脂联素对巨噬细胞源性泡沫细胞ABCA1及胆固醇含量的影响及机制

目的探讨脂联素对RAW264.7巨噬细胞源性泡沫细胞ABCA1及胆固醇含量的影响及其可能的机制。方法体外培养RAW264.7细胞,加入20 mg/L氧化低密度脂蛋白(ox-LDL)共同孵育48 h,将其诱导成泡沫细胞,加入不同浓度(0、1、5、10μg/mL)的脂联素干预24 h,RT-PCR测定ABCA1 mRNA的表达,高效液相色谱测定细胞内胆固醇含量。观察脂联素对泡沫细胞中ABCA1表达的影响。结果脂联素显著增加RAW264.7巨噬细胞源性泡沫细胞ABCA1 mRNA的表达(P<0.05),并增加细胞内胆固醇含量,且呈浓度依赖性(P<0.05)。结论脂联素可以增加巨噬源性泡沫细胞ABCA1转录水平,促进胆固醇流出,延缓AS的发生发展。     

EGCG通过miR-33a上调ABCA1表达减少巨噬细胞脂质蓄积

目的研究EGCG是否通过影响miR-33a的表达,进而调控ABCA1表达,促进巨噬细胞内胆固醇流出。方法先建立THP~(-1)巨噬细胞源性泡沫细胞,然后用EGCG处理,Real time PCR检测细胞miR-33a表达。细胞随机分为3组:空白对照组、50μmol·L~(-1)EGCG处理组、50μmol·L~(-1)EGCG+80 nmol·L~(-1)miR-33a mimic处理组,Real time PCR和Werstern blot检测细胞ABCA1 mRNA和蛋白表达,油红O染色和高效液相色谱法检测细胞内脂质含量,[3H]法检测细胞内胆固醇流出。结果在不影响细胞活性状况下,EGCG呈浓度和时间依赖性下调miRNA33a表达;EGCG能明显上调ABCA1 mRNA和蛋白的表达,但能被转染miRNA33 mimic抑制;EGCG可减少THP~(-1)巨噬细胞源性泡沫细胞中的脂质蓄积,但能被细胞中转染miRNA33 mimic所弱化;EGCG减少细胞内胆固醇蓄积是与其促进细胞内胆固醇流出有关,细胞中转入过量miRNA33a可以抑制胆固醇流出。结论 EGCG可通过减少miRNA33a的生成,进而上调ABCA1表达,促进巨噬细胞中胆固醇流出,这可能是EGCG抗动脉粥样硬化作用的分子机制之一。     

二氢杨梅素对巨噬细胞源性泡沫细胞胆固醇流出的影响

摘要：目的 观察二氢杨梅素 （DMY） 对鼠源巨噬细胞性泡沫细胞胆固醇流出的影响并探讨其可能机制。方法 用 50 mg/L 的氧化低密度脂蛋白 （ox-LDL） 孵育鼠源性巨噬细胞 RAW264.7 经 48 h 诱导细胞泡沫化。将泡沫细胞分为对照组 （只加 RPMI 1640 的培养基培养） 和 DMY1~4 组 （分别用 10、 20、 40 和 80 μmol/L DMY 处理）, 培养 24 h。采用[ 3 H]标记的胆固醇检测细胞胆固醇的流出率, 高效液相色谱测定细胞内游离胆固醇 （FC）、 胆固醇酯 （CE） 和总胆固醇 （TC） 的水平, Western blot 检测细胞中三磷酸腺苷结合盒转运体 A1 （ABCA1） 的表达。结果 与对照组比较, 20、 40 和 80 μmol/L DMY 组细胞的胆固醇流出率均显著增加, 细胞内 FC、 TC 和 CE 水平及 CE/TC 的比值均显著减少, ABCA1 的表达显著上调, 均呈剂量依赖性 （均 P < 0.05）。与对照组比较, DMY （10 μmol/L） 组细胞胆固醇流出率,细胞内 FC、 TC 和 CE 水平,ABCA1 的表达差异均无统计学意义 （均 P > 0.05）。结论 DMY 促进了鼠源巨噬细胞性泡沫细胞胆固醇流出, 其机制可能与上调泡沫细胞中 ABCA1的表达有关。 ,     

