Urolithin-C Suppresses Inflammation by Blocking NF-κB Signaling Pathway in LPS-Induced RAW 264.7 Macrophages

In the current study, the evaluation of anti-inflammatory (in vitro) activity of chemically synthesized Urolithin-C was examined. The synthesis of Urolithin-C (3,8,9-trihydroxy-6H-benzo[c]chromen-6-one) was carried out by chemical method and it was characterized using various techniques. The anti-inflammatory efficacy of synthesized Urolithin-C was studied by membrane stabilization, protein denaturation and protease inhibition assays. In addition, MTT (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide) assay was employed to evaluate the cytotoxic effect of Urolithin-C. The anti-inflammatory property of Urolithin-C was further examined using LPS (Lipopolysaccharide) induced RAW 264.7 (Mouse macrophage) cells. Furthermore, the anti-inflammatory properties of Urolithin-C was studied by quantifying pro/anti-inflammatory cytokines using ELISA (enzyme-linked immunosorbent assay). The mechanism of action of Urolithin-C on NF-κB (Nuclear Factor-kappa B) translocation was studied using CLSM (confocal laser scanning microscopy). While gene expression pattern was analyzed using RT-qPCR (Reverse Transcription quantitative Polymerase Chain Reaction). In comparison to the positive control aspirin, Urolithin-C showed a strong anti-inflammatory effect by preventing lysosomal degradation, protein denaturation and inhibition of protease. Furthermore, at the higher dose (200 µg/mL), Urolithin-C was found to be toxic to the mouse macrophages; however, at lower concentration (25 µg/mL) it did not cause toxicity to the said. Thus, 25 µg/mL of Urolithin-C was used to assess the anti-inflammatory activity. Interestingly, Urolithin-C efficiently reduced the expression of pro-inflammatory inducible enzyme (Cox-2), cytokines (IL-2, IL-6, and TNF-alpha) and increased the anti-inflammatory cytokine (TGF-beta1), compared to positive control diclofenac (DFC). Urolithin-C effectively abrogated the NF-κB p65 phosphorylation and its translocation to the nucleus as well. Most importantly, Urolithin-C efficiently suppressed the expression of pro-inflammatory genes and elevated the expression of anti-inflammatory gene. Urolithin-C exhibited anti-inflammatory properties by regulating the expression of pro-inflammatory inducible enzyme, cytokines and the translocation of NF-κB p65 to the nucleus.