尿石素A对动脉硬化进程中细胞功能及代谢的调控和作用机制研究

发布时间：2018-05-18 06:54

本文选题：尿石素A + microRNA　；参考：《西北农林科技大学》2016年博士论文

【摘要】：动脉粥样硬化(Atherosclerosis,AS),是一种最常见的和最具有危害性的心血管疾病,以中等和大动脉内膜经历脂质沉积、平滑肌和纤维组织增生及动脉硬化斑块形成为主要病变特征,易导致血栓、中风、心肌梗死等严重后果。发病人群多见于40岁以上的男性和绝经期后的女性,而无症状动脉粥样硬化早在儿童时期就已经存在。动脉粥样硬化严重危害人体健康,因此预防和控制动脉粥样硬化发生发展是提高我国人民健康水平的重要医疗手段。尿石素(Urolithin)是富含鞣花单宁的食物(石榴、草莓、核桃、花生等)经肠道微生物代谢生成的一类具有不同酚羟基的二苯并喃-6-酮衍生物。其中,尿石素A(Urolithin A,Uro-A)率先从喂食鞣花酸的小鼠粪便和尿液中被分离鉴定出。研究表明,尿石素A具有调控雌激素分泌、抗氧化、抗炎以及抗癌等生物活性,但其防治动脉粥样硬化的作用机制少有报道。因此,本课题主要研究尿石素A对内皮细胞功能紊乱及巨噬细胞源性泡沫细胞胆固醇外流(动脉粥样硬化启动环节)的影响,从分子水平深入探讨其中的作用机制,以此阐明尿石素A具有阻断动脉粥样硬化启动环节的重要作用,并揭示石榴等富含鞣花单宁的植物是潜在、可开发的治疗动脉粥样硬化的资源。本文的主要研究内容和结果如下:(1)研究尿石素A对氧化低密度脂蛋白(Oxidized low-density lipoprotein,ox-LDL)诱导的单核-内皮细胞粘附的作用。结果表明,低浓度的尿石素A(0.5~5μM)能够促进人动脉内皮细胞增殖,高浓度的尿石素A(25~100μM)则梯度降低了细胞存活率;尿石素A的最佳作用时间为24 h。尿石素A能够显著抑制50μg/mL ox-LDL诱导的乳酸脱氢酶漏出率上升,保护细胞结构完整性。尿石素A能够提高一氧化氮和内皮型一氧化氮合成酶的水平,并能抑制ox-LDL诱导的内皮素1表达上调,使内皮素1和一氧化氮水平处于平衡状态,从而维持内皮收缩-舒张功能的稳定。孟加拉红染色和荧光探针标记结果显示尿石素A能够减弱内皮细胞粘附单核细胞的能力,并下调细胞间粘附因子1和单核趋化蛋白1的基因表达,推测尿石素A可能通过调控粘附因子的表达进而抑制单核-内皮细胞粘附。(2)研究尿石素A通过调控MAPK信号通路对ox-LDL诱导的人动脉内皮细胞炎症反应的影响。尿石素a能够降低炎性因子白介素6和肿瘤坏死因子α的浓度,但ox-ldl和尿石素a均对干扰素γ含量没有显著性影响。尿石素a可以显著抑制ox-ldl诱导的micrornas(10,27,125a,126,155)表达上调,并能促进过氧化物酶体增殖物激活受体γ(peroxisomeproliferators-activatedreceptorgamma,ppar-γ)的基因表达;此外,转染实验证明尿石素a通过抑制microrna-27表达实现调控ppar-γ的转录水平。尿石素a能够明显抑制ox-ldl激活的mapk信号通路关键蛋白erk1/2、sapk/jnk和p38磷酸化,并且和通路抑制剂u0126及sp600125具有一定的协同作用。此外,尿石素a还可以通过erk/ppar-γ信号转导途径调控细胞间黏附因子-1基因表达,进而抑制炎症反应,实现维持内皮细胞功能稳定的作用。(3)研究尿石素a对巨噬细胞极化分型及巨噬-泡沫细胞形成的影响。低浓度的尿石素a(0.5~25μm)与小鼠巨噬细胞raw264.7共同孵育3h时,对细胞活性没有显著性抑制作用。尿石素a浓度为20μm时,可以显著抑制m1型巨噬细胞标志分子白介素1β、白介素6和肿瘤坏死因子α基因表达;尿石素a浓度为10和20μm时,可以明显提高m2型巨噬细胞标志分子白介素10和转化生长因子β的转录水平,表明尿石素a促进自然状态巨噬细胞向m2型极化。不同浓度的尿石素a能够明显抑制脂多糖诱导的m1型巨噬细胞标志分子的表达,且呈明显的浓度-剂量效应,因此抑制raw264.7向促炎的m1型巨噬细胞极化。不同浓度的尿石素a能够显著降低泡沫细胞的数量,减少巨噬细胞泡沫化程度;并且能够梯度降低胆固醇合成基因羟甲基戊二酸单酰辅酶a还原酶和脂肪酸合成酶的转录水平,显著上调三磷酸腺苷结合盒转运蛋白a1和g1的表达。(4)研究尿石素a通过介导载脂蛋白a-Ⅰ、erk/ampk信号通路和microrna-33途径对巨噬细胞源性泡沫细胞胆固醇外流的影响。不同浓度的尿石素a在载脂蛋白a-Ⅰ介导作用下可以明显减少巨噬细胞源性泡沫细胞内胆固醇累积,并提高胞外胆固醇的含量。尿石素a一方面能够显著抑制ox-ldl激活的erk1/2及其磷酸化蛋白和固醇调节元件结合蛋白-1的蛋白表达,且呈剂量-效应关系;另一方面促进ampkα和磷酸化ampkα的蛋白表达,表现出和erk1/2通路抑制剂u0126一致的作用。并且抑制erk1/2通路的激活可以提高尿石素a干预的实验组ampkα磷酸化水平。此外,采用ampk通路抑制剂dorsomorphin可以显著提高固醇调节元件结合蛋白-1蛋白含量,几乎消除了尿石素a抑制ox-ldl诱导的固醇调节元件结合蛋白-1表达上调的作用。尿石素a还可以通过microrna-33途径显著上调三磷酸腺苷结合盒转运蛋白a1和g1的基因表达,并且加强胞内胆固醇逆转运出细胞。(5)基于分子对接技术研究石榴多酚及其代谢物(鞣花酸、尿石素a、b、c、d)与心血管疾病蛋白标志物相互作用。共有27种心血管疾病蛋白标志物可以分别与鞣花酸和尿石素对接良好。其中,鞣花酸有12个靶标蛋白,尿石素a、b、c、d则分别有9个、7个、15个和19个靶标蛋白,推测尿石素c和d由于具有更多的羟基数量可以结合的靶标蛋白数量也更多。与靶标蛋白特定的激动剂或抑制剂相比,鞣花酸及尿石素与靶标蛋白的结合部位氨基酸残基种类更多;结合位点的氨基酸残基数量也与尿石素分子结构中的羟基数量呈正相关。石榴多酚与靶标蛋白间的相互作用主要依靠氢键作用,结合位点的氨基酸残基主要包括:丙氨酸(Ala)、精氨酸(Arg)、天冬酰胺(Asn)、谷氨酰胺(Gln)、谷氨酸(Glu)、组氨酸(His)、苯丙氨酸(Phe)和苏氨酸(Thr)。由于结构相近,不同多酚小分子与同一靶标蛋白结合的作用方式大体一致。而且,与鞣花酸和尿石素分子对接具有较高打分结果的靶标蛋白多具有与血栓、动脉粥样硬化相关的功能,支持了本课题前期实验尿石素A抗炎及维持内皮细胞功能稳定的结论。
