Patrignani P, Panara M R, Greco A, Fusco O, Natoli C, Iacobelli S, Cipollone F, Ganci A, Créminon C, Maclouf J
Division of Clinical Pharmacology, University of Chieti G. D'Annunzio, School of Medicine, Italy.
J Pharmacol Exp Ther. 1994 Dec;271(3):1705-12.
The aim of our study was to characterize a model of human prostaglandin endoperoxide synthase-2 (PGHS-2) expression allowing the assessment of pharmacological inhibition in vitro and ex vivo. Heparinized human whole blood samples were incubated with lipopolysaccharide (LPS, 0.1-50 micrograms/ml) for 0 to 24 hr at 37 degrees C. The contribution of platelet PGHS-1 was suppressed by either pretreating the subjects with aspirin (300 mg 48 hr before sampling) or adding aspirin (10 micrograms/ml) in vitro at time 0. PGE2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 micrograms/ml LPS, PGE2 production averaged 12.1 +/- 6.2 ng/ml (mean +/- S.D., n = 7). Cyclooxygenase activity increased in parallel with the mass of a monocyte protein doublet analyzed by Western blot using antibodies directed against the carboxyl-terminal portion of human PGHS-2. Dexamethasone (2 microM) inhibited LPS-induced PGE2 production by 96 +/- 4% (mean +/- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGH-2 and thrombin-stimulated platelet PGHS-1. IC50 values (microM) for inhibition of PGHS-1 and PGHS-2 were: indomethacin, 0.70 +/- 0.20 vs 0.36 +/- 0.10 (P < .05); S-indobufen, 0.64 +/- 0.22 vs. 14.9 +/- 8 (P < .05), R-indobufen, 38 +/- 18 vs. 230 +/- 68 (P < .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +/- 96 vs. 187 +/- 96.(ABSTRACT TRUNCATED AT 250 WORDS)
我们研究的目的是建立一种人前列腺素内过氧化物合酶-2（PGHS-2）表达模型，用于评估体外和离体情况下的药物抑制作用。将肝素化的人全血样本与脂多糖（LPS，0.1 - 50微克/毫升）在37℃孵育0至24小时。通过在采样前48小时给受试者服用阿司匹林（300毫克）或在时间0时在体外添加阿司匹林（10微克/毫升）来抑制血小板PGHS-1的作用。通过放射免疫测定法测量PGE2。LPS以时间和浓度依赖性方式诱导环氧化酶活性表达。在10微克/毫升LPS作用24小时后，PGE2产量平均为12.1±6.2纳克/毫升（平均值±标准差，n = 7）。使用针对人PGHS-2羧基末端部分的抗体通过蛋白质印迹分析，环氧化酶活性与单核细胞蛋白质双峰的质量平行增加。地塞米松（2微摩尔）抑制LPS诱导的PGE2产生达96±4%（平均值±标准差，n = 3）。在体外测试了四种不同的抑制剂对LPS诱导的单核细胞PGH-2和凝血酶刺激的血小板PGHS-1的环氧化酶活性的影响。抑制PGHS-1和PGHS-2的IC50值（微摩尔）分别为：吲哚美辛，0.70±0.20对0.36±0.10（P <.05）；S-吲哚布芬，0.64±0.22对14.9±8（P <.05），R-吲哚布芬，38±18对230±68（P <.01），6-甲氧基-萘乙酸（萘普生的活性代谢物），278±96对187±96。（摘要截断于250字）
