Mechanistic action of the acute toxicity of Bajitian (Morinda officinalis) in zebrafish embryos

Objectives: To investigate acute toxicity of Bajitian (Morinda officinalis) in zebrafish embryos. 
Methods: Zebrafish embryos at 48-h post fertilization (hpf) were exposed to Bajitian ethanol extract for 72 h. The causative action of a delay in yolk sac absorption by Bajitian was investigated by RT-PCR analysis of lipid metabolism-related microsomal triglyceride transfer protein (MTP), apolipoprotein C-II (ApoC2) and lipogenesis-related liver x receptor (LXR) genes. The effect of Bajitian eliciting an inflammatory response was studied by exposing 72 hpf myeloperoxidase (MPO): GFP transgenic zebrafish embryos to Bajitian extract for 4 h. Assessment was done by TUNEL, caspase-3/7, and RT-PCR analysis of the apoptosis related pathway B-cell lymphoma 2 associated X protein (Bax), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) genes, neutrophil development-related stem cell leukaemia (SCL) and transcription factor PU.1 genes, to reveal the causative action of Bajitian reducing neutrophils. 
Results: RT-PCR analysis found that Bajitian extract had no effect on the expression of MTP or ApoC2 genes, but upregulated LXR gene, which might explain the delay in yolk sac absorption. Analysis of the inflammatory response showed that compared with negative controls, Bajitian extract significantly (P < .05) reduced the number of neutrophils in MPO: GFP embryos. TUNEL, caspase-3/7, and RT-PCR analysis of Bax and NF-κB genes found that Bajitian extract did not trigger the cell apoptosis. Further RT-PCR analysis found that Bajitian extract did not affect SCL expression, but did lead to down-regulation of PU.1. The inhibition of neutrophil development/differentiation may explain the decline in the total number of neutrophils following Bajitian treatment, which could be attributed to the anti-inflammatory effects found clinically for this drug. 
Conclusions: This study demonstrated that Bajitian caused a delay in yolk sac absorption and a decrease neutrophil in zebrafish embryos, which may be related to the inhibition of neutrophil development.