| Atherosclerosis(AS) remains a major cause of mortality and morbidity in the world, and it causes acute cardiovascular events and chronic damage. Cholesterol accumulation in macrophage and the foam cell formation underneath the intima are the main hallmark pathological features in the early stage atherosclerosis. Uncontrolled cholesterol uptake infiltrating into arterial walls finally triggers the collapse of the foam cells, and next the release of intracellular cholesterol that forms the dominating chemical components of atherosclerotic lesions, and accelerates the development of atherosclerosis. Thus, to facilitate the macrophage cholesterol efflux and the reverse cholesterol transport(RCT), then inhibiting the lipid deposition and the foam cell formation in arterial wall, is of great significance in the prevention and treatment of atherosclerotic diseases.The ATP binding cassette transporter A1(ABCA1) is a integral membrane protein who transports free cholesterols and phospholipids from intracellular compartments to the lateral aspect of cell membranes by using ATP as the source of energy. Apoprotein AI(apo AI) removes lipidic cargos. ABCA1-mediated cholesterol efflux to apo AI, necessary for HDL formation, is defined as the first and rate-controlling step for the cholesterol efflux in macrophage and RCT. Therefore, ABCA1 mediated HDL formation is a key mediator regulating macrophage cholesterol homeostasis and this process plays a critically important role in the initiation of early atherosclerotic lesion development. In macrophages, the expression and function of ABCA1 are precisely regulated at several levels, including the transcriptional and posttranscriptional processes.Omentin is a novel adipokine, which is expressed in and released from omental adipose tissue. Notably, the gene was found to regulate insulin-stimulated glucose uptake in human adipocytes. In patients with obesity or obesity-linked disorders, including type 2 diabetes, endothelial dysfunction, coronary artery disease and carotid atherosclerosis, the plasma Omentin levels were found decreased. Furthermore, it was demonstrated that Omentin was positively correlated with high-density lipoprotein cholesterol(HDL-c), and inversely with glucose and triglyceride levels. The abnormal circulating Omentin levels in nascent metabolic syndrome(Met S) could play a higher risk for diabetes and cardiovascular diseases(CVD). Therefore, Omentin is closely related to Met S, and might confer an important role in atherosclerosis of patients with Met S.The differentially expression of Omentin-1 in RCT, lipid metabolism, and As, may provide us with new approaches and perspectives in CVD treatment. Omentin-1, therefore, is expected to become one of the cardiovascular protective factor and intervention target. However, few studies have investigated the role of Omentin-1 in the expression of ABCA1 which leads to enhanced HDL function and RCT. In the present study, the effect of Omentin-1 on the expression of ABCA1 and the underlying molecular mechanisms in macrophages were investigated.The antiatherogenic mechanism of Omentin-1 is still largely unknown. Accordingly, we speculate that Omentin-1 up-regulates macrophage ABCA1 expression, enhances the intracellular cholesterol efflux and the RCT, subsequently restrains the macrophage cholesterol accumulation and lipid deposition, which plays the role of antiatherosclerosis. To confirm the hypothesis of Omentin-1’s antiatherogenic effect, firstly, our study was to investigate the effect of Omentin-1 on ABCA1 expression and cholesterol efflux in THP-1 macrophages. Secondly, we observed the effect of Omentin-1 on the expression of ABCA1 and LXRα, the intracellular cholesterol efflux and lipid content in macropahges. In the third part, we observed the effect of Omentin-1 on the plasma lipid profile, RCT, and the aortic atherosclerosis development in vivo. Finally, we investigated the effect of Tanshinone IIA(Tan), which had confirmed to have sensational