摘要
目的探讨咖啡酸是否通过抑制氧化应激诱导的5-脂氧酶（5-LOX）激活而减轻细胞损伤。方法稳定转染绿荧光蛋白（GFP）-5-LOX的PC12细胞,预先给予咖啡酸0.001~10μmol.L-1和对照药MK886,30min后观察缺氧缺糖/恢复（OGD/R）及过氧化氢（H2O2）160μmol.L-1处理后的变化。MTT法和碘化丙啶染色法分析细胞存活率和死亡率;荧光显微镜观察OGD 2 h/R2 h和H2O2处理40 min时5-LOX的核膜移位;ELISA法测定OGD 2 h/R 3 h时5-LOX代谢产物的生成;DCF法检测OGD 2 h/R 0.5 h细胞内活性氧（ROS）的产生。结果 OGD 2 h/R 24 h GFP-5-LOX转染和GFP-转染PC12细胞的存活率分别为（63.1±6.6）%和（70.7±6.9）%;H2O2处理24 h细胞存活率分别为（62.5±7.7）%和（75.7±9.5）%。在GFP-5-LOX转染的PC12细胞中,咖啡酸和对照药MK886可使OGD/R细胞存活率从（63.1±6.6）%分别增加到（87.3±2.0）%和（89.9±6.3）%,细胞坏死率从（31.4±1.5）%降低到（10.1±2.0）%和（11.7±1.3）%（P〈0.01）;使H2O2处理细胞存活率从（62.51±7.65）%增加到（92.59±4.02）%和（75.31±6.60）%;使OGD/R细胞CysLTs的生成从261.1±33.7降低到108.5±16.7和（90.6±19.5）ng.g-1蛋白（P〈0.01）。此外,咖啡酸抑制OGD/R诱导PC12细胞的ROS产生,IC50值为8.021μmol.L-1;抑制OGD/R诱导的GFP-5-LOX转染的PC12细胞5-LOX核膜移位,IC50值为0.974μmol.L-1;抑制H2O2诱导的GFP-5-LOX转染的PC12细胞5-LOX核膜移位,IC50值为0.501μmol.L-1;MK886无上述作用。结论咖啡酸可抑制氧化应激诱导的PC12细胞5-LOX激活,对缺血损伤的PC12细胞具有保护作用。
OBJECTIVE To determine whether caffeic acid attenuates cell injury by inhibiting oxidative stress-induced 5-lipoxygenase（5-LOX） activation.METHODS In PC12 cells steadily expressed by green fluorescence protein（GFP）-5-LOX,caffeic acid or the 5-LOX activating protein inhibitor MK886 0.001-10 μmol·L-1 were added to cells for 30 min.The changes were observed after exposure to 2-h oxygen-glucose deprivation/2-h recovery（OGD 2 h/R 2 h） and hydrogen peroxide（H2O2） 160 μmol·L-1.Cell viability and cell death were assessed by MTT reduction assay and propidium iodide staining after treatment for 24 h.5-LOX translocation to nuclear envelope was determined by fluorescence microscopy after OGD 2 h/R 2 h or 40-min exposure to（H2O2）.5-LOX metabolites were evaluated by ELISA after OGD 2 h/R 3 h recovery,and production of reactive oxygen spices（ROS） was measured by DCF assay after 2-h OGD and 0.5-h recovery.RESULTS Cell viabilities were（63.1±6.6）% and（70.7±6.9）% after OGD 2 h/R 24 h;（62.5±7.7）% and（75.7±9.5）% after H2O2 treatment 24 h in GFP-5-LOX transfected and GFP transfected PC12 cells,respectively.In GFP-5-LOX transfected PC12 cells,caffeic acid and MK886 attenuated reduction of cell viability from（63.1±6.6）% to（87.3±2.0） % and（89.9±6.3）%,and reduced rate of necrotic cells from（31.4±1.5） % to（10.1±2.0） % and（11.7±1.3） % after oxygen-glucose deprivation（OGD） treatment.Caffeic acid and MK886 attenuated reduction in cell viability induced by H2O2 from（62.51±7.65）% to（92.59±4.02）% and（75.31±6.60）%.Both agents also reduced production of 5-LOX metabolites from 261.1±33.7 to 108.5±16.7 and（90.6±19.5）ng·g-1 protein after OGD/R in GFP-5-LOX transfected PC12 cells.Meanwhile,caffeic acid inhibited production of ROS after OGD/R in wild-type PC12 cells;IC50 was 8.021 μmol·L-1.Caffeic acid also inhibited 5-LOX translocation to the nuclear envelope induced by OGD/R and H2O2 in GFP-5-LOX transfected PC12 cells,respectively;IC50 was 0.97
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2011年第1期45-51,共7页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金资助项目(30772561)
浙江省自然科学基金资助项目(Y204210)
杭州市科技计划项目(20080333B21)~~
