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Inhibition of IL-1 β -induced inflammatory and catabolic pathways by the essential oil of Eryngium duriae subsp. juresianum in human chondrocytes Purpose: Osteoarthritis is a disease characterized by an imbalance between anabolic and catabolic processes and upregulation of inflammatory responses, leading to extracellular matrix degradation and joint destruction. The increased expression of inflammatory factors and matrix metalloproteinases is mediated by various signal transduction pathways, in particular the nuclear factor-kappaB (NF-kB) and the mitogen-activated protein kinase (MAPK)-mediated pathways. Nitric oxide (NO), specially resulting from the activity of the inducible isoform of the NO synthase (iNOS), is increased in osteoarthritic chondrocytes and is also one of the factors involved in the inhibition of matrix synthesis and induction of matrix degradation, consequently leading to cartilage loss and joint destruction. These intracellular signalling pathways are modulated by pro-inflammatory cytokines, like IL-1β, in articular chondrocytes. Thus, NF-kB and MAPK pathways, as well as inhibition of NO production by iNOS, are considered important targets for the development of novel anti-osteoarthitic therapies (1,2). Essential oils are complex mixtures of low molecular weight lipophilic molecules, obtained by distillation, whose chemical diversity and favourable pharmacokinetic properties make them important collections for drug screening. This work reports on the ability of the essential oil of Eryngium duriae subsp. juresianum (M Lainz) (an Apiaceae species endemic in the Iberian Peninsula) to decrease IL-1β-induced inflammatory and catabolic pathways in human chondrocytes. Methods: Knee cartilage samples were obtained from multi-organ donors (n=8) at the University Hospital of Coimbra with approval by the Ethics Committee. After enzymatic digestion, chondrocytes were cultured under non proliferating conditions to avoid dedifferentiation. The essential oil of the aerial parts of E. duriae subsp. juresianum (Ej) was diluted in DMSO to achieve final concentrations ranging from 5 to 200 μg/mL and added to the chondrocyte cultures 30 min before addition of IL-1β (10 ng/mL) for 5 min, 30 min or 18 h. The MTT reduction assay was used to rule out cytotoxic effects. NO levels were measured by a colorimetric method based on the Griess reaction. Protein levels of iNOS, IkB-α and the phosphorylated forms of IkB-α and of the MAPKs, p38, JNK and ERK1/2, were assessed by western blot. The results were considered statistically significant for P<0.05 using the Student t-test. Results: Ej significantly decreased IL-1-induced NO production and iNOS protein levels in human chondrocytes in a concentration-dependent manner. The greatest inhibition of IL-1-induced NO (38.4 ± 8.4%, P<0.001) and iNOS protein (53.1 ± 6.3%, P<0.001) levels was achieved with a concentration of 25 μg/mL. However, 200 μg/mL were required for maximal inhibition of IκB-α (70.9 ± 5.6%, P<0.001), JNK (36.4 ± 0.6%, P<0.001), p38 (57.1 ± 4.4%, P<0.001) and ERK1/2 (24.4 ± 3.3%, P<0.001) phosphorylation. Conclusions: These results suggest that by acting on various signalling pathways, Ej elicits the synergistic inhibition of iNOS expression and show the potential of Ej as a promising source of compounds with anti-inflammatory and anti-catabolic properties that may be useful as anti-osteoarthritic drugs. Future work will focus on the fractionation and identification of those compounds followed by further pharmacologic characterization. 1) Marcu, K.B., et al., Curr Drug Targets, 2010. 11(5): 599-613. 2) Berenbaum, F. Arthritis ResTher, 2008. 10(Suppl 2): p. S1. This work was supported by grants PTDC/EME-PME/103578/2008 and PTDC/EME-TME/113039/2009 from FCT.
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