Nrf2 overexpression protects against paraquat‑induced A549 cell injury primarily by upregulating P‑glycoprotein and reducing intracellular paraquat accumulation

Wu,Bin; Li,Hai‑Xiao; Lian,Jie; Guo,Yong‑Jie; Tang,Ya‑Hui; Chang,Zi‑Juan; Hu,Lu‑Feng; Zhao,Guang‑Ju; Hong,Guang‑Liang; Lu,Zhong‑Qiu

doi:10.3892/etm.2018.7044

Nrf2 overexpression protects against paraquat‑induced A549 cell injury primarily by upregulating P‑glycoprotein and reducing intracellular paraquat accumulation

Authors:
- Bin Wu
- Hai‑Xiao Li
- Jie Lian
- Yong‑Jie Guo
- Ya‑Hui Tang
- Zi‑Juan Chang
- Lu‑Feng Hu
- Guang‑Ju Zhao
- Guang‑Liang Hong
- Zhong‑Qiu Lu
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Affiliations: Emergency Center, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China, Department of Intensive Care Unit, Jiaxing Maternal and Child Health‑Care Center, Jiaxing, Zhejiang 314000, P.R. China, Emergency Center, Yantai Yuhuangding Hospital, Yantai, Shandong 264000, P.R. China, Department of Pharmacy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
Published online on: December 4, 2018 https://doi.org/10.3892/etm.2018.7044
Pages: 1240-1247
Copyright: © Wu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Paraquat (PQ) intoxication causes thousands of mortalities every year, worldwide. Its pulmonary‑targeted accumulation and the acute lung injury it subsequently causes, remain a challenge for detoxification treatment. A previous study has demonstrated that the upregulation of nuclear factor erythroid‑2 related factor 2 (Nrf2) prevents PQ toxicity in cell line and murine models. As Nrf2 target genes include a group of membrane transporters, the current study assessed the protective mechanism exerted by Nrf2 against PQ toxicity and intracellular PQ accumulation via its effects on P‑glycoprotein (P‑gp), a downstream transporter of Nrf2. Adenovirus vectors containing the Nrf2 gene were transfected into A549 cells. Cell proliferation was assessed by Cell Counting Kit‑8. The levels of LDH, MDA, SOD, TNF‑α, IL‑6 levels were detected using their respective ELISA kits. In addition, the levels of Nrf2 and P‑gp protein expression were detected by western blot analysis. The concentration of PQ was measured by HPLC. The results revealed that overexpressed Nrf2 significantly increased P‑gp protein levels, decreased the intracellular accumulation of PQ and attenuated PQ‑induced toxicity. However, the protective effects of Nrf2 overexpression on PQ‑challenged A549 cells were abrogated following cyclosporine A treatment, a competitive inhibitor of P‑gp, which also increased intracellular PQ levels. These data indicated that Nrf2 gene overexpression prevented PQ toxicity in A549 cells, potentially via the upregulation of P‑gp activity and the inhibition of intracellular PQ accumulation. Thus, Nrf2 and P‑gp may serve as potential therapeutic targets for the treatment of PQ‑induced injury.