[Abstract]:Atherosclerosis (AS) is one of the most common and most dangerous cardiovascular diseases. Lipid deposition is experienced in the middle and large artery intima. Smooth muscle and fibrous tissue hyperplasia and atherosclerotic plaque are the main features of the disease, which can lead to thrombosis, stroke, myocardial infarction and other serious consequences. The majority of the patients are found in 40. Male and postmenopausal women over the age of age and postmenopausal women have already existed early in childhood. Atherosclerosis is a serious harm to human health. Therefore, prevention and control of the development of atherosclerosis is an important medical means to improve the health of our people. Urinary stone (Urolithin) is rich in tannin tannin. Food (pomegranate, strawberry, walnut, peanut, etc.) produced by intestinal microbial metabolism, a class of two benzo -6- ketone derivatives with different phenolic hydroxyl groups. Among them, urinary stone A (Urolithin A, Uro-A) was isolated and identified in the feces and urine of mice fed tannic acid. The study showed that urinolite A has the regulation of estrogen secretion and antioxidant activity. The anti-inflammatory and anticancer activity, but its mechanism of prevention and control of atherosclerosis is rarely reported. Therefore, we mainly study the effect of urinary stone A on endothelial dysfunction and cholesterol efflux of macrophage derived foam cells (the starting link of atherosclerosis), and explore the mechanism of action from molecular level. This clarifies that urinary stone A plays an important role in blocking the initiation of atherosclerosis, and reveals that pomegranates and other plants rich in tannin are potential and developed resources for the treatment of atherosclerosis. The main contents and results of this study are as follows: (1) the study of urinary stone A (Oxidized low-density lipoprotei) N, ox-LDL) induced adhesion of mononuclear endothelial cells. The results showed that the low concentration of urinary stone A (0.5~5 mu M) could promote the proliferation of human arterial endothelial cells, and the high concentration of urinary calculus A (25~100 mu M) decreased the cell survival rate; the optimal action time of urinary calculus A was 24 h. urinary stone A could significantly inhibit the 50 micron g/mL induced milk. The leakage rate of acid dehydrogenase increases and protects the structural integrity of cells. Urinary stone A can increase the level of nitric oxide and endothelial nitric oxide synthase, inhibit the up regulation of endothelin 1 induced by ox-LDL, make the level of endothelin 1 and nitric oxide in a balanced state, and maintain the stability of endothelin systolic and diastolic function. Bengal red dye Color and fluorescence probe labeling results show that urinary stone A can weaken the ability of endothelial cells to adhere to mononuclear cells and down regulate the expression of intercellular adhesion factor 1 and mononuclear chemoattractant protein 1. It is suggested that urinary stone A may inhibit mononuclear endothelial