The role of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by PAHs in the calm Ruditapes philippinarum

The NF-E2-related factor 2 (Nrf2) is a master regulator of cellular responses against environmental stresses. In this study we cloned the full-length cDNAs of the RpNrf2 encompassed 2823 bp from the clam Ruditapes philippinarum (R. philippinarum). Sequences alignment and phylogenetic analysis showed that Nrf2 was highly specific in the clams. RpNrf2 expression was detected in gill, digestive gland, mantle and adductor, which the highest transcription level was observed in gill and digestive gland. The gene expressions of RpNrf2, Kelch-like-ECH-associated Protein 1 (Keap1), Cul3-based E3 Ubiquitin Ligase (E3), Glutathione S-transferase (GST-pi), Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in digestive gland was evaluated by real-time PCR after being exposed to benzo(a)pyrene (BaP) (0.25, 1and 4 μg/L) for 15 days, which showed that the expression of Nrf2 significantly increased at day 1 and day 6 after exposure (p < 0.05), and there was a negative relationship between the mRNA levels of Nrf2 and Keap1 that indicates the enhancement of Keap1 expression stimulating Nrf2 degradation. RNA interference experiments were conducted to examine the expression profiles of RpNrf2, antioxidant and detoxification genes (GST-pi, Cu/Zn-SOD, CAT and GPx) and Lipid Peroxidase (LPO) level in digestive gland exposed to BaP. The results showed that the mRNA level of Nrf2 was significantly decreased by 63.2%, and the changes of antioxidant and detoxification genes expression were consistent with the Nrf2 gene suggesting that Nrf2 is required for the induction of antioxidant and detoxification genes. Besides, the LPO levels expressed by malondialdehyde (MDA) contents were significant higher compared with the control group at 72 h post dsRNA-Nrf2 injection. In conclusion, our data demonstrated that Keap1 can sense nucleophilic or oxidative stress factors to regulate the Nrf2 signaling pathway together with E3 and Nrf2 signaling pathway plays an important role in modulating gene expression of antioxidant enzymes in bivalve mollusks.