AUTHOR=Pan Mingzhu , Liu Jiahuan , Huang Dong , Guo Yanlin , Luo Kai , Yang Mengxi , Gao Weihua , Xu Qiaoqing , Zhang Wenbing , Mai Kangsen 

TITLE=FoxO3 Modulates LPS-Activated Hepatic Inflammation in Turbot (Scophthalmus maximus L.)

JOURNAL=Frontiers in Immunology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.679704

DOI=10.3389/fimmu.2021.679704

ISSN=1664-3224

ABSTRACT=<p>In mammals, forkhead box O3 (<italic>foxo3</italic>) plays important roles in liver immune system. The <italic>foxo3</italic> can regulate cell cycle, DNA repair, hypoxia, apoptosis and so on. However, as such an important transcription factor, few studies on <italic>foxo3</italic> in fish have been reported. The present study characterized the <italic>foxo3</italic> in turbot (<italic>Scophthalmus maximus</italic> L.). Lipopolysaccharide (LPS) incubated <italic>in vitro</italic> (hepatocytes) and injected <italic>in vivo</italic> (turbot liver) were used to construct inflammatory models. The <italic>foxo3</italic> was interfered and overexpressed to investigate its functions in liver inflammation. The open reading frame (ORF) of <italic>foxo3</italic> was 1998 bp (base pair), encoding 665 amino acids. Sequence analysis showed that <italic>foxo3</italic> of turbot was highly homologous to other fishes. Tissue distribution analysis revealed that the highest expression of <italic>foxo3</italic> was in muscle. Immunofluorescence result showed that <italic>foxo3</italic> was expressed in cytoplasm and nucleus. Knockdown of <italic>foxo3</italic> significantly increased mRNA levels of tumor necrosis factor-α (<italic>tnf-α</italic>), interleukin-1β (<italic>il-1β)</italic>, interleukin-6 (<italic>il-6)</italic>, myeloid-differentiation factor 88 (<italic>myd88)</italic>, <italic>cd83</italic>, toll-like receptor 2 (<italic>tlr-2)</italic> and protein level of c-Jun N-terminal kinase (JNK) in si<italic>foxo3</italic> + LPS (siRNA of <italic>foxo3</italic>+ LPS) group compared with NC + LPS (negative control + LPS) group in turbot hepatocytes. Overexpressed <italic>foxo3</italic> significantly decreased mRNA levels of <italic>tnf-α</italic>, <italic>il-6</italic>, nuclear transcription factor-kappa B (<italic>nf-κb)</italic>, <italic>cd83</italic>, <italic>tlr-2</italic> and the protein level of JNK <italic>in vitro</italic>. In <italic>vivo</italic> analysis, <italic>foxo3</italic> knockdown significantly increased levels of GOT in serum after LPS injection compared with NC+LPS group. Overexpressed <italic>foxo3</italic> significantly decreased levels of GPT and GOT in pcDNA3.1-<italic>foxo3</italic>+LPS group compared with pcDNA3.1+LPS group <italic>in vivo</italic>. <italic>Foxo3</italic> knockdown significantly increased mRNA levels of <italic>tnf-α</italic>, <italic>il-1β</italic>, <italic>il-6</italic>, <italic>nf-κb</italic>, <italic>myd88</italic> and protein level of JNK <italic>in vivo</italic> in si<italic>foxo3</italic>+LPS group compared with NC+LPS group in turbot liver. Overexpressed <italic>foxo3</italic> significantly decreased mRNA levels of <italic>il-1β</italic>, <italic>il-6</italic>, <italic>myd88</italic>, <italic>cd83, jnk</italic> and protein level of JNK in pcDNA3.1-<italic>foxo3</italic>+LPS group compared with pcDNA3.1+LPS group in turbot liver. The results indicated that <italic>foxo3</italic> might modulate LPS-activated hepatic inflammation in turbot by decreasing the proinflammatory cytokines, the levels of GOT and GPT as well as activating JNK/caspase-3 and <italic>tlr-2/myd88/nf-κb</italic> pathways. Taken together, these findings indicated that FoxO3 may play important roles in liver immune responses to LPS in turbot and the research of FoxO3 in liver immunity enriches the studies on immune regulation, and provides theoretical basis and molecular targets for solving liver inflammation and liver injury in fish.</p>