Figure 4

Fisetin inhibits LPS-induced inflammatory response in zebrafish larvae. Zebrafish larvae at 1 day post fertilization (dpf) were cultured in 0.003% PTU containing E3 embryo media. Briefly, 2 nL of 0.5 mg/mL LPS was microinjected into the yolk at 3 dpf. Zebrafish larvae were immediately immersed in E3 embryo media containing different concentrations of fisetin. (A) In LPS-microinjected conditions, 10 zebrafish were euthanized at the indicated time points and subjected to RT-PCR for evaluating the expression of iNOS, COX-2a, IL-6, and TNF-α. (B) At 18 h post injection (hpi), 20 zebrafish larvae from each treatment were euthanized and the expression of iNOS, COX-2a, IL-6, and TNF-α was measured by RT-PCR. (C) Neutral red staining of macrophages and (D) sudan black staining of the neutrophils were performed at 24 hpi. Each value indicates the mean ± standard error median (SEM) and is representative of the results obtained from three independent experiments. Significant differences among the groups were determined using the Student’s t-test (^###p < 0.001, ^##p < 0.01, and ^#p < 0.05 vs. untreated zebrafish larvae) and one-way ANOVA with Bonferroni correction (***p < 0.001, **p < 0.01, and *p < 0.05 vs. LPS-treated zebrafish larvae).