Protein expressions in macrophage-derived foam cells：comparative analysis by two-dimensional gel electrophoresis

目的:研究氧化低密度脂蛋白诱导的U937泡沫细胞中蛋白质组的差异表达。方法:泡沫细胞模型由人类单核细胞系白血病U937细胞经氧化低密度脂蛋白诱导而成,将对照U937细胞与U937泡沫细胞的蛋白提取物分别进行双向凝胶电泳(2-DE)分离,凝胶经银染后,于PDquest 2-DE图像分析软件中进行图像处理,利用ExPASy蛋白质组学网站中U937细胞的蛋白表达谱信息,对所得的U937泡沫细胞2-DE图谱作进一步鉴定。结果:通过对2-DE蛋白图谱的分析,共150点与参考胶相匹配(匹配率:75％),与U937细胞图谱相比,泡沫细胞中有37个蛋白点表达量有显著性变化(P<0.05),与对照U937细胞相比,在U937泡沫细胞中28个蛋白点表达量上调,9个点表达量下调,其中,8个U937细胞中表达的蛋白点,在U937泡沫细胞中未检测到;而11个在泡沫细胞中表达的蛋白点,未在对照U937细胞中表达。结论:本研究首次建立经氧化低密度脂蛋白诱导的U937泡沫细胞的蛋白差异表达谱,从而提供了对动脉粥样硬化病理过程中巨噬源性泡沫细胞功能性分析研究的新思路。     

Relationship between G-proteins associated transmembrane cascades and MAPK phosphorylation induced by recombinant interleukin-α in fibroblast-like synoviocytes from rats with adjuvant arthritis

AIM: To elucidate the relationship between G protein-associ-ated transmembrane cascade and mitogen-activated protein kinases (MAPKs) phosphorylation in recombinant rat IL-1α(rIL-1α)-induced fibroblast-like synoviocytes (FLS) from rats with adjuvant arthritis (AA). METHODS: The expressions of MAPKs phosphorylation, the stimulatory subunit of G alpha     

Curcumin inhibits oxLDL-induced CD36 expression and foam cell formation through the inhibition of p38 MAPK phosphorylation

The uptake of oxidized low density lipoprotein (oxLDL) via scavenger receptors transforms macrophages into foam cells, which are a hallmark of atherosclerosis. OxLDL markedly increases the expression of the CD36 scavenger receptor. Here, we investigated whether curcumin modulate CD36 expression in oxLDL-treated RAW 264.7 murine macrophages. Our results showed that curcumin dramatically inhibits CD36 expression and foam cell formation. Furthermore, oxLDL-induced expression and activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ), which is involved in CD36 expression, is also blocked in curcumin-treated cells. OxLDL activates the mitogen-activated protein kinase (MAPK) signaling transduction pathway, and p38 MAPK is associated with oxLDL-induced CD36 and PPAR-γ expression. Overexpression of dominant negative p38 MAPK blocks oxLDL-induced CD36 and PPAR-γ expression. Furthermore, curcumin markedly inhibits p38 MAPK phosphorylation. Taken together, our results suggest that curcumin modulates oxLDL-induced CD36 expression and foam cell formation via the inhibition of p38 MAPK phosphorylation in RAW 264.7 murine macrophages.     

Anti-atherosclerotic potential of gossypetin via inhibiting LDL oxidation and foam cell formation

Gossypetin, a flavone originally isolated from Hibiscus species, has been shown to possess antioxidant, antimicrobial, and antimutagenic activities. Here, we investigated the mechanism(s) underlying the anti-atherosclerotic potential of gossypetin. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay showed that the addition of > 50 μM of gossypetin could scavenge over 50% of DPPH radicals. The inhibitory effects of gossypetin on the lipid and protein oxidation of LDL were defined by thiobarbituric acid reactive substance (TBARS) assay, the relative electrophoretic mobility (REM) of oxidized LDL (ox-LDL), and fragmentation of apoB in the Cu^2 +-induced oxidation of LDL. Gossypetin showed potential in reducing ox-LDL-induced foam cell formation and intracellular lipid accumulation, and uptake ability of macrophages under non-cytotoxic concentrations. Molecular data showed that these influences of gossypetin might be mediated via peroxisome proliferator-activated receptor α (PPARα)/liver-X receptor α (LXRα)/ATP-binding cassette transporter A1 (ABCA1) and PPARγ/scavenger receptor CD36 pathways, as demonstrated by the transfection of PPARα siRNA or PPARγ expression vector. Our data implied that gossypetin regulated the PPAR signals, which in turn led to stimulation of cholesterol removal from macrophages and delay atherosclerosis. These results suggested that gossypetin potentially could be developed as an anti-atherosclerotic agent.     