脂多糖对B类I型清道夫受体表达的调节

目的观察脂多糖（LPS）对THP-1巨噬细胞源性泡沫细胞B类I型清道夫受体（sR-BI）表达和胆固醇流出的影响,并探讨核因子-κB（NF-κB）信号途径在此过程中的作用。方法THP-1巨噬细胞源性泡沫细胞以LPS单独或NF-κB抑制剂对甲苯磺酰-L-苯丙氨酸氯甲基甲酮（TPCK）预处理后再加入LPS处理。Western blot检测sR．BI及核内NF-κBp65蛋白质的表达,液体闪烁计数器检测细胞内胆固醇流出,高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量。结果LPS抑制sR-BI蛋白质的表达,而增加核内NF-K-κB65蛋白质的表达,LPS使泡沫细胞细胞内胆固醇流出减少,细胞总胆固醇、游离胆固醇与胆固醇酯增加。TPCK预处理后,LPS的这种作用被部分抑制。结论NF-κB信号途径介导LPS对sR—BI表达及细胞内胆固醇流出的抑制作用。     

坎地沙坦对泡沫细胞胆固醇代谢的影响

目的观察坎地沙坦(CAN)对氧化低密度脂蛋白(ox-LDL)诱导急性单核白血病细胞(THP-1)源性泡沫细胞及对三磷腺苷结合盒转运子A1(ABCA1)表达水平的作用。方法 THP-1源性细胞加入160 nmol.L-1佛波酯,在RPMI-1640液中孵育48 h,将其诱导分化为巨噬细胞后随机分为3组,对照组加入50 mg.L-1ox-LDL培养液孵育48 h;血管紧张肽Ⅱ(AngⅡ)组加入1×10-6mol.L-1AngⅡ后再加入50 mg.L-1ox-LDL培养液孵育48 h;CAN组加入1×10-6mol.L-1CAN后再加入1×10-6mol.L-1AngⅡ孵育,然后以50 mg.L-1ox-LDL处理48 h。分别采用油红O染色观察细胞内脂滴变化,酶化学法测定总胆固醇和胆固醇酯含量,Western blotting法检测ABCA1蛋白表达水平。结果 AngⅡ作用后,细胞内脂滴明显增多,细胞内总胆固醇和胆固醇酯含量及胆固醇酯/总胆固醇比值明显升高,ABCA1蛋白表达水平降低(P<0.05)。CAN能减少泡沫细胞内脂滴形成,导致细胞内总胆固醇和胆固醇酯含量及胆固醇酯/总胆固醇比值明显降低,一定程度上调泡沫细胞转运蛋白ABCA1表达(P<0.05),促进细胞内胆固醇酯流出。结论 AngⅡ减少泡沫细胞中ABCA1的表达,CAN可通过阻断AngⅡ1型受体促进ABCA1的表达及胆固醇酯流出。     

载脂蛋白嵌合模拟肽对巨噬细胞胆固醇流出的影响

目的观察载脂蛋白嵌合模拟肽Ac-hE-18A-NH2对RAW264.7巨噬细胞胆固醇流出的影响,并探讨其机制。方法 RAW264.7巨噬细胞种植于24孔板,用0.5μCi/孔3H-胆固醇和含50mg·mL-1氧化低密度脂蛋白共同孵育24h之后,给予不同浓度的Ac-hE-18A-NH2(0～100mg·mL-1)干预24h,收集细胞用液体闪烁计数法检测胆固醇流出。采用ELISA测定细胞内cAMP含量,采用实时荧光定量PCR及Westernblot检测ABCA1、LXRα和PPARγ的mRNA及蛋白表达。结果 Ac-hE-18A-NH2以浓度依赖方式介导胆固醇流出;50mg·mL-1Ac-hE-18A-NH2干预不同时间,其介导的胆固醇流出率分别为(10.86±1.46)%(6h),(13.43±1.55)%(12h),(20.58±1.34)%(18h),和(26.93±4.37)%(24h)。同时,Ac-hE-18A-NH2还以浓度依赖方式增加细胞内cAMP水平,上调ABCA1、LXRα和PPARγmR-NA和蛋白表达。加用cAMP刺激剂8-Br-cAMP,Ac-hE-18A-NH2介导的胆固醇流出率由26.93±4.37增加至35.81±2.73,ABCA1mRNA表达增加了66.67%。而加用PPARγ特异性抑制剂预处理细胞后,PPARγ的表达几乎完全抑制,ABCA1和LXRα的表达也受到一定程度抑制,Ac-hE-18A-NH2介导的胆固醇流出率明显减少。结论模拟肽Ac-hE-18A-NH2可以明显促进巨噬细胞胆固醇流出,其机制可能与cAMP-ABCA1和PPARγ-LXRα-ABCA1两种途径有关。     

