| Atherosclerosis has become one of the cardiovascular diseases with the highest incidence and mortality rate worldwide. Atherosclerosis is a complex disease process involving both excessive lipid storage and chronic inflammation. Artery located within regions where are susceptible to atherosclerosis, preferentially develops into thrombus and stroke as well as myocardial infarction, typically accompanied by lipid accumulation, endothelial dysfunction, smooth muscle cell proliferation and atherosclerotic plaque formation. Men aged over 40 and postmenopausal women are more prone to atherosclerosis, and early atherosclerosis has existed in children and adolescents. Therefore, atherosclerosis has become a serious health-threatening disease for human beings, preventing and controlling atherosclerosis will be one of the indispensable ways to improve the national health status and the quality of life.Urolithins are a kind of gut microbial metabolites from ellagitannin-rich foods(such as pomegranate, strawberry, walnut and peanut), which belong to the family of molecules of 6-H-dibenzo-[b,d]pyran-6-one structure with different phenolic hydroxylation patterns. Urolithin A is first isolated and characterized from rat urine and feces after oral administration of ellagic acid. Studies have demonstrated that urolithin A possess anti-oxidant, anti-inflammatory and anti-cancer bioactivities, but little is known about the anti-atherogenic activity and mechanism of urolithin A. The dissertation is designed to explore the beneficial effect of urolithin A on endothelial dysfunction and cholesterol metabolism in macrophage-derived foam cell, as well as the corresponding mechanisms. The findings demonstrated that urolithin A played an important role in the prevention and treatment of atherosclerosis, and uncovered ellagitannin-rich food was potential to develop the drug for the atherosclerosis prevention.Human aortic endothelial cells(HAECs) and RAW264.7 cell line were used in this study. The main results were as follows:(1) Urolithin A inhibited ox-LDL induced monocytes adhesion to HAECs. It was found that low concentrations of urolithin A(0.5 ~ 5 μM) promoted HAECs proliferation while high concentrations of urolithin A(25 ~ 100 μM) reduced cells survival rate. And the optimal incubation time was 24 h. Urolithin A could markedly inhibit the increase of lactate dehydrogenase induced by 50 μg/m L ox-LDL, indicating urolithin A has protective effect on cells structural integrity. Moreover, urolithin A could significantly elevate the levels of nitric oxide and endothelial nitric oxide synthase. The results of rose bengal staining and fluorescent probe method revealed that urolithin A enabled to inhibit monocytes adhesion to HAECs via down-regulating the m RNA expressions of intercellular adhesion molecule 1(ICAM-1) and monocyte chemotactic protein 1(MCP-1).(2) Urolithin A inhibited the inflammatory response induced by ox-LDL in HAECs via modulating MAPK signal pathway. The results showed that urolithin A decreased the concentrations of inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, and did not alter the concentration of interferon γ. Interestingly, urolithin A dramatically suppressed the micro RNAs expressions, including micro RNA-10, 27, 125 a, 126 and 155. And urolithin A could up-regulate the decreased gene expression of peroxisome proliferators-activated receptor gamma(PPAR-γ) induced by 50 μg/m L ox-LDL via inhibiting micro RNA-27 expression. Besides, urolithin A dose-dependently attenuated the phosphorylation of ERK 1/2, SAPK/JNK and p38. Moreover, urolithin A regulated the expression of ICAM-1 partly through ERK/PPAR-γ signal transduction pathway.(3) Urolithin A had an effect on macrophage polarization and the formation of macrophage-derived foam cells. Low concentrations of urolithin A(0.5 ~ 25 μM) co-incubated with RAW264.7 for 3 h, the cell viability was not altered. It was found that 20 μM of urolithin A was capable to decrease the expressions of M1 macrophage biomarkers, such as IL-1β, IL-6 and TNF-α, while 10 and 20 μM of urolithin A could improve the levels of M2 macrophage biomarkers(IL-10 and TGF-β), indicating that urolithin A promoted the macrophage polarization from natural status to alternatively activated M2 type. Besides, urolithin A dose-dependently depressed the expressions of IL-1β, IL-6 and TNF-α induced by lipopolysaccharide(LPS), showing an inhibition effect on the RAW264.7 polarization to form pro-inflammatory M1 macrophages. Moreover, urolithin A inhibited the formation of macrophage-derived foam cells via decreasing the m RNA levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-Co AR) and fatty acid synthase(FAS) whereas significantly increasing ABCA1 and ABCG1 expressions in a dose-dependent manner.(4) Urolithin A promoted the cholesterol efflux in macrophage-derived foam cells. The combination treatment of urolithin A and Apolipprotein A-Ⅰ(apo A-Ⅰ) resulted in less accumulation of cholesterol in foam cells and raised blood cholesterol levels. On one hand, urolithin A could dose-dependently decrease the protein levels of ERK 1/2 and P-ERK 1/2 as well as SREBP-1. On the other hand, urolithin A activated AMPKα and P-AMPKα. Blocking ERK 1/2 signal pathway might increase the phosphorylation of AMPKα, and the effect was strengthened by the addtion of urolithin A. Moreover, Dorsomorphin, the inhibitor of AMPK signal pathway, greatly improved SREBP-1 level and even impaired the inhibition effect of urolithin A on the expression of SREBP-1. Besides, urolithin A might modulate the m RNA expressions of ABCA1 and ABCG1 via micro RNA-33 pathway.(5) Screening out the potential target proteins of ellagic acid and its metabolites urolithins based on molecular docking. The screening database constituted 84 of cardiovascular disease related proteins, in which there were 27 kinds of proteins docking higher than 5.00 with ellagic acid and urolithins by SYBYL X software. The numbers of target proteins of ellagic aicd and urolithin A, B, C, D were 12, 9, 7, 15 and 19, respectively. Moreover, it is also indicated that urolithin C and D had more effective anti-atherosclerotic property than ellagic acid because of the larger quantities of hydroxyl groups. The number and type of amino acid residues in bonding sites between the target protein and polyphenol were more than the ones between the target proteins and their specific agonists or inhibitors, which proved a closely bonding of polyphenols. The interaction of ellagic acid and urolithins relied mainly on hydrogen-bonding interaction. The amino acid residues of different binding sites were Ala, Arg, Asn, Gln, Glu, His, Phe and Thr. As with the similar structures of the ligands, the mode of interaction between different small molecules and the same target protein was also nearly the same. In addition, most of the target proteins had direct or indirect connection to atherosclerosis. This study provided theoretical basis for pomegranate polyphenol ellagic acid and its metabolites to show effective activity of resistance to atherosclerosis, and it is feasible that urolithins had direct effect against atherosclerosis in vivo. |
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