CDK5介导的PPARγ磷酸化在动脉粥样硬化泡沫细胞形成过程中的作用

摘要：目的 探讨细胞周期素依赖蛋白激酶5（CDK5）介导的过氧化物酶体增殖物激活受体γ（PPARγ）磷酸化在 动脉粥样硬化中的作用。方法 常规培养小鼠Raw264.7巨噬细胞,实验设对照组（C组）、氧化低密度脂蛋白（oxLDL）组（O组,50 mg/L ox-LDL）、Roscovitine+ox-LDL组（R组,15 μmol/L Roscovitine+50 mg/L ox-LDL）。待细胞融合 至70%左右,R组加入15 μmol/L Roscovitine预处理30 min,之后O组和R组分别加入50 mg/L ox-LDL继续培养24 h, 使其转化为泡沫细胞;C组不作处理。利用Western blot检测各组pPPARγ、tPPARγ、p35和CDK5蛋白表达的变化,油 红O染色和异丙醇萃取实验分析各组细胞内脂质聚积情况,酶法测定细胞内胆固醇含量,反转录PCR（RT-PCR）检 测各组ox-LDL摄取相关基因CD36、SR-A1和胆固醇外流相关基因ABCA1、ABCG1的mRNA表达水平。结果 oxLDL诱导后,O组pPPARγ/tPPARγ比值、p35/CDK5比值、细胞内脂质聚积、总胆固醇含量、游离胆固醇含量、胆固醇 酯/总胆固醇比值均较 C 组明显升高（P＜0.05）;CDK5 抑制剂干预后,R 组上述指标均较 O 组降低（P＜0.05）。RTPCR结果显示,ox-LDL诱导后,O组ox-LDL摄取相关基因CD36、SR-A1的mRNA表达水平升高,而胆固醇外流相关 基因ABCA1、ABCG1的mRNA表达水平降低（P＜0.05）;CDK5抑制剂干预后,上述指标变化与O组呈相反趋势（P＜ 0.05）。结论 CDK5/pPPARγ途径参与动脉粥样硬化泡沫细胞的形成。     

Inhibition of macrophage‐derived foam cell formation by ezetimibe via the caveolin‐1/MAPK pathway

Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, effectively reduces plasma cholesterol, but its effect on atherosclerosis is unclear. Foam cell formation has been implicated as a key mediator during the development of atherosclerosis. The purpose of this study was to investigate the effects of ezetimibe on foam cell formation and explore the underlying mechanism. The results presented here show that ezetimibe reduces atherosclerotic lesions in apolipoprotein E deficient (apoE‐/‐) mice by lowering cholesterol levels. Treatment of macrophages with Chol:MβCD resulted in foam cell formation, which was concentration‐dependently inhibited by the presence of ezetimibe. Mechanically, ezetimibe treatment downregulated the expression of CD36 and scavenger receptor class B1 (SR‐B1), but upregulated the expression of apoE and caveolin‐1 in macrophage‐derived foam cells, which kept consistent with our microarray results. Moreover, treatment with ezetimibe abrogated the increase of phospho‐extracellular signal regulated kinase (ERK) 1/2 and their nuclear accumulation in foam cells. Inhibition of the MAPK pathway by the MEK inhibitor PD98059 attenuated the inhibitory effect of ezetimibe on the expression of p‐ERK1/2 and caveolin‐1. Taken together, these results showed that ezetimibe suppressed foam cell formation via the caveolin‐1/MAPK signalling pathway, suggesting that inhibition of foam cell formation might be a novel mechanism underlying the anti‐atherosclerotic effect of ezetimibe.     

巨噬源性泡沫细胞中p62蛋白上调作用和机制的研究

目的 探讨巨噬源性泡沫细胞中p62蛋白对脂质代谢相关的自噬以及炎症因子表达的影响,为p62在抗动脉粥样硬化中的应用提供参考。方法 利用Ox-LDL刺激RAW264.7细胞的方法模拟巨噬源性泡沫细胞的形成,通过Western blot及实时荧光定量PCR检测巨噬源性泡沫细胞中p62蛋白和mRNA水平。通过Western blot比较p62 siRNA组和对照组中Ox-LDL诱导的LC3剪切、脂滴相关蛋白Plin2和过氧化物酶体相关蛋白PEX2的蛋白水平,通过实时荧光定量PCR比较TNFα和IL-6 mRNA表达水平。结果 Ox-LDL对RAW264.7细胞中p62蛋白以及mRNA水平均有上调作用。干扰p62的表达之后,LC3-Ⅱ蛋白水平降低,Plin2的蛋白水平并无明显变化,PEX2的蛋白水平升高,TNFα和IL-6 mRNA表达升高。进一步研究发现,干扰Nrf2后能明显抑制Ox-LDL对p62的上调作用。结论 巨噬源性泡沫细胞中Ox-LDL通过Nrf2介导p62蛋白的上调,p62可能具有抗动脉粥样硬化的作用。     