antiatherogenic features, on Omentin-1 and ABCA1 expression, and lipid metabolism in vitro, and on the plasma lipid profile, RCT, and the aortic atherosclerosis development in vivo. Maybe, our findings provide new insights to reveal the potential effects, mechanisms and therapeutic value of Omentin-1 about the atherosclerosis progression in the future.Part I: Effect of Omentin-1 on ABCA1 Expression and Cholesterol Efflux in THP-1 MacrophagesAims: To investigate the effect of Omentin-1 on ABCA1 expression and cholesterol efflux in THP-1 macrophages.Methods: THP-1 macrophage was cultured with and Omentin-1 or LXRα si RNA. The m RNA expression and protein of ABCA1 were examined by real-time quantitative PCR and western blot, separately. Intracellular 3H-cholesterol efflux was measured with the liquid scintillator. Intracellular lipid droplet was stained by oil red O. The levels of free cholesterol(FC), cholesterol ester(CE) and cellular total cholesterol(TC) were assayed by HPLC.Results: We found that Omentin-1 up-regulated ABCA1 in both protein and m RNA levels in a time and dose dependent manner in THP-1 macrophages. Omentin-1obviously facilitated macrophage cholesterol efflux,resulting in a decrease in intracellular level of TC,FC,CE and lipid droplet, and the foam cell formation. Furthermore, the action of Omentin-1 facilitating cholesterol efflux was almost entirely abrogated in THP-1 derived macrophage coincubated with ABCA1 si RNA, causing excessive lipid accumulation and increase formation of foam cells.Conclusion: The data suggested that :(1) Omentin-1 enhances ABCA1 m RNA and protein expression in THP-1 macrophages.(2) Omentin-1 promotes macrophage cholesterol efflux, which suppresses intracellular lipid accumulation and foam cell formation.Part II: Mechanism of Omentin-1 on ABCA1 Expression and Cholesterol Efflux in THP-1 MacrophagesAims: To observe the effect of Omentin-1 on ABCA1 expression, lipid accumulation and cholesterol efflux in THP-1 macrophages.Methods: THP-1 macrophages were cultured with medium 1640 and treated with Omentin-1 or/and LXRα si RNA, PI3 K si RNA, Akt si RNA, PI3 K inhibitor LY2940029 and Akt inhibitor HIMO. The m RNA and protein expressions of ABCA1 and LXRα were detected with real-time quantitative PCR and western blot, respectively. Intracellular 3H-cholesterol efflux was tested with liquid scintillator. Intracellular lipid droplet was stained by oil red O. The levels of TC, FC and CE were assayed by HPLC.Results: The m RNA and protein levels of ABCA1 and LXRα were increased in the THP-1 macrophages treated with Omentin-1. However, the m RNA and protein levels of ABCA1 were decreased in THP-1 macrophages transfected with LXRα si RNA. The m RNA and protein levels of ABCA1 and LXRα were decreased in THP-1 macrophage transfected with PI3 K si RNA, Akt si RNA, or treated with LY2940029 and HIMO. Accordingly, comparared with the Ome group, the Apo AI–mediated(ABCA1 dependent) cholesterol efflux was dramatically suppressed, leading to the increases in the contents of TC, TC and CE, and a lot of droplets densely presented all over the intracellular regions of in the macrophages.Conclusion: Omentin-1 promotes macrophage cholesterol efflux and inhibits lipid accumulation, and that PI3K/Akt signaling pathway may play an important effect in this process.Part III: Effect of Omentin-1 on Atherosclerosis in Apolipoprotein E Knockout MiceAims: To study the effect of Omentin-1 on the plasma lipid profile, RCT, the aortic lipid deposition and the atherosclerotic lesion in apo E knockout(apo E-/-) mice.Methods: Forty five of eight-week-old male apo E-/- mice were fed with the Western diet and randomly divided into 3 groups: the control(Con) group, the Omentin-1(Ome) group, Omentin-1+LY294002(Ome+LY) group. Every 2 weeks, Ome group and Ome+LY group were received tail vein injection of adenovirus Omentin-1(Ad-Ome) at a dose of 1×108 plaque-forming units, or added the phosphoinositide 3-kinase(PI3K) inhibitor