cell adhesion by regulating the expression of adhesion factors. (2) the study of urinary stone A by regulating MAPK letter The impact of ox-LDL induced inflammatory response in human arterial endothelial cells. Urinary stone a can reduce the concentration of il-c 6 and TNF - alpha, but both ox-LDL and urinary stone a have no significant effect on interferon gamma content. Urinary calculus a can significantly inhibit the expression of microRNAs (10,27125a, 126155) induced by ox-LDL. And it can promote the gene expression of peroxisome proliferator activated receptor gamma (peroxisomeproliferators-activatedreceptorgamma, ppar- gamma). In addition, the transfection experiment shows that urinary stone a regulates the transcription level of ppar- gamma by inhibiting the expression of microrna-27. Urinary stone a can clearly inhibit the key protein ERK1 of MAPK signaling pathway activated by ox-LDL The phosphorylation of /2, sapk/jnk and p38 has a synergistic effect with the pathway inhibitors, U0126 and sp600125. In addition, urinary calculus a can also regulate the expression of intercellular adhesion factor -1 gene through erk/ppar- gamma signal transduction pathway, and then inhibit the inflammatory response and achieve the role of maintaining endothelial cell function stability. (3) the study of urinary calculus a against giant macrophages The effects of cell polarization and macrophage foam cell formation. When low concentration of urinary stone a (0.5~25 m) and mouse macrophage RAW264.7 co incubate 3h, there is no significant inhibitory effect on cell activity. When urinary stone a concentration is 20 u m, it can significantly inhibit the interleukin 1 beta, IL-6 and tumor necrosis factor of M1 type macrophages. When the concentration of urinary stone a is 10 and 20 mu m, the transcription level of interleukin 10 and transforming growth factor beta of the M2 macrophage marker molecule can be significantly increased, indicating that urinary stone a promotes the M2 polarization in natural macrophages. The urinary stone a of different concentrations can obviously inhibit the M1 type macrophage markers induced by lipopolysaccharide. The expression, with an obvious concentration dose effect, inhibits the polarization of RAW264.7 to the proinflammatory M1 type macrophages. Different concentrations of urinary stone a can significantly reduce the number of foam cells, reduce the degree of macrophage foam, and reduce the gradient of the cholesterol synthesis gene hydroxymethylglutaric acid monoyl coenzyme A reductase and fatty acids. The transcriptional level of ATP significantly up-regulated the expression of ATP binding cassette transporter A1 and G1. (4) study the effect of urinary stone a through apolipoprotein a- I, erk/ampk signaling pathway and microrna-33 pathway on the cholesterol efflux of macrophage derived foam cells. Urinary calculus a at different concentrations can be