Potent Anti-Inflammatory Activity of Ursolic Acid, a Triterpenoid Antioxidant, Is Mediated through Suppression of NF-kB, AP-1 and NF-AT Rahul Checker1, Santosh K. Sandur1, Deepak Sharma 1 , Raghavendra S. Patwardhan1, S. Jayakumar1, Vineet Kohli2, Gautam Sethi3, Bharat B. Aggarwal4, Krishna B. Sainis1* 1 Radiation Biology and Health Sciences Division, Bio-Medical Group, Bhabha Atomic Research Centre, Mumbai, India, 2 Medical Division, Bio-Medical Group, Bhabha Atomic Research Centre, Mumbai, India, 3 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 4 Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America Abstract Background: Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-kB (nuclear factor kappa B) activation. Since NF-kB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. Methodology/Principal Findings: The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-kB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-kB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-c. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. Conclusions/Significance: The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders. Citation: Checker R, Sandur SK, Sharma D, Patwardhan RS, Jayakumar S, et al. (2012) Potent Anti-Inflammatory Activity of Ursolic Acid, a Triterpenoid Antioxidant, Is Mediated through Suppression of NF-kB, AP-1 and NF-AT. PLoS ONE 7(2): e31318. doi:10.1371/journal.pone.0031318 Editor: Pierre Bobe´, Institut Jacques Monod, France Received August 23, 2011; Accepted January 5, 2012; Published February 20, 2012 Copyright: 2012 Checker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Department of Atomic Energy, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kbsainis@barc.gov.in Introduction Inflammation involves the activation and recruitment of phagocytes (macrophages, neutrophils), NK cells, complement system and secretion of cytokines like IL-1b, IL-6, TNF-a by activated cells which are essential for the host defence system. The chronic inflammation that persists even after elimination of pathogen(s) has been associated with several diseases such as cancer [1,2], neoplasms, inflammatory bowel disease, ulcerative colitis [3,4], atherosclerosis, rheumatoid arthritis [5], asthma and Alzheimer’s disease [6]. The damaging responses resulting from chronic inflammation can be controlled by altering the molecular mediators of an inflammatory response. The key targets are proinflammatory cytokines and cytokine receptors (tumor necrosis factor (TNF-a) and TNF-RII, IL-12, IL-6, interferon-c), enzymes (COX-2, inosine monophosphate dehydrogenase), cell surface molecules required for intercellular interactions and leukocyte activation. Dysregulation of these cytokines and enzymes may contribute to the pathogenesis of many chronic inflammatory diseases [7,8]. Transcription factors (NF-kB, NF-AT and AP1) along with mitogen-activated protein kinases (ERK, JNK, and p38) are known to regulate these inflammatory cytokines and enzymes and are being targeted by several investigators to ameliorate chronic inflammation [9,10,11,12,13,14,15]. There are several reports showing the involvement of reactive oxygen species (ROS) and glutathione (GSH) in modulating these immunologically important kinases and transcription factors and resulting in altered immune responses [16,17]. The treatment approaches for these inflammatory disorders are addressed by administration of steroids and non-steroidal anti-inflammatory drugs (NSAID) like azathioprine. All of these drugs are accompanied by an array of side effects [18,19,20,21] restricting their continuous usage and complicating treatment modalities. Thus there is a need to identify safe and non-toxic anti- PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31318