本文選題：尿石素A + microRNA　； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：動脈粥樣硬化(Atherosclerosis,AS),是一種最常見的和最具有危害性的心血管疾病,以中等和大動脈內(nèi)膜經(jīng)歷脂質(zhì)沉積、平滑肌和纖維組織增生及動脈硬化斑塊形成為主要病變特征,易導(dǎo)致血栓、中風(fēng)、心肌梗死等嚴(yán)重后果。發(fā)病人群多見于40歲以上的男性和絕經(jīng)期后的女性,而無癥狀動脈粥樣硬化早在兒童時期就已經(jīng)存在。動脈粥樣硬化嚴(yán)重危害人體健康,因此預(yù)防和控制動脈粥樣硬化發(fā)生發(fā)展是提高我國人民健康水平的重要醫(yī)療手段。尿石素(Urolithin)是富含鞣花單寧的食物(石榴、草莓、核桃、花生等)經(jīng)腸道微生物代謝生成的一類具有不同酚羥基的二苯并喃-6-酮衍生物。其中,尿石素A(Urolithin A,Uro-A)率先從喂食鞣花酸的小鼠糞便和尿液中被分離鑒定出。研究表明,尿石素A具有調(diào)控雌激素分泌、抗氧化、抗炎以及抗癌等生物活性,但其防治動脈粥樣硬化的作用機(jī)制少有報道。因此,本課題主要研究尿石素A對內(nèi)皮細(xì)胞功能紊亂及巨噬細(xì)胞源性泡沫細(xì)胞膽固醇外流(動脈粥樣硬化啟動環(huán)節(jié))的影響,從分子水平深入探討其中的作用機(jī)制,以此闡明尿石素A具有阻斷動脈粥樣硬化啟動環(huán)節(jié)的重要作用,并揭示石榴等富含鞣花單寧的植物是潛在、可開發(fā)的治療動脈粥樣硬化的資源。本文的主要研究內(nèi)容和結(jié)果如下:(1)研究尿石素A對氧化低密度脂蛋白(Oxidized low-density lipoprotein,ox-LDL)誘導(dǎo)的單核-內(nèi)皮細(xì)胞粘附的作用。結(jié)果表明,低濃度的尿石素A(0.5~5μM)能夠促進(jìn)人動脈內(nèi)皮細(xì)胞增殖,高濃度的尿石素A(25~100μM)則梯度降低了細(xì)胞存活率;尿石素A的最佳作用時間為24 h。尿石素A能夠顯著抑制50μg/mL ox-LDL誘導(dǎo)的乳酸脫氫酶漏出率上升,保護(hù)細(xì)胞結(jié)構(gòu)完整性。尿石素A能夠提高一氧化氮和內(nèi)皮型一氧化氮合成酶的水平,并能抑制ox-LDL誘導(dǎo)的內(nèi)皮素1表達(dá)上調(diào),使內(nèi)皮素1和一氧化氮水平處于平衡狀態(tài),從而維持內(nèi)皮收縮-舒張功能的穩(wěn)定。孟加拉紅染色和熒光探針標(biāo)記結(jié)果顯示尿石素A能夠減弱內(nèi)皮細(xì)胞粘附單核細(xì)胞的能力,并下調(diào)細(xì)胞間粘附因子1和單核趨化蛋白1的基因表達(dá),推測尿石素A可能通過調(diào)控粘附因子的表達(dá)進(jìn)而抑制單核-內(nèi)皮細(xì)胞粘附。(2)研究尿石素A通過調(diào)控MAPK信號通路對ox-LDL誘導(dǎo)的人動脈內(nèi)皮細(xì)胞炎癥反應(yīng)的影響。尿石素a能夠降低炎性因子白介素6和腫瘤壞死因子α的濃度,但ox-ldl和尿石素a均對干擾素γ含量沒有顯著性影響。尿石素a可以顯著抑制ox-ldl誘導(dǎo)的micrornas(10,27,125a,126,155)表達(dá)上調(diào),并能促進(jìn)過氧化物酶體增殖物激活受體γ(peroxisomeproliferators-activatedreceptorgamma,ppar-γ)的基因表達(dá);此外,轉(zhuǎn)染實(shí)驗(yàn)證明尿石素a通過抑制microrna-27表達(dá)實(shí)現(xiàn)調(diào)控ppar-γ的轉(zhuǎn)錄水平。尿石素a能夠明顯抑制ox-ldl激活的mapk信號通路關(guān)鍵蛋白erk1/2、sapk/jnk和p38磷酸化,并且和通路抑制劑u0126及sp600125具有一定的協(xié)同作用。此外,尿石素a還可以通過erk/ppar-γ信號轉(zhuǎn)導(dǎo)途徑調(diào)控細(xì)胞間黏附因子-1基因表達(dá),進(jìn)而抑制炎癥反應(yīng),實(shí)現(xiàn)維持內(nèi)皮細(xì)胞功能穩(wěn)定的作用。