Cytotoxicity and Pro-/Anti-inflammatory Properties of Cinnamates, Acrylates and Methacrylates Against RAW264.7 Cells

Periodontitis is a chronic inflammatory disease linked to various systemic age-related conditions. It is known that α,β-unsaturated carbonyl compounds such as dietary cinnamates (β-phenyl acrylates) and related (meth)acrylates can have various positive and negative health effects, including cytotoxicity, allergic activity, pro-and anti-inflammatory activity, and anticancer activity. To clarify the anti-inflammatory properties of α,β-unsaturated carbonyl compounds without a phenolic group in the context of periodontal tissue inflammation and alveolar bone loss, we investigated the cytotoxicity and up-regulatory/down-regulatory effect of three trans-cinnamates (trans-cinnamic acid, methyl cinnamate, trans-cinnamaldehyde), two acrylates (ethyl acrylate, 2-hydroxyethyl acrylate), and three methacrylates (methyl methacrylate, 2-hydroxyethyl methacrylate, and triethyleneglycol dimethacrylate) using RAW264.7 cells.

Cytotoxicity was determined using a cell counting kit (CCK-8) and mRNA expression was determined using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Pro-inflammatory and anti-inflammatory properties were assessed in terms of expression of mRNAs for cyclo-oxygenase-2 (Cox2), nitric oxide synthase 2 (Nos2), tumor necrosis factor-alpha (Tnfa) and heme oxygenase 1 (Ho1).

The most cytotoxic compound was 2-hydroxyethyl acrylate, followed by ethyl acrylate and cinnamaldehyde (50% lethal cytotoxic concentration, LC₅₀=0.2-0.5 mM). Cox2 mRNA expression was up-regulated by cinnamaldehyde and 2-hydroxyethyl acrylate, particularly by the former. In contrast, the up-regulatory effect on Nos2 mRNA expression was in the order: cinnamaldehyde >> ethyl acrylate ≈ triethyleneglycol dimethacrylate >> methyl methacrylate ≈ methyl cinnamate. On the other hand, cinnamic acid and 2-hydroxyethyl methacrylate had no effect on gene expression. The two acrylates, but not cinnamates and methacrylates, up-regulated the expression of Ho1 mRNA at a non-cytotoxic concentration of 0.1 mM. Expression of Cox2, Nos2 and Tnfa mRNAs induced by Porphyromonas gingivalis lipopolysaccharide was greatly suppressed by cinnamaldehyde, methyl cinnamate and the two acrylates at 0.1 mM (p<0.05), and slightly, but significantly suppressed by cinnamic acid and methacrylates at 0.1-1 mM (p<0.05).

Cinnamaldehyde and acrylates exhibited both anti-inflammatory and pro-inflammatory properties, possibly due to their marked ability to act as Michael reaction acceptors, as estimated from the beta-carbon ¹³C-nuclear magnetic resonance spectra. Methyl cinnamate exhibited potent anti-inflammatory activity with less cytotoxicity and pro-inflammatory activity, suggesting that this compound may be useful for treatment of periodontal disease and related systemic diseases.