【作者】 姚瑞；

【导师】 廖玉华；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 第一部分非洛地平抑制高胆固醇饮食ApoE KO小鼠动脉粥样硬化形成机制的研究目的:氧化应激和炎症参与了动脉粥样硬化(As)的发生与发展。研究发现部分钙通道阻断剂(CCB)有抗As的作用,然而其具体机制尚未明了。本研究旨在探讨非洛地平对载脂蛋白E基因敲除(ApoE KO)小鼠As斑块发生发展的影响及其分子机制。方法:ApoE KO小鼠随机分为普食组(ND组)、高胆固醇饮食组(HCD组)、高胆固醇饮食+非洛地平组(HCD+Fel组),每组15只,分别予蒸馏水、蒸馏水、非洛地平5mg/kg/d灌胃12周,内眦取血检测血清总胆固醇、甘油三酯水平;冰冻切片光镜下定位主动脉根部,油红O染色计算斑块面积;Western Blot检测胞浆胞核核因子-кB(NF-кB)p50,p65和I-кB的表达;实时定量PCR和Western Blot方法检测主动脉烟酰胺腺嘌呤二核苷酸[NAD(P)H]氧化酶亚基p47phox和Rac-1及炎症因子肿瘤坏死因子-α(TNF-α)、单核细胞趋化因子-1(MCP-1)和血管细胞黏附分子-1(VCAM-1)的表达。结果:(1)高胆固醇饮食的ApoE KO小鼠血脂水平明显高于普食的ApoE KO小鼠。(2)无论普食还是高胆固醇饮食的ApoE KO小鼠均有明显的斑块形成;且HCD组斑块面积明显高于ND组;与HCD组相比,HCD+Fel组斑块面积明显减小。(3)非洛地平可以降低斑块内NF-κB的激活。(4)非洛地平降低斑块内NAD(P)H氧化酶亚基p47phox和Rac-1以及炎症因子TNF-α、MCP-1、VCAM-1的表达。结论:CCB非洛地平可以抑制As进展,其机制可能为通过抑制NF-κB的激活,阻断氧化应激和炎症信号通路激活后级联反应,从而降低炎症因子的表达,抑制炎症反应,阻止了As进一步发展。第二部分厄贝沙坦抑制高胆固醇饮食ApoE KO小鼠动脉粥样硬化斑块形成的机制研究目的:As是一种慢性炎症性疾病,肾素-血管紧张素系统(RAS)在其发生发展中扮演重要的角色。研究发现血管紧张素Ⅱ受体1拮抗剂(ARB)有抗As的作用,然而其具体机制尚未明了。本研究旨在探讨ARB厄贝沙坦对ApoE KO小鼠As斑块发生发展的影响及其分子机制。方法:ApoE KO小鼠随机分为普食组(ND组)、高胆固醇饮食组(HCD组)、高胆固醇饮食+厄贝沙坦组(HCD+Irb组),每组15只,分别予蒸馏水、蒸馏水、厄贝沙坦10mg/kg/d灌胃12周。内眦动脉取血检测血清胆固醇和甘油三脂水平;冰冻切片光镜下定位主动脉根部,油红O染色评估斑块大小;Western blot方法检测信号转导通路蛋白细胞外信号调节激酶1/2(ERK1/2)、Janus激酶2(JAK2)、信号传导子和激活子3(STAT3)、I-кB和胞浆胞核NF-κB的表达,实时定量PCR和Western blot方法检测主动脉NAD(P)H氧化酶亚基p47phox和Rac-1及炎症因子TNF-α、MCP-1、白介素-6(IL-6)和VCAM-1的表达。结果:(1)高胆固醇饮食的ApoE KO小鼠血脂水平明显高于普食的ApoE KO小鼠。(2)无论普食还是高胆固醇饮食的ApoE KO小鼠均有明显的斑块形成;HCD组斑块面积明显高于ND组;与HCD组相比,HCD+Irb组斑块面积明显减小。(3)厄贝沙坦可以降低信号转导通路蛋白ERK1/2、JAK2、STAT3和NF-κB的激活;(4)厄贝沙坦显著降低p47phox和Rac-1以及炎症因子TNF-α、IL-6、MCP-1和VCAM-1的表达。结论:ARB厄贝沙坦有显著的抑制As进展作用,其机制可能为抑制NAD(P)H氧化酶生成,降低信号通路蛋白ERK1/2、JAK2、STAT3和NF-κB的激活,阻断氧化应激和炎症信号通路,降低炎症因子的表达,从而达到抑制As发生发展的目的。第三部分低剂量非洛地平和厄贝沙坦联合治疗动脉粥样硬化的作用与机制目的:探讨低剂量CCB非洛地平和ARB厄贝沙坦联合使用对高胆固醇饮食ApoE KO小鼠As形成的影响及机制。方法:ApoE KO小鼠随机分为普食组(ND组)、高胆固醇饮食组(HCD组)、高胆固醇饮食+非洛地平+厄贝沙坦组(HCD+Fel+Irb组),每组15只,分别予蒸馏水、蒸馏水、非洛地平2.5mg/kg/d+厄贝沙坦5mg/kg/d灌胃12周。内眦动脉取血检测血清胆固醇和甘油三脂水平;冰冻切片光镜下定位主动脉根部,油红O染色评估斑块大小;实时定量PCR和Western blot方法检测主动脉中NAD(P)H氧化酶亚基p47phox和Rac-1及炎症因子TNF-α、IL-6、MCP-1和VCAM-1的表达。结果:(1)高胆固醇饮食的ApoE KO小鼠血脂水平明显高于普食的ApoE KO小鼠。(2)无论普食还是高胆固醇饮食的ApoE KO小鼠均有明显的斑块形成;且HCD组斑块面积明显高于ND组;与HCD组相比,HCD+Fel+Irb组斑块面积明显减小。(3)低剂量非洛地平+厄贝沙坦联合使用可以明显降低NAD(P)H氧化酶亚基p47phox和Rac-1及炎症因子TNF-α、IL-6、MCP-1、VCAM-1的表达。结论:低剂量非洛地平和厄贝沙坦联合使用同样可以抑制As进展,其机制可能是通过干预氧化应激和降低炎症因子的表达,抑制炎症反应,阻止了As进一步发展。更多还原

【Abstract】 Part I Molecular Mechanisms of Felodipine Suppressing Atherosclerosis in High Cholesterol-diet Apolipoprotein E Knock-out MiceObjective: Oxidative stress and inflammation processes are key components of atherosclerosis (As), from fatty streak formation to plaque rupture and thrombosis. Evidence has revealed that calcium channel blockers (CCBs) could retard atherogenesis, but the exact mechanisms have not been fully elucidated. The present study was undertaken to investigate the potential effects and molecular