格列本脲对巨噬细胞载脂蛋白E分泌的影响

目的： 通过研究格列本脲对巨噬细胞载脂蛋白E(apo E)分泌的影响，评价格列本脲在2型糖尿病治疗中的应用价值。方法：培养THP 1单核细胞并诱导其分化成为THP 1巨噬细胞；通过负载胆固醇制备巨噬细胞源性泡沫细胞。将格列本脲与THP 1巨噬细胞和泡沫细胞一起孵育， 孵育结束时分别通过酶联免疫测定和Northern blot测定格列本脲对细胞apo E的产生、分泌以及apo E基因表达的影响。结果：格列本脲明显减少THP 1巨噬细胞的apo E分泌(P＜0.01)，对巨噬细胞源性泡沫细胞亦表现出同样作用，同时不改变巨噬细胞和泡沫细胞内apo E含量和apo E mRNA表达水平。 结论：格列本脲影响巨噬细胞apo E的净分泌，这可能不利于2型糖尿病患者血管壁的脂质代谢，尤其不宜于治疗已经存在血脂异常的糖尿病患者。     

罗格列酮对人单核巨噬细胞THP-1中胆固醇代谢的影响机制研究

目的:研究罗格列酮(RG)对氧化低密度脂蛋白(oxLDL)诱导的人单核巨噬细胞THP-1源性泡沫细胞中胆固醇代谢的影响机制。方法:将THP-1巨噬细胞分为空白对照组、oxLDL(100 mg.L-1)组和oxLDL(100 mg.L-1)+RG(10μmol.L-1)组,后2组先加oxLDL培养48 h,最后1组再加RG培养48 h,采用高效液相色谱法检测每组细胞内游离胆固醇(FC)和胆固醇酯(CE)的含量,逆转录-聚合酶链式反应(RT-PCR)、蛋白质印迹法分别检测每组细胞中三磷酸腺苷结合盒转运蛋白(ABC)A1、ABCG1 mRNA及蛋白的表达情况。结果:与空白对照组比较,oxLDL组FC、CE含量明显升高,ABCA1、ABCG1 mRNA及蛋白表达均明显降低(P<0.01);与oxLDL组比较,oxLDL+RG组FC、CE含量明显降低,ABCA1、ABCG1 mRNA及蛋白表达均明显升高(P<0.01)。结论:RG可能通过上调THP-1巨噬细胞ABCA1、ABCG1的表达,减少FC在细胞内的蓄积,促进细胞内胆固醇代谢,进而抑制动脉粥样硬化的形成。     

PPARγ激动剂对RAW264.7巨噬细胞源性泡沫细胞胆固醇流出的影响

目的观察PPARγ激动剂吡格列酮对RAW264.7巨噬细胞源性泡沫细胞胆固醇流出调节蛋白三磷酸腺苷结合核转运蛋白G1(ABCG1)、肝X受体α(LXRα)表达的影响。方法 (1)体外培养RAW 264.7巨噬细胞,用50 mg/L的氧化型低密度脂蛋白胆固醇(ox-LDL)孵育48 h诱导成泡沫细胞,油红O染色并在光镜下鉴定泡沫细胞形态及变化。(2)以不同浓度吡格列酮(0、5、10、20、30μmol/L)作用泡沫细胞24 h后,酶法检测泡沫细胞内胆固醇酯的含量。用反转录-聚合酶链反应(RT-PCR)及免疫印迹法测定ABCG1、LXRα的mRNA及蛋白的表达。结果 PPARγ激动剂吡格列酮可显著减少泡沫细胞内胆固醇酯含量,并呈浓度依赖性增加RAW264.7巨噬细胞源性泡沫细胞ABCG1、LXRα的mRNA和蛋白的表达。结论 PPARγ激动剂吡格列酮减少泡沫细胞内胆固醇酯的含量可能是通过上ABCG1、LXRα的mRNA及蛋白表达来实现的。     

