β-榄香烯对RAW264.7巨噬细胞源性泡沫细胞作用的研究

目的:研究β-榄香烯抗巨噬细胞源性泡沫细胞的形成及抑制巨噬细胞炎症因子分泌的作用。为探讨β-榄香烯抗动脉粥样硬化(AS)的作用提供依据。方法:采用氧化低密度脂蛋白(ox-LDL)诱导小鼠单核/巨噬细胞(RAW264.7)建立巨噬细胞源性泡沫细胞模型,采用油红O染色鉴定泡沫细胞形成。给予不同浓度(0.5,5,50μM)β-榄香烯干预后,ELISA方法检测巨噬细胞源性泡沫细胞内胆固醇含量和肿瘤坏死因子-α(TNF-α),白介素-6(IL-6)分泌量的变化。结果:β-榄香烯可降低巨噬细胞源性泡沫细胞内总胆固醇(P0.05或P0.01),胆固醇酯含量(P0.01),减少炎症因子TNF-α,IL-6的分泌(P0.05或P0.01),并且呈现出一定的浓度依赖性。结论:β-榄香烯抑制巨噬细胞对ox-LDL的摄取,降低细胞内胆固醇的含量,抑制泡沫细胞的形成,同时改善巨噬细胞的炎症状态从而发挥抗动脉粥样硬化的作用。     

beta- 榄香烯对RAW264.7 巨噬细胞源性泡沫细胞作用的研究

目的:研究β-榄香烯抗巨噬细胞源性泡沫细胞的形成及抑制巨噬细胞炎症因子分泌的作用。为探讨β-榄香烯抗动脉粥样硬化(AS)的作用提供依据。方法:采用氧化低密度脂蛋白(ox-LDL)诱导小鼠单核/巨噬细胞(RAW264.7)建立巨噬细胞源性泡沫细胞模型,采用油红O染色鉴定泡沫细胞形成。给予不同浓度(0.5,5,50μM)β-榄香烯干预后,ELISA方法检测巨噬细胞源性泡沫细胞内胆固醇含量和肿瘤坏死因子-α(TNF-α),白介素-6(IL-6)分泌量的变化。结果:β-榄香烯可降低巨噬细胞源性泡沫细胞内总胆固醇(P<0.05或P<0.01),胆固醇酯含量(P<0.01),减少炎症因子TNF-α,IL-6的分泌(P<0.05或P<0.01),并且呈现出一定的浓度依赖性。结论:β-榄香烯抑制巨噬细胞对ox-LDL的摄取,降低细胞内胆固醇的含量,抑制泡沫细胞的形成,同时改善巨噬细胞的炎症状态从而发挥抗动脉粥样硬化的作用。     

肝X受体α在泡沫细胞胆固醇流出中的调控作用

以THP-1巨噬细胞源性泡沫细胞为研究对象,观察肝X受体α(LXRα)在THP-1巨噬细胞源性泡沫细胞胆固醇流出中的调控作用.结果发现,22(R)-羟基胆固醇剂量依赖性增加THP-1巨噬细胞源性泡沫细胞胆固醇流出, 而DIDS剂量依赖性减少THP-1巨噬细胞源性泡沫细胞胆固醇流出.逆转录聚合酶链反应显示, 22(R)-羟基胆固醇可增加THP-1巨噬细胞源性泡沫细胞LXRα mRNA的表达, DIDS可抑制THP-1巨噬细胞源性泡沫细胞LXRα mRNA的表达.结果提示,LXRα在巨噬细胞源性泡沫细胞胆固醇流出中起着重要的调控作用,这为开发和寻找抗动脉粥样硬化药物提供了新的思路.     

