Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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shRNA-interfered of Nrf2 reveals a critical role for Keap1-Nrf2 signaling pathway during effects of zearalenone induced oxidative stress in IPEC-J2 cells

  • Qun Cheng (Department of Animal Sciences and Technology, Qingdao Agricultural University) ;
  • Shu Zhen Jiang (Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Department of Animal Sciences and Technology, Shandong Agricultural University) ;
  • Li Bo Huang (Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Department of Animal Sciences and Technology, Shandong Agricultural University) ;
  • Wei Ren Yang (Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Department of Animal Sciences and Technology, Shandong Agricultural University)
  • Received : 2024.05.28
  • Accepted : 2024.07.30
  • Published : 2025.02.01
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Abstract

Objective: This study aims to verify the protective effect of the Kelch-like ECH-associated protein1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways by studying the effect of plasmids containing Nrf2-small hairpin RNA (shRNA) interference down-regulation of Nrf2 on zearalenone (ZEA)-induced intestinal porcine epithelial cells (IPEC-J2) oxidative stress. Methods: We constructed an IPEC-J2 model that interferes with Nrf2 expression, set blank (control), negative control group (Sh-control), positive control group (Sh-Nrf2), and added 10, 20, and 40 μmol/L ZEA experimental group (Sh-Nrf2+ZEA10, Sh-Nrf2+ZEA20, and Sh-Nrf2+ZEA40). Results: The study results showed that, compared with the Sh-Nrf2 group, ZEA significantly increased the apoptosis rate of IPEC-J2 in a time- and dose-dependent manner. Compared with the Sh-Nrf2 group, the activities of total superoxide dismutase and glutathione peroxidase and relative expressions of Keap1 at mRNA and protein level in the Sh-Nrf2+ZEA20 and Sh-Nrf2+ZEA40 groups were significantly reduced, the malondialdehyde level, and the fluorescence intensity around and within the nucleus of reactive oxygen species and Nrf2, and the relative expressions of Nrf2, quinone oxidoreductase 1, and hemeoxygenase 1 at mRNA and protein level significantly increased. Conclusion: These results further prove that interfering with the expression of Nrf2 in IPEC-J2 cells affected the activation of the Keap1-Nrf2 signaling pathway and reduced the ability of cells to resist ZEA-induced oxidative stress. Therefore, the Keap1-Nrf2 signaling pathway had an important protective effect in ZEA-induced intestinal oxidative stress.

Keywords

Acknowledgement

This research was financed in part by the Natural Science Foundation Youth Project of Shandong Province (grant no. ZR2022QC030); the Natural Science Foundation of Shandong Province (grant no. ZR2024MC124); the Qingdao Agricultural University Doctoral Start-Up Fund (grant no. XJ2023000401); and the Founding of Shandong Agriculture Research System in Shandong Province (grant no. SDAIT-08-04 and SDAIT-08-05).

References

  1. Stob M, Baldwin RS, Tuite J, et al. Isolation of an anabolic, uterotrophic compound from corn infected with Gibberella zeae. Nature 1962;196:1318. https://doi.org/10.1038/1961318a0
  2. Zinedine A, Soriano JM, Moltó JC, Mañes J. Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food Chem Toxicol 2007;45:1-18. https://doi.org/10.1016/j.fct.2006.07.030
  3. Gajecka M, Zielonka L, Dabrowski M, Gajecki M. Threats resulting from the presence of zearalenone in water. Med Weter 2011;67:643-6.
  4. Bi K, Zhang W, Xiao Z, Zhang D. Characterization, expression and application of a zearalenone degrading enzyme from Neurospora crassa. AMB Express 2018;8:194. https://doi.org/10.1186/s13568-018-0723-z
  5. Zhou M, Yang LJ, Chen YH, et al. Comparative study of stress response, growth and development of uteri in post-weaning gilts challenged with zearalenone and estradiol benzoate. J Anim Physiol Anim Nutr 2019;103:1885-94. https://doi.org/10.1111/jpn.13195
  6. Ahamed S, Foster JS, Bukovsky A, Wimalasena J. Signal transduction through the Ras/Erk pathway is essential for the mycoestrogen zearalenone-induced cell-cycle progression in MCF-7 cells. Mol Carcinog 2001;30:88-98. https://doi.org/10.1002/1098-2744(200102)30:2<88::AID-MC1017>3.0.CO;2-E
  7. Silva EO, Bracarense AP, Oswald IP. Mycotoxins and oxidative stress: where are we? World Mycotoxin J 2018;11:113-33. https://doi.org/10.3920/WMJ2017.2267
  8. Ben Salem I, Boussabbeh M, Prola A, et al. Crocin protects human embryonic kidney cells (HEK293) from α- and β-zearalenol-induced ER stress and apoptosis. Environ Sci Pollut Res Int 2016;23:15504-14. https://doi.org/10.1007/s11356-016-6741-y
  9. Cheng Q, Jiang SZ, Huang LB, et al. Zearalenone exposure affects the keap1–nrf2 signaling pathway and glucose nutrient absorption related genes of porcine jejunal epithelial cells. Toxins 2022;14:793. https://doi.org/10.3390/toxins14110793
  10. Boeira SP, Filho CB, Del'Fabbro L, et al. Possible role for glutathione-S-transferase in the oligozoospermia elicited by acute zearalenone administration in Swiss albino mice. Toxicon 2012;60:358-66. https://doi.org/10.1016/j.toxicon.2012.04.353
  11. Kraehenbuhl JP, Pringault E, Neutra MR. Review article: intestinal epithelia and barrier functions. Aliment Pharmacol Ther 1997;11:3-9. https://doi.org/10.1111/j.1365-2036.1997.tb00803.x
  12. Lewczuk B, Przybylska-Gornowicz B, Gajęcka M, et al. Histological structure of duodenum in gilts receiving low doses of zearalenone and deoxynivalenol in feed. Exp Toxicol Pathol 2016;68:157-66. https://doi.org/10.1016/j.etp.2015.11.008
  13. Chen XX, Yang CW, Huang LB, Niu QS, Jiang SZ, Chi F. Zearalenone altered the serum hormones, morphologic and apoptotic measurements of genital organs in post-weaning gilts. Asian-Australas J Anim Sci 2015;28:171-9. https://doi.org/10.5713/ajas.14.0329
  14. Zheng JQ, Zhang GR, Li J, Bi HW. Neutrophil elastase inhibitor suppresses oxidative stress in obese asthmatic rats by activating Keap1/Nrf2 signaling pathway. Eur Rev Med Pharmacol Sci 2019;23:361-9. https://doi.org/10.26355/eurrev_201901_16784
  15. Jain P, Sudandira DC. Identification of potential andrographolide-based drug candidate against Keap1-Nrf2 pathway through rigorous cheminformatics screening. Mol Divers 2023;27:341-56. https://doi.org/10.1007/s11030-022-10435-3
  16. Shanmugam G, Challa AK, Litovsky SH, et al. Enhanced Keap1-Nrf2 signaling protects the myocardium from isoproterenol-induced pathological remodeling in mice. Redox Biol 2019;27:101212. https://doi.org/10.1016/j.redox.2019.101212
  17. Hong C, Cao J, Wu CF, et al. The Chinese herbal formula Free and Easy Wanderer ameliorates oxidative stress through KEAP1-NRF2/HO-1 pathway. Sci Rep 2017;7:11551. https://doi.org/10.1038/s41598-017-10443-6
  18. Xiao D, Yuan D, Tan B, Wang J, Liu Y, Tan B. The Role of Nrf2 Signaling Pathway in Eucommia ulmoides Flavones Regulating Oxidative Stress in the Intestine of Piglets. Oxid Med Cell Longev 2019;2019:9719618. https://doi.org/10.1155/2019/9719618
  19. Ma X, Zhang J, Liu S, Huang Y, Chen B, Wang D. Nrf2 knockdown by shrna inhibits tumor growth and increases efficacy of chemotherapy in cervical cancer. Cancer Chemother Pharmacol 2012;69:485-94. https://doi.org/10.1007/s00280-011-1722-9
  20. Song TT, Yang WR, Huang LB, Yang Z, Jiang S. Zearalenone exposure affects the Wnt/β-catenin signaling pathway and related genes of porcine endometrial epithelial cells in vitro. Anim Biosci 2021;34:993-1005. https://doi.org/10.5713/AJAS.20.0292
  21. Jiang SZ, Yang ZB, Yang WR, et al. Effect of purified zearalenone with or without modified montmorillonite on nutrient availability, genital organs and serum hormones in postweaning piglets. Livest Sci 2012;144:110-8. https://doi.org/10.1016/j.livsci.2011.11.004
  22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method. Methods 2001;25:402-8. https://doi.org/10.1006/meth.2001.1262
  23. Karaman EF, Zeybel M, Ozden S. Evaluation of the epigenetic alterations and gene expression levels of HepG2 cells exposed to zearalenone and α-zearalenol. Toxicol Lett 2020;326:52-60. https://doi.org/10.1016/j.toxlet.2020.02.015
  24. Wang JJ, Li MM, Zhang W, et al. Protective effect of n-acetylcysteine against oxidative stress induced by zearalenone via mitochondrial apoptosis pathway in SIEC02 cells. Toxins 2018;10:407. https://doi.org/10.3390/toxins10100407
  25. Ji XJ, Chen SH, Zhu L, et al. Knockdown of NF-E2-related factor 2 inhibits the proliferation and growth of U251MG human glioma cells in a mouse xenograft model. Oncol Rep 2013;30:157-64. https://doi.org/10.3892/or.2013.2476
  26. Morito N, Yoh K, Itoh K, et al. Nrf2 regulates the sensitivity of death receptor signals by affecting intracellular glutathione levels. Oncogene 2003;22:9275-81. https://doi.org/10.1038/sj.onc.1207024
  27. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 2007;47:89-116. https://doi.org/10.1146/annurev.pharmtox.46.120604.141046
  28. Armstrong BK, Kricker A. The epidemiology of UV induced skin cancer. J Photochem Photobiol B 2001;63:8-18. https://doi.org/10.1016/s1011-1344(01)00198-1
  29. Zhang B, Xie C, Zhong J, et al. A549 cell proliferation inhibited by RNAI mediated silencing of the nrf2 gene. Biomed Mater Eng 2014;24:3905-16. https://doi.org/10.3233/BME-141222
  30. Tatay E, Espín S, García-Fernández AJ, Ruiz M. Oxidative damage and disturbance of antioxidant capacity by zearalenone and its metabolites in human cells. Toxicol In Vitro 2017;45:334-9. https://doi.org/10.1016/j.tiv.2017.04.026
  31. Zheng WL, Wang BJ, Wang L, et al. ROS-mediated cell cycle arrest and apoptosis induced by zearalenone in mouse sertoli cells via er stress and the ATP/AMPK pathway. Toxins 2018; 10:24. https://doi.o rg/10.3390/toxins10010024
  32. Chaudhary M, Rao PVL. Brain oxidative stress after dermal and subcutaneous exposure of T-2 toxin in mice. Food Chem Toxicol 2010;48:3436-42. https://doi.org/10.1016/j.fct.2010.09.018
  33. Huang D, Cui L, Liu X, et al. Protective mechanisms involving enhanced mitochondrial functions and mitophagy against T-2 toxin-induced toxicities in GH3 cells. Toxicol Lett 2018; 295:41-53. https://doi.org/10.1016/j.toxlet.2018.05.041
  34. Chan K, Lu R, Chang JC, Kan YW. NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci USA 1996;93:13943-8. https://doi.org/10.1073/pnas.93.24.13943
  35. Williamson TP, Johnson DA, Johnson JA. Activation of the Nrf2-ARE pathway by siRNA knockdown of Keap1 reduces oxidative stress and provides partial protection from MPTPmediated neurotoxicity. Neurotoxicology 2012;33:272-9. https://doi.org/10.1016/j.neuro.2012.01.015
  36. Yeager RL, Reisman SA, Aleksunes LM, Klaassen CD. Introducing the "tcdd-inducible ahr-nrf2 gene battery". Toxicol Sci 2009;111:238-46. https://doi.org/10.1093/toxsci/kfp115
  37. Zhang DD. Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug Metab Rev 2006;38:769-89. https://doi.org/10.1080/03602530600971974
  38. Li S, Song Z, Liu T, et al. Polysaccharide from Ostrea rivularis attenuates reproductive oxidative stress damage via activating Keap1-Nrf2/ARE pathway. Carbohydr Polym 2018;186:321-31. https://doi.org/10.1016/j.carbpol.2018.01.075
  39. Hayashi A, Suzuki H, Itoh K, et al. Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts. Biochem Biophys Res Commun 2003;310:824-9. https://doi.org/10.1016/j.bbrc.2003.09.086