Epigallocatechin-3-Gallate suppresses neutrophil accumulation and the IL-1 beta pathway in a transgenic zebrafish model of inflammation

Abstract

Abstract Background The zebrafish represents the simplest and most convenient vertebrate model for undertaking large screens because of its small size, rapid reproduction time and transparency of the body. Here, we adopt a well-established model of inflammation in zebrafish to assess the impact of Epigallocatechin-3-Gallate (EGCG), the main catechin in green tea. Method Tg (MPX:GFP) or Tg (MPX:mCHERRY) transgenic zebrafish were used, bearing GFP- or mCherry-tagged neutrophils, respectively. 4dpf embryos were anesthetized, and inflammation was induced by sterile tailfin-resection. The inflamed groups were separated into those that were non-treated or exposed to EGCG from 0 – 600 uM for 6 hours. 10 fish per group were then mounted alive in methyl cellulose for wide-field fluorescence imaging and analysis was done using ImageJ. Animation of neutrophil migration or accumulation was recorded using an inverted fluorescence confocal microscope till 6-hours after resection. To examine the mechanistic basis of EGCG’s effect, 37 zebrafish inflammatory genes were studied using qPCR. Results The static accumulation of neutrophils at the resection site was highly elevated after wounding (n=32, p<0.0001), and this accumulation was reduced significantly by 31% and 43%, respectively, after exposure to EGCG 300 uM (p<0.05) and 600 uM (p<0.01). Dynamic neutrophil tracking studies are in progress. qPCR studies revealed 6 signature genes in the IL-1 beta pathway to be significantly down-regulated at least 2-fold after EGCG treatment. Conclusion EGCG suppresses inflammation in zebrafish through similar biochemical pathways as in other species. This novel animal model allows facile screening of inflammatory modulators in real-time.