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.

Figure 1. LPS-enhanced leukocyte migration assay in 4 dpf zebrafish larvae.

All larvae (nacre) are four days post-fertilization (4 dpf), with anterior to the left, scale bar = 10 µm. A, tail of alive larva without tail cut; B, tail of alive larva with tail cut; C–D, whole-mount MPO staining in uncut tails of zebrafish larvae; C, without lipopolysaccharides (−LPS) and D, with lipopolysaccharides (+LPS); E–F, whole-mount MPO staining in cut tails of zebrafish larvae; E, without the inclusion of LPS; F, with the inclusion of LPS. Dark-spots (marked by arrows) represent the migrating leukocytes, which are semi-quantified in the region to the right of the dashed red arc.

Figure 2. Scoring of leukocyte migration in the transected tails of zebrafish larvae.

All larvae (nacre) are four days post-fertilization (4 dpf), with anterior to the left, scale bar = 10 µm. Dark spots (marked by arrows) represent leukocytes migrating to the injured zone in the transected tails. Migrating leukocytes were counted in the region to the right of the dashed red arc. A, tail of an uncut larva; B, tail-cut with score 0; C, tail-cut with score 1; D, tail-cut with score 2; E tail-cut with score 3; F, tail-cut with score 4. ^aExperimental values obtained for each larvae are normalized to a relative value expressed as relative leukocyte migration (RLM); +LPS: with inclusion of lipopolysaccharides. ^bPercentage of anti-inflammatory activity is obtained as (1−RLM)x100.

Figure 3. Inhibition of the leukocyte migration by known anti-inflammatory drugs and non anti-inflammatory compounds.

After exposing the larvae to mechanical and biological damages, anti-inflammatory drugs and non anti-inflammatory compounds were evaluated for their activity in the LPS-enhanced leukocyte migration assay. Migrating MPO-positive cells were counted in the tail tip and the results expressed as relative leukocyte migration (RLM) values. Results for NSAIDs (upper panel) and SAIDs (middle panel) show a significant inhibition of leukocytes migration in a concentration-dependent manner while non anti-inflammatory drugs (lower panel) indicate no significant effect on leukocyte migration. In each case, values are plotted as RLM (SEM, n = 3 replicates with 10 larvae each) and a value of 0.5 was established as a cut-off for anti-inflammatory activity. ***p<0.001, **0.001

Figure 4. Anti-inflammatory effect of aqueous and methanolic plan extracts.

Anti-inflammatory activity of aqueous and methanolic plant extracts was evaluated in the LPS-enhanced leukocyte migration assay. A concentration-dependent inhibitory trend was observed for Capraria biflora (A), Chamaemelum nobile (B), Harpagophytum procumbens (C), and Schefflera chimbuensis (D). Values are plotted as relative leukocyte migration, RLM (SEM, n = 3 replicates with 10 larvae each) and a value of 0.5 was established as a cut-off for anti-inflammatory activity. ***p<0.001, **0.001<p<0.01, *0.01<p<0.05.