Butylated Hydroxyanisole-Dimer Inhibits P.gingivalis Fimbria-Stimulated NF-kB activation in Macrophages

Objectives: Porphyromonas gingivalis fimbriae are important bacterial components involved in the initiation and development of chronic periodontal diseases via the production of inflammatory cytokines. Butylated hydroxyanisole, BHA (a mixture of 2- and 3-BHA) is widely used as an potent antioxidant; but its adverse effects such as promotion of carcinogenesis and proinflammatory activity have been reported. This is possibly due to the prooxidant property of this compound. We recently demonstrated that the dimer of 2-methoxyphenols exhibits cyclooxygenase-2 inhibition, because of lessening of its prooxidant property by dimerization. In the present study, in our search for a chemopreventive agent for chronic periodontal diseases, we examined whether 2-BHA (2-tert-butyl-4-methoxyphenol) and its synthetic ortho dimer, bis-BHA (3,3'-di-tert-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) could inhibit fimbria-stimulated nuclear factor kappa B (NF-kB) activation and the expression of an inflammatory cytokine. Methods: bis-BHA was synthesized by oxidative cross-linking BHA monomers with Cu²⁺. Porphyromonas gingivalis ATCC 33277 fimbriae were prepared and purified from cell washings by the method of Yoshimura et al. The fimbria-stimulated NF-kB activity was evaluated by conducting gel mobility shift and NF-kB-promoted luciferase assays. IkB-a protein was detected by Western blotting. IL-1b mRNA and protein levels were analyzed by Northern blotting assay and ELISA, respectively. Results: Fimbria-induced gene expression and production of IL-1b in RAW264.7 cells was strongly inhibited by bis-BHA. In contrast, 2-BHA caused only slight inhibition. bis-BHA also significantly inhibited the fimbria-stimulated phosphorylation-dependent proteolysis of IkB-a and transcriptional activity of NF-kB in the cells. Conclusion: bis-BHA inhibited fimbria-stimulated NF-kB activation. bis-BHA might be applicable to the chemoprevention of oral diseases, particularly chronic periodontal diseases; because this compound exhibited an inhibitory effect on NF-kB activation and/or inflammatory responses.