mediated by apolipoprotein a- I To significantly reduce the accumulation of cholesterol in macrophage derived foam cells and increase the content of extracellular cholesterol. Urinary stone a, on the one hand, can significantly inhibit the expression of ox-LDL activated erk1/2 and its phosphorylated protein and sterol regulator binding protein -1 protein expression, and in a dose effect relationship; on the other hand, it promotes AMPK alpha and phosphorylated AMPK alpha. Protein expression is consistent with the erk1/2 pathway inhibitor U0126. And inhibition of the activation of the erk1/2 pathway can increase the level of AMPK alpha phosphorylation in the experimental group of urinary stone a intervention. In addition, the use of AMPK pathway inhibitor dorsomorphin can significantly increase the content of the egg white -1 protein binding to the egg white -1 protein and almost eliminate the urinary calculus a suppression. Ox-LDL induced up regulation of sterol regulator binding protein -1 expression. Urinary stone a also significantly up-regulated gene expression of adenosine triphosphate binding cassette transporter A1 and G1 through microrna-33 pathway, and enhanced intracellular cholesterol reversal and transport of cells. (5) pomegranate polyphenols and their metabolites (tanning flowers) were studied based on molecular docking. Acid, urinary stone a, B, C, d) interact with the protein markers of cardiovascular disease. There are 27 kinds of cardiovascular disease protein markers that can be butted with tannic acid and urinary stone respectively. Among them, tannin acid has 12 target proteins, urinary stone a, B, C, D, respectively, 9, 7, 15 and 19 target proteins, speculates that urinary stone C and d have more The number of hydroxyl groups can be combined with the number of target proteins. Compared with the specific activator or inhibitor of the target protein, the amino acid residues in the binding site of ellagic acid and urorocin and the target protein are more species; the number of amino acid residues in the binding site is also positively related to the number of hydroxyl groups in the urinary stone molecular structure. Pomegranate polyphenols and targets The interaction between the proteins mainly depends on the hydrogen bond, and the amino acid residues of the binding sites mainly include: alanine (Ala), arginine (Arg), asparagine (Asn), glutamine (Gln), glutamic acid (Glu), histidine (His), phenylalanine (Phe) and threonine (Thr). In addition, the target proteins associated with tannic acid and urinary stone molecules with high scoring results are mostly associated with thrombus and atherosclerosis, which supports the conclusion of the anti inflammation of urinary stone A and the maintenance of endothelial cell function stability in the early experiment.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5

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