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1	Yao R, Cao Y, He YR, Lau WB, Zeng Z and Liang ZA: Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat. PLoS One. 10:e01251692015. View Article : Google Scholar : PubMed/NCBI
2	Cherukuri H, Pramoda K, Rohini D, Thunga G, Vijaynarayana K, Sreedharan N, Varma M and Pandit V: Demographics, clinical characteristics and management of herbicide poisoning in tertiary care hospital. Toxicol Int. 21:209–213. 2014. View Article : Google Scholar : PubMed/NCBI
3	Seok SJ, Gil HW, Jeong DS, Yang JO, Lee EY and Hong SY: Paraquat intoxication in subjects who attempt suicide: Why they chose paraquat. Korean J Intern Med. 24:247–251. 2009. View Article : Google Scholar : PubMed/NCBI
4	Senarathna L, Eddleston M, Wilks MF, Woollen BH, Tomenson JA, Roberts DM and Buckley NA: Prediction of outcome after paraquat poisoning by measurement of the plasma paraquat concentration. QJM. 102:251–259. 2009. View Article : Google Scholar : PubMed/NCBI
5	Gil HW, Hong JR, Jang SH and Hong SY: Diagnostic and therapeutic approach for acute paraquat intoxication. J Korean Med Sci. 29:1441–1449. 2014. View Article : Google Scholar : PubMed/NCBI
6	Koepsell H: Polyspecific organic cation transporters: Their functions and interactions with drugs. Trends Pharmacol Sci. 25:375–381. 2004. View Article : Google Scholar : PubMed/NCBI
7	He X, Wang L, Szklarz G, Bi Y and Ma Q: Resveratrol inhibits paraquat-induced oxidative stress and fibrogenic response by activating the nuclear factor erythroid 2-related factor 2 pathway. J Pharmacol Exp Ther. 342:81–90. 2012. View Article : Google Scholar : PubMed/NCBI
8	She X, Hong G, Tan J, Zhao G, Li M and Lu Z: The protective effect of ulinastatin on paraquat-induced injury in HK-2 cells and the underlying mechanisms. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 33:501–506. 2015.(In Chinese). PubMed/NCBI
9	Yao R, Zhou Y, He Y, Jiang Y, Liu P, Ye L, Zheng Z, Lau WB, Cao Y and Zeng Z: Adiponectin protects against paraquat-induced lung injury by attenuating oxidative/nitrative stress. Exp Ther Med. 9:131–136. 2015. View Article : Google Scholar : PubMed/NCBI
10	Kimura M, Yamamoto T, Zhang J, Itoh K, Kyo M, Kamiya T, Aburatani H, Katsuoka F, Kurokawa H, Tanaka T, et al: Molecular basis distinguishing the DNA binding profile of Nrf2-Maf heterodimer from that of Maf homodimer. J Biol Chem. 282:33681–33690. 2007. View Article : Google Scholar : PubMed/NCBI
11	Aleksunes LM and Manautou JE: Emerging role of Nrf2 in protecting against hepatic and gastrointestinal disease. Toxicol Pathol. 35:459–473. 2007. View Article : Google Scholar : PubMed/NCBI
12	Zhou SF: Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition. Xenobiotica. 38:802–832. 2008. View Article : Google Scholar : PubMed/NCBI
13	Bosquillon C: Drug transporters in the lung-do they play a role in the biopharmaceutics of inhaled drugs? J Pharm Sci. 99:2240–2255. 2010. View Article : Google Scholar : PubMed/NCBI
14	Lacher SE, Gremaud JN, Skagen K, Steed E, Dalton R, Sugden KD, Cardozo-Pelaez F, Sherwin CM and Woodahl EL: Absence of P-glycoprotein transport in the pharmacokinetics and toxicity of the herbicide paraquat. J Pharmacol Exp Ther. 348:336–345. 2014. View Article : Google Scholar : PubMed/NCBI
15	Huang H, Lu-Bo Y and Haddad GG: A Drosophila ABC transporter regulates lifespan. PLoS Genet. 10:e10048442014. View Article : Google Scholar : PubMed/NCBI
16	Lehmann T, Kohler C, Weidauer E, Taege C and Foth H: Expression of MRP1 and related transporters in human lung cells in culture. Toxicology. 167:59–72. 2001. View Article : Google Scholar : PubMed/NCBI
