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Identification of the Nrf2 in the fathead minnow muscle cell line: role for a regulation in response to H2O2 induced the oxidative stress in fish cell

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Abstract

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 H2O2. 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 H2O2 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 H2O2 exposure, leading to oxidative damage and apoptosis. These findings provided an insight to understand the mechanisms of Nrf2 against oxidative stress in fish.

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References

  • Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126

    CAS  PubMed  Google Scholar 

  • Akalin PP, Bucak MN, Güngör S, Başpinar N, Çoyan K, Dursun S (2016) Influence of lycopene and cysteamine on sperm and oxidative stress parameters during liquid storage of ram semen at 5 °C. Small Rumin Res 137:117–123

    Google Scholar 

  • Babich H, Borenfreund E (1991) Cytotoxicity and genotoxicity assays with cultured fish cells: a review. Toxicol in Vitro 5(1):91–100

    CAS  PubMed  Google Scholar 

  • Bayele HK, Debnam ES, Srai KS (2015) Nrf2 transcriptional derepression from Keap1 by dietary polyphenols. Biochem Biophys Res Commun 469(3):521–528

    PubMed  Google Scholar 

  • Buendia I, Michalska P, Navarro E, Gameiro I, Egea J, León R (2015) Nrf2-ARE pathway: an emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacol Ther 157:84–104

    PubMed  Google Scholar 

  • Burgess JT, Bolderson E, Adams MN, Baird AM, Zhang SD, Gately KA, Umezawa K, O’Byrne KJ, Richard DJ (2016) Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response. Cell Death Dis 7(11):e2469–e2469

    CAS  PubMed  PubMed Central  Google Scholar 

  • Carocho M, Ferreira ICFR (2013) A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food Chem Toxicol 51:15–25

    CAS  PubMed  Google Scholar 

  • Chui DH, Tang W, Orkin SH (1995) Cdna cloning of murine Nrf 2 gene, coding for a p45 NF-E2 related transcription factor. Biochem Biophys Res Commun 209(1):40–46

    CAS  PubMed  Google Scholar 

  • Damm J, Roth J, Gerstberger R, Rummel C (2017) The use of sirna as a pharmacological tool to assess a role for the transcription factor NF-IL6 in the brain under in vitro and in vivo conditions during LPS-induced inflammatory stimulation. J Basic Clin Physiol Pharmacol 28(6):563–571

    CAS  PubMed  Google Scholar 

  • Devlin EW, Clary B (1998) In vitro, toxicity of methyl mercury to fathead minnow cells. Bull Environ Contam Toxicol 61(4):527–533

    CAS  PubMed  Google Scholar 

  • Dröge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82(1):47–95

    PubMed  Google Scholar 

  • Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, O'Connor T (2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of are-regulated drug metabolizing enzymes and antioxidant genes. Toxicol Sci 59(1):169–177

    CAS  PubMed  Google Scholar 

  • Feng Y, Qin ZD, Dai YJ, Zhang YL, Liu XL, Zhou Y (2017) Cloning and analysis of nuclear factor E2-related factor 2 (Nrf2) and its function in the regulation of respiratory burst in grass carp (Ctenopharyngodon idella). J Fish China 42(11):161–177

    Google Scholar 

  • Giuliani ME, Regoli F (2014) Identification of the Nrf2-Keap1 pathway in the European eel Anguilla anguilla: role for a transcriptional regulation of antioxidant genes in aquatic organisms. Aquat Toxicol 150(160):117–123

    CAS  PubMed  Google Scholar 

  • Gravell M, Malsberger RG (2010) A permanent cell line from the fathead minnow (Pimephales promelas). Ann N Y Acad Sci 126(1):555–565

    Google Scholar 

  • Halliwell B (1996) Antioxidants in human health and disease. Annu Rev Nutr 16(1):33–50

    CAS  PubMed  Google Scholar 

  • Irshad M, Chaudhuri PS (2002) Oxidant-antioxidant system: role and significance in human body. Indian J Exp Biol 40(11):1233–1239

    CAS  PubMed  Google Scholar 

  • Itoh K, Igarashi K, Hayashi N, Nishizawa M, Yamamoto M (1995) Cloning and characterization of a novel erythroid cell-derived cnc family transcription factor heterodimerizing with the small maf family proteins. Mol Cell Biol 15(8):4184–4193

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang WD, Liu Y, Hu K, Jiang J, Li SH, Feng L (2014) Copper exposure induces oxidative injury, disturbs the antioxidant system and changes the Nrf2/ARE(CuZnSOD) signaling in the fish brain: protective effects of myo -inositol. Aquat Toxicol 155(4):301–313

    CAS  PubMed  Google Scholar 

  • Kensler TW, Wakabayashi N (2010) Nrf2: friend or foe for chemoprevention? Carcinogenesis 31(1):90–99

