Effects of n‐3 polyunsaturated fatty acids on COX‐2 and PGE2 protein levels in articular cartilage chondrocytes

Introduction  Previous studies within our laboratory have shown that supplementation with n-3 polyunsaturated fatty acids (PUFAs), but not other fatty acids, has a beneficial effect on reducing the expression and activity of degradative and inflammatory factors known to cause damage and destruction of cartilage in arthritic diseases (Curtis et al. 2000,2002). Cyclooxygenase (COX), also known as prostaglandin H synthase, catalyses the rate-limiting step in the formation of inflammatory prostaglandins (PGs) (Hla & Neilson 1992). PG synthesis requires conversion of arachidonic acid to PGH2 by either the constitutive COX-1 or by COX-2, which is induced by inflammatory and mitogenic stimuli (Smith et al. 2000). Prostaglandin E2 (PGE2) is produced from PGH2 and has been shown to have a number of functions including both anti- and pro-inflammatory actions (Christman et al. 1991). The aim of this project was to investigate the effects of n-3 PUFAs on cyclooxygenase-2 and prostaglandin E2 protein levels in articular cartilage chondrocytes. Materials and methods  Articular cartilage was obtained both from 7-day-old bovine metacarpo-metatarsophalangeal joints and from human patients undergoing total knee replacement surgery for osteoarthritis (Llandough Hospital, S.Wales, UK). Both explant and monolayer cultures were set up in DMEM with or without 10–300 µg/ml n-3[eicosapentaenoic acid (EPA)] or n-6[arachidonic acid (AA)] PUFAs (minimum 8 h, at 37°C, in 5%CO2) and in the absence or presence of IL-1 (10 ng/ml) for a further 4 days. Results  Total RNA was extracted and RT-PCR performed using oligonucleotide primers specific to COX-2 (Invitrogen, UK). COX-2 mRNA was found to be absent or only present at very low levels in both bovine and human control cultures. After treatment with IL-1, this expression greatly increased. However, supplementation of IL-1-treated cultures with n-3 PUFA (EPA) resulted in a loss of COX-2 mRNA expression. In contrast, supplementation with n-6 PUFA (AA) had no effect. Western blot analysis, using a polyclonal antibody specific to COX-2 (Santa Cruz Biotechnology Inc., USA), showed that COX-2 protein was absent in all control samples and the IL-1 induction of bovine COX-2 protein could also be reduced when supplemented with n-3 PUFAs but not n-6 PUFAs. Using a commercially available PGE2 ELISA Immunoassay kit, it was possible to analyse the PGE2 levels present in explant media from bovine or human samples. In one example, in a 74-year-old female patient, PGE2 protein was very low in the cartilage cultured without IL-1 treatment or PUFA supplementation (control). When the cartilage was treated with IL-1, there was a huge induction of PGE2 levels by as much as 60 fold. This induction is in turn reduced greatly with the supplementation of n-3 PUFA (EPA) but not with n-6 PUFA (AA). Discussion  It has long been accepted that COX-2 plays an important role in inflammation. COX-2 has been found in joints affected by arthritic diseases (Hla & Neilson 1992) and in cultures induced by IL-1. The current study has shown that both the IL-1-induced COX-2 message and protein levels can be reduced with supplementation by n-3 PUFAs but not n-6 PUFAs. This work has also shown that with a decrease in COX-2 protein levels, there is also a corresponding decrease in prostaglandin E2 levels caused by n-3 PUFA supplementation.