The defensive effect of phellodendrine against AAPH-induced oxidative stress through regulating the AKT/NF-κB pathway in zebrafish embryos

This study is to investigate the effect of phellodendrine (PHE) against AAPH-induced oxidative stress and find out the biological mechanism of PHE by using the zebrafish embryo model. After treatments by AAPH or PHE, the mortality and heartbeat of zebrafish embryos were recorded and the production of reactive oxygen species (ROS), lipid-peroxidation and the rate of cell death were detected by fluorescence spectrophotometry respectively. Whereafter, the pathways of PHE against AAPH-induced oxidative stress were screened by inhibitors to explore its biological mechanism. The related genes and proteins expressions were analyzed by real-time quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) and western blotting. The PHE obviously improved the decreased survival rate and abnormally elevated heart-beating rate of zebrafish embryos caused by AAPH. Especially 200 μg/mL of PHE make the survival rate increased to 90.26 ± 1.40% at 72 hfp and the heartbeat back to normal. Besides, AAPH caused a significant increase in the production of reactive oxygen species (ROS), lipid-peroxidation and cell death rate, all of which could be decreased after PHE treatment dose-dependently. And PHE exerted the protective activity against AAPH-induced oxidative stress through down-regulating AKT phosphorylation and NF-kB3 expression, which associate with modulation of IKK phosphorylation in zebrafish embryos. The PHE showed a good antioxidant effect in vivo, and the mechanism has been stated that the PHE can down-regulating AKT, IKK, NF-kB phosphorylation and COX-2 expression induced by AAPH. Moreover, the PHE also ameliorated the ROS-mediated inflammatory response.