PLA₂ antagonists suppress inducible nitric oxide synthase and inducible cyclooxygenase in lipopolysaccharide-induced Raw264.7 cells

Phospholipase A₂ (PLA₂) regulates eicosanoids and platelet activating factor production and plays an important role in regulating critical mediators in inflammatory diseases. PLA₂ activity is significantly enhanced during sepsis and multiple organ failure and therefore offers an intriguing target in developing anti-inflammatory drugs. We have identified several kinds of biflavonoids with inhibition of PLA₂ activity, which are isolated from plant sources, as potential putative anti-inflammatory and anti-septic agents. Two of them (bilobetin and ginkgetin) potently inhibit several kinds of type II 14 kDa PLA₂ but exhibits a weaker inhibition of type I 14 kDa PLA₂ using 2-linol-[1-¹⁴C]PE as substrate. These inhibitors have been tested for their ability to inhibit the production of TNF-α and the formation of two enzymes, inducible NO synthase (iNOS) and inducible cyclooxygenase (COX-2) using LPS-stimulated Raw264.7 macrophages as assay systems. In the Raw264.7 cells, bacterial LPS induced the protein of COX-2 and iNOS as well as TNF-α release. The inhibitors consistently inhibited the production of TNF-α in a dose-dependent manner. The inhibitory effect of TNF-α was observed at concentrations similar to those related by PLA₂. Moreover, treatment of cells with bilobetin and ginkgetin inhibited nitrite production, one of the stable end products of NO production measured in culture supernatants. The inhibition of NO products is caused by decreased iNOS protein levels as assessed by immunoblotting using a specific anti-iNOS antibody. The inhibitors treatment also reduce the expression of COX-2 protein level to about 80% in LPS-stimulated cells, which coincided with reduction of the iNOS protein. These results suggest that inhibition of PLA₂ and subsequent metabolism of arachidonic acid by COX-2 contribute to LPS-induced NOS pathway including TNF-α in Raw264.7 cells and these two inhibitors may develop as useful agents for anti-inflammation.