The Keap1-Nrf2/ARE signaling pathway regulates redox balance and apoptosis in the small yellow croaker (Larimichthys polyactis) under hypoxic stress.

Abstract

Hypoxic stress can result in redox imbalance and apoptosis in teleostean fishes; however, the precise molecular mechanisms underlying this process, including its regulation by the key signaling pathway Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 related factor (Nrf2)/antioxidant response element (ARE), remain elusive. Therefore, in this study, we chose the Keap1-Nrf2/ARE signaling pathway as the entry point and a combination of in vivo (target organ liver) and in vitro (small yellow croaker fry [SYCF] cell line) experiments to investigate the molecular mechanism by which Larimichthys polyactis (L. polyactis) adapts to hypoxic stress by regulating redox balance and apoptosis. As our previous study found that hypoxic stress could lead to redox imbalance and apoptosis in L. polyactis. First, we observed significant alterations in the expression of key genes Lpkeap1, Lpnrf2, Lpho-1, and Lpnqo1 within the Keap1-Nrf2/ARE signaling pathway in both liver tissue and SYCF cells of L. polyactis under hypoxic stress, indicating activation of this pathway in response to hypoxia. Subsequently, we elucidated the mechanism by which hypoxia activates this pathway, that is, hypoxia weakened the interaction between LpNrf2 and LpKeap1, promoting the nuclear translocation of LpNrf2 and enhancing its binding activity to ARE, thereby activating the transcription of target genes. Furthermore, we found that significant changes occurred in the redox balance and apoptosis-related indicators after LpNrf2 knockdown and exposure to hypoxic stress for 24 h in SYCF cells, indicating that this pathway can regulate redox balance and apoptosis regulation under hypoxic stress in L. polyactis. Additionally, we used DNA affinity purification sequencing (DAP-seq) to identify the ARE sequence (ATGATTTAGC) that bound to LpNrf2 and its target genes. Finally, we conducted a combined analysis of DAP-seq and RNA-seq to identify six key target genes involved in the process: haeme oxygenase-1 (Ho-1), B-cell lymphoma-2 (Bcl2), pituitary homeobox 2 isoform X1 (Pitx2), aquaporin-4 isoform X1 (Aqp4), stress-induced phosphoprotein 1-like isoform X1 (Stip1), and guanine nucleotide-binding protein G (i) subunit alpha-2-like (Gnai2). In summary, hypoxic stress induced by weakening LpNrf2 and LpKeap1 interaction promoted LpNrf2 nuclear entry and enhanced its binding activity to ARE, thereby activating the transcription of multiple target genes to regulate redox balance and apoptosis. The research results not only help deepen our understanding of the adaptive mechanisms of L. polyactis and even marine fish to hypoxic stress and its survival strategies but also provide new ideas and potential targets for breeding new hypoxia-tolerant strains.