Investigation of the Nrf2-Keap1 signaling pathway's response to oxidative stress in Meretrix meretrix

The Keap1-Nrf2 signaling pathway primarily regulates cytoprotective responses to oxidative and electrophilic stress. This pathway involves two key proteins: Nuclear factor erythroid 2-related factor 2 (Nrf2), a crucial regulator of cellular defenses, and Kelch ECH associating protein 1 (Keap1), a repressor protein. Keap1 interacted with Nrf2, leading to the degradation of Nrf2 via the ubiquitin-proteasome pathway. In this study, we cloned these proteins from Meretrix meretrix, obtaining full-length cDNA sequences of 2172 bp and 1791 bp. Sequence alignment and phylogenetic analysis revealed that Nrf2 and Keap1 were highly conserved among Molluscan species, yet distinct in M. meretrix. MmNrf2 and MmKeap1 were expressed in multiple tissues, with the highest expression levels in the digestive gland and gill, respectively. We conducted RNA interference experiments to analyze the mRNA transcription profiles of key genes in the Keap1-Nrf2 signaling pathway (Nrf2, Keap1, Maf, p62), xenobiotic metabolism genes (CYP3A3 and Pgp), and antioxidant and detoxification genes (SOD, NQO1, CAT, and GCLM). We also measured ROS, T-AOC, malondialdehyde (MDA), and GSH levels in the digestive gland after exposure to 10 μg/L benzo[a]pyrene (BaP) for 6 h. Results showed a 60.95% decrease in MmNrf2 mRNA levels. Changes in the Keap1-Nrf2 signaling pathway and BaP-induced oxidative stress gene expression aligned with MmNrf2, suggesting its important role in activating antioxidant and detoxification genes. Additionally, ROS, T-AOC, and GSH levels in the digestive gland significantly increased after 10 μg/L BaP exposure but decreased 6 h post-siNrf2 injection. We also evaluated the effects of 10 μM Sulforaphane on the Keap1-Nrf2 signaling pathway and BaP-induced oxidative stress indicators in M. meretrix using real-time PCR after 24 h of exposure to 1 μg/L BaP. The combined exposure to 10 μM SFN and 1 μg/L BaP led to decreased ROS levels compared with BaP single treatment, while T-AOC and GSH levels increased significantly; all genes showed induced expression after 24 h, though slightly decreased after adding 10 μM SFN. In conclusion, our data imply that MmNrf2 responds to oxidative stress factors, regulated the Keap1-Nrf2 signaling pathway, and played a crucial role in modulating antioxidant enzyme gene expression in bivalve mollusks.