人参皂苷Rb1通过促进巨噬细胞自噬稳定动脉粥样硬化斑块的研究

发布时间：2018-01-22 18:44

  本文关键词： 动脉粥样硬化 易损斑块 脂质代谢 人参皂苷Rb1 自噬　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景动脉粥样硬化(atherosclerosis,AS)斑块破裂和血栓形成是导致急性心脑血管事件的主要病理机制,对可能发生破裂的易损斑块(vulnerable plaque)进行早期积极干预是预防和减低急性心脑血管事件的发生率的重要方法。AS是一种脂质驱动的慢性炎症性疾病。其主要病理表现为脂质堆积和炎症细胞浸润,巨噬细胞在这一过程中发挥重要作用。血液中的低密度脂蛋白胆固醇被巨噬细胞吞噬后以胆固醇酯的形式存在于脂滴中,蓄积了大量脂质的巨噬细胞变为泡沫细胞。如何减少泡沫细胞中脂质的蓄积,对于降低斑块脂负荷、促进斑块稳定具有重要意义。人参皂苷Rb1是人参当中含量最多的活性成分。目前的研究发现,Rb1具有降低血脂、降低体重、对抗炎症、抑制血管形成和增强胰岛素敏感性等生理作用。但是,Rb1在动脉粥样硬化中的作用尚没有研究。本课题拟通过构建AS小鼠模型和体外培养巨噬细胞,探讨人参皂苷Rb1通过调控巨噬细胞自噬对动脉粥样硬化斑块稳定性的作用及其相关机制。目的通过构建体外泡沫细胞模型及动脉粥样硬化小鼠易损斑块模型,研究人参皂苷Rb1调控巨噬细胞自噬、影响脂质代谢及稳定AS易损斑块的分子机制。方法在体外实验中,我们提取C57BL/6小鼠腹腔原代巨噬细胞,给予ox-LDL(100μg/ml)刺激24小时诱导成泡沫细胞,然后分别给予不同浓度Rb1处理24小时。油红O染色检测各组细胞脂质沉积情况,western blot检测自噬蛋白LC3Ⅱ和SQSTM1含量,电镜观察细胞中脂滴及自噬体膜形成数量;通过慢病毒转染Atg5 siRNA抑制细胞自噬过程,观察干预自噬前后,Rb1对巨噬细胞脂质代谢的影响;给予AMPK抑制剂Compond C,研究AMPK磷酸化在Rb1介导的自噬中的作用。体内实验中,通过高脂喂养ApoE-/-小鼠20周构建动脉粥样硬化晚期斑块模型。然后随机分为两个组,分别通过灌胃给予生理盐水和人参皂苷Rb1(50 mg/kg/d),隔天一次给药。干预7周后,病理学方法检测斑块脂质沉积、巨噬细胞浸润、平滑肌数量以及胶原含量,并通过分子学检测斑块中自噬蛋白LC3B和SQSTM1表达及AMPK磷酸化,探究Rb1影响斑块稳定性的可能机制。结果1.Rb1在20-80 μ范围内浓度依赖性地降低了泡沫细胞内的脂质沉积。2.Rb1处理后细胞LC3Ⅱ/LC3Ⅰ比例增加、SQSTM1降低,并且细胞中自噬双层膜结构增多,提示Rb1促进了细胞的自噬水平。3.经慢病毒转染Atg5 siRNA抑制自噬后,Rb1降低泡沫细胞脂质沉积的作用明显减弱。4.Rb1促进了 AMPK磷酸化,加入AMPK抑制剂compound C后,Rb1激活自噬的作用减弱。5.与对照组相比,Rb1处理组小鼠斑块自噬蛋白LC3Ⅱ/LC3Ⅰ比例增加、SQSTM1降低,并且AMPK磷酸化增加。6.与对照组相比,Rb1处理组小鼠斑块脂质沉积和巨噬细胞浸润减少,胶原和平滑肌数量增多,斑块稳定性增加。结论Rb1通过激活巨噬细胞自噬促进了动脉粥样硬化斑块脂质代谢和斑块稳定;AMPK通路在Rb1激活自噬中发挥重要作用。
[Abstract]:Background Atherosclerotic plaque rupture and thrombosis are the main pathological mechanisms leading to acute cardiovascular and cerebrovascular events. Vulnerable plaques for potentially ruptured plaques. Early and active intervention is an important method to prevent and reduce the incidence of acute cardiovascular and cerebrovascular events. As is a chronic inflammatory disease driven by lipid. Its main pathological manifestations are lipid accumulation and inflammatory cell infiltration. Macrophages play an important role in this process. Low density lipoprotein cholesterol in the blood is swallowed by macrophages and is found in the form of cholesterol ester in lipid droplets. Macrophages that accumulate large amounts of lipid become foam cells. How to reduce lipid accumulation in foam cells, for reducing plaque lipid load. It is important to promote plaque stability. Ginsenoside Rb1 is the most active ingredient in ginseng. Current studies have found that Rb1 can reduce blood lipids, reduce body weight and resist inflammation. Inhibition of angiogenesis and enhancement of insulin sensitivity and other physiological effects. However, the role of Rb1 in atherosclerosis has not been studied. This study intends to construct as mouse model and culture macrophages in vitro. To investigate the effect of ginsenoside Rb1 on the stability of atherosclerotic plaque by macrophage autophagy and its related mechanism. Objective to establish a foam cell model in vitro and a vulnerable plaque model in atherosclerotic mice. . To study the molecular mechanism of ginsenoside Rb1 regulating macrophage autophagy, affecting lipid metabolism and stabilizing as vulnerable plaque. Methods in vitro, we extracted primary peritoneal macrophages from C57BL / 6 mice. The foam cells were induced by ox-LDL(100 渭 g / ml for 24 hours, then treated with different concentrations of Rb1 for 24 hours. Oil red O staining was used to detect lipid deposition in each group. The contents of autophagy protein LC3 鈪，

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