地高辛对动脉粥样硬化形成以及斑块稳定性的影响

发布时间：2018-06-10 13:51

本文选题：动脉粥样硬化 + 地高辛　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分地高辛对动脉粥样硬化形成的影响目的：体外实验证实地高辛可以显著抑制炎症反应,同时影响脂质代谢。然而,地高辛对动脉粥样硬化是否有影响,一直以来缺少相关的研究报道。本研究的目的是通过ApoE-/-动脉粥样硬化小鼠来研究地高辛对动脉粥样硬化斑块形成的影响。方法：随机分组,每组8只ApoE-/-小鼠,分为对照组、低剂量地高辛组、高剂量地高辛组,给予高脂饲料Western-type diet (WD,含有0.15%胆固醇和21%脂肪)。每天监测小鼠体重,根据小鼠的体重给予相应剂量的地高辛干预。其中对照组腹腔注射相应体积的PBS；第二组为低剂量组,腹腔注射注射(i.p.)地高辛接近20μg(1mg/kg perday);第三组为高剂量组,腹腔注射(i.p.)地高辛接近40μg (2 mg/kg per day),高脂喂养12周。在12周结束时,同一天将所有小鼠戊巴比妥钠(50 mg/kg)腹腔注射麻醉、眶静脉采血后,快速颈椎脱臼法执行安乐死,分离脾脏用于流式实验,主动脉、肝脏、血样本保存在-80℃的冰箱中进一步分析。通过地高辛干预12周后,通过主动脉大体油红O染色以及主动脉瓣油红O染色斑块定量分析,评价地高辛对动脉粥样硬化斑块形成的影响。检测血浆中总胆固醇、低密度脂蛋白胆固醇、甘油三脂、高密度脂蛋白胆固醇的水平以及炎症因子的表达,为相关机制研究提供理论依据。结果：地高辛剂量依赖性的降低了动脉粥样化斑块和血浆中脂质水平(总胆固醇减少41%,甘油三酯降低了54%,低密度胆固醇降低了20%,对照组与高剂量地高辛组相比)。另外,地高辛显著降低了白介素(IL)-17A的水平以及1L-17A相关的炎症反应,同时增加调节性T细胞(Tregs)的比例。结论：我们的实验数据证实,地高辛作为维甲酸相关核孤儿受体γ的阻断剂,通过降低血脂和IL-17A相关的炎症反应,显著抑制了动脉粥样硬化斑块的形成。第二部分地高辛对动脉粥样硬化斑块稳定性的影响目的：动脉粥样硬化斑块的的稳定性在冠心病事件的发生中起着重要作用,其稳定性易受免疫反应和炎症反应的影响,前期研究证实地高辛影响着白介素(IL)-17A的水平以及IL-17A相关炎症反应,因此本研究的目的,评价地高辛对动脉粥样斑块的稳定性的影响。方法：随机分组每组8只ApoE-/-小鼠，分为对照组、低剂量地高辛组、高剂量地高辛组,给予Western-type diet (WD,含有0.15%胆固醇和21%脂肪),三组分别进行不同的干预,其中对照组腹腔注射相应体积的PBS,低剂量组每天腹腔注射1 mg/kg地高辛,高剂量组每天腹腔注射2mg/kg地高辛,12周后通过免疫组化对斑块内巨噬细胞、平滑肌细胞、胶原以及CD4+T细胞的变化检测,对动脉粥样硬化斑块稳定性做出评估。采用RT-PCR检测主动脉中IL-17A相关因子TNF-α,IL-1β, MCP-1, IFN-γ,MMP-2和MMP-9的mRNA水平变化。结果：地高辛剂量依赖性的加强了动脉粥样硬化斑块的稳定性,显著降低CD68+阳性的巨噬细胞,斑块纤维帽中α-SMA+阳性的平滑肌细胞和Collagen+显著增加与对照组相比,同时在地高辛治疗组斑块中CD4+T细胞显著降低。采用RT-PCR检测IL-17A相关因子mRNA的表达,发现在主动脉中地高辛治疗组剂量依赖性的降低了TNF-α,IL-1β,MCP-1,IFN-γ,MMP-2和MMP-9的mRNA水平。结论：与对照组相比,地高辛治疗组通过抑制斑块中IL-17A相关的炎症反应以及增加了斑块纤维帽中胶原、平滑肌细胞,增强动脉粥样硬化斑块的稳定性。
[Abstract]:Part 1 the effect of digoxin on the formation of atherosclerosis: in vitro experiments have demonstrated that digoxin can significantly inhibit inflammatory response and affect lipid metabolism. However, the effect of digoxin on atherosclerosis has been lacking related research reports. The aim of this study was to pass ApoE-/- atherosclerosis. The effects of digoxin on the formation of atherosclerotic plaque. Methods: randomly divided into groups of 8 ApoE-/- mice in each group, divided into control group, low dose digoxin group, high dose digoxin group, high fat diet Western-type diet (WD, containing 0.15% cholesterol and 21% fat). The control group was treated with digoxin. The control group was intraperitoneally injected with the corresponding volume of PBS; the second group was the low dose group, the intraperitoneal injection (i.p.) of digoxin was close to 20 g (1mg/kg perday); the third group was the high dose group, the peritoneal injection (i.p.) of digoxin was close to 40 mu (2 mg/kg per day), and the high fat was fed for 12 weeks. At the end of 12 weeks, all of them would be on the same day. The mice were intraperitoneally injected with pentobarbital sodium (50 mg/kg). After the orbital vein was collected, the method of rapid cervical dislocation was performed to perform euthanasia and the spleens were separated for flow test. The aorta, liver and blood samples were further analyzed in the refrigerator of -80 C. After 12 weeks of digoxin intervention, the aorta was stained with gross oil red O and the aortic valve oil