Jingqian Wang, Li Wang, Yang Liu, Congcong Hou, Qingping Xie, Daojun Tang, Feng Liu, Bao Lou, Junquan Zhu
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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.</p>","PeriodicalId":422,"journal":{"name":"Science of the Total Environment","volume":" ","pages":"177396"},"PeriodicalIF":8.2000,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"The Keap1-Nrf2/ARE signaling pathway regulates redox balance and apoptosis in the small yellow croaker (Larimichthys polyactis) under hypoxic stress.\",\"authors\":\"Jingqian Wang, Li Wang, Yang Liu, Congcong Hou, Qingping Xie, Daojun Tang, Feng Liu, Bao Lou, Junquan Zhu\",\"doi\":\"10.1016/j.scitotenv.2024.177396\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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. 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引用次数: 0
摘要
缺氧应激可导致跃层鱼类体内氧化还原失衡和细胞凋亡;然而,这一过程的确切分子机制,包括其受关键信号通路 Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 related factor (Nrf2)/antioxidant response element (ARE) 的调控,仍然难以捉摸。因此,本研究选择Keap1-Nrf2/ARE信号通路为切入点,结合体内(靶器官肝脏)和体外(小黄鱼鱼苗[SYCF]细胞系)实验,探讨小黄鱼通过调节氧化还原平衡和细胞凋亡来适应缺氧胁迫的分子机制。我们之前的研究发现,缺氧应激可导致多刺鱼体内氧化还原失衡和细胞凋亡。首先,我们观察到在缺氧胁迫下,L. polyactis肝组织和SYCF细胞中Keap1-Nrf2/ARE信号通路中的关键基因Lpkeap1、Lpnrf2、Lpho-1和Lpnqo1的表达发生了显著变化,表明该通路在缺氧条件下被激活。随后,我们阐明了缺氧激活该通路的机制,即缺氧削弱了LpNrf2与LpKeap1之间的相互作用,促进了LpNrf2的核转位,增强了其与ARE的结合活性,从而激活了靶基因的转录。此外,我们还发现,LpNrf2敲除并暴露于缺氧胁迫24 h后,SYCF细胞的氧化还原平衡和细胞凋亡相关指标发生了显著变化,表明该通路可调控多杀菌素在缺氧胁迫下的氧化还原平衡和细胞凋亡调控。此外,我们利用DNA亲和纯化测序(DAP-seq)确定了与LpNrf2及其靶基因结合的ARE序列(ATGATTTAGC)。最后,我们对 DAP-seq 和 RNA-seq 进行了综合分析,确定了参与这一过程的六个关键靶基因:它们是:血红蛋白加氧酶-1(Ho-1)、B细胞淋巴瘤-2(Bcl2)、垂体同工酶2同工酶X1(Pitx2)、水光素-4同工酶X1(Aqp4)、应激诱导磷蛋白1样同工酶X1(Stip1)和鸟嘌呤核苷酸结合蛋白G(i)亚基α-2样(Gnai2)。综上所述,缺氧应激诱导的LpNrf2与LpKeap1相互作用减弱,促进了LpNrf2进入核内,并增强了其与ARE的结合活性,从而激活多个靶基因的转录,调控细胞氧化还原平衡和凋亡。这些研究成果不仅有助于加深我们对多刺鱼乃至海水鱼对缺氧胁迫的适应机制及其生存策略的理解,而且为培育耐缺氧新品系提供了新的思路和潜在靶标。
The Keap1-Nrf2/ARE signaling pathway regulates redox balance and apoptosis in the small yellow croaker (Larimichthys polyactis) under hypoxic stress.
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.
期刊介绍:
The Science of the Total Environment is an international journal dedicated to scientific research on the environment and its interaction with humanity. It covers a wide range of disciplines and seeks to publish innovative, hypothesis-driven, and impactful research that explores the entire environment, including the atmosphere, lithosphere, hydrosphere, biosphere, and anthroposphere.
The journal's updated Aims & Scope emphasizes the importance of interdisciplinary environmental research with broad impact. Priority is given to studies that advance fundamental understanding and explore the interconnectedness of multiple environmental spheres. Field studies are preferred, while laboratory experiments must demonstrate significant methodological advancements or mechanistic insights with direct relevance to the environment.