Omentin-1对巨噬细胞ABCA1介导胆固醇流出及抗动脉粥样硬化的作用及机制

发布时间：2018-05-20 06:17

本文选题：Omentin-1 + 丹参酮IIA　；参考：《南华大学》2016年博士论文

【摘要】：动脉粥样硬化(Atherosclerosis,As)在全球的罹患率以及致死率都名居前列,严重危害人类的健康。在动脉粥样硬化早期,其标志性病理特征是血管内皮下大量的巨噬细胞内脂质蓄积以及泡沫细胞的形成。泡沫细胞最终坏死崩解,释放出细胞内的胆固醇,是构成动脉粥样硬化斑块的最主要成分,促进动脉粥样硬化的进一步进展。因此,促进巨噬细胞内胆固醇流出及体内胆固醇逆向转运(reverse cholesterol transport,RCT),从而抑制泡沫细胞形成和血管壁内脂质沉积,对动脉粥样硬化性的防治具有重要意义。三磷酸腺苷结合盒转运体A1(ATP binding cassette transporter A1,ABCA1)是一个膜整合蛋白,其以ATP作为能源将胞内游离的胆固醇及磷脂转运至胞膜的外侧面,最终与贴附在细胞表面的载脂蛋白AI(apoprotein AI,apoAI)结合,从而形成新生的高密度脂蛋白(high density lipoprotein,HDL)。这是HDL生成所必需的过程,也是促进巨噬细胞内过多的胆固醇外流以及rct的关键步骤。abca1在巨噬细胞中的表达以及其活性往往受到转录及转录后水平的精细调控。网膜素(omentin)是一类新发现的脂肪细胞因子,主要由网膜脂肪组织表达和释放。值得注意的是,研究发现这种基因可调节葡萄糖摄取。肥胖或肥胖相关疾病患者,包括2型糖尿病、内皮功能障碍、颈动脉粥样硬化和冠状动脉疾病,发现其血浆omentin-1水平降低了。研究证明,omentin-1与高密度脂蛋白胆固醇(hdl-c)呈正相关,与葡萄糖和甘油三酸酯水平呈负相关。代谢综合征(mets)初期患者循环omentin-1水平异常者其罹患糖尿病和心血管疾病的风险更高。因此,omentin-1与代谢综合征密切相关,在代谢综合征患者的动脉粥样硬化发生发展中产生重要作用。omentin-1在rct、脂质代谢和as中差异表达的发现为我们提供了新的途径和视角。因此,omentin-1有望成为心血管保护因子和干预靶点之一。然而,omentin-1在abca1表达及导致提高hdl功能和胆固醇逆向转运中的作用研究尚少。本研究中,我们研究omentin-1对巨噬细胞abca1表达的作用和潜在的分子机制。为了探讨omentin-1抗动脉粥样硬化的相关作用机制,我们首先在第一部分实验中观察omentin-1对thp-1巨噬细胞abca1表达、胞内胆固醇流出和脂质含量的影响。在第二部分实验中我们探索了omentin-1介导thp-1巨噬细胞abca1表达及胆固醇流出的作用机制。接着,我们在第三部分实验中,观察了omentin-1对apoe-/-小鼠的血脂水平、胆固醇逆向转运和动脉粥样硬化病变的影响。最后,我们在第四部分实验中,观察了具有较强调脂作用的丹参酮iia(tanshinoneiia,tan)对巨噬细胞的omentin-1和abca1的表达、脂质代谢,以及对apoe-/-小鼠的动脉粥样硬化病变的影响,研究tan通过上调omentin-1发挥抗动脉粥样硬化的作用机制。本研究揭示了omentin-1抗动脉粥样硬化的作用以及机制,也许为动脉粥样硬化性疾病提供新的防治靶点。第1章omentin-1对thp-1巨噬细胞abca1表达及胆固醇流出的影响目的:观察omentin-1对thp-1巨噬细胞abca1mrna及蛋白表达水平、胞内胆固醇流出和脂质含量的影响。方法:用omentin-1和/或lxrαsirna处理thp-1源性巨噬细胞后,用rt-pcr方法检测各个实验组细胞的abca1mrna的表达,westernblot检测abca1蛋白水平,hplc检测胞内总胆固醇(tc)游离胆固醇(fc)及胆固醇酯(ce)含量,液体闪烁计数仪检测胞内胆固醇流出,油红o染色观察胞内脂滴情况。结果:omentin-1呈时间和浓度依赖性上调巨噬细胞abca1的表达,促进胞内胆固醇流出,降低胞内tc、fc和ce含量,抑制胞内脂质蓄积和泡沫细胞形成。而abca1sirna与omentin-1共处理细胞后,明显抑制omentin-1促胆固醇流出的作用,胞内脂质蓄积和泡沫细胞形成明显加剧。结论:(1)omentin-1增加thp-1巨噬细胞abca1mrna和蛋白表达。(2)omentin-1促进abca1介导的thp-1巨噬细胞脂质流出,抑制巨噬细胞内脂质蓄积和泡沫细胞形成。第2章omentin-1介导thp-1巨噬细胞abca1表达及胆固醇流出的作用机制目的:探索omentin-1介导thp-1巨噬细胞abca1表达及胆固醇流出的作用机制。方法:分别用omentin-1或和lxrαsirna、pi3ksirna、aktsirna、pi3k抑制剂ly2940029及akt抑制剂himo处理thp-1源性巨噬细胞24h后,westernblot检测abca1lxrα蛋白水平,hplc检测胞内总胆固醇(tc)游离胆固醇(fc)及胆固醇酯(ce)含量,液体闪烁计数仪检测胞内胆固醇流出,油红o染色观察胞内脂滴情况。结果:omentin-1上调巨噬细胞abca1的表达,促进胞内胆固醇流出,降低胞内tc、fc和ce含量,抑制胞内脂质蓄积和泡沫细胞形成。而分别加用lxrαsirna、pi3ksirna、aktsirna、pi3k抑制剂ly2940029及akt抑制剂himo与omentin-1共处理细胞后,明显抑制abca1的表达及其促胆固醇流出的作用,胞内脂质蓄积和泡沫细胞形成明显加剧。结论:(1)omentin-1通过pi3k/akt途径增加人thp-1巨噬细胞lxrα和abca1表达,促进脂质流出,抑制巨噬细胞内脂质蓄积和泡沫细胞形成。第3章omentin-1对apoe-/-小鼠动脉粥样硬化的影响目的:探讨omentin-1对apoe-/-小鼠体内rct、血脂水平、主动脉壁的脂质沉积,以及对粥样硬化病变的影响和机制。方法:采用高脂饮食(含15%猪油+0.25%胆固醇)喂饲的45只apoe-/-小鼠,并随机地分为三组,分别为:高脂对照组、omentin-1组、omentin-1+ly2940029组。omentin-1组腺病毒转染omentin-1以1×108pfu/kg(pfu:空斑形成单位),ome+ly组小鼠再加以20mg/kgpi3k抑制剂ly2940029,溶入0.2ml的生理盐水中进行尾静脉注射,每2周注射一次,都用基础饮食加上高脂高胆固醇(15%猪油+0.25%胆固醇)喂养,直至实验结束,共处理8周。采用液体闪烁计数以测定mpm来源的rct效率;采用酶氧化法检测甘油三酯(tg)、总胆固醇、高密度脂蛋白胆固醇(hdl-c)及低密度脂蛋白胆固醇(ldl-c)等血脂水平;采用苏丹Ⅳ染色以显示主动脉内膜的as病变情况;采用he染色显示主动脉窦的as病变情况;采用油红o染色以观察主动脉窦处脂质沉积的情况;采用masson三色染色以显示斑块中的胶原纤维;采用westernblot检测小鼠的主动脉abca1等蛋白的表达。结果:输注了转染omentin-1的质粒明显增强了apoe-/-小鼠粪便中3h固醇的放射活性,并增高血浆的hdl-c水平,降低了ldl-c水平,使主动脉内膜的as病变面积减小,减少主动脉窦处的脂质沉积,增强as斑块的稳定性,增加了主动脉abca1的蛋白表达。注射ly2940029则明显减少粪便中的固醇水平,增加主动脉壁内脂质沉积,促进动脉粥样硬化病变的发展,下调abca1蛋白表达。