氧化型低密度脂蛋白诱导巨噬细胞凋亡

氧化型低密度脂蛋白诱导巨噬细胞凋亡牛喜林，张修武，郭兆贵（湖南医科大学分子药理研究室，长沙４１００７８，中国）关键词ＤＮＡ片断；细胞凋亡；低密度脂蛋白；腹腔巨噬细胞目的：研究氧化型低密度脂蛋白（ｏｘＬＤＬ）诱导巨噬细胞凋亡．方法：超速离心法分离人血...     

Flavonoids inhibit myelin phagocytosis by macrophages; a structure-activity relationship study

Demyelination is a characteristic hallmark of the neuro-inflammatory disease multiple sclerosis. During demyelination, macrophages phagocytose myelin and secrete inflammatory mediators that worsen the disease. Here, we investigated whether flavonoids, naturally occurring immunomodulating compounds, are able to influence myelin phagocytosis by macrophages in vitro. The flavonoids luteolin, quercetin and fisetin most significantly decreased the amount of myelin phagocytosed by a macrophage cell line without affecting its viability. IC(50) values for these compounds ranged from 20 to 80 microM. The flavonoid structure appeared to be essential for observed effects as flavonoids containing hydroxyl groups at the B-3 and B-4 positions in combination with a C-2,3 double bond were most effective. The capacity of the various flavonoids to inhibit phagocytosis correlated well with their potency as antioxidant, which is in line with the requirement of reactive oxygen species for the phagocytosis of myelin by macrophages. Our results implicate that flavonoids may be able to limit the demyelination process during multiple sclerosis.     

Mild autoxidation of low density lipoproteins

The autoxidation of low density lipoproteins was investigated in a closed system, with limited amount of oxygen. The concentrations of several components were monitored: oxygen, aldehydic oxidation products and a-tocopherol. The concentration of lipid radicals generated in the processes of lipid peroxidation was monitored by the EPR spin trapping method. It was observed that the consumption of oxygen starts quickly after the onset of incubation at physiological temperature. After prolonged incubation, several types of trapped radicals were formed. At the same time, no consumption of a-tocopherol and no formation of aldehydic products of oxidation took place, indicating that the oxidation process is rather mild. The dynamics of oxidation processes were simulated by a mathematical model in which the oxidation is initiated by the degradation products of the pre-existing lipid peroxides. The best agreement between the theoretical predictions and experimental results was obtained with the rate constants which are several orders of magnitude smaller than the corresponding rate constants in ‘neat’ lipids. The possible reasons for the behaviour observed are discussed.     

Mulberry leaf extract inhibits the oxidative modification of rabbit and human low density lipoprotein

In a previous study, we demonstrated that the intake of mulberry leaves or their 1-butanol extract (MLBE) reduced the concentration of serum lipids and atheromatous thickening of arterial intima in hypercholesterolemic rabbits. In the present study, we investigated the antioxidative activity of MLBE and isoquercitrin, the main component of MLBE. First, we determined the effect on a stable radical agent, finding that quercetin, isoquercitrin and MLBE scavenged the DPPH radical. We then determined the copper-induced oxidative modification of rabbit and human low-density lipoprotein (LDL). Oxidation of LDL was spectrophotometrically monitored by changes in absorbance at 234 nm accompanied by the formation of conjugated dienes, and measured the formation of thiobarbituric acid reacting substances (TBARS). Quercetin, an aglycone of isoquercitrin, inhibited the formation of conjugated dienes and TBARS by copper-induced oxidative modification of rabbit and human LDLs. MLBE and isoquercitrin also inhibited the oxidation of LDL. These results indicate that mulberry leaves inhibit the oxidative modification of LDL and suggest that mulberry leaves may had prevent atherosclerosis.     