胰岛素在巨噬细胞泡沫化过程中对Toll样受体4表达的影响及其机制

目的:观察胰岛素对巨噬细胞破泡沫化过程中Toll样受体4(TLR4)表达及IL-6、TNF-α分泌的影响.方法:采用体外培养小鼠巨噬细胞系RAW264.7,氧化低密度脂蛋白(ox-LDL)诱导建立泡沫细胞模型,分为对照组、ox-LDL组、用胰岛素组、PI3K-AKT抑制剂组.油红O染色观察泡沫细胞模型的建立,取细胞上清用ELISA法检测白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的水平;流式细胞术检测膜蛋白TLR4表达量,Western-blot检测TLR4、核因子-κB (NF-κB)的表达水平.结果:与对照组相比,ox-LDL处理过的巨噬细胞可向泡沫细胞转换,同时TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著增加(P＜0.05);而使用胰岛素干预后ox-LDL的作用显著减弱,TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著降低(P ＜0.05 vs ox-LDLgroup);而使用PI3K-AKT抑制剂干预后,抑制剂显著降低胰岛素的作用,TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著升高(P ＜0.05 vs ox-LDL+ insulin).结论:ox-LDL可诱导巨噬细胞向泡沫细胞转化,同时上调TLR4及NF-κB蛋白表达,增加炎性因子分泌,促进了AS进程,而胰岛素可使ox-LDL的作用显减弱,减少TLR4、NF-κB蛋白表达及炎性因子分泌,从而减轻AS进程,其机制可能与胰岛素通过PI3K-AKT抑制TLR4-NF-κB通路有关.     

氧化低密度脂蛋白诱导巨噬细胞内质网应激及CD36的可能作用

本文旨在研究在鼠源巨噬细胞泡沫化过程中氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)对巨噬细胞内质网应激(endoplasmic reticulum stress,ERS)的诱导作用及其机制。体外培养RAW264.7巨噬细胞,分别给予ox-LDL(25、50和100mg/L)、抗CD36抗体+ox-LDL和衣霉素(tunicamycin,TM)等不同处理。采用油红O染色观察细胞内脂质蓄积情况,酶比色法测定细胞内总胆固醇含量,免疫细胞化学法检测ERS标志分子糖调节蛋白94(glucose-regulated protein94,GRP94)表达,免疫印迹法检测GRP94及未折叠蛋白反应关键分子p-IRE1(phosphorylated inositol-requiring enzyme1)和X盒结合蛋白1(X box binding protein1,XBP1)蛋白表达水平。结果显示,不同浓度(25、50和100mg/L)ox-LDL处理细胞24h后,胞浆内可见大量油红O染色阳性脂质颗粒,细胞内总胆固醇含量明显增加,分别为空白对照组的2.1倍、2.8倍和3.1倍;使用抗CD36抗体阻断ox-LDL的摄入,可显著减少100mg/Lox-LDL所致的细胞内胆固醇蓄积。不同浓度ox-LDL和ERS诱导剂TM均可显著增加GRP94及其上游信号分子p-IRE1和XBP1蛋白表达,且表达强度随着ox-LDL诱导浓度的增加而增强;抗CD36抗体显著抑制100mg/Lox-LDL所致的上述3种蛋白表达上调。上述结果提示,ox-LDL可呈剂量依赖性诱导RAW264.7巨噬细胞产生ERS,激活未折叠蛋白反应信号通路;该过程可能由清道夫受体CD36所介导。     

肺炎衣原体抑制LXRα-ABCA1途径促进巨噬细胞脂质蓄积

为探讨肝X受体α(LXRα)-三磷酸腺苷结合盒转运体A1(ABCA1)途径在肺炎衣原体(C.pneumoniae)促巨噬细胞脂质蓄积中的作用和机制,以THP-1巨噬细胞源性泡沫细胞为模型,采用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,液体闪烁计数器检测细胞内胆固醇流出,RT-PCR检测ABCA1和LXRαm RNA的表达,蛋白质印迹检测ABCA1和LXRα的蛋白质表达;使用LXRα的特异性激动剂T0901317对细胞进行预处理,再观察上述指标的变化.结果显示,C.pneumoniae可促进THP-1巨噬细胞源性泡沫细胞内总胆固醇、游离胆固醇和胆固醇酯含量增加,抑制胆固醇外流,降低细胞ABCA1和LXRα的表达;使用ABCA1激动剂8-溴-环磷酸腺苷预处理细胞或LXR激动剂T0901317预处理细胞后,可明显减弱C.pneumoniae对THP-1细胞ABCA1的表达抑制,促进细胞胆固醇流出,降低细胞内胆固醇的含量.结果提示,C.pneumoniae促进巨噬细胞脂质蓄积及胆固醇流出障碍,其机制可能与LXRα-ABCA1途径有关.     