17	Silva R, Palmeira A, Carmo H, Barbosa DJ, Gameiro M, Gomes A, Paiva AM, Sousa E, Pinto M, Bastos Mde L and Remião F: P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity. Arch Toxicol. 89:1783–1800. 2015. View Article : Google Scholar : PubMed/NCBI
18	Dinis-Oliveira RJ, Remiao F, Duarte JA, Ferreira R, Sanchez Navarro A, Bastos ML and Carvalho F: P-glycoprotein induction: An antidotal pathway for paraquat-induced lung toxicity. Free Radic Biol Med. 41:1213–1224. 2006. View Article : Google Scholar : PubMed/NCBI
19	Itoh K, Tong KI and Yamamoto M: Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles. Free Radic Biol Med. 36:1208–1213. 2004. View Article : Google Scholar : PubMed/NCBI
20	Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD and Yamamoto M: Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13:76–86. 1999. View Article : Google Scholar : PubMed/NCBI
21	Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, Igarashi K and Yamamoto M: Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol. 24:7130–7139. 2004. View Article : Google Scholar : PubMed/NCBI
22	Hong GL, Liu JM, Zhao GJ, Wang L, Liang G, Wu B, Li MF, Qiu QM and Lu ZQ: The reversal of paraquat-induced mitochondria-mediated apoptosis by cycloartenyl ferulate, the important role of Nrf2 pathway. Exp Cell Res. 319:2845–2855. 2013. View Article : Google Scholar : PubMed/NCBI
23	Li S, Zhao G, Chen L, Ding Y, Lian J, Hong G and Lu Z: Resveratrol protects mice from paraquat-induced lung injury: The important role of SIRT1 and NRF2 antioxidant pathways. Mol Med Rep. 13:1833–1838. 2016. View Article : Google Scholar : PubMed/NCBI
24	Lu H, Chang Z, Han W, Wang L and Hong G: Curcumin reduces paraquat-induced oxidative injury in A549 cells by activation of the Nrf2-ARE pathway. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 32:44–49. 2014.(In Chinese). PubMed/NCBI
25	Adachi T, Nakagawa H, Chung I, Hagiya Y, Hoshijima K, Noguchi N, Kuo MT and Ishikawa T: Nrf2-dependent and -independent induction of ABC transporters ABCC1, ABCC2, and ABCG2 in HepG2 cells under oxidative stress. J Exp Ther Oncol. 6:335–348. 2007.PubMed/NCBI
26	Cen J, Zhang L, Liu F, Zhang F and Ji B: Long-term alteration of reactive oxygen species led to multidrug resistance in MCF-7 cells. Oxid Med Cell Longev. 2016:70534512016. View Article : Google Scholar : PubMed/NCBI
27	Wu J, Zhu Y, Li F, Zhang G, Shi J, Ou R, Tong Y, Liu Y, Liu L, Lu L and Liu Z: Spica prunellae and its marker compound rosmarinic acid induced the expression of efflux transporters through activation of Nrf2-mediated signaling pathway in HepG2 cells. J Ethnopharmacol. 193:1–11. 2016. View Article : Google Scholar : PubMed/NCBI
28	Ghanem CI, Rudraiah S, Bataille AM, Vigo MB, Goedken MJ and Manautou JE: Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice. Biochem Pharmacol. 94:203–211. 2015. View Article : Google Scholar : PubMed/NCBI
29	Gawarammana IB and Buckley NA: Medical management of paraquat ingestion. Br J Clin Pharmacol. 72:745–757. 2011. View Article : Google Scholar : PubMed/NCBI
30	Bai Y, Wang X, Zhao S, Ma C, Cui J and Zheng Y: Sulforaphane protects against cardiovascular disease via Nrf2 activation. Oxid Med Cell Longev. 2015:4075802015. View Article : Google Scholar : PubMed/NCBI
31	de Oliveira MR, Nabavi SF, Habtemariam S, Erdogan Orhan I, Daglia M and Nabavi SM: The effects of baicalein and baicalin on mitochondrial function and dynamics: A review. Pharmacol Res. 100:296–308. 2015. View Article : Google Scholar : PubMed/NCBI
32	de Oliveira MR: Phloretin-induced cytoprotective effects on mammalian cells: A mechanistic view and future directions. Biofactors. 42:13–40. 2016.PubMed/NCBI
33	de Oliveira MR, Nabavi SF, Manayi A, Daglia M, Hajheydari Z and Nabavi SM: Resveratrol and the mitochondria: From triggering the intrinsic apoptotic pathway to inducing mitochondrial biogenesis, a mechanistic view. Biochim Biophys Acta. 1860:727–745. 2016. View Article : Google Scholar : PubMed/NCBI