绿茶多酚和桑色素对低密度脂蛋白氧化修饰的抑制作用(英文)

许多研究报告表明,低密度脂蛋白(LDL)可经多种途径被氧化修饰成ο-LDL,巨噬细胞对ο-LDL的降解速率较LDL快3～10倍,动脉粥样硬化损伤部位的泡沫细胞是由巨噬细胞聚集了大量的胆固醇而形成的,LDL的氧化修饰以及由此导致的巨噬细胞吞噬的增加,对泡沫细胞的形成起到重要作用,本项研究采用紫外线照射和Cu~(2+)-PBS系统分别对LDL进行氧化修饰并且观察了绿茶多酚(10μg·ml~(-1))和黄酮类化合物桑色素(8.5μg·ml~(-1))对这一化学过程的抑制作用,其结果表明,两者均对LDL的氧化修饰具有显著抑制作用,主要实验证据为,它们可使琼脂糖电泳迁移率,硫代巴比妥酸反应物和激发光为360 nm,发射光为420 nm时的荧光强度等的增加量抑制50％左右并使小鼠腹腔巨噬细胞降解o-LDL的速率抑制70％,绿茶多酚和桑色素分别存在于绿茶和蔬菜中,因此它们防止LDL的氧化修饰对于动脉粥样硬化的预防和治疗具有一定的临床意义。     

新型α-氨基腈及α-氨基膦酸酯类衍生物对人低密度脂蛋白氧化修饰的影响

生物体通常暴露在活性氧等引起的氧化应激中 ,同时又具备与之相对应的防御系统而处于氧化 抗氧化的平衡状态。一旦平衡打破 ,即内源性抗氧化剂耗竭或抗氧化酶发生改变 ,过多的活性氧可影响细胞的信号传递系统 ,氧化损伤生物膜脂质、蛋白质和ＤＮＡ ,进而影响基因表达与细胞的生长和死亡 ,而启动如动脉粥样硬化等一系列病变。已证明自由基可引起或加剧的疾病尚有脑卒中、心脏病、早老性痴呆以及老化等 ,而且此类疾病的名单还在不断扩大。抗氧化剂作为一种小分子化合物通过直接或间接捕捉、灭活自由基 ,对抗其对生物大分子的损伤所导致的一系…     

Flavonoids protect U-937 macrophages against tert-butylhydroperoxide induced oxidative injury.

The study was carried out to determine the relative efficacies of polyphenolic flavonoids, quercetin, catechin and epicatechin against tert-BOOH induced oxidative stress in human macrophage, U-937 cell line. Exposure of the cells to tert-BOOH oxidative stress resulted in a significant increase in cytotoxicity and reactive oxygen species (ROS) generation. Further, a significant decrease in mitochondrial membrane potential and increase in lipid peroxidation and DNA damage was observed in cells exposed to tert-BOOH. Pretreatment of cells with quercetin, catechin and epicatechin significantly inhibited tert-BOOH induced cytotoxicity by inhibiting ROS generation. The flavonoids inhibited DNA damage induced by tert-BOOH and preserved the mitochondrial transmembrane potential significantly. Epicatechin and catechin were found to be more efficient than quercetin in inhibiting tert-BOOH induced cellular damage.     

氨氯地平对糖基化低密度脂蛋白诱导的血管内皮功能障碍的修复作用

目的研究氨氯地平(Am)对糖基化低密度脂蛋白(AGE-LDL)诱导的内皮功能障碍的修复作用。方法以AGE-LDL于体外模拟糖尿病合并脂代谢异常环境,将人脐静脉内皮细胞(HEC,来自EA.hy926细胞系)分别在AGE-LDL及AGE-LDL+Am环境中孵育,并以天然LDL(nLDL)孵育组为对照,应用实时PCR检测细胞中单核细胞趋化蛋白-1(MCP-1)、血管细胞粘附分子-1(VCAM-1)的基因表达水平变化,应用WesternBlot检测培养液中MCP-1蛋白量的变化。结果与nLDL组细胞相比,AGE-LDL组细胞中MCP-1、VCAM-1基因表达水平明显升高,培养液中MCP-1蛋白含量亦明显增多;在AGE-LDL中加入Am处理后,与AGE-LDL组细胞相比,细胞中MCP-1、VCAM-1基因表达水平明显下降,培养液中MCP-1蛋白含量亦明显减少。结论氨氯地平在糖尿病合并动脉粥样硬化所致内皮细胞功能障碍方面具有修复及保护作用。     