LY2940029(20mg/kg; dissolved in dimethyl sulfoxide) was intraperitoneally injected into the abdomen of 8-week-old apo E-/-mice 1 day before first injection of Ad-Ome. At the same time, the control group was received intravenous injections with the same volume of PBS. Mouse peritoneal macrophages(MPM) were radiolabeled by 3H-cholesterol and intraperitoneally injected into the apo E-/- mice at two days before sacrificed. All animals were euthanized after 8 weeks. Blood, hepatic tissues and feces were collected to exam RCT efficiency by liquid scintillation counting. Plasma lipids were detected with commercially enzymatic methods. Atherosclerotic areas of aortic intima were stained with Sudan IV. Atherosclerotic lesions in aortic sinus were detected by HE stain. Lipid accumulation in aortic sinus was evaluated with Oil Red O stain. Collagen contents were evaluated by Masson’s staining. Protein expression of LXRα and ABCA1 in aorta were detected with western blot.Results: The excretion of 3H-cholesterol originating from cholesterol-laden MPM into feces and plasma HDL-C level were increased in apo E-/- mice injected with Omentin-1. Omentin-1 reduced atherosclerosis areas and lipid deposition in aortic intima, and increased the stability of atherosclerosis lesions and aortic LXRα and ABCA1 expressions. PI3 K inhibitor LY294002 obviously reduced fecal 3H-sterol and plasma HDL-C level. Lipid deposition and atherosclerosis areas were increased, LXRα and ABCA1 expression were decreased in aortas of apo E-/- mice treated by Omentin-1 LY294002.Conclusion:(1) Omentin-1 promotes apo E-/- mice RCT and elevates plasma HDL-c level in apo E-/- mice.(2) Omentin-1 inhibits aorta lipid deposition and delays the progress of the atherosclerosis progression in apo E-/- mice.(3) Omentin-1 increases aortic tissue ABCA1 expression, and it may be through the PI3K/Akt signaling pathway in apo E-/- mice. Part Ⅳ: Effect and Mechanism of Tanshinone IIA on Macrophage Omentin-1/ABCA1 Expression and Atherosclerosis in Apo E-/- MiceAims: To investigate the role and mechanism of Tanshinone IIA(Tan) on Omentin-1/ABCA1 expressions in THP-1 macrophages, and the RCT and the aortic atherosclerosis in apo E-/- mice.Methods: THP-1 macrophages were treated with Tan, alone or with Omentin-1 si RNA. The m RNA and protein expressions of ABCA1 and LXRα and Omentin-1 were examined with real-time quantitative PCR and western blot, respectively. Liquid scintillator was for detecting intracellular 3H-cholesterol efflux. The contents of intracellular lipid were assayed by HPLC. Forty-five eight-week-old male apo E-/- mice were fed by Western diet, and randomly divided into 3 groups: the Control group, the Tan group, the Tan+Omentin-1 si RNA group. Liquid scintillation counting was for RCT efficiency, in vivo. Plasma lipids were detected with commercially enzymatic methods. Atherosclerotic lesions in aortic sinus were tested with HE stain. Atherosclerotic areas of aortic intima were stained by Sudan IV. Lipid accumulation in aortic sinus was evaluated by Oil Red O stain. Collagen contents were evaluated by Masson’s staining.Results: Tan increased Omentin-1 and ABCA1 expressions in time- and dose- manner in macropahges. Tan significantly reduced macrophage intracellular content of FC, CE and TC. Tan immensely increased fecal 3H-sterol and the plasma HDL-C level, and decreased plasma LDL-C level. Tan enhanced aortic LXRα and ABCA1 expressions, decreased aortic atherosclerotic areas, reduced lipid accumulation in aortic sinus and enhanced the stability of atherosclerotic lesions. However, Omentin-1 inhibition abrogated the antiatherogenic effect of Tan.Conclusion:(1) Effect of Tan on antiatherosclerosis is for the facilitation of cholesterol efflux in macrophages and in vivo RCT.(2) Partially, mechanism of Tan is that Tan enhances LXRα and ABCA1 expressions by increasing Omentin-1 level, and it may be through the PI3K/Akt signaling pathway in apo E-/- mice. |
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