title: Optimization and pharmacological validation of a leukocyte migration assay in zebrafish larvae for the rapid in vivo bioactivity analysis of anti-inflammatory secondary metabolites.
abstract: Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts.
authors.affiliation: Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Leuven, Belgium ; Faculty of Chemical Sciences, School of Biochemistry and Pharmacy, University of Cuenca, Cuenca, Ecuador ; Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
authors.forename: María Lorena
authors.initials: ML
authors.lastname: Cordero-Maldonado
authors.forename: Dany
authors.initials: D
authors.lastname: Siverio-Mota
authors.forename: Liliana
authors.initials: L
authors.lastname: Vicet-Muro
authors.forename: Isabel María
authors.initials: IM
authors.lastname: Wilches-Arizábala
authors.forename: Camila V
authors.initials: CV
authors.lastname: Esguerra
authors.forename: Peter A M
authors.initials: PA
authors.lastname: de Witte
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Crawford
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D054441:Cell Migration Assays, Leukocyte / Q000379:methods*; D007249:Inflammation / Q000139:chemically induced / Q000188:drug therapy*; D007814:Larva / Q000166:cytology* / Q000187:drug effects; D008070:Lipopolysaccharides / Q000633:toxicity; D015027:Zebrafish
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides