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


METHODS
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.


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.


CONCLUSIONS
Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.
Altavilla
D
D
Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy.
Squadrito
F
F
Bitto
A
A
Polito
F
F
Burnett
B P
BP
Di Stefano
V
V
Minutoli
L
L
D000818:Animals; D002392:Catechin / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D015232:Dinoprostone / Q000378:metabolism; D004338:Drug Combinations; D004797:Enzyme-Linked Immunosorbent Assay; D005419:Flavonoids / Q000494:pharmacology*; D020672:I-kappa B Proteins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016859:Lipoxygenase Inhibitors; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D000072000:NF-KappaB Inhibitor alpha; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016861:Cyclooxygenase Inhibitors; D004338:Drug Combinations; D005419:Flavonoids; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016859:Lipoxygenase Inhibitors; C000606206:Nfkbia protein, rat; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C038044:baicalin; D002392:Catechin; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D015232:Dinoprostone
Pharmacol Res. 2001 May;43(5):429-36
11394934
Oncogene. 1999 Nov 22;18(49):6853-66
10602461
Circ Res. 2002 Jul 26;91(2):120-6
12142344
J Clin Invest. 2001 Jan;107(1):3-6
11134170
Nature. 1980 Jul 17;286(5770):264-5
6250050
Gut. 1993 Jun;34(6):742-7
8390958
Science. 1980 Nov 28;210(4473):978-84
6254151
Rheumatology (Oxford). 2004 Feb;43 Suppl 1:i16-20
14752171
Mol Cancer Ther. 2007 Nov;6(11):3039-48
18025287
Int J Clin Pract. 2003 May;57(4):301-4
12800462
Nature. 1997 Jun 5;387(6633):620-4
9177352
Arthritis Res Ther. 2006;8(6):R181
17156456
FASEB J. 2001 Jul;15(9):1622-4
11427506
Mol Pharmacol. 1997 Sep;52(3):465-72
9281609
Life Sci. 2006 Jul 17;79(8):801-7
16564553
Curr Pharm Des. 2005;11(26):3431-47
16250846
Postepy Hig Med Dosw (Online). 2006;60:129-41
16552393
Mol Med. 2000 May;6(5):347-73
10952018
FEBS Lett. 2001 Jun 29;500(1-2):52-5
11434925
Comp Biochem Physiol C Toxicol Pharmacol. 2006 Mar-Apr;142(3-4):317-327
16380298
Prostaglandins Other Lipid Mediat. 2003 Jul;71(3-4):147-62
14518558
Curr Atheroscler Rep. 2008 Jun;10(3):244-51
18489853
Mini Rev Med Chem. 2006 Aug;6(8):945-51
16918500
Curr Top Med Chem. 2007;7(3):265-75
17305569
Ann Rheum Dis. 2003 Jun;62(6):501-9
12759283
Anticancer Res. 2000 Sep-Oct;20(5A):2861-5
11062694
J Neurochem. 2001 Nov;79(4):777-86
11723170
Pharmacology. 2006;77(2):71-7
16636611
Curr Allergy Asthma Rep. 2008 Jul;8(4):367-73
18606091
Br J Pharmacol. 1992 Nov;107(3):660-4
1472964
Med Res Rev. 2000 Sep;20(5):323-49
10934347
J Immunol Methods. 1983 Dec 16;65(1-2):55-63
6606682
Cardiovasc Hematol Disord Drug Targets. 2006 Jun;6(2):85-100
16787194
Mol Immunol. 2002 Feb;38(10):781-91
11841838
J Med Food. 2007 Sep;10(3):442-51
17887937
Anesth Analg. 2003 Dec;97(6):1724-1729
14633550
J Ethnopharmacol. 1993 Mar;38(2-3):105-12
8510457
J Leukoc Biol. 1994 Nov;56(5):576-82
7525816
Evid Based Complement Alternat Med. 2006 Mar;3(1):117-24
16550232
Pharmacol Res. 2007 Feb;55(2):148-57
17187986
Curr Top Med Chem. 2007;7(3):311-40
17305573
Jpn J Cancer Res. 2000 Jul;91(7):686-91
10920275
Life Sci. 2004 Jun 25;75(6):639-53
15172174
Adv Immunol. 1995;58:1-27
7741027
J Physiol Pharmacol. 2006 Nov;57 Suppl 5:113-24
17218763
Proc Soc Exp Biol Med. 2000 Oct;225(1):32-8
10998196
Biochem Pharmacol. 2008 Feb 15;75(4):914-22
18191816
Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9730-4
7692452
Curr Med Chem. 2007;14(18):1940-4
17691936
In Vivo. 2005 Sep-Oct;19(5):895-909
16097445
Curr Drug Targets Cardiovasc Haematol Disord. 2005 Apr;5(2):177-84
15853750
J Pharmacol Exp Ther. 2002 Feb;300(2):367-75
11805193
Antioxid Redox Signal. 2006 Sep-Oct;8(9-10):1791-806
16987032
Int J Oncol. 2004 Mar;24(3):703-10
14767556
J Am Soc Nephrol. 1999 Sep;10(9):1991-6
10477152
Dev Immunol. 2002 Sep;9(3):135-41
12885154
Annu Rev Immunol. 1996;14:649-83
8717528
Am J Kidney Dis. 2000 May;35(5):937-40
10793030