(3)研究尿石素a對巨噬細(xì)胞極化分型及巨噬-泡沫細(xì)胞形成的影響。低濃度的尿石素a(0.5~25μm)與小鼠巨噬細(xì)胞raw264.7共同孵育3h時,對細(xì)胞活性沒有顯著性抑制作用。尿石素a濃度為20μm時,可以顯著抑制m1型巨噬細(xì)胞標(biāo)志分子白介素1β、白介素6和腫瘤壞死因子α基因表達(dá);尿石素a濃度為10和20μm時,可以明顯提高m2型巨噬細(xì)胞標(biāo)志分子白介素10和轉(zhuǎn)化生長因子β的轉(zhuǎn)錄水平,表明尿石素a促進(jìn)自然狀態(tài)巨噬細(xì)胞向m2型極化。不同濃度的尿石素a能夠明顯抑制脂多糖誘導(dǎo)的m1型巨噬細(xì)胞標(biāo)志分子的表達(dá),且呈明顯的濃度-劑量效應(yīng),因此抑制raw264.7向促炎的m1型巨噬細(xì)胞極化。不同濃度的尿石素a能夠顯著降低泡沫細(xì)胞的數(shù)量,減少巨噬細(xì)胞泡沫化程度;并且能夠梯度降低膽固醇合成基因羥甲基戊二酸單酰輔酶a還原酶和脂肪酸合成酶的轉(zhuǎn)錄水平,顯著上調(diào)三磷酸腺苷結(jié)合盒轉(zhuǎn)運(yùn)蛋白a1和g1的表達(dá)。(4)研究尿石素a通過介導(dǎo)載脂蛋白a-Ⅰ、erk/ampk信號通路和microrna-33途徑對巨噬細(xì)胞源性泡沫細(xì)胞膽固醇外流的影響。不同濃度的尿石素a在載脂蛋白a-Ⅰ介導(dǎo)作用下可以明顯減少巨噬細(xì)胞源性泡沫細(xì)胞內(nèi)膽固醇累積,并提高胞外膽固醇的含量。尿石素a一方面能夠顯著抑制ox-ldl激活的erk1/2及其磷酸化蛋白和固醇調(diào)節(jié)元件結(jié)合蛋白-1的蛋白表達(dá),且呈劑量-效應(yīng)關(guān)系;另一方面促進(jìn)ampkα和磷酸化ampkα的蛋白表達(dá),表現(xiàn)出和erk1/2通路抑制劑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干預(yù)的實(shí)驗(yàn)組ampkα磷酸化水平。此外,采用ampk通路抑制劑dorsomorphin可以顯著提高固醇調(diào)節(jié)元件結(jié)合蛋白-1蛋白含量,幾乎消除了尿石素a抑制ox-ldl誘導(dǎo)的固醇調(diào)節(jié)元件結(jié)合蛋白-1表達(dá)上調(diào)的作用。尿石素a還可以通過microrna-33途徑顯著上調(diào)三磷酸腺苷結(jié)合盒轉(zhuǎn)運(yùn)蛋白a1和g1的基因表達(dá),并且加強(qiáng)胞內(nèi)膽固醇逆轉(zhuǎn)運(yùn)出細(xì)胞。(5)基于分子對接技術(shù)研究石榴多酚及其代謝物(鞣花酸、尿石素a、b、c、d)與心血管疾病蛋白標(biāo)志物相互作用。共有27種心血管疾病蛋白標(biāo)志物可以分別與鞣花酸和尿石素對接良好。其中,鞣花酸有12個靶標(biāo)蛋白,尿石素a、b、c、d則分別有9個、7個、15個和19個靶標(biāo)蛋白,推測尿石素c和d由于具有更多的羥基數(shù)量可以結(jié)合的靶標(biāo)蛋白數(shù)量也更多。與靶標(biāo)蛋白特定的激動劑或抑制劑相比,鞣花酸及尿石素與靶標(biāo)蛋白的結(jié)合部位氨基酸殘基種類更多;結(jié)合位點(diǎn)的氨基酸殘基數(shù)量也與尿石素分子結(jié)構(gòu)中的羥基數(shù)量呈正相關(guān)。石榴多酚與靶標(biāo)蛋白間的相互作用主要依靠氫鍵作用,結(jié)合位點(diǎn)的氨基酸殘基主要包括:丙氨酸(Ala)、精氨酸(Arg)、天冬酰胺(Asn)、谷氨酰胺(Gln)、谷氨酸(Glu)、組氨酸(His)、苯丙氨酸(Phe)和蘇氨酸(Thr)。由于結(jié)構(gòu)相近,不同多酚小分子與同一靶標(biāo)蛋白結(jié)合的作用方式大體一致。而且,與鞣花酸和尿石素分子對接具有較高打分結(jié)果的靶標(biāo)蛋白多具有與血栓、動脈粥樣硬化相關(guān)的功能,支持了本課題前期實(shí)驗(yàn)?zāi)蚴谹抗炎及維持內(nèi)皮細(xì)胞功能穩(wěn)定的結(jié)論。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R543.5

