In vitro and in vivo anti-inflammatory effects of IMMLG5521, a coumarin derivative Zhipeng Li, Jinfeng Hu, Mingna Sun, Xiuyun Song, Gang Li, Yan Liu, Guangyan Li, Haijie Ji, Gang Liu, Naihong Chen ⁎ State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China abstract article info Article history: Received 19 March 2013 Received in revised form 3 June 2013 Accepted 3 June 2013 Available online 10 July 2013 Keywords: Polymorphonuclear leukocytes β-Glucuronidase Inflammation Swelling Granuloma Several coumarin derivatives have been reported to present multiple biological activities. In this study, in vitro the compound IMMLG5521 (0.1, 1, 10 μM) can inhibit the release of β-glucuronidase from PAF- stimulated polymorphonuclear leukocytes, the compound IMMLG5521 (0.1, 1, 10 μM) can inhibit NO pro- duction and decrease TNF-α and IL-β release from LPS-stimulated RAW264.7 cells. In vivo, we evaluated the effect of IMMLG5521 on acute and chronic inflammation models. Our data showed that IMMLG5521 (6 mg/kg, 12 mg/kg) could inhibit xylene-induced ear swelling and cotton pellet-induced granuloma forma- tion in mice. Taken together, the compound IMMLG5521 inhibited the release of inflammatory factors and mediators in vitro, decreased inflammation response in mice. The compound IMMLG5521 can inhibit inflam- mation in vitro and in vivo. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Coumarins comprise a very large class of compounds which were naturally found in plants [1]. Simple coumarins and analogs are a large class of compounds exhibiting a wide spectrum of pharmaco- logical activities. It has been already reported that several coumarin derivatives have significantly antiinflammatory activities [2–5]. We have already reported that 3-piperazine substituted coumarin compound 41 could reduce asthmatic pathologic changes in lung tissue of human CKLF1-transfected mice [6]. IMMLG5521 is a 3-piperazine substituted coumarin compound. We have reported that the coumarin compound IMMLG5521 inhibited inflammation on carrageenan- induced pleurisy and suppressed sephadex-induced lung inflammation in rats [7,8]. In this study, we examined the potential effect of IMMLG5521 in vitro and in vivo. We examined the effect of IMMLG5521 on polymorphonuclear leukocytes and RAW264.7 cell acti- vation in vitro. The xylene-induced ear swelling model and cotton pellet-induced granuloma model were adopted to evaluate compound IMMLG5521 in mice. 2. Materials and methods 2.1. Chemicals The compound IMMLG5521 was synthesized in our laboratory. The compound IMMLG5521 was suspended in 0.5% sodium carboxymethyl cellulose and administered p.o. at a volume of 10 mL/kg. The compound IMMLG5521 structure is shown in (Fig. 1). 2.2. PAF-induced polymorphonuclear leukocytes activation The suspension of rat polymorphonuclear leukocytes (245 μL) at a density of 2.5 × 10 6 cells mL -1 and compound IMMLG5521 (0.1, 1,10 μM) were incubated at 37 °C for 20 min. 2.5 μL of 1 mM cytochala- sin B was added for further 5 min, followed by 0.2 μM PAF. After 10 min, cells were placed immediately on ice to terminate any further reaction. The supernatant was obtained by centrifugation at 4000 rpm for 5 min. Then, 25 μL of the supernatant and 2.5 mM phenolphthalein glucuronic acid (25 μL) were incubated with 100 μL of 0.1 M acetic acid buffer (pH 4.6) at 37 °C for 18 h. The reaction was stopped by addition of 0.3 M NaOH (150 μL). The absorbance was read at 550 nm. 2.3. LPS-induced RAW264.7 cells activation 2.3.1. Cell culture and treatment The RAW264.7 cell line was grown at 37 °C in DMEM medium supplemented with 10% FBS in a humidified atmosphere of 5% CO 2 . International Immunopharmacology 17 (2013) 400–403 ⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No.2 Nanwei road, Beijing 100050, China. Tel./fax: +86 10 63165177. E-mail address: chennh@imm.ac.cn (N. Chen). 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.06.007 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp