Grass carp (Ctenopharyngodon idella) NRF2 alleviates the oxidative stress and enhances cell viability through upregulating the expression of HO-1

摘要

As a member of the Cap 'n' Collar (CNC) family, NRF2 contains a basic leucine zipper (bZip) and can regulate the downstream target gene heme oxygenase 1 (HO-1) in response to oxidative stress. In the present study, a grass carp (Ctenopharyngodon idella) NRF2 ORF was cloned and identified. The largest ORF (1782 bp) encodes a polypeptide of 593 amino acids. The deduced amino acid sequence of grass carp NRF2 (CiNRF2) contains a well-conserved DNA-binding domain (BRLZ domain). Phylogenetic tree analysis revealed that CiNRF2 has a closer evolutionary relationship with other fish counterparts. After CIK (C. idellus kidney) cells were persistently stimulated with tunicamycin (TM), CiNRF2 was significantly upregulated from 12 to 36 h. Then, the expression was dropped at 48 h post-infection. Additionally, when TM or TG (thapsigargin) stimulated CIK cells, overexpression of CiNRF2 in cells downregulated the expression of Bip mRNA, a marker protein of oxidative stress, suggesting that fish NRF2 can alleviate the oxidative stress level induced by TM or TG. To study the protective mechanism of fish NRF2, the DNA sequences of CiNRF2 and CiATF4 (grass carp ATF4) were separately sub-cloned into the expression vectors pEGFP and pCMV-Flag for co-immunoprecipitation and GST pull-down assays. These assays showed that CiNRF2 can combine with CiATF4 through its Neh1 domain. Meanwhile, we cloned grass carp HO-1 promoter sequence and constructed the recombinant plasmid of pGL3-HO-1. Soon afterwards, pGL3-HO-1 was co-transfected into grass carp ovary (CO) cells with pcDNA3.1-CiNRF2 or pcDNA3.1-CiATF4, respectively. The results showed that the luciferase activity of pGL3-HO-1 in the overexpressed CiNRF2 plus CiATF4 cells was significantly increased, along with the increase of cell viability ( 133%). However, when HO-1 was knocked down in cells, CiNRF2 was unable to perform its function. These results demonstrated that CiNRF2 was effective in protecting grass carp against the oxidative stress induced by TM and increasing cell viability by upregulating HO-1 expression.