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http://dspace.ucuenca.edu.ec/handle/123456789/22105
Title:
Optimization and pharmacological validation of a leukocyte migration assay in zebrafish larvae for the rapid In vivo bioactivity analysis of anti-inflammatory secondar metabolitesy
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 antiinflammatory
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 antiinflammatory
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 bioassayguided
isolation of anti-inflammatory secondary metabolites from complex NP extracts.
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