title: Dilodendron bipinnatum Radlk. inhibits pro-inflammatory mediators through the induction of MKP-1 and the down-regulation of MAPKp38/JNK/NF-κB pathways and COX-2 in LPS-activated RAW 264.7 cells.
abstract: BACKGROUND
  The stem bark of Dilodendron bipinnatum Radlk. (Sapindaceae), a tree native to Pantanal of Mato Grosso, Brazil, popularly known as "mulher-pobre" (poor woman), has been historically used by the locals, after decoction and maceration, for the treatment of inflammatory conditions. We have recently shown that these preparations indeed possess anti-inflammatory properties, which are mediated by the inhibition of cell migration and the modulation of Th1 and Th2 cytokines. The NO pathway was not affected.
  The aim of the present study was to further investigate the mechanisms responsible for the anti-inflammatory properties of the hydroethanolic extract of the stem bark of Dilodendron bipinnatum (HEDb).
  
  
  METHODS
  HEDb was obtained by maceration of the stem bark of D. bipinnatum as previously described. The corresponding effects on a macrophage-like cell line, RAW 264.7, were investigated. The apoptosis of RAW 264.7 upon treatment with LPS, HEDb and N-acetyl-L-cysteine (NAC) was assessed by flow cytometry, using an Annexin V-PE kit. The production of inflammatory cytokines (TNF-α, IL-1β and IL-10) and PGE2were evaluated by ELISA, after cell challenge with LPS. The intracellular redox state and changes in mitochondrial membrane potential were also assessed by flow cytometry, using DCFH-DA and JC-1 as probes. The protein expression levels of MAPK p-p38, p-ERK, p-JNK, MKP-1 and COX-2 were analysed by western blotting. Nuclear translocation of NF-қB was assessed by immunofluorescence microscopy. The quantified results are presented as a nuclear:cytoplasmic ratio.
  
  
  RESULTS
  LPS, HEDb and NAC did not appear to decrease the number of viable cells in comparison to control treatment. HEDb attenuated the production of pro-inflammatory cytokines (IL-1β and TNF-α) and PGE2induced by LPS but did not affect IL-10. The production of ROS was also inhibited by HEDb (1, 5 or 20µg/mL), even at the lowest concentration; at 20µg/mL, HEDb was more effective than NAC, which was used as a positive control (74.1% and 66.2% inhibition of LPS's effect, respectively). LPS induced an increase in ΔΨm (19.2%, p<0.001), while HEDb inhibited the change in ΔΨm (7.7% at 20µg/mL, p<0.001). The results of western blotting showed that HEDb inhibited the expression of MAPK p-p38, p-JNK and COX-2, while the expression of MKP-1 was increased. p-ERK was not affected. LPS promoted the nuclear translocation of NF-қB p65 (67%, p<0.01) in RAW 264.7 cells, in comparison to baseline (33%). Pre-treatment with HEDb inhibited this translocation of NF-κB p65 (58% at 20µg/mL, p<0.001).
  
  
  CONCLUSIONS
  HEDb has a potent anti-inflammatory activity and acts on multiple targets and biological pathways of potential therapeutic relevance.
authors.affiliation: Department of Basic Health Sciences, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Coxipó, Cuiabá, Mato Grosso 78060-900, Brazil; Faculdade de Farmácia, Universidade de Cuiabá, Avenida Beira Rio, no. 3100, CEP: 78025-190, Cuiabá, MT, Brazil.
authors.forename: Ruberlei Godinho
authors.initials: RG
authors.lastname: de Oliveira
authors.affiliation: Department of Basic Health Sciences, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Coxipó, Cuiabá, Mato Grosso 78060-900, Brazil.
authors.forename: Geovane Roberto
authors.initials: GR
authors.lastname: de Campos Castilho
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Unifesp, São Paulo, SP 04044-020, Brazil.
authors.forename: André Luiz
authors.initials: AL
authors.lastname: da Cunha
authors.affiliation: Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3GL, UK; Group of Neuropharmacology, Drug Research and Development Center, Federal University of Ceara (NPDM-UFC), Fortaleza, CE, Brazil.
authors.forename: Fábio
authors.initials: F
authors.lastname: Miyajima
authors.affiliation: Department of Basic Health Sciences, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Coxipó, Cuiabá, Mato Grosso 78060-900, Brazil. Electronic address: taba@terra.com.br.
authors.forename: Domingos Tabajara
authors.initials: DT
authors.lastname: de Oliveira Martins
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D001938:Brazil; D016207:Cytokines / Q000096:biosynthesis; D015232:Dinoprostone / Q000096:biosynthesis; D006801:Humans; D018836:Inflammation Mediators / Q000037:antagonists & inhibitors*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008519:Medicine, Traditional; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects; D024301:Plant Bark / Q000737:chemistry; D010936:Plant Extracts / Q000494:pharmacology*; D018547:Plant Stems / Q000737:chemistry; D000067996:RAW 264.7 Cells; D029631:Sapindaceae / Q000737:chemistry*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000975:Antioxidants; D016207:Cytokines; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D010936:Plant Extracts; D015232:Dinoprostone