Role of oxidatively modified low density lipoproteins and anti-oxidants in atherothrombosis

Retrospective studies have demonstrated an association between coronary artery disease (CAD) and increased plasma levels of oxidised low density lipoproteins (LDL). A very recent prospective study in heart transplant patients has demonstrated that oxidised LDL is an independent risk factor for transplant CAD, thus further supporting the hypothesis that oxidised LDL is actively involved in the development of CAD. The increase of circulating oxidised LDL is most probably caused by back-diffusion from the atherosclerotic arterial wall in the blood, independent of plaque rupture. Indeed, plasma levels of oxidised LDL were very similar in patients with stable CAD and in patients with acute coronary syndromes. These were, however, associated with increased release of malondialdehyde (MDA)-modified LDL. Oxidised LDL may be generated by radical-mediated or by lipoxygenase or phospholipase catalysed lipid oxidation, and by myeloperoxidase catalysed protein and lipid oxidation. Prostaglandin synthesis by endothelial cells under oxidative stress and platelet activation are associated with the release of aldehydes; these induce the oxidative modification of the apolipoprotein B-100 moiety of LDL in the absence of lipid peroxidation, and thus generate MDA-modified LDL. Efficient prevention of in vivo oxidation may involve efficient cholesterol lowering, improving the anti-oxidative status of LDL by increasing the anti-oxidant content and increasing the oleate content of LDL, and by shifting the LDL away from phenotype B (characterised by small dense LDL particles). Anti-oxidative and anti-inflammatory enzymes associated with HDL may inhibit the oxidation of LDL or reverse the atherothrombotic effects of LDL.     

Role of oxidatively modified low density lipoproteins and anti-oxidants in atherothrombosis

Retrospective studies have demonstrated an association between coronary artery disease (CAD) and increased plasma levels of oxidised low density lipoproteins (LDL). A very recent prospective study in heart transplant patients has demonstrated that oxidised LDL is an independent risk factor for transplant CAD, thus further supporting the hypothesis that oxidised LDL is actively involved in the development of CAD. The increase of circulating oxidised LDL is most probably caused by back-diffusion from the atherosclerotic arterial wall in the blood, independent of plaque rupture. Indeed, plasma levels of oxidised LDL were very similar in patients with stable CAD and in patients with acute coronary syndromes. These were, however, associated with increased release of malondialdehyde (MDA)-modified LDL. Oxidised LDL may be generated by radical-mediated or by lipoxygenase or phospholipase catalysed lipid oxidation, and by myeloperoxidase catalysed protein and lipid oxidation. Prostaglandin synthesis by endothelial cells under oxidative stress and platelet activation are associated with the release of aldehydes; these induce the oxidative modification of the apolipoprotein B-100 moiety of LDL in the absence of lipid peroxidation, and thus generate MDA-modified LDL. Efficient prevention of in vivo oxidation may involve efficient cholesterol lowering, improving the anti-oxidative status of LDL by increasing the anti-oxidant content and increasing the oleate content of LDL, and by shifting the LDL away from phenotype B (characterised by small dense LDL particles). Anti-oxidative and anti-inflammatory enzymes associated with HDL may inhibit the oxidation of LDL or reverse the atherothrombotic effects of LDL.     

Effects of low density lipoprotein and oxidized low density lipoprotein on the cytotoxic activity of Asp-hemolysin to murine macrophages

We examined the effects of human low density lipoprotein (LDL) and oxidized LDL (Ox-LDL) on the cytotoxic activity of Asp-hemolysin from Aspergillus fumigatus Fresenius-Muramatsu strain to mouse peritoneal macrophages (M phi). The inhibitory effects of LDL and Ox-LDL on the cytotoxic activity of Asp-hemolysin to M phi increased in a dose-dependent manner, and the effect of Ox-LDL was greater than the inhibitory effect of LDL. Furthermore, the binding of Asp-hemolysin to LDL or Ox-LDL was observed by western blot analysis of the culture medium. These results suggest that the inhibition by LDL or Ox-LDL on the cytotoxic activity of Asp-hemolysin to M phi was due to the binding of LDL or Ox-LDL to Asp-hemolysin in the culture medium.     

