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RESEARCH PAPER Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-in?ammatory phenotype activation in endotoxin-stimulated macrophages D Altavilla1 , F Squadrito1 , A Bitto1 , F Polito1 , BP Burnett2 , V Di Stefano1 and L Minutoli1

1 Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy, and

2 Primus Pharmaceuticals, Inc.

, Scottsdale, AZ, USA Background and purpose: The ?avonoids, 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-in?ammatory activity, measured by protein and gene expression of in?ammatory markers, of ?avocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. Experimental approach: LPS-stimulated (1 mg・mL-1 ) peritoneal rat macrophages were co-incubated with different concen- trations of ?avocoxid (32C128 mg・mL-1 ) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein kB-a (IkB-a) levels were evaluated by Western blot analysis. Nuclear factor kB (NF-kB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-a (TNF-a) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respec- tively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. Key results: LPS stimulation induced a pro-in?ammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 mg・mL-1 ) signi?cantly inhibited COX-2 (LPS =

18 ? 2.1;

?avocoxid = 3.8 ? 0.9 integrated intensity), 5-LOX (LPS =

20 ? 3.8;

?avocoxid = 3.1 ? 0.8 integrated intensity) and iNOS expression (LPS =

15 ? 1.1;

?avocoxid = 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 IkB-a protein (LPS = 1.9 ? 0.2;

?avocoxid = 7.2 ? 1.6 integrated intensity), blunted increased NF-kB binding activity (LPS = 9.2 ? 2;

?avocoxid = 2.4 ? 0.7 integrated intensity) and the enhanced TNF-a mRNA levels (LPS =

8 ? 0.9;

?avocoxid = 1.9 ? 0.8 n-fold/b-actin) induced by LPS. Finally, ?avocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages. Conclusion and implications: Flavocoxid might be useful as a potential anti-in?ammatory agent, acting at the level of gene and protein expression. British Journal of Pharmacology (2009) 157, 1410C1418;

doi:10.1111/j.1476-5381.2009.00322.x Keywords: ?avocoxid;

Limbrel;

medical food;

COX;

5-LOX;

iNOS;

NF-kB;

TNF-a;

dual inhibitor;

in?ammation Abbreviations: COX, cyclooxygenase;

IkB-a, inhibitory protein kB-a;

iNOS, inducible nitric oxide synthase;

LOX, lipoxyge- nase;

LTB4, leukotriene B4;

MAL, malondialdehyde;

NFkB, nuclear factor kB;

PGE2, prostaglandin E2;

TNF-a, tumour necrosis factor-a Introduction In?ammation is a bene?cial host response to external chal- lenge or cellular injury that leads to the release of a complex array of in?ammatory mediators, promoting the restoration of tissue structure and function. However, prolonged in?am- mation can be harmful, contributing to the pathogenesis of many diseases. During in?ammation, fatty acid production is considerably increased (Kuehl and Egan, 1980). Arachidonic acid (AA) is the main precursor of fatty acid metabolites, which regulate functions of various organs and systems. It is released from cellular membrane phospholipids by phospho- lipase A2 or indirectly by phospholipases C and D (Burdan et al., 2006), and subsequently transformed by cyclooxyge- nase (COX) and lipoxygenase (LOX) enzymes to prostaglan- dins (PGs), thromboxane and leukotrienes (LTs) (Khanapure et al., 2007) Correspondence: Prof. Francesco Squadrito, Department of Clinical and Experi- mental Medicine and Pharmacology, Section of Pharmacology Torre Biologica 5th ?oor, '