Flavocoxid, a dual inhibitor of cyclooxygenase and 5‐lipoxygenase, blunts pro‐inflammatory phenotype activation in endotoxin‐stimulated macrophages

Abstract

Background and purpose: The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5‐lipoxygenase (LOX) enzymes. The anti‐inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. Experimental approach: LPS‐stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co‐incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX‐2, 5‐LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB‐α (IκB‐α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF‐κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor‐α (TNF‐α) gene and protein expression were measured by real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. Key results: LPS stimulation induced a pro‐inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX‐2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5‐LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX‐1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB‐α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF‐κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF‐α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n‐fold/β‐actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS‐stimulated macrophages. Conclusion and implications: Flavocoxid might be useful as a potential anti‐inflammatory agent, acting at the level of gene and protein expression.