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

Vani Karadi Manjappa,1,2,* Manjula Mavathuru Venkatappa,3,* Deepadarshan Urs,4 Suliphuldevara Mathada Basavarajaiah,5 Shivakumar Venkataramaiah,3 Sanjana Bai Shivramsingh Mahendranathsingh,1 Sushma Mohan,1 Hunase Rajaiah Pushpavathi,4 Dharmappa Kattepura Krishnappa,4,* Devaraja Sannaningaiah1,* 1Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, Karnataka, 572103, India; 2Department of Biochemistry, Maharani Lakshmi Ammanni College for Women, Autonomous, Bangalore, Karnataka, 560012, India; 3Department of Studies and Research in Food Science and Nutrition, Tumkur University, Tumkur, Karnataka, 572103, India; 4Inflammation Research Laboratory, Department of Studies and Research in Biochemistry, Mangalore University, Jnana Kaveri Postgraduate Centre, Kodagu, 571232, India; 5P.G. Department of Chemistry, Vijaya College, Bangalore, 560004, India*These authors contributed equally to this workCorrespondence: Devaraja Sannaningaiah, DOSR in Biochemistry, Tumkur University, Tumkur, Karnataka, 572103, India, Email [email protected] Dharmappa Kattepura Krishnappa, Department of Biochemistry, Mangalore University, Karnataka, India, Email [email protected]: In the current study, the evaluation of anti-inflammatory (in vitro) activity of chemically synthesized Urolithin-C was examined.Methods: 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).Results: 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.Conclusion: 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. Keywords: anti-inflammatory, urolithin-C, cytokines, LPS, macrophage