Pirfenidone inhibits the induction of iNOS stimulated by interleukin-1β at a step of NF-κB DNA binding in hepatocytes

Background/Aims Pirfenidone has antiinflammatory effects in animals with endotoxemia. We reported that pirfenidone inhibits the enhancement of inflammatory cytokines and inducible nitric oxide synthase (iNOS) in liver of endotoxin-treated rats, leading to the prevention of hepatic injury. However, the mechanisms involved in suppression of these gene inductions are obscure. Studies were performed to investigate whether pirfenidone directly influences iNOS induction in hepatocytes. Methods Cultured hepatocytes were treated with interleukin-1β (IL-1β) in the presence and absence of pirfenidone, and iNOS induction and its signal including NF-κB were analyzed. Results Pirfenidone inhibited the induction of iNOS mRNA and protein, resulting in the decrease of nitric oxide production. Gel shift assay demonstrated that pirfenidone inhibited the activation of NF-κB. Consistent with this observation, transfection experiments revealed that pirfenidone decreased transcriptional activation of iNOS gene promoter. In contrast, pirfenidone had no effect on the degradation of IκB, and could not prevent nuclear translocation of p50/p65. Finally, pirfenidone inhibited the activation of Akt and up-regulation of IL-1 receptor stimulated by IL-1β. Conclusions Results indicate that pirfenidone inhibits the induction of iNOS gene expression at a step of NF-κB DNA binding, but not its nuclear translocation, partly through the inhibition of IL-1 receptor induction in hepatocytes.