    CAS  PubMed  Google Scholar 

  • Kivrak E, Yurt K, Kaplan A, Alkan I, Altun G (2017) Effects of electromagnetic fields exposure on the antioxidant defense system. J Microsc Ultrastruct 5(4):167–176

    PubMed  PubMed Central  Google Scholar 

  • Kobayashi M, Itoh K, Suzuki T, Osanai H, Nishikawa K, Katoh Y (2002) Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system. Genes Cells 7(8):807–820

    CAS  PubMed  Google Scholar 

  • Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T (2004) Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol 24(16):7130–7139

    CAS  PubMed  PubMed Central  Google Scholar 

  • Leblanc GA, Mastone JD, Paradice AP, Wilson BF, Lockhart HB Jr, Robillard KA (2010) The influence of speciation on the toxicity of silver to fathead minnow (Pimephales promelas). Environ Toxicol Chem 3(1):37–46

    Google Scholar 

  • Li HT, Feng L, Jiang WD, Liu Y, Jiang J, Li SH, Zhou XQ (2013) Oxidative stress parameters and anti-apoptotic response to hydroxyl radicals in fish erythrocytes: protective effects of glutamine, alanine, citrulline and proline. Aquat Toxicol 126:169–179

    CAS  PubMed  Google Scholar 

  • Lushchak VI (2011) Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol 101(1):13–30

    CAS  PubMed  Google Scholar 

  • Lyakurwa FS, Yang X, Li X, Qiao X, Chen J (2014) Development of in silico models for predicting lser molecular parameters and for acute toxicity prediction to fathead minnow (Pimephales promelas). Chemosphere 108:17–25

    CAS  PubMed  Google Scholar 

  • Mcmahon M, Itoh K, Yamamoto M, Chanas SA, Henderson CJ, Mclellan LI (2001) The cap'n'collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res 61(8):3299–3307

    CAS  PubMed  Google Scholar 

  • Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7(9):405–410

    CAS  PubMed  Google Scholar 

  • Moi P, Chan K, Asunis I, Cao A, Kan YW (1994) Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci U S A 91(21):9926–9930

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mylroie H, Dumont O, Bauer A, Thornton CC, Mackey J, Calay D, Hamdulay SS, Choo JR, Boyle JJ, Samarel AM, Randi AM, Evans PC, Mason JC (2015) PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis. Cardiovasc Res 106(3):509–519

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ogasawara T (2013) Cell cycle control factors and skeletal development. Jpn Dent Sci Rev 49(2):79–87

    Google Scholar 

  • Petitjean Q, Jean S, Gandar A, Côte J, Laffaille P, Jacquin L (2019) Stress responses in fishes: from molecular to evolutionary processes. Sci Total Environ 684:371–380

    CAS  PubMed  Google Scholar 

  • Qin S, Deng F, Wu W, Jiang L, Yamashiro T, Yano S (2014) Baicalein modulates Nrf2/Keap1 system in both Keap1-dependent and Keap1-independent mechanisms. Arch Biochem Biophys 559(5):53–61

    CAS  PubMed  Google Scholar 

  • Sahin K, Orhan C, Yazlak H, Tuzcu M, Sahin N (2014) Lycopene improves activation of antioxidant system and Nrf2/HO-1 pathway of muscle in rainbow trout (Oncorhynchus mykiss) with different stocking densities. Aquaculture 430(430):133–138

    CAS  Google Scholar 

  • Santilli F, Guagnano MT, Vazzana N, La BS (2015) Oxidative stress drivers and modulators in obesity and cardiovascular disease: from biomarkers to therapeutic approach. Curr Med Chem 22(5):582–595

    CAS  PubMed  Google Scholar 

  • Sarkar S, Mukherjee S, Chattopadhyay A (2014) Low dose of arsenic trioxide triggers oxidative stress in zebrafish brain: expression of antioxidant genes. Ecotoxicol Environ Saf 107(9):1–8

    CAS  PubMed  Google Scholar 

  • Satoh H, Moriguchi T, Taguchi K, Takai J, Maher JM, Suzuki T (2010) Nrf2-deficiency creates a responsive microenvironment for metastasis to the lung. Carcinogenesis 31(10):1833–1843

    CAS  PubMed  Google Scholar 

  • Su Z, Yang Z, Xu Y, Chen Y, Yu Q (2015) Apoptosis, autophagy, necroptosis, and cancer metastasis. Mol Cancer 14(1):48–61

    PubMed  PubMed Central  Google Scholar 

  • Suzuki T, Yamamoto M (2015) Molecular basis of the keap1-Nrf2 system. Free Radic Biol Med 88(Pt B):93–100

