Nitric oxide synthase regulates coelomocytes apoptosis through the NF-κB signaling pathway in the sea cucumber Apostichopus japonicus

Abstract

Nitric oxide synthase (NOS) was initially discovered to participate in the generation of nitric oxide as a defense mechanism against pathogenic infections. In recent years, it has been found that NOS plays a pivotal role in regulating apoptosis and inflammation in mammals. However, the mechanisms underlying NOS-mediated apoptosis in invertebrates remain largely unclear. In this study, we found that the Apostichopus japonicus NOS (AjNOS) expression levels were upregulated by 2.20-fold and 3.46-fold after being challenged with Vibrio splendidus at concentrations of 107 CFU mL−1 and 108 CFU mL−1 for 12 h compared to the control group, respectively. Under these conditions, the rates of coelomocytes apoptosis were increased from 14.7% to 32.7% and 45.4%, respectively. Treatment with NOS inhibitor (l-NAME) resulted in a reduction of coelomocytes apoptosis rates from 32.6% to 26.5% in V. splendidus (107 CFU mL−1) groups and from 42.3% to 33.3% in V. splendidus (108 CFU mL−1) groups, respectively. NOS has been reported to regulate apoptosis through IκBα phosphorylation. Simultaneously, exposure to V. splendidus in conjunction with l-NAME resulted in down-regulation of AjIκBα phosphorylation levels compared to the group infected solely with V. splendidus. Furthermore, immunofluorescence analysis revealed that treatment with l-NAME or interference of AjNOS using siRNA inhibited translocation of AjNF-κB/p65 (RelA) into the nucleus. Previous studies have shown that NF-κB can down-regulate expression levels of Bcl-2 family members, which is an important pathway for regulating apoptosis. In the present study, treatment with l-NAME was found to promote anti-apoptotic AjBcl-2 mRNA increase to 1.41-fold and protein expression increase to 1.86-fold at 12 h post V. splendidus challenge. However, these effects were suppressed by PMA (an NF-κB activator). Overall, our findings demonstrate that AjNOS regulates coelomocytes apoptosis induced by V. splendidus through activation of the AjNF-κB signaling pathway and down-regulation of AjBcl-2 in A. japonicus.