Activation of membrane outward currents by human low density lipoprotein in mouse peritoneal macrophages

Summary The aim of the present study was to search for electrophysiological effects of human lipoproteins on membrane currents in mouse peritoneal macrophages which had been cultured for 5 to 20 days. Whole-cell currents were recorded by using a voltage-clamp technique.Low density lipoprotein (LDL, 100 g/ml) increased a slowly activating nonspecific cation current (i_so) in the positive potential range to 244 ± 23% of the reference (test potential + 55 mV, n = 13, P < 0.005). Augmentation of current resulted out of a negative shift of the activation curve along the voltage axis (–22 mV) and an increase of maximally available current.Furthermore, LDL increased a rapidly activating outward current (i_fo) at test potentials positive to the potassium equilibrium potential. At +55 mV i_fo-amplitude increasedto 165 ± 14% ofreference (n = 16, P < 0.005). LDL-induced effects on i_fo-current could be mimicked by application of the calcium ionophore A 23187 (1 mol/l) which led to an increase of i_fo-current to 161 ± 25% of the reference (test potential + 55 mV, n = 11, P < 0.005).Acetylated-LDL (100 g/ml, 5–15 min) produced no significant effect on the membrane currents under investigation. Correspondence to U. Borchard at the above address     

Flavonoids and a sesquiterpene lactone from Tanacetum microphyllum inhibit anti-inflammatory mediators in LPS-stimulated mouse peritoneal macrophages

In our ongoing research into anti-inflammatory compounds from Tanacetum microphyllum, four naturally occurring flavonoids (santin, ermanin, centaureidin and 5,3'-dihydroxy-4'-methoxy-7-methoxycarbonylflavonol) and one sesquiterpene lactone (hydroxyachillin) isolated from this plant, were evaluated as potential inhibitors of some macrophage functions involved in the inflammatory process. These five compounds significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) in a concentration-dependent manner. In the tumour necrosis factor-alpha (TNF-alpha) assay, only centaureidin and hydroxyachillin significantly inhibited the accumulation of TNF-alpha. These results indicate that these compounds may contribute to the anti-inflammatory properties of T. microphyllum.     

Oxidized low-density lipoproteins induce apoptosis in aortic and endocardial endothelial cells

目的：研究氧化型低密度脂蛋白（ｏｘＬＤＬ）诱导血管和心内膜内皮细胞凋亡．方法：用超速离心法分离健康人血浆低密度脂蛋白（ＬＤＬ），以ＣｕＳＯ４１０μｍｏｌ·Ｌ－１氧化．观察ｏｘＬＤＬ对培养新生小牛主动脉内皮细胞及心内膜细胞的损伤作用．琼脂糖凝胶电泳和Ｈｏｅｃｈｓｔ３３２５８荧光密度法定性与定量分析ＤＮＡ降解．结果：ｏｘＬＤＬ诱导血管内皮细胞及心内膜细胞典型凋亡形态学改变，ＤＮＡ降解呈时间和剂量依赖性．环己米特和硫酸葡聚糖对此作用无影响．ＢＨＴ２０μｍｏｌ·Ｌ－１可取消ＤＮＡ降解．溶血性磷脂酰胆碱５０μｍｏｌ·Ｌ－１无诱导凋亡作用．ｏｘＬＤＬ诱导的ＤＮＡ降解可被依他酸取消．结论：ｏｘＬＤＬ诱导血管内皮细胞及心内膜细胞凋亡．     

Effects of policosanol treatment on the susceptibility of low density lipoprotein (LDL) isolated from healthy volunteers to oxidative modification in vitro

