A Novel Compound C12 Inhibits Inflammatory Cytokine Production and Protects from Inflammatory Injury In Vivo Yi Wang1., Congcong Yu1., Yong Pan1, Jianling Li1, Yali Zhang2, Faqing Ye1, Shulin Yang 2 , Hui Zhang 3 , Xiaokun Li1,4, Guang Liang1,2* 1 Bioorganic and Medicinal Chemistry Research Center, School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou, People’s Republic of China, 2 Institute of Bioengineering, Nanjing University of Science and Technology, Nanjing, People’s Republic of China, 3 Department of Pharmacy, The First Affiliated Hospital, Wenzhou Medical College, Wenzhou, People’s Republic of China, 4 Norman Bethune College of Medical Science, Jilin University, Changchun, People’s Republic of China Abstract Inflammation is a hallmark of many diseases. Although steroids and cyclooxygenase inhibitors are main anti-inflammatory therapeutical agents, they may cause serious side effects. Therefore, developing non-steroid anti-inflammatory agents is urgently needed. A novel hydrosoluble compound, C12 (2,6-bis(4-(3-(dimethylamino)-propoxy)benzylidene)cyclohexanone), has been designed and synthesized as an anti-inflammatory agent in our previous study. In the present study, we investigated whether C12 can affect inflammatory processes in vitro and in vivo. In mouse primary peritoneal macrophages, C12 potently inhibited the production of the proinflammatory gene expression including TNF-a, IL-1b, IL-6, iNOS, COX-2 and PGE synthase. The activity of C12 was partly dependent on inhibition of ERK/JNK (but p38) phosphorylation and NF-kB activation. In vivo, C12 suppressed proinflammatory cytokine production in plasma and liver, attenuated lung histopathology, and significantly reduced mortality in endotoxemic mice. In addition, the pre-treatment with C12 reduced the inflammatory pain in the acetic acid and formalin models and reduced the carrageenan-induced paw oedema and acetic acid-increased vascular permeability. Taken together, C12 has multiple anti-inflammatory effects. These findings, coupled with the low toxicity and hydrosolubility of C12, suggests that this agent may be useful in the treatment of inflammatory diseases. Citation: Wang Y, Yu C, Pan Y, Li J, Zhang Y, et al. (2011) A Novel Compound C12 Inhibits Inflammatory Cytokine Production and Protects from Inflammatory Injury In Vivo. PLoS ONE 6(9): e24377. doi:10.1371/journal.pone.0024377 Editor: Aimin Xu, University of Hong Kong, China Received May 16, 2011; Accepted August 5, 2011; Published September 8, 2011 Copyright: 2011 Wang 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: Financial support was provided by the National Natural Science Funding of China (20802054 & 30872308 & 30871434), High-level Innovative Talent Funding of Zhejiang Department of Health (G. L.), Project of Wenzhou Sci&Tech Bureau (Y20090009 & Y20100006 & Y20100078), Zhejiang Natural Science Funding (Y2090680 & Y20101108 & Y2090358) and China Postdoctoral Science Foundation funded project (20090461121 & 201003591). 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: wzmcliangguang@163.com . These authors contributed equally to this work. Introduction Inflammation is a complex biological response. A number of reports have suggested that a chronic or acute inflammatory state is tightly associated with, and even constitutes a crucial part of the pathogenesis of a variety of diseases, including atherosclerosis [1], diabetic complications [2], obstructive pulmonary disease [3], asthma [4], arthritis [5], infectious diseases [6], and cancer [7]. In inflammatory diseases and ischemic processes, large amounts of cytokines are produced causing oedema, cellular metabolic stress, and finally, tissue necrosis [8]. Cytokines also induce vasodilatation and transient increase in capillary permeability producing extravasation of plasma proteins [9]. The proinflammatory cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1, and IL-6, and the proinflammatory enzymes cycloxygenase (COX)-2, prostaglandin E synthase (PGES) and inducible NO synthase (iNOS) which produce hormone-like mediators such as NO, are primarily involved in promoting inflammatory processes, and they also play an important role in those disorders [10,11]. Steroids and cyclooxygenase inhibitors have long been used as the main therapeutic anti-inflammatory agents, but they are frequently associated with significant detrimental effects in patients [12,13]. Thus, there is an urgent need for the development of unique anti-inflammatory agents. Non-steroid anti-inflammatory candidates targeting proinflammatory cytokines or inhibiting the overexpression of cytokines become a major focus of current drug development [14]. Our previous study has demonstrated that a novel compound, 2,6-bis(4-(3-(dimethylamino)-propoxy)benzylide- ne)cyclohexanone (C12), is able to dose-dependently inhibit LPS- induced TNF-a and IL-6 release in RAW264.7 macrophages [15]. In addition, C12 is a hydrosoluble compound when combined with HCl to form a quaternary ammonium salt. These in vitro advantages render C12 as a potential anti-inflammatory agent. The aim of the present work is to evaluate the anti-inflammatory effects of C12 and investigate its pharmacological mechanism in macrophages and mouse models. Herein, we showed that C12 inhibited ERK/JNK and NF-kB dependent inflammatory re- sponses in vitro. The anti-inflammatory effects of C12 contribute to PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24377