肾康灵调控系膜细胞炎性反应因子的机制研究

目的：观察氧化低密度脂蛋白（Oxidized Low-Density Lipoprotein,ox-LDL）对系膜细胞（Mesangial Cells,MCs）分泌炎性反应递质功能的影响,并从细胞分子生物学水平阐明肾康灵的作用机制。方法：采用肾康灵干预增殖的系膜细胞,并利用分子生物学技术检测CXCL16、CD36、IFN-γ、IL6、TNF-α含量或基因水平。结果：利用ox-LDL诱导大鼠系膜细胞增殖并加入CXCR6受体后,CXCL16、CD36、IFN-γ、IL6、TNF-α的含量或基因水平显著升高,肾康灵呈浓度依赖性降低其表达水平。结论：ox-LDL诱导系膜细胞增殖时通过CXCR6介导,可促进CXCL16、CD36、IFN-γ、IL6、TNF-α等炎性反应递质的释放,中药肾康灵可通过抑制炎性反应递质的释放,保护系膜细胞的功能。     

激活多巴胺Ⅰ类受体对氧化性低密度脂蛋白诱导的人单核细胞THP-1分泌NO/NOS的影响

目的:探讨激活多巴胺Ⅰ类受体(DR1)对氧化型低密度脂蛋白(ox-LDL)诱导的人单核细胞(THP-1)分泌一氧化氮/一氧化氮合酶(NO/NOS)的影响及可能机制。方法:THP-1细胞经佛波酯PMA诱导分化,分为正常对照组(control),氧化型低密度脂蛋白处理组(ox-LDL),DR1激动剂干预组(SKF),DR1阻断剂干预组(SCH),ERK阻断剂干预组(PD98059);应用油红O染色法鉴定泡沫细胞;硝酸还原法检测NO、NOS的变化情况;免疫荧光和Western blot检测各组细胞蛋白表达情况。结果:ox-LDL刺激48 h可形成泡沫细胞;DR1在THP1细胞上表达,oxLDL刺激后,DR1蛋白表达降低(P0.01);激活DR1受体能够明显抑制由ox-LDL引起的NO、i NOS增多(P0.01);在MAPK阻断剂PD98059存在的情况下,SKF的作用部分丧失。结论:激活DR1受体可抑制ox-LDL引起的THP-1细胞NO的大量产生,此过程可能由ERK信号通路所介导。     

动脉粥样硬化早期病变过程CD36的表达与oxLDL的摄取作用

清道夫受体CD36最初被认为是一种血小板膜糖蛋白和一种血栓反应蛋白受体(TSP-1)。近来,CD36也被认为是单核细胞产生的活性氮物质修饰的低密度脂蛋白(LDL)的主要受体,参与包括动脉粥样硬化(AS)在内的许多病理生理过程。该文介绍CD36的生物学特性及其在巨噬细胞源性泡沫细胞形成和AS形成中的作用,以及CD36表达的调控机制。     

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux

The NOD-like receptor family, pyrin domain–containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively.     

Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL

Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.     

