Inhibition of lipopolysaccharide-induced I-κB degradation and tumor necrosis factor-α expression by acriflavine, an antimicrobial agent

Abstract

Acriflavine neutral (ACF) has been used for treatment of microbial infections for humans and fishes. Effects of ACF on the nuclear factor-κB (NF-κB) activation and tumor necrosis factor-α (TNF-α) production by lipopolysaccharide (LPS), an endotoxin, were examined in rat and RAW264.7 cells. Gel retardation analysis revealed that LPS (1 μg/kg) activated NF-κB in the liver, whereas pretreatment of rats with ACF (10 mg/kg) completely prevented the NF-κB activation. Selectivity of the NF-κB DNA binding was confirmed by immunodepletion with anti-p65 and anti-p50 antibodies. Translocation of NF-κB to the nucleus is preceded by phosphorylation and proteolytic degradation of inhibitor-κBα (I-κBα) subunit. Whereas the level of I-κBα protein was rapidly decreased after treatment of rats with LPS (1 μg/kg), ACF treatment prior to LPS attenuated the decrease in I-κBα protein level. LPS-induced increase in the production of TNF-α, the principal inflammatory mediator, was prevented by ACF pretreatment by 80%. Stimulation of RAW264.7 cells with 1 μg/ml of LPS caused an increase in DNA binding activity of NF-κB, which was 80% inhibited by 1 μg/ml of ACF. LPS reduced I-κBα level in RAW264.7 cells by 77%. ACF attenuated LPS-induced decrease in I-κBα protein in a concentration-dependent manner. Production of TNF-α by LPS from RAW264.7 cells was decreased by 84% in the presence of ACF. Data showed that ACF inhibited LPS-induced NF-κB activation through inhibition of I-κBα degradation and TNF-α production in both rat and RAW264.7 cells. Inhibition of NF-κB activation and TNF-α production may be associated with the anti-inflammatory activity of ACF.