结论:(1)omentin-1促进apoe-/-鼠体内rct和升高血浆hdl-c水平。(2)omentin-1抑制apoe-/-鼠主动脉脂质沉积,延缓粥样硬化病变的进展。(3)omentin-1增强apoe-/-鼠主动脉壁组织的abca1表达,并且其可能是通过pi3k/akt信号通路而起调节作用的。第4章丹参酮iia对巨噬细胞omentin-1/abca1水平及apoe-/-小鼠主动脉病变的影响及其机制目的:观察丹参酮iia(tanshinoneiia,tan)对thp-1巨噬细胞中omentin-1和abca1表达,以及对apoe-/-小鼠体内血脂水平、rct、主动脉壁as病变的影响,并探讨tan抗as的作用机制。方法:用tan处理thp-1巨噬细胞,并结合omentin-1sirna转染thp-1巨噬细胞后,采用实时荧光定量pcr以检测omentin-1和abca1水平,采用westernblot检测omentin-1和abca1蛋白表达;采用液体闪烁计数以测定荷脂THP-1巨噬细胞内的胆固醇流出情况,采用HPLC检测细胞内脂质的含量。高脂饮食(含15%猪油+0.25%胆固醇)喂饲的45只apoE-/-小鼠被随机分为三组,分别为:高脂对照组、丹参酮IIA组和丹参酮IIA+Omentin-1 siRNA组。采用液体闪烁计数以测定MPM来源的RCT效率,采用酶氧化法以检测TG、TC、HDL-c和LDL-c等的血脂水平,采用苏丹Ⅳ染色以显示主动脉内膜的As病变情况,采用HE染色以显示主动脉窦As病变情况,采用油红O染色以显示主动脉窦处脂质的沉积情况,采用Masson三色染色以显示As斑块中的胶原纤维情况。结果:观察到Tan可以呈剂量和时间依赖性地增强了THP-1巨噬细胞中Omentin-1和ABCA1蛋白的表达,增强细胞内胆固醇流出,并减少细胞内的FC、CE和TC含量。Tan可增强apoE-/-小鼠粪便中3H胆固醇的放射活性,并增高血浆HDL-c水平,降低LDL-c水平,增强主动脉Omentin-1和ABCA1表达,减少了主动脉内膜的As变面积,减轻了主动脉窦处的脂质沉积,增强了斑块的稳定性。抑制Omentin-1表达后,则明显削弱Tan的上述作用。结论:(1)Tan抗As的作用与增强巨噬细胞内胆固醇外流和体内的RCT功能密切相关。(2)Tan抗As的作用机制与升高Omentin-1水平并进而增加ABCA1表达有关,并且可能是通过PI3K/Akt信号通路而起调节作用的。
[Abstract]:Atherosclerosis (As) is in the forefront of the global morbidity and mortality rate, which seriously endangers human health. In the early stage of atherosclerosis, its marked pathological feature was the accumulation of lipid and the formation of foam cells in a large number of macrophages under vascular endothelium. Internal cholesterol, the most important component of atherosclerotic plaques, promotes the further progress of atherosclerosis. Therefore, it promotes cholesterol outflow in macrophages and reverse cholesterol transport (RCT) in vivo, which inhibits the formation of foamy cells and the lipid deposition in the blood vessel wall. Adenosine triphosphate binding cassette transporter A1 (ATP binding cassette transporter A1, ABCA1) is a membrane integrin that transships intracellular free cholesterol and phospholipids to the outer side of the cell membrane with ATP as a source of energy and eventually combines with the apolipoprotein AI (apoprotein AI, apoAI) attached to the surface of the cell. The formation of high density lipoprotein (HDL), which is a necessary process for HDL production, is also a key step in promoting excessive cholesterol Exodus in macrophages and the key step in RCT, the expression of.Abca1 in macrophages and its activity often finely regulated by the transcriptional and post transcriptional levels. The omentin (omentin) is a HDL. A newly discovered class of adipocyte factors, mainly expressed and released from omentum adipose tissue. It is worth noting that this gene has been found to regulate glucose uptake. Patients with obesity or obesity related diseases, including type 2 diabetes, endothelial dysfunction, carotid atherosclerosis, and coronary artery disease, have been found to be in the plasma omentin-1 level. Lower. Studies have shown that omentin-1 is positively related to high density lipoprotein cholesterol (HDL-C) and is negatively correlated with glucose and triglyceride levels. Patients with abnormal circulating omentin-1 levels in the early stage of metabolic syndrome (MetS) have a higher risk of developing diabetes and cardiovascular disease. Therefore, omentin-1 