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張秀云,王松凌,李金華;尿石排出觀察過濾器的制作和使用[J];醫(yī)學(xué)理論與實(shí)踐;2000年04期

2 陳寶國,劉能俊,胡振義,饒淑華,彭仁才,楊光華;尿石通顆粒劑治療尿石癥的臨床和實(shí)驗(yàn)研究[J];江西中醫(yī)學(xué)院學(xué)報;2003年01期

3 石錦明;尹巧英;;尿石鎮(zhèn)痛湯配合西藥治療尿石引起腰腹痛40例[J];中國中醫(yī)藥現(xiàn)代遠(yuǎn)程教育;2012年13期

4 麥冠民;尿石四法[J];新中醫(yī);1978年03期

5 謝叔良,金浩祥,喬衛(wèi)東,眭元庚,連保羅;尿石患者草酸代謝異常的初步研究[J];徐州醫(yī)學(xué)院學(xué)報;1984年01期

6 李浩然;;談治尿石[J];山東中醫(yī)雜志;1984年06期

7 胡龍珠;;尿石成份的顯微重結(jié)晶鑒別法[J];臨床檢驗(yàn)雜志;1985年04期

8 金杰，沈紹基;有關(guān)制動綜合征尿石形成危險因素及其防治[J];中華泌尿外科雜志;1995年10期

9 孫曉青，謝叔良;膽固醇與尿石形成的關(guān)系[J];徐州醫(yī)學(xué)院學(xué)報;1995年02期

10 王劍松，，楊德林;含鈣尿石的預(yù)防[J];云南醫(yī)藥;1995年06期

相關(guān)會議論文 前7條

1 鄺荔;王鳳新;謝瑜珊;柳一鳴;歐陽健明;;尿石癥患者的尿微晶組分與尿石類型的關(guān)系[A];中國化學(xué)會2008年中西部地區(qū)無機(jī)化學(xué)、化工學(xué)術(shù)交流會會議論文集[C];2008年

