Aliskiren对动脉粥样硬化斑块内新生血管形成的影响及机制研究

发布时间：2018-05-23 08:56

本文选题：Aliskiren + 动脉粥样硬化　；参考：《青岛大学》2016年博士论文

【摘要】：研究背景动脉粥样硬化(AS)是缺血性心脑血管疾病主要的病理生理基础。临床研究发现不稳定性AS斑块内出血或斑块发生破裂是引起缺血性血管事件急性发作的主要原因。AS斑块内有病理性新生血管,这些血管的渗透性高、脆性大,易发生破裂,从而导致斑块内出血,引起急性心脑血管事件。研究证实炎症与斑块内新生血管的形成关系密切,炎症因子刺激斑块内部的血管新生。TLR4/NF-κB信号传导通路可通过刺激炎症细胞分泌炎症因子来介导炎症反应。有研究显示TLR4/NF-κB途径可能会诱导MMPs表达,从而参与AS的病理过程。研究表明AS内的多种MMPs含量升高。AS的动脉壁中MMP-2、MMP-9水平明显增加,且增加程度与病变严重程度正相关。Aliskiren是新发现的肾素抑制剂,可明显减少AS病变。研究证实RAAS激活后可以通过TLRs依赖的信号途径诱导Apo E-/-小鼠AS斑块的血管炎症,但其作用机制与途径尚不明确。目的:观察Aliskiren对动脉粥样硬化斑块内新生血管形成和斑块稳定性的影响,探讨Aliskiren是否通过抑制TLR4/NF-κB信号通路介导的炎症反应及MMP-2、MMP-9的生成来影响新生血管的形成,增强斑块的稳定性。方法:取30只健康雄性、8周龄的载脂蛋白E基因敲除(Apo E-/-)小鼠,将其随机分为3组:模型组10只、Aliskiren低剂量组10只、Aliskiren高剂量组10只,另取C57BL/6小鼠10只作为对照组。Aliskiren低剂量组、高剂量组分别给予Aliskiren25mg/kg/d,50mg/kg/d灌胃;C57BL/6小鼠及模型组给予生理盐水灌胃1m L/100g/d,灌胃时间为12周。制作主动脉根部切片,免疫组化染色观察分析斑块内新生血管的密度;HE染色分析斑块形态,计算血管斑块面积、管腔面积;油红O染色观察斑内脂质成分含量;Masson染色观察斑块中胶原含量;ELISA检测血清中炎症因子TNF-α、IL-6、IL-1β、MCP-1水平;RT-PCR法检测主动脉组织中MMP-2、MMP-9、TLR4 m RNA水平变化;Western Blot法检测主动脉组织中MMP-2、MMP-9、TLR4及NF-κB蛋白水平变化。结果:1.血脂测定:模型组小鼠血清TC、TG、LDL-C浓度较对照组明显升高(P0.05);Aliskiren低剂量组和高剂量组小鼠血清TC、TG、LDL-C浓度与模型组无明显变化(P0.05)。2.HE染色:模型组小鼠主动脉AS斑块形成显著,管腔狭窄较重,动脉内膜不连续、不光滑,内皮细胞部分缺失。与模型组相比,Aliskiren低剂量组和高剂量组斑块面积、斑块面积/管腔面积比值明显降低(P0.05);与Aliskiren低剂量组相比,Aliskiren高剂量组降低更显著(P0.05)。3.油红O染色:模型组小鼠主动脉AS斑块内红染的沉积的脂质量大。Aliskiren低剂量组和高剂量组斑块内脂质含量较模型组明显减少(P0.05);与Aliskiren低剂量组相比,Aliskiren高剂量组降低更显著(P0.05)。4.Masson染色:模型组小鼠主动脉斑块内蓝染胶原量少。与模型组相比,Aliskiren低剂量组和高剂量组斑块内胶原含量增加明显(P0.05);与Aliskiren低剂量组相比,Aliskiren高剂量组增加更显著(P0.05)。5.斑块内新生血管的密度:模型组可见大量的棕褐色的颗粒,CD34阳性表达多。Aliskiren低剂量组和高剂量组新生血管较模型组显著减少(P0.05);与Aliskiren低剂量组相比,Aliskiren高剂量组新生血管减少更显著(P0.05)。6.TNF-α、IL-6、IL-1β、MCP-1水平:与对照组相比,模型组TNF-α、IL-6、IL-1β、MCP-1水平显著升高(P0.05);与模型组相比,Aliskiren低剂量组和高剂量组模型组TNF-α、IL-6、IL-1β、MCP-1水平明显降低(P0.05),Aliskiren高剂量组降低更明显(P0.05)。7.主动脉组织中MMP-2、MMP-9、TLR4 m RNA表达水平:与对照组相比,模型组MMP-2、MMP-9、TLR4 m RNA表达明显升高(P0.05);与模型组相比,Aliskiren低剂量组和高剂量组模型组MMP-2、MMP-9、TLR4 m RNA表达明显降低(P0.05),Aliskiren高剂量组降低更明显(P0.05)。8.主动脉组织中MMP-2、MMP-9、TLR4及NF-κB蛋白表达水平:与对照组相比,模型组MMP-2、MMP-9、TLR4及NF-κB蛋白表达显著升高(P0.05);与模型组相比,Aliskiren低剂量组和高剂量组MMP-2、MMP-9、TLR4及NF-κB蛋白表表达明显降低(P0.05),Aliskiren高剂量组降低更明显(P0.05)。结论:1.Aliskiren能够减少AS斑块内新生血管的形成,增强斑块的稳定性。2.Aliskiren可能是通过抑制TLR4/NF-κB信号通路介导的炎症反应及MMP-2、MMP-9的生成来影响新生血管的形成,增强斑块稳定性。
[Abstract]:Background atherosclerosis (AS) is the main pathophysiological basis of ischemic cardio cerebrovascular disease. Clinical studies have found that bleeding or plaque rupture in unstable AS plaques is the main cause of acute ischemic vascular events in.AS, and there are pathological new blood tubes in plaque. These vessels have high permeability, large brittleness and easy onset. The rupture, which leads to hemorrhage in plaque, causes acute cardiovascular and cerebrovascular events. Studies have confirmed that inflammation is closely related to the formation of neovascularization in the plaque. Inflammatory factors stimulate the angiogenesis.TLR4/NF- kappa B signal transduction pathway within the plaque within the plaque to mediate inflammatory reactions by stimulating inflammatory cells to secrete inflammatory factors. Research shows that TLR4/NF- The kappa B pathway may induce the expression of MMPs and thus participate in the pathological process of AS. The study shows that a variety of MMPs levels in the AS increase the MMP-2, MMP-9 level in the arterial wall of.AS, and the degree of increase is positively related to the severity of the lesion, which is a newly discovered renin inhibitor, which can significantly reduce the AS lesion. TLRs dependent signaling pathway induces vascular inflammation in the Apo E-/- mouse AS plaque, but its mechanisms and pathways are not yet clear. Objective: To observe the effect of Aliskiren on the formation of neovascularization and plaque stability in atherosclerotic plaques and to explore whether Aliskiren is mediated by the TLR4/ NF- kappa B signaling pathway and MMP-2, MMP -9 formation to influence the formation of new blood vessels and enhance plaque stability. Methods: 30 healthy males and 8 weeks old apolipoprotein E gene knockout (Apo E-/-) mice were randomly divided into 3 groups, 10 in the