red O staining. Quantitative analysis of color plaques to evaluate the effect of digoxin on the formation of atherosclerotic plaque. The detection of plasma total cholesterol, low density lipoprotein cholesterol, glycerol three fat, high density lipoprotein cholesterol level and the expression of inflammatory factors provide the rationale for the study of related mechanisms. Results: the dose dependence of digoxin is reduced. Lipid levels in atherosclerotic plaque and plasma (total cholesterol decreased by 41%, triglycerides decreased by 54%, low density cholesterol decreased by 20%, compared with the high dose digoxin group). In addition, digoxin significantly decreased the level of IL -17A and the inflammatory response in 1L-17A phase, and increased the ratio of regulatory T cells (Tregs). Conclusion: our experimental data confirm that digoxin, as a blocker of retinoic acid related nuclear orphan receptor gamma, significantly inhibits the formation of atherosclerotic plaque by lowering blood lipid and IL-17A related inflammatory responses. Second the effects of partial digoxin on atherosclerotic plaque stability: atherosclerotic plaques Stability plays an important role in the occurrence of coronary heart disease. Its stability is susceptible to the effects of immune response and inflammatory response. Previous studies have confirmed that digoxin affects the level of IL -17A and the IL-17A related inflammatory response. Method: 8 ApoE-/- mice in each group were divided into two groups: control group, low dose digoxin group, high dose digoxin group, Western-type diet (WD, 0.15% cholesterol and 21% fat). The control group was injected with the corresponding volume of PBS, and the low dose group was injected with 1 mg/kg digoxin every day, high dose. The dose group was intraperitoneally injected with 2mg/kg digoxin every day. After 12 weeks, the changes of macrophages, smooth muscle cells, collagen and CD4+T cells were detected by immunohistochemistry, and the stability of atherosclerotic plaque was evaluated. RT-PCR was used to detect the mRNA levels of IL-17A related factors TNF- alpha, IL-1 beta, MCP-1, IFN- gamma, MMP-2, and MMP-9. Results: the dose-dependent manner of digoxin enhanced the stability of atherosclerotic plaques, significantly reduced CD68+ positive macrophages, and a significant increase in the -SMA+ positive smooth muscle cells and Collagen+ in the plaque fibrous cap compared with the control group, while the CD4+T cells in the plaque of the digoxin treatment group were significantly reduced. RT-PCR was used to detect I. The expression of L-17A related factor mRNA showed that the dose-dependent reduction of TNF- alpha, IL-1 beta, MCP-1, IFN- gamma, MMP-2 and MMP-9 in the aortic digoxin treatment group was in a dose-dependent manner. Conclusion: compared with the control group, the digoxin treatment group could increase the collagen and smooth muscle of the plaque in the plaque of the plaque by inhibiting the IL-17A related inflammation in the plaque. Cells enhance the stability of atherosclerotic plaque.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5

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