Pentraxin-3 regulates the inflammatory activity of macrophages

Background and aimsPentraxin-3 (PTX3) reportedly has protective roles in atherosclerosis and myocardial infarction, and is a useful biomarker of vascular inflammation. However, the detailed functions of PTX3 in inflammation are yet to be elucidated. This study aimed to investigate the function of PTX3 in macrophages.MethodsPMA-treated THP-1 cell line (THP-1 macrophage) and monocyte-derived human primary macrophages were treated with recombinant PTX3. Cytokine and chemokine levels in the THP-1 culture medium were measured as well as monocyte chemoattractant protein (MCP-1) concentrations in the Raw 264.7 cell culture medium. PTX3-silenced apoptotic macrophages (THP-1 cell line) were generated to investigate the roles of PTX3 in phagocytosis.ResultsIn the presence of PTX3, macrophage interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and MCP-1 levels were reduced significantly (?39%, P=0.007; ?21%, P=0.008; and ?67%, P=0.0003, respectively), whilst activated transforming growth factor-β (TGF?β) was detected in the THP-1 macrophages (P=0.0004). Additionally, PTX3 induced Akt phosphorylation and reduced nuclear factor-kappa B (NF-κB) activation by 35% (P=0.002), which was induced by TNF-α in THP-1 macrophages. Furthermore, silencing of PTX3 in apoptotic cells resulted in increased macrophage binding, elevated expression rate of HLA-DR (+30%, P=0.015) and CD86 (+204%, P=0.004) positive cells, and induction of IL-1β (+36%, P=0.024) production. Conversely, adding recombinant PTX3 to macrophages reduced CD86 and HLA-DR expression in a dose-dependent manner.ConclusionsWe identified PTX3 as a novel regulator of macrophage activity, and this function suggests that PTX3 acts to resolve inflammation.     

Macrophage iron retention aggravates atherosclerosis: Evidence for the role of autocrine formation of hepcidin in plaque macrophages

Iron accumulation has been frequently found in atherosclerotic lesions, especially in macrophages/foam cells, but the exact mechanisms by which hepcidin induces iron retention in plaque macrophages and its roles in atherogenesis remain unknown. Double immunofluorescence staining showed colocalization of hepcidin-positive macrophages with ox-LDL, TLR4, p-p65 and ferritin light chain (ferritin-L) both in human and murine atherosclerotic lesions. RAW264.7 macrophages incubated with ox-LDL showed elevated expression of TLR4, p-p65, hepcidin, ferritin-L/H, CYP27A1, CD36, PPARγ, liver X receptor α (LXRα), and ATP binding cassette transporter A1/G1 (ABCA1/G1), as well as increased intracellular labile iron pool level and lipid accumulation. Ox-LDL-induced iron retention and lipid accumulation were aggravated by lipopolysaccharide but blocked by TAK-242, an antagonist of TLR4. Moreover, macrophage TLR4/NF-κB pathway activation and foaming triggered by ox-LDL was enhanced by ferric ammonium citrate or exogenous hepcidin but attenuated by hepcidin silencing or the use of iron chelator. Meanwhile, the addition of hepcidin stimulated CD36-mediated Dil-labeled-ox-LDL uptake and inhibited the LXRα-ABCA1/G1 pathway-dependent cholesterol efflux in macrophages, which was significantly reversed by 27-hydroxycholesterol but further exacerbated by cyclosporin A, a selective inhibitor of CYP27A1. Our study provided the evidence that iron trapped in atherosclerosis plaque macrophages contributes to cholesterol disequilibrium-initiated foam cell formation, which is provoked by the unique but largely unknown autocrine formation of hepcidin in plaque macrophages via activating the TLR4/NF-κB pathway when exposed to ox-LDL. Such findings, considering the intricate vicious cycle between macrophage hepcidin autocrine-triggered iron retention and cholesterol disequilibrium, may shed new light on the “iron hypothesis” of atherosclerosis.     

