Cytoprotective roles of “E3 ubiquitin ligases-NF-κB-autophagy” axis in Pacific oysters Crassostrea gigas exposed to phenanthrene

Abstract Phenanthrene (PHE), as one of the most frequently found polycyclic aromatic hydrocarbons can induce immunotoxicity, oxidative stress, and endocrine disruption in marine organisms. However, whether autophagy can be induced by PHE and the regulatory mechanism and cytoprotective roles of autophagy under PHE stress condition have not been unveiled. Our data first unveil a “E3 ubiquitin ligases-NF-κB-autophagy” axis, which play cytoprotective roles in Pacific oysters Crassostrea gigas exposed to PHE. The results of confocal laser scanning microscope, flow cytometry and transmission electron microscope confirmed that PHE could induce autophagy in the haemocytes of Pacific oysters, and the presence of autophagosomes was also confirmed. The proteomics results showed that the expression of the E3 ubiquitin ligase HUWE1, TRIM36, and autophagy-related protein 7 (ATG7) were significantly upregulated. The expression of genes of the “axis” were significantly upregulated, and the expression of genes of autophagy was downregulated after the inhibition of the NF-κB, indicating that the expression of the “axis”-related genes can be stimulated by PHE, and thus autophagy is activated. The upregulation of the expression of “axis”-related genes in mouse macrophages, further demonstrating the existence of the “axis” proposed by this study and the “axis” can be activated by PHE. Incorporating with changes of cell number, apoptosis rate, phagocytic capacity, and ROS levels of lymphocytes, we demonstrated that autophagy plays a cytoprotective role in cellular defence against PHE. This study proposed a novel pathway and supplied a comprehensive understanding of the protective role of autophagy in Pacific oysters to cope with pollutants. Competing Interest Statement The authors have declared no competing interest. Abbreviations - PAHs - Polycyclic aromatic hydrocarbons - POPs - persistent organic pollutant - AMPK - AMP-activated protein kinase - PI3K - phosphatidylinositol 3-kinase - KK - IκB kinase - κB - NF-κB inhibitor - UPS - ubiquitin-proteasome system - ALP - autophagy-lysosomal pathway - CLSM - confocal laser scanning microscopy - GO - Gene Ontology - THC - total number of blood lymphocyte - C. gigas - Pacific oysters - DMEM - Dulbecco’s modified Eagle’s medium - PBS - phosphate-buffered saline - CAPE - caffeic acid phenyl ether - FITC - fluorescein isothiocyanate - PR - phagocytic rate.