Ketoconazole-induced JNK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

This study examined the effect of ketoconazole on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca²⁺ levels in MG63 osteosarcoma cells. Ketoconazole at 20–200 μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that ketoconazole induced phosphorylation of ERK and JNK, but not p38, MAPKs. Ketoconazole-induced cell death and apoptosis were partially reversed by the selective JNK inhibitor SP600125, but not by the selective ERK inhibitor PD98059, suggesting that ketoconazole’s cytotoxic action was via JNK, but not via ERK and p38 MAPKs. Ketoconazole at a concentration of 100 μM induced [Ca²⁺]_i increases. Chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) totally inhibited ketoconazole-induced [Ca²⁺]_i increases without reversing ketoconazole-induced cell death. Collectively, in MG63 cells, ketoconazole induced cell death and apoptosis via evoking JNK phosphorylation in a Ca²⁺-independent manner.     

Angiotensin II-induced MAPK phosphorylation mediated by Ras and/or phospholipase C-dependent phosphorylations but not by protein kinase C phosphorylation in cultured rat vascular smooth muscle cells

Angiotensin II (Ang II) induces a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMCs). In the present study, we examined the effects of the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor GF109203X, and the Ras inhibitor farnesylthiosalicylic acid (FTS) on Ang II-induced activation of p42/p44 MAPKs in cultured VSMCs. Phosphorylation was shown using the Western blot technique with specific phospho-antibodies against MAPK proteins. The PLC inhibitor U73122 abolished the Ang II-induced MAPK activity, while the PKC inhibitor GF109203X only decreased it. There was also an inhibition observed with the Ras inhibitor, FTS on Ang II-induced MAPK activity. These data suggest that Ang II-induced MAPK phosphorylation through the Ang II type 1 receptor could be mediated by Ras and/or PLC-dependent phosphorylations but not by PKC phosphorylation.     

Involvement of p38 MAPK phosphorylation and nitrate formation in aristolochic acid-mediated antiplatelet activity

Aristolochic acid (AsA) is produced from Aristolochia fangchi, and has been used as a Chinese herbal medicine. AsA possesses various biological activities including antiplatelet, antifungal, and anti-inflammatory properties. The aim of this study was to examine the mechanisms of AsA in inhibiting platelet aggregation. AsA (75 - 150 microM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen (1 microg/mL) than other agonists. AsA (115 and 150 microM) inhibited collagen-induced platelet activation accompanied by [Ca+2)]i mobilization, thromboxane A2 (TxA2) formation and phosphoinositide breakdown. On the other hand, AsA also markedly increased levels of NO/cyclic GMP, and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. AsA inhibited p38 MAPK but not ERK1/2 phosphorylation in washed platelets. In conclusion, the most important findings of this study suggest that the inhibitory effects of AsA possibly involve the (1) inhibition of the p38 MAPK-cytosolic phospholipase A2-arachidonic acid-TxA2-[Ca+2)]i cascade, and (2) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C. These results imply that Aristolochia fangchi treatment alone or in combination with other antiplatelet drugs, may result in alteration of hemostasis in vivo.     

Effect of IL-10 on formation of foam cell induced by ox-LDL

Atherosclerosis is a chronic disease that causes various cardiovascular complications. It has been realized that cellular and humoral immunity plays crucial roles in atherogenic lesion formation. In this study the effects of lipopolysaccharide (LPS) and interleukin-10 (IL-10) on the formation of foam cells during the early stages of atherosclerosis have been investigated. Macrophage was induced by phorbol myristate acetate (PMA) treatment on THP-1 cells. The cells were further stimulated by ox-LDL, ox-LDL plus LPS, ox-LDL plus IL-10 and LPS. By using an oil red O staining technique, the formation of foam cells was evaluated by lipid granules formation in the cells. The ratio of foam cell formation was increased from (9.77 ± 1.70)% to (16.27 ± 2.27)% after 24 h stimulation with ox-LDL, and the increase was observed with incubating time. The foam cells were significantly increased in the presence of LPS in a dose-dependent manner. The maximum increase of about 40% was observed. However, the significant elevation by LPS was abrogated when IL-10 was added. These results indicated that IL-10 can effectively prevent the formation of foam cells induced by ox-LDL with or without LPS. This study demonstrates that ox-LDL can cause foam cell formation from macrophages in vitro. LPS can significantly accelerate this event. IL-10, an anti-inflammatory cytokine, can inhibit the effect of ox-LDL and LPS. These results indicate that inflammatory effects in blood vessels can speed up foam cell formation. The inhibitive effect of IL-10 is an important factor for delaying atherosclerosis processes. __________ Translated from Journal of Tongji University (Medical Science), 2007, 28(1): 1–5 [译自: 同济大学学报(医学版)]