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 microg.mL(-1)) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32-128 microg.mL(-1)) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein kappaB-alpha (IkappaB-alpha) levels were evaluated by Western blot analysis. Nuclear factor kappaB (NF-kappaB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-alpha (TNF-alpha) 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 microg.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. PGE(2) and LTB(4) levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IkappaB-alpha protein (LPS = 1.9 +/- 0.2; flavocoxid = 7.2 +/- 1.6 integrated intensity), blunted increased NF-kappaB binding activity (LPS = 9.2 +/- 2; flavocoxid = 2.4 +/- 0.7 integrated intensity) and the enhanced TNF-alpha mRNA levels (LPS = 8 +/- 0.9; flavocoxid = 1.9 +/- 0.8 n-fold/beta-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.

Figure 1

Cell viability in macrophages stimulated for 24 h with 1 µg·mL⁻¹ of lipopolysaccharide (LPS) or its vehicle (1 mL of RPMI). LPS-stimulated macrophages were co-incubated with flavocoxid or RPMI alone. Bars represent the mean ± SD of seven experiments. *P < 0.001 versus LPS.

Figure 2

Western blot analysis of COX-1 (A), COX-2 (B) and 5-LOX expression (C) in macrophages stimulated for 24 h with 1 µg·mL⁻¹ of lipopolysaccharide (LPS) or its vehicle (1 mL of RPMI). LPS-stimulated macrophages were co-incubated with flavocoxid (32, 64 and 128 µg mL⁻¹) or RPMI alone. Bars represent the mean ± SD of seven experiments. *P < 0.05 versus control, ^#P < 0.01 versus LPS + RPMI, ^##P < 0.005 versus LPS + RPMI, ^§P < 0.001 versus LPS + RPMI.

Figure 3

LTB₄ (B) and PGE₂ (C) concentrations in macrophages stimulated for 24 h with 1 µg·mL⁻¹ of lipopolysaccharide (LPS) or its vehicle (1 mL of RPMI). LPS-stimulated macrophages were co-incubated with flavocoxid (32, 64 and 128 µg·mL⁻¹) or RPMI alone. Bars represent the mean ± SD of seven experiments. *P < 0.05 versus control, ^#P < 0.01 versus LPS + RPMI, ^##P < 0.005 versus LPS + RPMI, ^§P < 0.001 versus LPS + RPMI.

Figure 4

Electrophoretic mobility shift assay (EMSA) of NF-κB binding activity in the nucleus (A) and Western blot analysis of IκB-α proteins levels (B) in the cytoplasm of macrophages stimulated for 1 h with 1 µg·mL⁻¹ of lipopolysaccharide (LPS) or its vehicle (1 mL of RPMI). LPS-stimulated macrophages were co-incubated with flavocoxid (32, 64 and 128 µg mL⁻¹) or RPMI alone. Bars represent the mean ± SD of seven experiments. *P < 0.05 versus control, ^#P < 0.01 versus LPS + RPMI, ^##P < 0.005 versus LPS + RPMI, ^§P < 0.001 versus LPS + RPMI.

Figure 5

Western blot analysis of inducible nitric oxide synthase (iNOS) activity (A) and nitrite concentration (B) in macrophages stimulated for 24 h with 1 µg·mL⁻¹ of lipopolysaccharide (LPS) or its vehicle (1 mL of RPMI). LPS-stimulated macrophages were co-incubated with flavocoxid (32, 64 and 128 µg·mL⁻¹) or RPMI alone. Bars represent the mean ± SD of seven experiments. *P < 0.05 versus control, ^#P < 0.005 versus LPS + RPMI, ^§P < 0.001 versus LPS + RPMI.

Figure 6

Tumour necrosis factor-α (TNF-α) mRNA (A) and TNF-α levels (B) in macrophages stimulated for 4 h with 1 µg·mL⁻¹ of lipopolysaccharide (LPS) or its vehicle (1 mL of RPMI). LPS-stimulated macrophages were co-incubated with flavocoxid (32, 64 and 128 µg·mL⁻¹) or RPMI alone. Bars represent the mean ± SD of seven experiments. *P < 0.05 versus control, ^#P < 0.01 versus LPS + RPMI, ^##P < 0.005 versus LPS + RPMI, ^§P < 0.001 versus LPS + RPMI.

Figure 7

Malondialdehyde (MAL) levels in macrophages stimulated for 4 h with 1 µg·mL⁻¹ of lipopolysaccharide (LPS) or its vehicle (1 mL of RPMI). LPS-stimulated macrophages were co-incubated with flavocoxid (32, 64 and 128 µg·mL⁻¹) or RPMI alone. Bars represent the mean ± SD of seven experiments. *P < 0.05 versus control, ^#P < 0.01 versus LPS + RPMI, ^##P < 0.005 versus LPS + RPMI, ^§P < 0.001 versus LPS + RPMI.