NG701通过减少CD36 mRNA表达抑制泡沫细胞形成

目的:观察NG701对泡沫细胞形成及CD36mRNA表达的影响。方法:利用大鼠腹腔提取的巨噬细胞与氧化低密度脂蛋白共培养得到巨噬细胞源性泡沫细胞,加入不同浓度的NG701,观察泡沫细胞内胆固醇含量的变化,并利用RT-PCR法对CD36mRNA表达情况进行检测。结果:NG701能够显著减少泡沫细胞内总胆固醇和胆固醇酯的含量,降低CE/TC的值。并剂量依赖性的降低CD36mRNA表达。结论:NG701能通过减少CD36表达抑制巨噬细胞源性泡沫细胞的形成。     

Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1 macrophages

INTRODUCTIONOxidized lipid signaling in macrophages is centralto the pathogenesis of atherosclerosis[1]. Exposure ofmacrophages and other vascular cells to oxidized low-density lipoprotein (ox-LDL) leads to complex changesin gene expression that are collectively thought to influ-ence the development of the atherosclerotic lesion[2].Using two-dimensional gel electrophoresis, the overallprotein map in U937 control cells and U937 foam cellswas obtained. Compared with U937 cells, 37 spots…     

Ibrolipim increases ABCA1/G1 expression by the LXRα signaling pathway in THP-1 macrophage-derived foam cells

Aim:

To determine the effects and potential mechanisms of ibrolipim on ATP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1) expression from human macrophage foam cells, which may play a critical role in atherogenesis.

Methods:

Human THP-1 cells pre-incubated with ox-LDL served as foam cell models. Specific mRNA was quantified using real-time RT-PCR and protein expression using Western blotting. Cellular cholesterol handling was studied using cholesterol efflux experiments and high performance liquid chromatography assays.

Results:

Ibrolipim 5 and 50 μmol/L significantly increased cholesterol efflux from THP-1 macrophage-derived foam cells to apoA-I or HDL. Moreover, it upregulated the expression of ABCA1 and ABCG1. In addition, LXRα was also upregulated by the ibrolipim treatment. In addition, LXRα small interfering RNA completely abolished the promotion effect that was induced by ibrolipim.

Conclusion:

Ibrolipim increased ABCA1 and ABCG1 expression and promoted cholesterol efflux, which was mediated by the LXRα signaling pathway.     

Angiotensin‐(1–7) upregulates expression of adenosine triphosphate‐binding cassette transporter A1 and adenosine triphosphate‐binding cassette transporter G1 through the Mas receptor through the liver X receptor alpha signalling pathway in THP‐1 macrophages treated with angiotensin‐II

Adenosine triphosphate‐binding cassette transporter A1 (ABCA1) and ABCG1 play crucial roles in reverse cholesterol transport, and have anti‐atherosclerosis effects, and liver X receptor alpha (LXRα) can stimulate cholesterol efflux through these transporters. Angiotensin (Ang)‐(1–7) can protect endothelial cells, inhibit smooth muscle cell growth, ameliorate inflammation and exert anti‐atherosclerotic effects. In the present study, we attempted to clarify the effect of Ang‐(1–7) on expression of ABCA1 and ABCG1, and explored the role of LXRα in the regulation of ABCA1 and ABCG1 in THP‐1 macrophages that had been incubated with angiotensin‐II (AngII). Ang‐(1–7) increased ABCA1 and ABCG1 expression in a concentration‐dependent manner at both the mRNA and protein levels, promoted cholesterol efflux, and decreased cholesterol content in THP‐1 macrophages treated with AngII. Furthermore, Ang‐(1–7) upregulated the expression of LXRα in a concentration‐dependent manner in these cells. LXRα small interfering RNA, as well as the Mas receptor antagonist A‐779, completely abolished these effects of Ang‐(1–7). In summary, Ang‐(1–7) upregulates ABCA1 and ABCG1 expression in THP‐1 macrophages treated with AngII through the Mas receptor, via the LXRα pathway. This novel insight into the molecular mechanism underlying Ang‐(1–7) and AngII interaction could prove useful for developing new strategies for treatment of cardiovascular diseases.     

