Methylxanthine derivative, pentoxifylline attenuates inflammation via NF-κB and AP-1 pathway inhibition in murine macrophages.

The prolonged use of current anti-inflammatory therapies has significant limitations for chronic inflammation management, requiring safer alternatives. In this study, we investigated the anti-inflammatory effect of methylxanthine derivative, pentoxifylline. MTT assay was used for the determination of the cytotoxicity of pentoxifylline. Lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells were used for the determination of the anti-inflammatory effect of pentoxifylline using Griess assay, qRT-PCR, Western blot, gene reporter assay and cell migration assay. Pentoxifylline exhibited a high level of safe therapeutic window with no cytotoxicity up to 20 μM in the MTT assay on RAW 264.7 cells. Pentoxifylline demonstrated concentration and time-dependent anti-inflammatory effects with significantly (p < 0.05) reduced NO production at 10 and 20 μM concentrations. Gene expression revealed significant downregulation of inflammatory mediator genes: Nos2, Tnfα, and Il1β (p < 0.05). Protein analysis confirmed these effects with reductions in iNOS, TNFα, IL1β, and IL6 (p < 0.05). Pentoxifylline caused a significant reduction (p < 0.05) of transcriptional activity of NF-κB and AP-1. Further, a significant reduction of nuclear translocation of NF-κB by pentoxifylline confirmed the transcriptional inhibition. Additionally, in the scratch assay, pentoxifylline exhibited a significant reduction (p < 0.05) in macrophage migration, supporting its role in inhibiting inflammation progression. These comprehensive multi-target anti-inflammatory effects of pentoxifylline support its promising potential for use as a therapeutic candidate for inflammatory diseases.