Nrf2 regulates the expression of NOX1 in TNF-α-induced A549 cells

Main Article Content

Weijing Wu
Jiamin Zhang
Xiaoshan Su
Xiaoping Lin
Li Zhu
Zesen Zhuang
Chennan Liu
Zhixing Zhu
Yiming Zeng


Acute Lung Injury, NOX1, Nrf2, Oxidative Stress, TNF-α


Acute lung injury causes severe inflammation and oxidative stress in lung tissues. In this study, we analyzed the potential regulatory role of nuclear factor erythroid-2-related factor 2 (Nrf2) on NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced inflammation and oxidative stress in human type II alveolar epithelial cells. In this study, A549 cells were transfected with Nrf2 siRNA and overexpression vectors for 6 h before being induced by TNF-α for 24 h. TNF-α upregulated the expression of NOX1 and Nrf2 in A549 cells. Furthermore, overexpression of Nrf2 could reduce TNF-α-induced NF-κB mRNA and protein expression after transfection with the Nrf2 siRNA vector, and the levels of IL-6, IL-8, ROS, and malondialdehyde (MDA) in TNF-α-induced A549 cells increased, while the level of total antioxidation capability (T-AOC) decreased. On the other hand, the overexpression of Nrf2 decreased the levels of IL-6, IL-8, ROS, and MDA, while increasing T-AOC. The mRNA and protein levels of NOX1 were dramatically increased by TNF-α, while those changes were notably suppressed by Nrf2 overexpression. Further studies demonstrated that Nrf2 suppressed NOX1 transcription by binding to the -1199 to -1189 bp (ATTACACAGCA) region of the NOX1 promoter in TNF-α-stimulated A549 cells. Our study suggests that Nrf2 may bind to and regulate NOX1 expression to antagonize TNF-α-induced inflammatory reaction and oxidative stress in A549 cells.

Abstract 104 | PDF Downloads 65 HTML Downloads 10 XML Downloads 2


1. Hayes M, Curley G, Ansari B, Laffey JG. Clinical review: Stem cell therapies for acute lung injury/acute respiratory distress syndrome—Hope or hype? Crit Care. 2012;16(2):205. 10.1186/cc10570

2. Mowery NT, Terzian WTH, Nelson AC. Acute lung injury. Curr Probl Surg. 2020;57(5):100777. 10.1016/j.cpsurg.2020.10077

3. Li X, Ma X. Acute respiratory failure in COVID-19: Is it “typical” ARDS? Crit Care. 2020;24(1):198. 10.1186/s13054-020-02911-9

4. Zhu Z, Lian X, Su X, Wu W, Marraro GA, Zeng Y. From SARS and MERS to COVID-19: A summary and comparison of severe acute respiratory infections caused by three highly pathogenic human coronaviruses. Respir Res. 2020;21(1):224. 10.1186/s12931-020-01479-w

5. Hughes KT, Beasley MB. Pulmonary manifestations of acute lung injury: More than just diffuse alveolar damage. Arch Pathol Lab Med. 2017;141(7):916–22. 10.5858/arpa.2016-0342-RA

6. Sarma JV, Ward PA. Oxidants and redox signaling in acute lung injury. Compr Physiol. 2011;1(3):1365–81. 10.1002/cphy.c100068

7. Sies H, Jones DP. Reactive oxygen species (ROS) as pleio-tropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21(7):363–83. 10.1038/s41580-020-0230-3

8. Pendyala S, Natarajan V. Redox regulation of Nox proteins. Respir Physiol Neurobiol. 2010;174(3):265–71. 10.1016/j.resp.2010.09.016

9. Mou Y, Wen S, Li YX, Gao XX, Zhang X, Jiang ZY. Recent progress in Keap1-Nrf2 protein-protein interaction inhibitors. Eur J Med Chem. 2020;202:112532. 10.1016/j.ejmech.2020.112532

10. Shaw P, Chattopadhyay A. Nrf2-ARE signaling in cellular protection: Mechanism of action and the regulatory mechanisms. J Cell Physiol. 2020;235(4):3119–30. 10.1002/jcp.29219

11. Zhang S, Jiang W, Ma L, Liu Y, Zhang X, Wang S. Nrf2 transfection enhances the efficacy of human amniotic mesenchymal stem cells to repair lung injury induced by lipopolysaccharide. J Cell Biochem. 2018;119(2):1627–36. 10.1002/jcb.26322

12. Liu Q, Gao Y, Ci X. Role of Nrf2 and its activators in respiratory diseases. Oxid Med Cell Longev. 2019;2019:7090534. 10.1155/2019/7090534

13. Johnson PA. Novel understandings of host cell mechanisms involved in chronic lung infection: Pseudomonas aeruginosa in the cystic fibrotic lung. J Infect Public Health. 2019;12(2):242–6. 10.1016/j.jiph.2018.10.014