2 齊勇;周永;周萍;徐峰;湯春波;;65例尿石的紅外光譜分析[A];2009年浙江省男科、泌尿外科學(xué)術(shù)年會論文匯編[C];2009年

3 包楚清;;尿石舒通飲液治療尿路結(jié)石100例[A];第七次中國中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會暨第二次廣東省中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會論文集[C];2009年

4 鐘海;秦曦;周玲;胡曉風(fēng);;Eswl配合腎石通沖劑治療尿石證680例的療效觀察[A];中國中西醫(yī)結(jié)合泌尿外科專業(yè)委員會第四次學(xué)術(shù)會議、浙江省中西醫(yī)結(jié)合泌尿外科男科專業(yè)委員會第三次學(xué)術(shù)會議暨泌尿外科男科疾病中西醫(yī)研究新進(jìn)展學(xué)習(xí)班資料匯編[C];2003年

5 李界訓(xùn);;尿石凈沖劑治療尿路結(jié)石108例[A];全國中醫(yī)藥科研與教學(xué)改革研討會論文集[C];2000年

6 石澤武;張立;張中發(fā);陳秀寧;;泌尿系統(tǒng)112例較大結(jié)石的非手術(shù)治療臨床觀察[A];第五次全國中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)會議論文匯編[C];2005年

7 張樹雄;;泌尿系CT成像(CTU)的技術(shù)[A];2009中華醫(yī)學(xué)會影像技術(shù)分會第十七次全國學(xué)術(shù)大會論文集[C];2009年

相關(guān)重要報紙文章 前9條

1 廣若;防治尿石有中藥[N];中國醫(yī)藥報;2003年

2 黃每裕;防治尿石 “三管齊下”[N];醫(yī)藥經(jīng)濟(jì)報;2004年

3 黃每裕;排出尿石并非萬事大吉[N];醫(yī)藥經(jīng)濟(jì)報;2004年

4 黃每裕;中藥治尿石 療效勝一籌[N];醫(yī)藥經(jīng)濟(jì)報;2004年

5 梅雨;一箭射三雕[N];醫(yī)藥經(jīng)濟(jì)報;2001年

6 黃每裕;亞洲雄風(fēng)[N];醫(yī)藥經(jīng)濟(jì)報;2001年

7 黃海裕;防治結(jié)合 一箭三雕[N];醫(yī)藥經(jīng)濟(jì)報;2004年

8 黃每裕;尿石通丸的組方和工藝特色[N];健康報;2004年

9 黃每裕;科研二十年 成果“三級跳”[N];健康報;2004年

相關(guān)博士學(xué)位論文 前2條

1 韓淇安;尿石素A對動脈硬化進(jìn)程中細(xì)胞功能及代謝的調(diào)控和作用機(jī)制研究[D];西北農(nóng)林科技大學(xué);2016年

2 張建偉;板栗殼斗鞣花單寧及其代謝產(chǎn)物鞣花酸和尿石素的生物活性研宄[D];北京林業(yè)大學(xué);2015年

相關(guān)碩士學(xué)位論文 前3條

1 郭昌華;半量尿石通對含鈣尿石形成患病因素的影響[D];華中科技大學(xué);2012年

2 曾建峰;尿石通丸預(yù)防雙J管鹽垢形成的臨床研究[D];廣州中醫(yī)藥大學(xué);2012年

3 張禮財;尿石通丸治療URSL術(shù)后雙J管相關(guān)并發(fā)癥的臨床觀察[D];廣州中醫(yī)藥大學(xué);2015年

本文編號：1904867