Screening of Five Essential Oils for Identification of Potential Inhibitors of IL-1-induced Nf-κB Activation and NO Production in Human Chondrocytes: Characterization of the Inhibitory Activity of α-Pinene

Abstract

Nuclear factor-κB is a key transcription factor activated by pro-inflammatory signals, like interleukin-1β (IL-1), being required for the expression of many inflammatory and catabolic mediators, such as nitric oxide (NO), that play an important role in arthritic diseases. This work aimed at screening and identifying natural inhibitors of IL-induced NF-κB activation and NO production in human articular chondrocytes. Five essential oils obtained from four plants of the Iberian flora, Mentha x piperita L. (Lamiaceae), Origanum virens L. (Lamiaceae), Lavandula luiseri L. (Lamiaceae), and Juniperus oxycedrus L. subsp. oxycedrus (Cupressaceae), were screened for their ability to prevent IL-1-induced NO production. The oil showing higher inhibitory activity was fractionated, concentrated, analyzed for composition elucidation and prepared for further assays. For this purpose, the human chondrocytic cell line C-28/I2 was used to evaluate NF-κB activation by determining the cytoplasmic levels of the total and phosphorylated forms of the inhibitory protein, IκB-α, and the NF-κB‐DNA binding activity. The essential oil from the leaves of J. oxycedrus in a concentration of 0.02 % (v/v) achieved the greatest inhibition (80 ± 8 %) of IL-1-induced NO production. Chemical analysis showed that this essential oil is predominantly composed of monoterpene hydrocabons, being α-pinene [2,6,6-trimethyl-bicyclo(3.1.1)hept-3-ene] the major constituent (76 %). Similarly to the effect of the whole oil, a fraction containing 93 % α-pinene reduced significantly IL-1-induced IκB-α degradation. Moreover, α-pinene also decreased IκB-α phosphorylation, NF-κB‐DNA binding activity, and NO production. Another fraction containing oxygenated mono- and sesquiterpenes was nearly as effective as α-pinene. The ability of the α-pinene-containing fraction to reduce IL-1-induced NF-κB activation and NO production warrants further studies to demonstrate the usefulness of α-pinene in the treatment of arthritic diseases and other conditions in which NF-κB and NO play pathological roles.