ATP-binding cassette transporters A1 and G1, HDL metabolism, cholesterol efflux, and inflammation: important targets for the treatment of atherosclerosis

Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries. Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. One central antiatherogenic role of HDL is believed to be its ability to remove excessive peripheral cholesterol back to the liver for subsequent catabolism and excretion, a physiologic process termed reverse cholesterol transport (RCT). Cholesterol efflux from macrophage foam cells, the initial step of RCT is the most relevant step with respect to atherosclerosis. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play crucial roles in the efflux of cellular cholesterol to HDL and its apolipoproteins. Moreover, ABCA1 and ABCG1 affect cellular inflammatory cytokine secretion by modulating cholesterol content in the plasma membrane and within intracellular compartments. In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excessive cholesterol in macrophages, and physiological manipulation of ABCA1 expression affects atherogenesis. Here we review recent advances in the role of ABCA1 and ABCG1 in HDL metabolism, macrophage cholesterol efflux, inflammation, and atherogenesis. Next, we summarize the structure, expression, and regulation of ABCA1 and ABCG1. Finally, we give an update on the progress and pitfalls of therapeutic approaches that target ABCA1 and ABCG1 to stimulate the flux of lipids through the RCT pathway.     

Atorvastatin inhibits ABCA1 expression and cholesterol efflux in THP-1 macrophages by an LXR-dependent pathway

The effect of atorvastatin on adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux remains controversial. In an effort to clarify this issue, ABCA1 expression and apolipoprotein AI (apoAI)-mediated cholesterol efflux after atorvastatin treatment were investigated in THP-1 macrophages. Atorvastatin from 2 microM to 40 microM dose-dependently inhibited ABCA1 expression in human monocyte-derived macrophages and phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 monocytes. ApoAI-mediated cholesterol efflux was reduced in PMA-stimulated THP-1 cells treated with atorvastatin, this effect was abolished with acetylated low-density lipoprotein (LDL) pretreatment. Atorvastatin treatment also dose-dependently reduced liver X receptor alpha (LXRalpha) expression and Rho activation. Rho activation by farnysylpyophosphate (FPP) and lysophosphatidic acid (LPA) did not salvage, but further depressed, the cholesterol efflux and ABCA1 expression in the presence of atorvastatin. Without atorvastatin, Rho activation by mevalonate, FPP, and LPA diminished apoAI-mediated cholesterol efflux, and Rho activation by GTPgammaS also decreased ABCA1 messenger ribonucleic acid (mRNA) by 16%. Furthermore, Rho inhibition by C3 exoenzyme increased ABCA1 mRNA by 48% despite a 17% decrease in apoAI-mediated cholesterol efflux. LXRalpha agonists (T01901317 and 22(R)-hydroxycholesterol) prevented any reductions in cholesterol efflux or ABCA1 expression associated with atorvastatin treatment. Furthermore, Western blot analysis demonstrated the reciprocal inhibition of Rho and LXRalpha. In conclusion, atorvastatin decreases ABCA1 expression in noncholesterol-loaded macrophages in an LXRalpha- but not Rho-dependent pathway; this effect can be compromised after acetylated LDL cholesterol loading.     

Tributyltin chloride induces ABCA1 expression and apolipoprotein A-I-mediated cellular cholesterol efflux by activating LXRalpha/RXR

Organotins, including tri-butyltin chloride (TBTC), are widely used in agricultural and chemical industries and cause persistent and widespread pollution. TBTC has been shown to activate nuclear receptor retinoid X receptor (RXR)/PPARγ signaling by interacting with RXR to modulate adipogenesis. However, whether TBTC affects liver X receptor (LXR)/RXR activity and subsequently the expression of cholesterol mobilizing genes is not known. In this study, we evaluated the ability of TBTC to activate LXR/RXR and ABC transporter A1 (ABCA1) expression. ABCA1 plays a critical role in HDL generation, maintaining cholesterol homeostasis, and cholesterol accumulation-induced diseases, such as atherosclerosis and pancreatic islet dysfunction. In a reporter gene assay, TBTC activated LXRα/RXR but not LXRβ/RXR. In mouse macrophage RAW264 cells, TBTC activated the ABCA1 promoter in an LXR-responsive element dependent manner and increased ABCA1 mRNA expression. TBTC augmented ABCA1 protein levels and apolipoprotein A-I-dependent cellular cholesterol efflux (HDL generation). The LXR-target fatty acid synthase and Spα mRNA levels were also increased by TBTC exposure. We conclude that TBTC has the ability to activate permissive LXRα/RXR signaling and thereby modulate cellular cholesterol efflux.