is closely related to metabolic syndrome, in metabolism. The discovery of.Omentin-1 in the occurrence and development of atherosclerosis in patients with RCT, the discovery of differential expression in lipid metabolism and as provides us with new ways and perspectives. Therefore, omentin-1 is expected to become one of the cardiovascular protective factors and intervention targets. However, omentin-1 is expressed in ABCA1 and leads to the improvement of HDL function and bile. In this study, we studied the role of omentin-1 on the expression of ABCA1 in macrophages and the potential molecular mechanism in this study. In order to explore the mechanism of omentin-1 anti atherosclerosis, we first observed the expression of ABCA1 and intracellular cholesterol in THP-1 macrophages in the first part of the experiment. The effect of outflow and lipid content. In the second experiment, we explored the mechanism of omentin-1 mediated THP-1 macrophage ABCA1 expression and cholesterol efflux. Then, in the third experiment, we observed the effects of omentin-1 on blood lipid levels, cholesterol reverse transport and atherosclerotic lesions in apoe-/- mice. In the fourth experiment, we observed the expression of omentin-1 and ABCA1 in macrophages, lipid metabolism, and the effect on atherosclerotic lesions of apoe-/- mice, which had strong lipid regulating effect, and studied the mechanism of the anti atherosclerosis mechanism of Tan by up regulation of omentin-1. This study was to study the mechanism of the anti atherosclerosis of Tan by up regulation of omentin-1. The anti atherosclerotic effect and mechanism of omentin-1 may provide new prevention and treatment targets for atherosclerotic disease. First the effect of omentin-1 on ABCA1 expression and cholesterol efflux of THP-1 macrophages is to observe the expression level of abca1mrna and protein in THP-1 macrophages, intracellular cholesterol efflux and lipids. Methods: after the treatment of THP-1 derived macrophages with omentin-1 and / or LXR alpha siRNA, the expression of abca1mrna in the cells of each experimental group was detected by RT-PCR, the level of ABCA1 protein was detected by Westernblot, and the content of intracellular total cholesterol (TC) free cholesterol (FC) and cholesteryl ester (CE) was detected by HPLC, and the liquid scintillation counting instrument was used to detect the internal gallbladder. Results: omentin-1 showed time and concentration dependent increase of ABCA1 expression in macrophages, promoted intracellular cholesterol efflux, reduced intracellular TC, FC and Ce content, inhibited intracellular lipid accumulation and foam cell formation, while abca1sirna and omentin-1 co treated cells significantly inhibited omentin-1 promoted by abca1sirna and omentin-1. The effect of cholesterol outflow, intracellular lipid accumulation and foam cell formation increased obviously. Conclusion: (1) omentin-1 increases the abca1mrna and protein expression of THP-1 macrophages. (2) omentin-1 promotes lipid efflux of ABCA1 mediated THP-1 macrophages and inhibits lipid accumulation and foam cell formation in macrophages. Second chapter omentin-1 mediates THP-1 meagage The mechanism of the