异丹叶大黄素在体外对Cu2+介导的人低密度脂蛋白过氧化的抑制作用

异丹叶大黄素在体外对Cu2+介导的人低密度脂蛋白过氧化的抑制作用

方亚南, 林茂, 刘耕陶^*

中国医学科学院, 中国协和医科大学药物研究所, 北京 100050

收稿日期:2003-11-21 修回日期:2004-02-10 出版日期:2004-03-15 发布日期:2004-03-15
通讯作者: 刘耕陶*

Inhibitory Effect of Isorhapontigenin on Copper-Mediated Peroxidation of Human Low-Density Lipoprotein in vitro

FANG Ya-nan, LIN Mao, LIU Geng-tao^*

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

Received:2003-11-21 Revised:2004-02-10 Online:2004-03-15 Published:2004-03-15
Contact: LIU Geng-tao*

摘要/Abstract

摘要： 目的研究异丹叶大黄素(Iso)对Cu²⁺介导的人低密度脂蛋白(LDL)过氧化及氧化LDL(ox-LDL)对小鼠巨噬细胞毒性的抑制作用. 方法用序贯超离心法分离血脂正常的供血者血清LDL, 分离的LDL 1 mg·mL^-1磷酸缓冲液(pH 7.4), 与10 μmol·L^-1CuSO4于37℃水浴温育10 h 以引起LDL过氧化, 给药组预先加入不同浓度Iso, 对照组加等量生理盐水,测定丙二醛(MDA)的生成量、维生素E的耗竭以及LDL电泳迁移率. 另外测定用ox-LDL处理小鼠腹腔巨噬细胞线粒体的膜电位、吞噬刚果红及释放NO的量. 结果 1-100 μmol·L^-1Iso 剂量依赖性地抑制Cu²⁺引起的人LDL氧化时MDA的生成量、维生素E的耗竭及电泳迁移率的升高.10 μmol·L^-1Iso能防止0.1 mg·mL^-1ox-LDL与小鼠腹腔巨噬细胞温育4 h 后线粒体膜电位的损伤、吞噬刚果红功能以及NO释放量的降低. 结论 Iso在体外对Cu²⁺介导的LDL过氧化以及ox-LDL对小鼠巨噬细胞的毒性有保护作用.

关键词: 异丹叶大黄素, 低密度脂蛋白, 氧化低密度脂蛋白, 巨噬细胞, 细胞吞噬, 线粒体膜电位

Abstract: Aim To study the effect of Isorhapontigenin (Iso) on copper-mediated peroxidation of human low-density lipoprotein (LDL) and on the toxicity of oxidized LDL (ox-LDL) to mouse macrophages in vitro. Methods Human LDL from sera of normal lipidemic donors was separated by sequential ultracentrifugation.The separated human LDL 1 mg·mL^-1 in phosphate buffer saline, pH 7.4, was incubated with cupric sulfate (10 μmol·L^-1) at 37 °C for 10 h in the presence or absence of various concentrations of Iso.Malondialdehyde (MDA) formation, vitamin E consumption, electrophoretic mobility of LDL, mitochondrial membrane potential of mouse peritoneal macrophages, phagocytosis of neutral red, and release of nitric oxide (NO) from macrophages were determined by various methods. Results Iso 1-100 μmol·L^-1 significantly inhibited the increase of MDA formation, vitamin E consumption and electrophoretic mobility of LDL induced by Cu²⁺ in a concentration-dependent manner.The injury of the mitochondrial membrane potential of mouse peritoneal macrophages due to incubation with ox-LDL (0.1 mg·mL^-1) at 37 °C for 12 h was markedly protected by 10 μmol·L^-1 Iso. After pretreatment of the macrophages with 10 μmol·L^-1 of Iso and then exposure to ox-LDL for 4 h, the reduction of phagocytosis of neutral red and release of NO in response to lipopolysaccharide (LPS) stimulation were significantly prevented. Conclusion Iso has protective action against Cu²⁺ mediated LDL peroxidation and ox-LDL induced toxicity to macrophages in vitro.

Key words: isorhapontigenin, isorhapontigenin, low-density lipoprotein, low-density lipoprotein, oxidized low-density lipoprotein, oxidized low-density lipoprotein, macrophages, macrophages, phagocytosis, phagocytosis, mitochondrial membrane potential, mitochondrial membrane potential

中图分类号:

R963

R972.6

Supporting: ^*Corresponding author. Tel.: 010-63165178; fax: 010-63017757

方亚南, 林茂, 刘耕陶^*. 异丹叶大黄素在体外对Cu2+介导的人低密度脂蛋白过氧化的抑制作用[J]. .

FANG Ya-nan, LIN Mao, LIU Geng-tao^*. Inhibitory Effect of Isorhapontigenin on Copper-Mediated Peroxidation of Human Low-Density Lipoprotein in vitro[J]. .

参考文献

[1] Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol: modulations of low-density lipoprotein that increases atherogenicity [J]. New Engl J Med, 1989, 320: 915-924.
[2] Esterbauer H, Dieber-Rotheneder M, Waeg G, et al. Biochemical structure and functional properties of oxidized low density lipoprotein [J]. Chem Res Toxicol, 1990, 3: 77-92.
[3] Kuzuya M, Naito M, Funaki C, et al. Lipid peroxide and transition metals are required for the toxicity of oxidized low density lipoprotein to cultured endothelial cells [J]. Biochem Biophys Acta, 1991, 1096: 155-160.
[4] Heinecke J. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis [J]. Atherosclerosis, 1998, 141: 1-15.
[5] Tribble DL. AHA Science advisor. Antioxidant consumption and risk of coronary heart disease: emphasis on vitamin C, vitamin E, andβ-carotene. A statement for healthcare professionals from the American Heart Association [J]. Circulation, 1999, 99 (2): 591-595.
[6] Fang YN, Liu GT. Effect of isorhapontigenin on respiratory burst of rat neutrophils [J]. Phytomedicine, 2002, 9: 734-738.
[7] Zhang LH, Liu BW. Separation of human serum lipoproteins by one-step ultracentrifugation [J]. Acta Biochim Biophys Sin, 1989, 21: 257-260.
[8] Lowry OH, Rosebrough NJ, Farr A, et al. Protein measurement with the Folin phenol reagent [J]. J Biol Chem, 1951, 193: 265-275.
[9] Liu SX, Zhou M, Chen Y. Comparative study of oxidative modification and malondialdehyde modification of low density lipoprotein [J]. Acta Biochim Biophys Sin, 1992, 24: 569-574.
[10] Wang KC, Chou FL, Chen HK. Agarose-gel electrophoresis and its application to the separation and classification of human serum lipoprotein [J]. Acta Biochim Biophys Sin, 1979, 11: 37-49.
[11] Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma [J]. Biochem Med, 1976, 15: 212-216.
[12] Taylor SL, Lamden MP, Tappel AL. Sensitive fluorometric method for tissue tocopherol analysis [J]. Lipids, 1976, 11: 530-538.
[13] Liu SX, Chen Y, Zhou M. Protective effect of PSKon the injury of mitochondrial membrane of mouse peritoneal macrophage induced by oxidative low density lipoprotein [J]. Acad J First Med Coll PLA, 1995, 15: 117-120.
[14] Wang GY, Lin ZB. Effects of 10-hydroxy-2-decenoic acid on phagocytosis and cytokines production of peritoneal macrophages in vitro [J]. Acta Pharm Sin, 1997, 18 (2): 180-182.
[15] Le Y, Zhou M, Chen Y. Effects of minimally modified and oxidized low density lipoprotein on lipopolysaccharide-induced tumor necrosis factor and nitric oxide production by peritoneal macrophages of mice [J]. Chin J Arterioscler,1994, 2 (2): 72-74.
[16] Chen Y, Zhou M. The oxidation of low density lipoprotein and the structure and composition of oxidized low density lipoprotein [J]. Acta Biophys Sin, 1993, 9: 334-339.
[17] Burghardt RC, Barhoumi R, Lewis EH, et al. Patulin-induced cellular toxicity: a vital fluorescence study [J]. Toxicol Appl Pharmacol, 1992, 112: 235-244.
[18] Stuehr DJ, Nathan CF. Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells [J]. J Exp Med, 1989, 169: 1543-1555.
[19] Chester AH, O’Neil GS, Moneada S, et al. Low basal and stimulated release of nitric oxide in atherosclerotic epicardial coronary arteries [J]. Lancet, 1990, 336: 897-900.