Blockade of NEAT1 represses inflammation response and lipid uptake via modulating miR-342-3p in human macrophages THP-1 cells

Atherosclerosis has been recognized as a chronic inflammation process induced by lipid of the vessel wall. Oxidized low-density lipoprotein (ox-LDL) can drive atherosclerosis progression involving macrophages. Recently, long noncoding RNAs (lncRNAs) have been reported to play critical roles in atherosclerosis development. In our current study, we focused on the biological roles of lncRNA NEAT1 in atherosclerosis progress. Here, we found that ox-LDL was able to trigger human macrophages THP-1 cells, a human monocytic cell line, apoptosis in a dose-dependent and time-dependent course. In addition, we observed that NEAT1 was significantly increased in THP-1 cells incubated with ox-LDL and meanwhile miR-342-3p was greatly decreased. Then, NEAT1 was silenced by transfection of small interfering RNA (siRNA) of NEAT1 into THP-1 cells. As exhibited, CD36, oil-red staining levels, total cholesterol (TC), total cholesterol (TG) levels and THP-1 cell apoptosis were obviously repressed by knockdown of NEAT1. Furthermore, inhibition of NEAT1 contributed to the repression of inflammation in vitro. Interleukin 6 (IL-6), IL-1β, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-α) protein levels were remarkably depressed by NEAT1 siRNA in THP-1 cells. By using bioinformatics analysis, miR-342-3p was predicted as a downstream target of NEAT1 and the correlation between them was confirmed in our study. Moreover, overexpression of miR-342-3p could also greatly suppress inflammation response and lipid uptake in THP-1 cells. Knockdown of NEAT1 and miR-342-3p mimics inhibited lipid uptake in THP-1 cells. In conclusion, we implied that blockade of NEAT1 repressed inflammation response through modulating miR-342-3p in human macrophages THP-1 cells and NEAT1 may offer a promising strategy to treat atherosclerotic cardiovascular diseases.     

Interleukin-10 inhibits the down-regulation of ATP binding cassette transporter A1 by tumour necrosis factor-alpha in THP-1 macrophage-derived foam cells

It is suggested that cholesterol efflux mediated by ATP binding cassette transporter A1 (ABCA1) plays an important role in anti-atherogenesis. However, the effects of inflammatory cytokines on ABCA1 expression and cholesterol accumulation in foam cells are little known. This study investigates the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells. ABCA1mRNA and protein levels were determined by RT-PCR and Western blot, respectively. The total cholesterol content in THP-1 macrophage-derived foam cells was detected by the zymochemistry method. Results revealed that TNF-alpha could increase cholesterol content by down-regulating ABCA1 expression in a time-dependent manner in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. In addition IL-10 time-dependently decreased cholesterol accumulation by up-regulating ABCA1 expression and inhibited the down-regulation of ABCA1 by TNF-alpha in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action.     

Phytosterols differentially influence ABC transporter expression, cholesterol efflux and inflammatory cytokine secretion in macrophage foam cells

Phytosterol supplements lower low-density lipoprotein (LDL) cholesterol, but accumulate in vascular lesions of patients and limit the anti-atherosclerotic effects of LDL lowering in apolipoprotein E (Apo E)-deficient mice, suggesting that the cholesterol-lowering benefit of phytosterol supplementation may not be fully realized. Individual phytosterols have cell-type specific effects that may be either beneficial or deleterious with respect to atherosclerosis, but little is known concerning their effects on macrophage function. The effects of phytosterols on ABCA1 and ABCG1 abundance, cholesterol efflux and inflammatory cytokine secretion were determined in cultured macrophage foam cells. Among the commonly consumed phytosterols, stigmasterol increased expression of ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein (Apo) AI and high-density lipoprotein (HDL). Campesterol and sitosterol had no effect on ABCA1 or ABCG1 levels. Sitosterol had no effect on cholesterol efflux to Apo AI or HDL, whereas campesterol had a modest but significant reduction in cholesterol efflux to HDL in THP-1 macrophages. Whereas stigmasterol blunted aggregated LDL (agLDL) induced increases in tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β secretion, sitosterol exacerbated these effects. The presence of campesterol had no effect on agLDL-induced inflammatory cytokine secretion from THP-1 macrophages. In conclusion, the presence of stigmasterol in modified lipoproteins promoted cholesterol efflux and suppressed inflammatory cytokine secretion in response to lipid loading in macrophage foam cells. While campesterol was largely inert, the presence of sitosterol increased the proinflammatory cytokine secretion.