Key words

arthritis - α‐pinene - IL‐1β - essential oil - NF‐κB - nitric oxide

References

• 1 Andreakos E, Sacre S, Foxwell B M, Feldmann M. The toll-like receptor-nuclear factor kappaB pathway in rheumatoid arthritis.  Front Biosci. 2005;  10 2478-2488
• 2 Berenbaum F. Signaling transduction: target in osteoarthritis.  Curr Opin Rheumatol. 2004;  16 616-622
• 3 Martel-Pelletier J, Boileau C, Pelletier J P, Roughley P J. Cartilage in normal and osteoarthritis conditions.  Best Practice Res Clin Rheumatol. 2008;  22 351-384
• 4 Palmer R M J, Hickery M S, Charles I G, Moncada S, Bayliss M T. Induction of nitric oxide synthase in human chondrocytes.  Biochem Biophys Res Commun. 1993;  193 398-405
• 5 Stadler J M, Stefanovic-Racic M, Billiar T R, Curran R D, McIntyre L A, Georgescu H I, Simmons R L, Evans C H. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide.  J Immunol. 1991;  147 3915-3920
• 6 Vuolteenaho K, Moilanen T, Al-Saffar N, Knowles R G, Moilanen E. Regulation of the nitric oxide production resulting from the glucocorticoid-insensitive expression of iNOS in human osteoarthritic cartilage.  Osteoarthritis Cartilage. 2001;  9 597-605
• 7 Rosa S C, Judas F, Lopes M C, Mendes A F. Nitric oxide synthase isoforms and NF-kappaB activity in normal and osteoarthritic human chondrocytes: regulation by inducible nitric oxide.  Nitric Oxide. 2008;  19 276-283
• 8 Förstermann U, Kleinert H. Nitric oxide synthase: expression and expressional control of the three isoforms.  Naunyn-Schemiedeberg's Arch Pharmacol. 1995;  352 351-364
• 9 Boileau C, Martel-Pelletier J, Moldovan F, Jouzeau J Y, Netter P, Manning P T, Pelletier J P. The in situ up-regulation of chondrocyte interleukin-1-converting enzyme and interleukin-18 levels in experimental osteoarthritis is mediated by nitric oxide.  Arthritis Rheum. 2002;  46 2637-2647
• 10 Cao M, Westerhausen-Larson A, Niyibizi C, Kavalkovich K, Georgescu H I, Rizzo C F, Hebda P A, Stefanovic-Racic M, Evans C H. Nitric oxide inhibits the synthesis of type-II collagen without altering Col2A1 mRNA abundance: prolyl hydroxylase as a possible target.  Biochem J. 1997;  324 305-310
• 11 Lo Y Y, Conquer J A, Grinstein S, Cruz T F. Interleukin-1β induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species.  J Cell Biochem. 1998;  69 19-29
• 12 Notoya K, Jovanovic D V, Reboul P, Martel-Pelletier J, Mineau F, Pelletier J P. The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2.  J Immunol. 2000;  165 3402-3410
• 13 Taskiran D, Stefanovic-Racic M, Georgescu H, Evans C H. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1.  Biochem Biophys Res Commun. 1994;  200 142-148
• 14 Largo R, Alvarez-Soria M A, Diez-Ortego I, Calvo E, Sanchez-Pernaute O, Egido J, Herrero-Beaumont G. Glucosamine inhibits IL-1beta-induced NFkappaB activation in human osteoarthritic chondrocytes.  Osteoarthritis Cartilage. 2003;  11 290-298
• 15 Mendes A F, Caramona M M, Carvalho A P, Lopes M C. Diacerhein and rhein prevent interleukin-1β-induced nuclear factor-κB activation by inhibiting the degradation of inhibitor κB-α.  Pharmacol Toxicol. 2002;  91 22-28
• 16 McIntyre K W, Shuster D J, Gillooly K M, Dambach D M, Pattoli M A, Lu P, Zhou X D, Qiu Y, Zusi F C, Burke J R. A highly selective inhibitor of I kappa B kinase, BMS-345541, blocks both joint inflammation and destruction in collagen-induced arthritis in mice.  Arthritis Rheum. 2003;  48 2652-2659
• 17 Pahl H L. Activators and target genes of Rel/NF-κB transcription factors.  Oncogene. 1999;  18 6853-6866
• 18 Mendes A F, Caramona M M, Carvalho A P, Lopes M C. Differential roles of hydrogen peroxide and superoxide in mediating IL-1-induced NF-κB activation and iNOS expression in bovine articular chondrocytes.  J Cell Biochem. 2003;  88 783-793
• 19 Ghosh S, Karin M. Missing pieces in the NF-κB puzzle.  Cell. 2002;  109 S81-S96
• 20 Li X, Massa P E, Hanidu A, Peet G W, Aro P, Savitt A, Mische S, Li J, Marcu K B. IKKα, IKKβ, and NEMO/IKKγ are each required for the NF-κB-mediated inflammatory response program.  J Biol Chem. 2002;  277 45129-45140
• 21 Conseil de l'Europe .Pharmacopée Européene, Vol. 1. Sainte Ruffine; Maisonneuve S.A. 1996
• 22 Adams R P. Identification of essential oil components by gas chromatography–quadrupole mass spectroscopy. Carol Stream; Allured Publ 2001
• 23 Joulain D, König W A. The atlas of spectral data of sesquiterpene hydrocarbons. Hamburg; E.B.-Verlag 1998
• 24 Green L C, Wagner D A, Glogowski J, Skipper P L, Wishnok J S, Tannenbaum S R. Analysis of nitrate, nitrite and [¹⁵ N]nitrate in biological fluids.  Anal Biochem. 1982;  126 131-138
• 25 Boti J B, Bighelli A, Cavaleiro C, Salgueiro L, Casanova J. Chemical variability of Juniperus oxycedrus subsp. oxycedrus leaf and berry oils from Corsica, analysed by combination of GC, GC/MS and 13C-NMR.  Flavour Fragrance J. 2006;  21 268-273
• 26 Cavaleiro C, Pinto E, Gonçalves M J, Salgueiro L. Antifungal activity of Juniperus essential oils against dermatophyte, Aspergillus and Candida strains.  J Appl Microbiol. 2006;  100 1333-1338
• 27 Martin S, Padilla E, Ocete M A, Galvez J, Jiménez J, Zarzuelo A. Anti-inflammatory activity of the essential oil of Bupleurum fruticescens.  Planta Med. 1993;  59 533-536
• 28 Zhou J Y, Tang F D, Mao G G, Bian R L. Effect of alpha-pinene on nuclear translocation of NF-kappa B in THP-1 cells.  Acta Pharmacol Sin. 2004;  25 480-484

1 These authors contributed equally to this work.