Potent anti-inflammatory activity of betulinic acid treatment in a model of lethal endotoxemia José Fernando Oliveira Costa a , José Maria Barbosa-Filho b , Gabriela Lemos de Azevedo Maia b , Elisalva Teixeira Guimarães a,c , Cássio Santana Meira a , Ricardo Ribeiro-dos-Santos a,d , Lain Carlos Pontes de Carvalho a , Milena Botelho Pereira Soares a,d, ⁎ a Laboratory of Tissue Engineering and Immunopharmacology Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Rua Waldemar Falcão, 121, 40296–750, Salvador, Bahia, Brazil b Laboratory of Pharmaceutical Technology, Federal University of Paraíba, João Pessoa, Cidade Universitária, s/n, 58051–900, João Pessoa, PB, Brazil c Department of Life Sciences, State University of Bahia, Rua Silveira Martins, 2555, 41150–000, Salvador, BA, Brazil d Center of Biotecnology and Cell Therapy, São Rafael Hospital, Av. São Rafael, 2152. São Marcos 41253–190, Salvador, BA, Brazil abstract article info Article history: Received 24 February 2014 Received in revised form 4 September 2014 Accepted 17 September 2014 Available online xxxx Keywords: Betulinic acid Endotoxemia Anti-inflammatory activity Macrophages Cytokines Betulinic acid (BA) is a lupane-type triterpene with a number of biological activities already reported. While potent anti-HIV and antitumoral activities were attributed to BA, it is considered to have a moderate anti- inflammatory activity. Here we evaluated the effects of BA in a mouse model of endotoxic shock. Endotoxemia was induced through intraperitoneally LPS administration, nitric oxide (NO) and cytokines were assessed by Griess method and ELISA, respectively. Treatment of BALB/c mice with BA at 67 mg/kg caused a 100% survival against a lethal dose of lipopolysaccharide (LPS). BA treatment caused a reduction in TNF-α production induced by LPS but did not alter IL-6 production. Moreover, BA treatment increased significantly the serum levels of IL-10 compared to vehicle-treated, LPS-challenged mice. To investigate the role of IL-10 in BA-induced protection, wild-type and IL-10 -/- mice were studied. In contrast to the observations in IL-10 +/+ mice, BA did not protect IL-10 -/- mice against a lethal LPS challenge. Addition of BA inhibited the production of pro-inflammatory mediators by macrophages stimulated with LPS, while promoting a significant increase in IL-10 production. BA-treated peritoneal exudate macrophages produced lower concentrations of TNF-α and NO and higher con- centrations of IL-10 upon LPS stimulation. Similarly, macrophages obtained from BA-treated mice produced less pro-inflammatory mediators and increased IL-10 when compared to non-stimulated macrophages obtained from vehicle-treated mice. In conclusion, we have shown that BA has a potent anti-inflammatory activity in vivo, protecting mice against LPS by modulating TNF-α production by macrophages in vivo through a mechanism dependent on IL-10. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid; BA), a C-30 carboxylic acid derivative of the ubiquitous triterpene betulin, is a member of the class of lupane-type triterpenes. The molecule is abundant in the plant kingdom and has been isolated from several plant species, including Zizyphus joazeiro [1], Syzigium clariflorum [2], and Doliocarpus schottianus [3]. A number of reports have shown diverse biological activities of BA, such as anticancer [4], anti-HIV, anti-HSV-1 [5], anti-HBV [6], antihelmintic [6], antinociceptive [7], and antiplasmodial [1,8]. Of particular interest, in view of the large prevalence of chronic inflammatory-degenerative diseases, is the BA anti-inflammatory activity [9,10]. However, in a number of in vitro and in vivo models of inflammation, the intensity of the BA anti-inflammatory activity has been considered only moderate (reviewed by [5]). Sepsis is one of the most frequent complications in surgical patients and one of the leading causes of mortality in intensive care units. Severe sepsis is an important cause of mortality worldwide, and is estimated as directly responsible for 9% of all deaths in the United States [11]. It is de- fined as an infection-induced syndrome characterized by a generalized inflammatory state and can be caused by infection with Gram- negative or Gram-positive bacteria, fungi, or viruses. Sepsis can also occur in the absence of detectable bacterial invasion and, in these cases, microbial toxins (lipopolysaccharide; LPS) and endogenous cytokines have been implicated as initiators and mediators of the condition [12]. Macrophage activation by LPS results in the release of several inflammatory mediators, including proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-8, and IL-12, International Immunopharmacology xxx (2014) xxx–xxx ⁎ Corresponding author at: Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, 121 – Candeal, Salvador, BA, 40296–710. Tel.: +55 71 3176 2260; fax: +55 71 3176 2272. E-mail address: milena@bahia.fiocruz.br (M.B.P. Soares). INTIMP-03412; No of Pages 6 http://dx.doi.org/10.1016/j.intimp.2014.09.021 1567-5769/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Please cite this article as: Oliveira Costa JF, et al, Potent anti-inflammatory activity of betulinic acid treatment in a model of lethal endotoxemia, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.09.021