花生四烯酸参与人晶状体上皮细胞中超氧阴离子的生成

Involvement of arachidonic acid in superoxide anion generation in human lens epithelial cells

目的以花生四烯酸为刺激因子,研究人晶状体上皮细胞中是否存在超氧阴离子生成体系。设计实验研究。研究对象人晶状体上皮细胞B3(HLE B3)。方法用花生四烯酸及其衍生物刺激HLE B3细胞,超氧阴离子用化学发光法定量检测。用超氧阴离子歧化酶和山梨醇与细胞预培养作为阴性对照;以3%乙醇(花生四烯酸及其衍生物的溶剂)刺激细胞作基线;测定超氧阴离子前,所有抑制剂先与细胞预培养30分钟。花生四烯酸对丝裂原激活蛋白激酶(MAPK)磷酸化影响的时间依赖实验和剂量依赖实验用蛋白质印迹杂交进行检测。主要指标超氧阴离子的含量,MAPK磷酸化的变化。结果30～150mM花生四烯酸能刺激HLE B3细胞迅速产生超氧阴离子,超氧阴离子歧化酶(SOD)和山梨醇抑制了该反应,尼克酰胺二磷酸腺苷(NADPH)氧化酶抑制剂DPI也抑制了超氧阴离子的产生。花生四烯酸的衍生物二十碳二烯酸、二十碳三烯酸以及硬脂酸、亚油酸不能引起超氧阴离子的产生。环氧合酶、细胞色素p450的抑制剂对超氧阴离子的产生无影响,提示花生四烯酸这两条代谢途径不参与超氧阴离子的生成。脂氧合酶,尤其是5-脂氧合酶的抑制剂完全阻断了该反应。30～150mM花生四烯酸能在2.5～30分钟瞬间激活ERK和JNK,并有剂量和时间依赖性。结论抑制超氧离子的生成可能是抑制品状体上皮细胞增殖、预防后发发障的新途径。

Objective This study is to identify the presence of superoxide anion-generating system in human lens epithelial cells using arachidonic acid （AA） as the stimulator. Design Experimental study. Participants Human lens epithelial cells B3 （HLE B3）. Methods Confluent human lens epithelial cells （HLE B3） were subjected to stimulation by AA and its derivatives. The generation of su- peroxide anion was quantified with a luminometer （LumiStar BMG） immediately upon AA and its derivatives addition. Cells preloaded with superoxide dismutase （SOD） or mannitol were used as negative controls and cells mixed with 3% ethanol （solvent for AA） were used as baseline. Cells were preloaded with inhibitors 30 minutes before luminometer measurement. A time- and concentration-dependent study on the AA-stimulated activation of mitogen-activated protein kinase （MAPK） was carried out using western blot analysis. Main Outcome Measures Superoxide generation, phosphorylation of MAPK. Results AA at dosage of 30-150 mM proportionally induced luminescence in HLE B3 cells, but was ineffective in cells preloaded with SOD or mannitol. DPI, a non-specific NADPH oxidase inhibitor eliminated AA-induced superoxide anion generation partially. Leinoleic acid, stearic acid, eicosa-llZ,14Z,17Z-trienoie acid （20：3） and eicosa-11Z,14Z-dienoic acid （20：2） were ineffective. The generation of superoxide anion was not contributed by cyclooxygenase or the cytochrome p450 pathway since indomethacin （inhibitor for cyclooxygenase） or ketoeonazole （inhibitor for eytochrome p450） could not eradicate the stimulatory effect of AA. While CDC, a specific inhibitor for lipoxygenase could eliminate superoxide generation partially. The specific inhibitor for 5-1ipoxygenase AA861 completely blocked the generation of superoxide anion. Western blot analysis of the cell lysate showed that AA at the concentrations of 30-150 mM progressively activated ERK and JNK. They were transiently activated between 2.5-30 minutes. The activations of ERK and JNK were dose-dependent and time-dependent. Conclusions Inhibition of superioxide anion generation may be a new approach to block lens epithelial cell proliferation and post-capsule opacification.