AIMS: The aim of this study was to investigate the effect of policosanol on the susceptibility of LDL-C to in vitro lipid peroxidation in human healthy volunteers. METHODS: The effect of policosanol (5 and 10 mg day(-1) on LDL-C oxidation was studied in a double-blind, randomized, placebo-controlled trial conducted in 69 subjects. LDL-C samples isolated at baseline and after 8 weeks were subjected to in vitro tests of LDL-C oxidation. We tested the susceptibility of LDL-C to lipid peroxidation in a cell-free system by the addition of copper ions as well as in a more physiological system, macrophage-mediated oxidation. RESULTS: At baseline all groups were well matched regarding all variables. After 8 weeks of therapy policosanol administered at 5 and 10 mg, significantly and in a dose-dependent manner increased the lag phase of conjugated diene generation (mean +/- s.d.) from 83.79+/-29.16 min to 94.90+/-25.50 min (5 mg day(-1)) and from 82.74+/-17.16 min to 129.89+/-35.71 min (10 mg day(-1)), while in the placebo group LDL-C oxidation did not change significantly. Policosanol (10 mg day(-1)), but not placebo, significantly decreased the rate of conjugated diene generation. Comparison with placebo after therapy also showed significant differences. Macrophage mediated-oxidation was also inhibited by policosanol as evident by measuring thiobarbituric acid reactive substances (TBARS). Policosanol (10 mg day(-1)) significantly lowered malondialdehyde (MDA) generation from 8.50+/-0.91 to 5.76+/- 1.01 nmol mg(-1) protein. Comparison with placebo after 5 and 10 mg day(-1) showed significant differences. Policosanol significantly lowered total cholesterol by 10.5% (5 mg day(-1)) and 12.4% (10 mg day(-1)) and LDL-C by 16.7% and 20.2%, respectively. Also, policosanol (10 mg day(-1)) increased HDL-C by 15.2%. Five subjects withdrew from the study, none because of adverse experiences. No clinical or blood biochemical drug-related disturbances were found. CONCLUSIONS: The present study demonstrated that policosanol administered within its therapeutic dosage for lowering cholesterol (5 and 10 mg day(-1)), decreased the susceptibility of LDL-C to lipid peroxidation in vitro.     

低密度脂蛋白对人血小板磷脂酰肌醇代谢产物及钙摄取的影响

观察了低密度脂蛋白(LDL)或高密度脂蛋白(HDL)加LDL对人血小板磷脂酰肌醇循环中的重要物质磷脂酰肌醇4,5-二磷酸(PIP_2),磷脂酸(PA),以及~(45)Ca~(2+)摄取的影响,同时还观察了LDL,HDL对猪肺微粒体酶催化前列腺素内过氧化物生成血栓素B_2(TXB_2)的影响.结果表明,LDL引起血小板PIP_2短暂下降,继之增加,同时伴有PA的增加,未观察到HDL拮抗LDL的作用,此外,LDL能刺激Ca~(2+)内流入血小板.HDL明显抑制TXB_2生成。     

Correlation between oxidation of low density lipoproteins and prostacyclin synthesis in cultured smooth muscle cells

The effect of antioxidants on the oxidation of low density lipoproteins in relation to prostacyclin synthesis was investigated in the prescence of rabbit smooth muscle cells (SMC) and Fe-containing culture medium. The lipid peroxidation of low density lipoproteins (LDL) assayed as thiobarbituric acid reactive substances was increased from 0.5 to 1.4 nmol malondialdehyde/mL by the presence of smooth muscle cells. Two potent antioxidants, nordihydroguairetic acid (NDGA) and butylated hydroxytoluene (BHT), inhibited lipoprotein oxidation by IC50 values of 0.2 and 0.8 microM, respectively. Inhibition of lipoprotein oxidation was associated with an increased prostacyclin synthesis by the SMC, the effect being more pronounced with nordihydroguairetic acid than with butylated hydroxytoluene. The stable metabolite of the lipid hydroperoxide, 15-hydroxyeicosatetraenoic acid (15-HETE), formed in the 15-lipoxygenase pathway was measured following antioxidant treatment and found to be eliminated or greatly reduced by both antioxidants. The results presented show that lipid hydroperoxides, formed as a consequence of lipoprotein oxidation and promoted by the smooth muscle cells through a lipoxygenase reaction, may regulate prostacyclin synthase, a process which may be influenced by the addition of antioxidants.