Expression of cyclooxygenase-2, nitric oxide synthase 2 and heme oxygenase-1 mRNA induced by bis-eugenol in RAW264.7 cells and their antioxidant activity determined using the induction period method

Abstract

Background/Aim: To clarify the mechanisms responsible for the anti-inflammatory/proinflammatory activities of eugenol-related compounds, we investigated the cytotoxicity and up-regulatory/down-refgulatory effects of the biphenols curcumin, bis-eugenol, magnolol and honokiol, and the monophenols eugenol and isoeugenol, on major regulators of cyclooxygenase-2 (Cox-2), nitric oxide synthase 2 (Nos2) and heme oxygenase-1 (HO-1) mRNA in RAW264.7 cells. Materials and Methods: mRNA expression was investigated using real-time reverse transcriptasepolymerase chain reaction (RT-PCR), and the theoretical parameters were calculated using the DFT/B3LYP/6-31, method. Also, the antioxidant activity of eugenol-related compounds in combination with 2-mercapto-1- methylimidazole (MMI, as a model for glutathione (GSH)) was investigated using the induction period method for polymerization of methyl methacrylate initiated by benzoyl peroxide (BPO). Results: The cytotoxicity of eugenol-related compounds showed a linear relationship with their softness (σ) and electrophilicity (ω). At a concentration of 50 μM, biphenols except for bis-eugenol elicited the expression of mRNA for both Cox-2 and Nos2, but monophenols did not. In contrast, bis-eugenol elicited Cox-2 gene expression, but down-regulated Nos2 gene expression. bis-Eugenol alone induced the expression of HO-1 mRNA, and when combined with MMI it showed a potent antagonistic effect on BPOinduced antioxidant activity. The ability of methoxyphenols to inhibit LPS-stimulated Cox-2 gene expression declined in the order curcumin ≫ isoeugenol > bis-eugenol ≫ eugenol, and the rank of ability was related to their ω value. Conclusion: Most eugenol-related compounds had proinflammatory activity at high concentrations. However, they had also anti-inflammatory activity at lower concentrations. Eugenol-related compounds may exert antioxidant and anti-inflammatory activity in LPS-stimulated RAW264.7 cells possibly by inhibiting the activation of nuclear factor-kappa B (Nf-κB), whereas bis-eugenol requires induction of HO-1 expression. bis-Eugenol as well as curcumin, may have anti-inflammatory and anticancer therapeutic applications.