action mechanism of ABCA1 expression and cholesterol efflux: To explore the mechanism of omentin-1 mediated ABCA1 expression and cholesterol efflux in THP-1 macrophages. Methods: omentin-1 or LXR alpha siRNA, pi3ksirna, aktsirna, PI3K inhibitor ly2940029 and Akt inhibitors were used to detect the derived macrophages. R alpha protein level, HPLC detection of intracellular total cholesterol (TC) free cholesterol (FC) and cholesteryl ester (CE) content, liquid scintillation counting apparatus to detect intracellular cholesterol efflux, oil red O staining to observe the intracellular lipid droplets. Results: omentin-1 increases the expression of ABCA1 in macrophages, promotes intracellular cholesterol efflux, reduces intracellular TC, FC and Ce content, inhibits intracellular. Lipid accumulation and foam cell formation. After adding LXR alpha siRNA, pi3ksirna, aktsirna, PI3K inhibitor ly2940029 and Akt inhibitor HimO and omentin-1 co processing cells, the expression of ABCA1 and the effect of cholesterol efflux were inhibited, and the accumulation of intracellular lipids and the formation of foam cells were obviously intensified. Conclusion: (1) omentin-1 through pi3k/akt. Diameter increases the expression of LXR alpha and ABCA1 in human THP-1 macrophages, promotes lipid outflow, inhibits lipid accumulation in macrophages and foams cell formation. Third the effects of omentin-1 on atherosclerosis in apoe-/- mice: the study of omentin-1 on RCT, lipid levels, lipid deposition in the aortic wall and atherosclerosis in apoe-/- mice Methods: 45 apoe-/- mice fed with high fat diet (containing 15% lard +0.25% cholesterol) were randomly divided into three groups: high fat control group, omentin-1 group, omentin-1+ly2940029 group.Omentin-1 adenovirus transfected omentin-1 with 1 x 108pfu/kg (pfu: empty plaque formation unit), ome+ly group mice then 20mg/kgpi3k The inhibitor ly2940029, injected into the normal saline of 0.2ml, injected the tail vein every 2 weeks, fed the basal diet plus high fat and high cholesterol (15% lard +0.25% cholesterol), until the end of the experiment, for 8 weeks. The liquid scintillation counting was used to determine the RCT efficiency of the MPM source, and the enzyme oxidation method was used to detect triglyceride (TG), general Cholesterol, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) and other blood lipid levels; Sultan IV staining was used to show the as lesion of the aortic intima; he staining was used to display the as lesion of the aortic sinus, and the oil red O staining was used to observe the lipid deposition in the aortic sinus; Masson trichromatic staining was used. The expression of ABCA1 and other proteins in the aorta of the mice was detected by Westernblot. Results: the transfection of omentin-1 transfected plasmid significantly enhanced the radioactivity of 3H sterol in the feces of apoe-/- mice, increased the level of HDL-C in plasma, reduced the LDL-C level, reduced the as lesion area of the aorta intima, and reduced the lesion area of the aorta intima. The lipid deposition at the sinus of aortic sinus enhanced the stability of as plaque and increased the protein expression of ABCA1 in the aorta. Injection of ly2940029 