Involvement of arachidonic acid in superoxide anion generation in human lens epithelial cells
- VernacularTitle:花生四烯酸参与人晶状体上皮细胞中超氧阴离子的生成
- Author:
Bing DONG
;
Ying AN
;
Wei ZHANG
;
Lou MARJORIE
;
- Publication Type:Journal Article
- Keywords:
arachidonic acid;
superoxide anion;
lens
- From:
Ophthalmology in China
2006;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
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-depen- dent 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 in- duced 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-11Z,14Z,17Z-trienoic acid (20:3) and eicosa-11Z,14Z-dienoic acid (20:2) were ineffective.The generation of superoxide anion was not contributed by cyclooxyge- nase or the cytochrome p450 pathway since indomethacin (inhibitor for cyclooxygenase) or ketoconazole (inhibitor for cytochrome 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-lipoxygenase 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 ac- tivated 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.
