College of Animal Science and Technology, Jilin Agricultural University, Changchun, 130118, Jilin, China.
Jilin Provincial Key Laboratory of Animal Nutrition and Feed Science, Jilin Agricultural University, Changchun, 130118, Jilin, China.
Fish Physiol Biochem. 2020 Oct;46(5):1699-1711. doi: 10.1007/s10695-020-00822-8. Epub 2020 Jul 3.
The Nrf2 (nuclear factor erythroid 2-related factor 2) plays a central role in cell protection against a wide variety of environmental stressors through the Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathway, but its involvement in modulation of antioxidant system of fish cell is still largely unexplored. The present study focused on the molecular cloning and silencing of the Nrf2 in the fathead minnow muscle cell line (FHM) in response to the oxidative stress induced by HO. A full-length cDNA of coding Nrf2 was cloned from FHM cells by RT-PCR and RACE approaches. The obtained cDNA covered 2578 bp with an open reading frame (1770 bp) of encoding 589 amino acids. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (51-86%) among 16 fishes. Based on the cloned Nrf2 sequence, the siRNA-242 of targeting Nrf2 with the best knocking down efficiency was designed and detected. Then, the mRNA levels of Keap1, Nrf2, Maf (musculoaponeurotic fibrosarcoma oncogene), and HO-1 (haemoxygenase-1); the activities of T-SOD (total superoxide dismutase), CAT (catalase), and GSH-PX (glutathione peroxidase); the levels of GSH (glutathione) and MDA (malonaldehyde); and the cell cycle and apoptosis were analyzed to investigate the molecular responses after HO exposure. These results showed a coordinated transcriptional regulation of Keap1, Maf, and HO-1 and antioxidants (T-SOD, GSH, CAT, and GSH-PX) and MDA levels after HO exposure, leading to oxidative damage and apoptosis. These findings provided an insight to understand the mechanisms of Nrf2 against oxidative stress in fish.
Nrf2（核因子红细胞 2 相关因子 2）通过 Nrf2-Keap1（Kelch 样 ECH 相关蛋白 1）途径在细胞对抗多种环境应激因子中发挥核心作用，但 Nrf2 对鱼类细胞抗氧化系统的调节作用在很大程度上仍未得到探索。本研究聚焦于 HO 诱导的氧化应激下，胖头鱼肌肉细胞系（FHM）中 Nrf2 的分子克隆和沉默。通过 RT-PCR 和 RACE 方法从 FHM 细胞中克隆出编码 Nrf2 的全长 cDNA。获得的 cDNA 覆盖 2578 bp，开放阅读框（1770 bp）编码 589 个氨基酸。序列比对和系统发育分析显示，16 种鱼类之间存在高度保守性（51-86%）。基于克隆的 Nrf2 序列，设计并检测了针对 Nrf2 的最佳敲低效率的 siRNA-242。然后，分析了 Keap1、Nrf2、Maf（肌筋膜纤维肉瘤癌基因）和 HO-1（血红素加氧酶-1）的 mRNA 水平；T-SOD（总超氧化物歧化酶）、CAT（过氧化氢酶）和 GSH-PX（谷胱甘肽过氧化物酶）的活性；GSH（谷胱甘肽）和 MDA（丙二醛）的水平；以及细胞周期和细胞凋亡，以研究 HO 暴露后分子反应。这些结果表明，HO 暴露后 Keap1、Maf 和 HO-1 以及抗氧化剂（T-SOD、GSH、CAT 和 GSH-PX）和 MDA 水平的转录调控协调一致，导致氧化损伤和细胞凋亡。这些发现为理解鱼类中 Nrf2 对抗氧化应激的机制提供了深入了解。