significantly reduced the level of sterol in the feces, increased the lipid deposition in the aortic wall, promoted the development of atherosclerotic lesions, and lowered the expression of ABCA1 protein. Conclusion: (1) omentin-1 promotes RCT in the body of apoe-/- mice. Increase the plasma HDL-C level. (2) omentin-1 inhibits the aortic lipid deposition in apoe-/- rats and delays the progression of atherosclerotic lesions. (3) ABCA1 expression in the aorta wall of apoe-/- rats is enhanced by omentin-1, and it may be regulated by the pi3k/akt signaling pathway. The fourth chapter of tanshinone IIA to macrophage omentin-1/abca1 level and apoe-/ Effect and mechanism of mouse aortic disease: To observe the expression of tanshinone IIA (tanshinoneIIA, tan) on omentin-1 and ABCA1 in THP-1 macrophages, as well as the effect on serum lipid level, RCT, as lesion of the aortic wall in apoe-/- mice, and to explore the mechanism of Tan as on tan. After transfection of RNA to THP-1 macrophages, real-time fluorescence quantitative PCR was used to detect the level of omentin-1 and ABCA1. The expression of omentin-1 and ABCA1 protein was detected by Westernblot; liquid scintillation counting was used to determine the cholesterol efflux in the lipid THP-1 macrophages. The content of intracellular lipids was detected by HPLC. High fat diet (containing 15% lard +0.25%) was used. 45 apoE-/- mice fed with cholesterol were randomly divided into three groups: the high fat control group, the tanshinone IIA group and the tanshinone IIA+Omentin-1 siRNA group. The liquid scintillation counting was used to determine the RCT efficiency of the MPM source. The lipid levels of TG, TC, HDL-c and LDL-c were detected by enzyme oxidation, and the Sultan IV staining was used to display the internal aorta. The As lesion of the membrane was used to display the As lesion of the aortic sinus by HE staining. The oil red O staining was used to show the deposition of lipid in the aortic sinus, and the Masson stain was used to show the collagen fiber in the As plaque. The results showed that Tan could be dosed and time dependent on the increase of Omentin-1 and A in THP-1 macrophages. The expression of BCA1 protein, the increase of intracellular cholesterol efflux, and the reduction of the intracellular FC, CE and TC content.Tan can enhance the radioactivity of 3H cholesterol in the apoE-/- mice, increase the level of HDL-c, reduce the LDL-c level, increase the expression of Omentin-1 and ABCA1 in the aorta, reduce the area of the intima intima of the aorta, and reduce the sinus of the aortic sinus. Lipid deposition enhanced the stability of plaque. After inhibiting the expression of Omentin-1, the effect of Tan was obviously weakened. Conclusion: (1) the anti As effect of Tan is closely related to the enhancement of cholesterol Exodus in macrophages and the function of RCT in the body. (2) the mechanism of the anti As action of Tan is related to the increase of Omentin-1 level and the increase of ABCA1 expression, and may be associated with As. It is regulated by the PI3K/Akt signaling pathway.
【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5

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