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

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