model group, 10 in the Aliskiren low dose group and 10 in the Aliskiren high dose group, and 10 in the C57BL/6 mice were used as the low dose of the control group as the low dose of the control group.Aliskiren. Group, high dose group were given Aliskiren25mg/kg/d, 50mg/kg/d gavage respectively; C57BL/6 mice and model group were given 1m L/100g/d with saline for 12 weeks. The density of the neovascularization in the aortic root was made by immunohistochemistry. The plaque morphology was analyzed by HE staining, and the area of plaque and the area of the lumen were calculated. The content of lipid components in the plaque was observed by oil red O staining, the content of collagen in the plaque was observed by Masson staining, and the levels of inflammatory factors TNF- a, IL-6, IL-1 beta and MCP-1 in the serum were detected by ELISA, and the levels of MMP-2, MMP-9, TLR4 m in aorta were detected by RT-PCR. Results: 1. blood lipid measurement: the serum levels of TC, TG and LDL-C in the model group were significantly higher than those in the control group (P0.05). The serum TC, TG, LDL-C concentration in the low dose group and the high dose group of the Aliskiren group had no significant changes (P0.05).2.HE staining (P0.05).2.HE staining: the aortic AS plaque in the model group was significant, the stenosis of the lumen was heavier, the intima of the artery was not continuous and unsmooth. Compared with the model group, the patch area of the Aliskiren low dose group and the high dose group decreased significantly (P0.05). Compared with the low dose group of Aliskiren, the Aliskiren high dose group decreased more significantly (P0.05).3. oil red O staining: the lipid quality of the red dye in the aorta AS plaque of the model mice. The lipid content in the plaque in the large.Aliskiren low dose group and the high dose group was significantly lower than that in the model group (P0.05). Compared with the low dose group of Aliskiren, the Aliskiren high dose group decreased more significantly (P0.05).4.Masson staining: the amount of blue stained collagen in the aortic plaque in the model group was less. Compared with the model group, the low dose group and the high dose group were within the plaque group. The increase of collagen content was significant (P0.05); compared with the low dose group of Aliskiren, the density of the neovascularization in the Aliskiren high dose group increased significantly (P0.05) in the.5. plaque: the model group showed a large number of brown brown particles, the CD34 positive expression of the multi.Aliskiren low dose group and the high dose group decreased significantly compared with the model group (P0.05), and the low level of Aliskiren (P0.05). Compared with the control group, the level of.6.TNF- alpha, IL-6, IL-1 beta, MCP-1 was significantly higher in the Aliskiren high dose group than in the control group. Compared with the control group, the level of TNF- alpha, IL-6, IL-1 beta and MCP-1 increased significantly (P0.05). Compared with the model group, the low dose group and the high dose group were significantly lower than the model group. Iskiren high dose group decreased the expression level of MMP-2, MMP-9, TLR4 m RNA in the aorta of (P0.05).7.: compared with the control group, the expression of MMP-2, MMP-9, TLR4 m was significantly higher than that in the control group. The expression level of MMP-2, MMP-9, TLR4 and NF- kappa B in the aorta of (P0.05).8. was significantly reduced in the dose group. Compared with the control group, the expression of MMP-2, MMP-9, TLR4 and NF- kappa B protein in the model group was significantly higher than that in the model group. The decrease of skiren in high dose group is more obvious (P0.05). Conclusion: 1.Aliskiren can reduce the formation of neovascularization in AS plaque and enhance the stability of plaque by inhibiting the inflammatory response mediated by TLR4/NF- kappa B signaling pathway and MMP-2, MMP-9 formation to influence the formation of neovascularization and enhance plaque stability.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5

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