14. Yan J, Li J, Zhang L, Sun Y, Jiang J, Huang Y, et al. Nrf2 protects against acute lung injury and inflammation by modulating TLR4 and Akt signaling. Free Radic Biol Med. 2018;121:78–85. 10.1016/j.freeradbiomed.2018.04.557

15. Malaviya R, Laskin JD, Laskin DL. Anti-TNFα therapy in inflammatory lung diseases. Pharmacol Ther. 2017;180:90–8. 10.1016/j.pharmthera.2017.06.008

16. Mukhopadhyay S, Hoidal JR, Mukherjee TK. Role of TNFalpha in pulmonary pathophysiology. Respir Res. 2006;7(1):125. 10.1186/1465-9921-7-125

17. Yang HM, Zhuo JY, Sun CY, Nie J, Yuan J, Liu YL, et al. Pogostone attenuates TNF-α-induced injury in A549 cells via inhibiting NF-κB and activating Nrf2 pathways. Int Immunopharmacol [Internet]. [cited 2021 Aug 25]. 2018;62:15–22. 10.1016/j.intimp.2018.06.029. Available from:

18. Malec V, Gottschald OR, Li S, Rose F, Seeger W, Hänze J. HIF-1 alpha signaling is augmented during intermittent hypoxia by induction of the Nrf2 pathway in NOX1-expressing adenocarcinoma A549 cells. Free Radic Biol Med. 2010;48(12):1626–35. 10.1016/j.freeradbiomed.2010.03.008

19. Kellner M, Noonepalle S, Lu Q, Srivastava A, Zemskov E, Black SM. ROS Signaling in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Adv Exp Med Biol. 2017;967:105–37. 10.1007/978-3-319-63245-2_8

20. Su LJ, Zhang JH, Gomez H, Murugan R, Hong X, Xu D, et al. Reactive oxygen species-induced lipid peroxidation in apoptosis, autophagy, and ferroptosis. Oxid Med Cell Longev. 2019;2019:5080843. 10.1155/2019/5080843

21. Schröder K. NADPH oxidases: Current aspects and tools. Redox Biol. 2020;34:101512. 10.1016/j.redox.2020.101512

22. Carnesecchi S, Deffert C, Pagano A, Garrido-Urbani S, Métrailler-Ruchonnet I, Schäppi M, et al. NADPH oxidase-1 plays a crucial role in hyperoxia-induced acute lung injury in mice. Am J Respir Crit Care Med. 2009;180(10):972–81. 10.1164/rccm.200902-0296OC

23. Carnesecchi S, Dunand-Sauthier I, Zanetti F, Singovski G, Deffert C, Donati Y, et al. NOX1 is responsible for cell death through STAT3 activation in hyperoxia and is associated with the pathogenesis of acute respiratory distress syndrome. Int J Clin Exp Pathol. 2014;7(2):537–51.

24. Feng YW, Li L, Xu H, Feng LW, Jie HW. [TNFR-Fc prevents lipopolysaccharide-induced acute lung injury in mice via oxidative stress inhibition]. Zhonghua Jie He He Hu Xi Za Zhi. 2012;35:435–9.

25. Oshima H, Ishikawa T, Yoshida GJ, Naoi K, Maeda Y, Naka K, et al. TNF-α/TNFR1 signaling promotes gastric tumorigenesis through induction of Noxo1 and Gna14 in tumor cells. Oncogene. 2014;33(29):3820–9. 10.1038/onc.2013.356

26. Gopallawa I, Kuek LE, Adappa ND, Palmer JN, Lee RJ. Small-molecule Akt-activation in airway cells induces NO production and reduces IL-8 transcription through Nrf-2. Respir Res. 2021;22(1):267. 10.1186/s12931-021-01865-y

27. Hu R, Xu H, Jiang H, Zhang Y, Sun Y. The role of TLR4 in the pathogenesis of indirect acute lung injury. Front Biosci (Landmark Ed). 2013;18(4):1244–55. 10.2741/4176

28. Morgan MJ, Liu Z gang. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 2011;21(1):103–15. 10.1038/cr.2010.17

29. Liu Y, Liu K, Huang Y, Sun M, Tian Q, Zhang S, et al. TRIM25 promotes TNF-α-induced NF-κB activation through potentiating the K63-linked ubiquitination of TRAF2. J Immunol. 2020;204(6):1499–507. 10.4049/jimmunol.1900482

30. de Almeida AJPO, de Almeida Rezende MS, Dantas SH, de Lima Silva S, de Oliveira JCPL, de Lourdes Assunção Araújo de Azevedo F, et al. Unveiling the role of inflammation and oxidative stress on age-related cardiovascular diseases. Oxid Med Cell Longev. 2020;2020:1954398. 10.1155/2020/1954398

31. Wardyn JD, Ponsford AH, Sanderson CM. Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochem Soc Trans. 2015;43(4):621–6. 10.1042/BST20150014