Main Article Content
To investigate the effects of COX-metabolites (PGI2, PGE2, PGF2α and TXA2) on COX-2 expressed in human umbilical vein endothelial cells (HUVEC) treated with IL-1β.
Materials & methods
Human umbilical vein endothelial cells (HUVEC) were obtained from babies born to normal pregnant women (HUVEC) and cultured in 96-well/6-well plates as standard techniques. Cells were grown to confluent and replaced with fresh medium containing no addition, IL-1β alone, COX-metabolites alone and IL-1β plus COX metabolites (0.001, 0.01, 0.1or1 .μg/ml) for 24h. Then, the medium was removed and replaced with fresh medium containing arachidonic acid (10 μM for 10 min). After which time, the medium was collected to measured COX activity by the production of 6-keto-PGF1α (stable metabolites of PGI2) using enzyme immunoassay. The remained cells were extracted and detected COX isoform expression by using immunoblotting.
PGI2, PGE2, PGF2α or TXA2, did not effect on basal COX activity in untreated HUVEC (24h incubation). Untreated HUVEC contained COX-1 protein but not COX-2 protein. When HUVEC were treated with IL-1β (1 ng/ml for 24h), COX activity and COX-2 protein was increased in a dose dependent manner. The increased COX activity in IL- 1β (1 ng/ml) treated HUVEC was inhibited with PGE2 (0.03, 0.3 or 3 μM), but not PGI2, PGF2a or TXA2 in a dose dependent manner. Similarly, COX- 2 protein expression in IL-1 p treated HUVEC was also inhibited with PGE2, but not PGI2, PGF2α or TXA2 in a dose dependent manner.
These results suggested that PGE2, but not PGI2, PGF2α or TXA2 is a key in feedback regulation of COX-metabolites produced in HUVEC.
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