    CAS  PubMed  Google Scholar 

  • Ting LY (2016) Critical roles of reactive oxygen species in age-related impairment in ischemia-induced neovascularization by regulating stem and progenitor cell function. Oxid Med Cell Longevity 2016:1–14

    Google Scholar 

  • Uruno A, Yagishita Y, Yamamoto M (2015) The Keap1-Nrf2 system and diabetes mellitus. Arch Biochem Biophys 566:76–84

    CAS  PubMed  Google Scholar 

  • Victor VM, Rocha M, De la Fuente M (2004) Immune cells: free radicals and antioxidants in sepsis. Int Immunopharmacol 4(3):327–347

    CAS  PubMed  Google Scholar 

  • Wan J, Ge X, Liu B, Xie J, Cui S, Zhou M (2014) Effect of dietary vitamin C on non-specific immunity and mrna expression of three heat shock proteins (HSPs) in juvenile Megalobrama amblycephala under pH stress. Aquaculture 434:325–333

    CAS  Google Scholar 

  • Wang L, Gallagher EP (2013) Role of Nrf2 antioxidant defense in mitigating cadmium-induced oxidative stress in the olfactory system of zebrafish. Toxicol Appl Pharmacol 266(2):177–186

    CAS  PubMed  Google Scholar 

  • Wang QJ, Ju X, Chen YK, Dong XQ, Luo S, Liu HJ, Zhang DM (2015) L-carnitine exerts a cytoprotective effect against H2O2-induced oxidative stress in the fathead minnow muscle cell line. Aquac Res 48:941–954

    Google Scholar 

  • Wang H, Pan L, Si L, Miao J (2018) The role of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by PAHs in the calm Ruditapes philippinarum. Fish Shellfish Immunol 80:325–334

    CAS  PubMed  Google Scholar 

  • Wang H, Pan L, Xu R, Si L, Zhang X (2019) The molecular mechanism of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by BaP in the scallop Chlamys farreri. Fish Shellfish Immunol 92:489–499

    CAS  PubMed  Google Scholar 

  • Yang HL, Lin MW, Korivi M, Wu JJ, Liao CH, Chang CT, Liao JW, Hseu YC (2016) Coenzyme Q0 regulates NFκB/AP-1 activation and enhances Nrf2 stabilization in attenuation of LPS-induced inflammation and redox imbalance: evidence from in vitro and in vivo studies. Biochim Biophys Acta Gene Regul Mech 1859(2):246–261

    CAS  Google Scholar 

  • Young IS, Woodside JV (2001) Antioxidants in health and disease. J Clin Pathol 54(3):176–186

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zheng JL, Zeng L, Shen B, Xu MY, Zhu AY, Wu CW (2016) Antioxidant defenses at transcriptional and enzymatic levels and gene expression of Nrf2-Keap1 signaling molecules in response to acute zinc exposure in the spleen of the large yellow croaker Pseudosciaena crocea. Fish Shellfish Immunol 52:1–8

    PubMed  Google Scholar 

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Funding

The research was supported by National Natural Science Foundation of China (30671621); industrial technology research and development project of Jilin Provincial Development and Reform Commission (2011002-1); foreign trade development project of northeast old industrial base, Jilin provincial department of science and technology key project (20160204019NY); and Scientific Research Foundation of Jilin Agricultural University (201704).

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Authors and Affiliations

  1. College of Animal Science and Technology, Jilin Agricultural University, Changchun, 130118, Jilin, China

    Xiumei Chen, Qiuju Wang, Sha Luo, Ting Yu, Dongming Zhang & Guiqin Wang

  2. Jilin Provincial Key Laboratory of Animal Nutrition and Feed Science, Jilin Agricultural University, Changchun, 130118, Jilin, China

    Xiumei Chen, Qiuju Wang, Dongming Zhang & Guiqin Wang

  3. Ministry of Education Laboratory of Animal Production and Quality Security, Jilin Agricultural University, Changchun, 130118, Jilin, China

    Xiumei Chen, Qiuju Wang, Dongming Zhang & Guiqin Wang

  4. Joint Laboratory of Modern Agricultural Technology International Cooperation, Ministry of Education, Jilin Agricultural University, Changchun, 130118, Jilin, China

    Xiumei Chen, Qiuju Wang, Dongming Zhang & Guiqin Wang

  5. College of life science, Tonghua Normal University, Tonghua, 134001, Jilin, China

    Zhixin Guo, Yunlong Zhao & Dongming Zhang

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  1. Xiumei Chen
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Correspondence to Dongming Zhang.

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Chen, X., Wang, Q., Guo, Z. et al. Identification of the Nrf2 in the fathead minnow muscle cell line: role for a regulation in response to H2O2 induced the oxidative stress in fish cell. Fish Physiol Biochem 46, 1699–1711 (2020). https://doi.org/10.1007/s10695-020-00822-8

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