Anti-inflammatory effect of procumbenoside B from Justicia spicigera on lipopolysaccharide-stimulated RAW 264.7 macrophages and zebrafish model

Background: Justicia spicigera is widely used in the Mexican traditional medicine for the treatment of inflammation. Objectives: The purpose of this study was to investigate the anti-inflammatory effect of procumbenoside B (PB) from J. spicigera on pro-inflammatory mediators. Materials and Methods: Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and Zebrafish model were used to assess the potential anti-inflammatory effects of PB; its structure was elucidated on the basis of spectroscopic data. The production of inflammatory mediators such as interferon-β, prostaglandin E2, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB) p65, interleukin-6 (IL-6), IL-1β, IL-12, cyclooxygenase (COX-2), tumor necrosis factor-α, and anti-inflammatory IL-10 was measured using enzyme-linked immunosorbent assay. NO production was measured using Griess reagent. Results: In LPS-induced-inflammatory response in RAW264.7 macrophage cells, PB strongly inhibited secretion of all pro-inflammatory mediators test and increased the production of IL-10 and blockade of NF-κB. In addition, PB suppressed LPS-stimulated nitric oxide and reactive oxygen species generation in a zebrafish model. Conclusion: These results indicated that the anti-inflammatory effects of PB may be attributed to the downregulation of iNOS and COX-2 through the suppression of NF-κB signaling pathway in RAW264.7 macrophages. Abbreviations used: 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM-DA), Cyclooxygenase-2 (COX-2), 2,7-dichlorofluorescein (DCH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethylsulphoxide (DMSO), Dulbecco's modified eagle's medium (DMEM), Fetal bovine serum (FBS), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interferon-β (IFN β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-1B (IL-1b), Layer chromatography (TLC), Lipopolysaccharide (LPS), Matrix metalloproteinases-3 (MMP-3), Matrix metalloproteinases-13 (MMP-13), Nitric oxide (NO), Nitric oxide synthase (iNOS), Nuclear factor-kappa B (NF-βB), Prostaglandin E2 (PGE2), Reactive oxygen species (ROS), Tumor necrosis factor-β (TNF-β).