The effects of two new antagonists of secretory PLA2 on TNF, iNOS, and COX-2 expression in activated macrophages

Phospholipase A(2) (PLA(2)) regulates eicosanoid and platelet-activating factor production. It also plays an important role in the regulation of critical mediators in inflammatory diseases in which PLA(2) activity is significantly enhanced during sepsis and multiple organ failure. Therefore, inhibitors of PLA(2) activity offer themselves as target substances in the development of anti-inflammatory drugs. We identified 2 biflavonoids, bilobetin and ginkgetin, that can inhibit PLA(2) activity. In experiments using 2-linol-[1-C-14]PE as substrate both substances potently inhibited several kinds of type II 14-kDa PLA(2) while inhibiting type I 14-kDa PLA(2) to a lesser extent. We tested these PLA(2) inhibitors for their ability to inhibit the production of tumor necrosis factor a (TNF alpha) and 2 enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in an assay system using lipopolysaccharide (LPS)-stimulated Raw264.7 macrophages. In Raw264.7cells, bacterial LPS induced the production of COX-2 and iNOS proteins as well as TNF alpha. The inhibitors consistently inhibited the production of TNF alpha in a dose-dependent manner. Moreover, treatment of the macrophages with bilobetin and ginkgetin shut down the production of nitrite, one of the stable end products of NO released into the culture supernatant. The decrease in NO products was accompanied by a decrease in iNOS protein level as assessed by Western blot probed with specific anti-iNOS antibody. Both inhibitors also reduced the expression of COX-2 protein in the LPS-stimulated cells, which coincided with the reduction in iNOS protein. These results, therefore, suggest that these two sPLA(2) inhibitors may be useful for inhibiting the production of inflammatory cytokine and NO production in inflammatory diseases.