staining positive cells and the contents of lipid droplet of foam cells. There was no significant differences between paraoxon (1μmol/l) group and control group.2.Compared with the control macrophages group, the contents of TC, FC, eholesterylester (CE) and CE/TC ratio in model group were significantly increased in foam cells model group. Paraoxon (10μmol/l, 100μmol/l) significantly increased the contents of TC, FC, eholesterylester (CE) and CE/TC ratio compared with foam cells model group. There was no significant differences between paraoxon (1μmol/l) group and control group.3. Compared with the control macrophages group, cholesterol efflux was markly increased in foam cell model group. Compared with the foam cell model group, cholesterol efflux was significant increased in paraoxon (10μmol/l,100μmol/l) group. Paraoxon (1μmol/l) did not influence cholesterol efflux.4. Expression of CD36 was markly up regulated in foam cells compared to the control cells. Compared with the foam cell, expression of CD36 was significantly increased in paraoxon (10μmol/l,100μmol/l) group. Paraoxon (1μmol/l) did not affect the expression of CD36.5. Compared with the control macrophages group, expression of ABCA1 was markly up regulated in foam cells group. Paraoxon (10μmol/l,100μmol/l) significantly down regulated the expression of ABCA1 compared to foam cells group. There was no significant differences between paraoxon (1μmol/l) group and control group.6. Expression of ACAT1 was markly increased in foam cells compared to the control cells. Compared with the foam cell, expression of ACAT1 was significantly up regulated in paraoxon (10μmol/l,100μmol/l) group. Paraoxon (1μmol/l) did not affect the expression of CD3Conclusion:Paraoxon accelerates the formation of foam cell induced by Ox-LDL, which the mechanism is related with up-regulation of expression CD36 and ACAT1 and down-regulation of expression ABCA1 induced by paraoxon.Objective:To investigated the effect of paraoxon on ABCA1 expression and ABCA1-dependent cholesterol efflux, and then examined the role of cyclic adenosine monophosphate (cAMP) signaling pathway in the regulation of ABCA1 expression and ABCA1-mediated cholesterol efflux by paraoxon in RAW 264.7 macrophage-derived foam cells.Methods:1. RAW 264.7 macrophages were cultured in the medium with 50μg/ml Ox-LDL to induce foam cells. Foam cells were divided into several groups:control group, paraoxon group:paraoxon (1,10 and 100μmol/1) for 24 h and paraoxon (100μmol/1) for 6,12,24 h.2. The cellular contents of total cholesterol (TC) and free cholesterol (FC) were deteeted by high performance liquid chromatography assays. Cholesterol efflux from macrophages was examined.3. The level of intracellular cAMP was measure by ELISA. The activities of adenylate cyclase(AC) and phosphodiesterase (PDE) were examined.4.The expressions of ATP-binding Cassette Transporter A1 (ABCA1) was detected by Real-time PCR and Western blot.Results:1.Paraoxn significantly down regulated ABCA1 expression and reduced ABCA1-dependent cholesterol efflux and increased the levels of the total, free and esterified cholesterols in a time-and dose-dependent manner.2.Paraoxon also markedly reduced cAMP level and decreased adenylate cyclase (AC) activity and increased cAMP-specific phosphodiesterase (PDE) activity.3.CAMP analogs dibutyryl cyclic adenosine monophosphate (dBcAMP) markedly compensated the down-regulation of ABCA1 expression and partly compensated the reduction of ABCA1-mediated cholesterol efflux induced by paraoxon.4.Both adenylate cyclase agonist forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) markedly compensated the suppression effect on cAMP level induced by paraoxon.Conclusion:Paraoxon down regulates ABCA1 expression and decreases ABCA1-mediated cholesterol efflux through cyclic AMP signaling pathway in RAW 264.7 macrophage-derived foam cells.Objective:To investigate the effect of ectogenic PON1 transfected on accelerated atherosclerosis effect of paraoxon in RAW 264.7 macrophage-derived foam cellsMethods:1. Experiments were divided into several groups:RAW 264.7 macrophages group; Ox-LDL+RAW 264.7 macrophages group; paraoxon (100μmol/1)+Ox-LDL+RAW 264.7 macrophages group; human PON1 gene transfection+paraoxon (100μmol/l)+Ox-LDL+ RAW 264.7 macrophages group. The plasmid DNA (human PON1 gene in pcDNA3.1+plasmid) was introduced into the RAW 264.7 macrophages. Foam cells were induced by 50μg/ml Ox-LDL and 100μmol/1 paraoxon was treated for 48 h.2. The cellular lipid accumulation was examined by oil red staining. The cellular contents of total cholesterol (TC) and free cholesterol (FC) were deteeted by high performance liquid chromatography assays. Cholesterol efflux from macrophages was examined.Results:1. The contents of TC, FC, eholesterylester (CE) and CE/TC ratio were significantly increased and cholesterol efflux was markly decreased in foam cells group compared with RAW macrophages group. The contents of TC, FC, eholesterylester (CE) and CE/TC ratio were significantly increased and cholesterol efflux was markly decreased in foam cells treated with paraoxon group compared with foam cells group. Compared with foam cells treated with paraoxon, the contents of TC, FC, eholesterylester (CE) and CE/TC ratio were significantly decreased and cholesterol efflux was markly increased in ectogenic PON1 transfected group.2. Cholesterol efflux was markly increased in foam cells group compared with RAW macrophages group. Cholesterol efflux was markly increased in foam cells treated with paraoxon group compared with foam cells group. Compared with foam cells treated with paraoxon, cholesterol efflux was markly increased in ectogenic PON1 transfected group.Conclusion:Ectogenic PON1 inhibits accelerated atherosclerosis effect of paraoxon in RAW 264.7 macrophage-derived foam cells.更多还原