mechanisms of a calcium channel blocker felodipine on the process of As in high cholesterol-diet (HCD) apolipoprotein E knock-out (ApoE KO) mice.Methods: Adult male ApoE KO mice were divided into three groups randomly (n=15 each): normal diet (ND) group, HCD group and HCD plus felodipine (HCD+Fel) group. They were given ND or HCD and randomized to no treatment or felodipine 5mg/kg/d for 12 weeks. The plasma total cholesterol and triglyceride concentration were measured by autoanalyzer. Atherosclerotic lesion area in arotic root was evaluated by oil red O staining. Nuclear factor-κB (NF-κB) and inhibitors ofκB (IκB) were measured by western blot. Nicotinamide-adeninedinucleotide phosphate [NAD(P)H] oxidase subunits and inflammatory cytokines were measured by real-time reverse-transcription polymerase chain reaction (PCR) and western blot.Results: (1) The ApoE KO mice with HCD were associated with a marked increase in plasma lipid levels, and felodipine treatment didn’t affect the plasma lipid levels. (2) Atherosclerotic lesion size was larger in HCD group than those in ND group or HCD+Fel group. (3) Felodipine treatment can decrease the activity of NF-κB in atherosclerotic lesion. (4) Felodipine treatment can decrease the expression of NADPH oxidase subunits (p47phox and Rac-1). (5) Felodipine treatment also decreased the expression of cytokines such as tumor necrosis-α(TNF-α), monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1).Conclusion: The results suggest that felodipine could inhibit As. This effect is partly related to the decrease of NF-κB activity, inhibition of oxidative stress and inflammatory signal transduction pathways, which leads to the decrease in the expression of inflammatory cytokines.Part II Molecular Mechanisms of Irbesartan Suppressing Atherosclerosis in High Cholesterol-diet Apolipoprotein E Knock-out MiceObjective: As is a chronic inflammatory disease in which the renin-angiotensin system (RAS) plays an important role. Evidences indicate that the angiotensin type 1 receptor blockers (ARBs) can suppress atherogenesis, but the exact mechanisms have not been fully elucidated. The present study was undertaken to investigate the potential effects and molecular mechanisms of an ARB irbesartan on the process of As in HCD ApoE KO mice.Methods: Adult male ApoE KO mice were divided into three groups randomly (n=15 each): normal diet (ND) group, HCD group and HCD plus irbesartan (HCD+Irb) group. Those mice were given ND or HCD and randomized to receive no treatment or irbesartan 10 mg/kg/d for 12 weeks. The plasma total cholesterol and triglyceride concentration were measured by autoanalyzer. Atherosclerotic lesion area in arotic root was evaluated by oil red O staining. NF-κB, IκB, extracellular signal-regulated kinase 1/2 (ERK1/2), janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. NAD(P)H oxidase subunits and inflammatory cytokines were measured by real-time reverse-transcription PCR and western blot.Results:(1) The ApoE KO mice with HCD were associated with a marked increase in plasma lipid levels, and irbesartan treatment didn’t affect the plasma lipid levels. (2) Atherosclerotic lesion size was larger in HCD group than those in ND group or HCD+Irb group. (3) Irbesartan treatment can decrease the activity of NF-κB as well as the activity of ERK1/2, JAK2, STAT3 in atherosclerotic lesion. (4) Irbesartan treatment attenuated the expression of NADPH oxidase subunits (p47phox and Rac-1) in the aortas. (5) Irbesartan treatment decreased the expression of cytokines such as TNF-α, interleukin 6 (IL-6), MCP-1 and VCAM-1 in the aortas.Conclusions: The results suggest that irbesartan can attenuate As. The effects maybe relate to the decrease of the activity of signal transcriptional factors such as NF-κB, ERK1/2, JAK2 and STAT3, thus leading to the inhibition of oxidative stress and inflammatory signal transduction pathways which eventually leads to the reduction of inflammatory cytokines expression.Part III Effect and Mechanism of Combination of Low Dose Felodipine and Irbesartan on Atherosclerosis in High Cholesterol-diet Apolipoprotein E Knock-out MiceObjective: To investigate the potential effect and mechanism of combination of low dose felodipine and irbesartan on the process of As in HCD ApoE KO mice.Methods: Adult male ApoE KO mice were divided into three groups randomly (n=15 each): normal diet (ND) group, HCD group and HCD plus felodipine and irbesartan (HCD+Fel+Irb) group. Those mice were given ND or HCD and randomized to receive no treatment or felodipine 2.5 mg/kg/d plus irbesartan 5mg/kg/d for 12 weeks. The plasma total cholesterol and triglyceride concentration were measured by autoanalyzer. Atherosclerotic lesion area in arotic root was evaluated by oil red O staining. NAD(P)H oxidase subunits and inflammatory cytokines were measured by real-time reverse- transcription PCR and western blot.Results:(1) The ApoE KO mice with HCD were associated with a marked increase in plasma lipid levels, and the drug treatment didn’t affect the plasma lipid levels. (2) Atherosclerotic lesion size was larger in HCD group than those in ND group or HCD+Fel+Irb group. (3) HCD feeding increased the expression of NADPH oxidase subunits (p47phox and Rac-1) and cytokines such as TNF-α, IL-6, MCP-1 and VCAM-1 in the aortas. These changes were suppressed in mice that were treated with low dose of felodipine and irbesartan.Conclusions: The results suggest that combination of low dose of felodipine and irbesartan could also inhibit As, and this effect maybe partly relates to the inhibition of oxidative stress and inflammatory cytokines expression.更多还原