34	Oliveira MR, Nabavi SF, Daglia M, Rastrelli L and Nabavi SM: Epigallocatechin gallate and mitochondria-A story of life and death. Pharmacol Res. 104:70–85. 2016. View Article : Google Scholar : PubMed/NCBI
35	Hichor M, Sampathkumar NK, Montanaro J, Borderie D, Petit PX, Gorgievski V, Tzavara ET, Eid AA, Charbonnier F, Grenier J and Massaad C: Paraquat induces peripheral myelin disruption and locomotor defects: Crosstalk with LXR and wnt pathways. Antioxid Redox Signal. 27:168–183. 2017. View Article : Google Scholar : PubMed/NCBI
36	de Oliveira MR, de Souza ICC and Furstenau CR: Carnosic acid induces anti-inflammatory effects in paraquat-treated SH-SY5Y cells through a mechanism involving a crosstalk between the Nrf2/HO-1 axis and NF-kB. Mol Neurobiol. 55:890–897. 2018. View Article : Google Scholar : PubMed/NCBI
37	Shinjo T, Tanaka T, Okuda H, Kawaguchi AT, Oh-Hashi K, Terada Y, Isonishi A, Morita-Takemura S, Tatsumi K, Kawaguchi M and Wanaka A: Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells. PLoS One. 13:e01961912018. View Article : Google Scholar : PubMed/NCBI
38	Hong GL, Cai QQ, Tan JP, Jiang XZ, Zhao GJ, Wu B, Li MF, Qiu QM and Lu ZQ: Mifepristone-inducible recombinant adenovirus attenuates paraquat-induced lung injury in rats. Hum Exp Toxicol. 34:32–43. 2015. View Article : Google Scholar : PubMed/NCBI
39	Ding YW, Zhao GJ, Li XL, Hong GL, Li MF, Qiu QM, Wu B and Lu ZQ: SIRT1 exerts protective effects against paraquat-induced injury in mouse type II alveolar epithelial cells by deacetylating NRF2 in vitro. Int J Mol Med. 37:1049–1058. 2016. View Article : Google Scholar : PubMed/NCBI
40	Xu C, Li CY and Kong AN: Induction of phase III and III drug metabolism/transport by xenobiotics. Arch Pharm Res. 28:249–268. 2005. View Article : Google Scholar : PubMed/NCBI
41	Maher JM, Dieter MZ, Aleksunes LM, Slitt AL, Guo G, Tanaka Y, Scheffer GL, Chan JY, Manautou JE, Chen Y, et al: Oxidative and electrophilic stress induces multidrug resistance-associated protein transporters via the nuclear factor-E2-related factor-2 transcriptional pathway. Hepatology. 46:1597–1610. 2007. View Article : Google Scholar : PubMed/NCBI
42	Jeddi F, Soozangar N, Sadeghi MR, Somi MH, Shirmohamadi M, Eftekhar-Sadat AT and Samadi N: Nrf2 overexpression is associated with P-glycoprotein upregulation in gastric cancer. Biomed Pharmacother. 97:286–292. 2018. View Article : Google Scholar : PubMed/NCBI
43	Zerin T, Kim YS, Hong SY and Song HY: Protective effect of methylprednisolone on paraquat-induced A549 cell cytotoxicity via induction of efflux transporter, P-glycoprotein expression. Toxicol Lett. 208:101–107. 2012. View Article : Google Scholar : PubMed/NCBI
44	She XR, Tian X, Fan XK, Hong GL, Zhao GJ, Li MF and Lu ZQ: The effects of P-glycoprotein expression induced by ulinastatin on HK-2 cells damage induced by paraquat. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 34:805–809. 2016.(In Chinese). PubMed/NCBI
45	Bartels AL, Kortekaas R, Bart J, Willemsen AT, de Klerk OL, de Vries JJ, van Oostrom JC and Leenders KL: Blood-brain barrier P-glycoprotein function decreases in specific brain regions with aging: A possible role in progressive neurodegeneration. Neurobiol Aging. 30:1818–1824. 2009. View Article : Google Scholar : PubMed/NCBI
46	Lee G and Bendayan R: Functional expression and localization of P-glycoprotein in the central nervous system: Relevance to the pathogenesis and treatment of neurological disorders. Pharm Res. 21:1313–1330. 2004. View Article : Google Scholar : PubMed/NCBI
47	Furuno T, Landi MT, Ceroni M, Caporaso N, Bernucci I, Nappi G, Martignoni E, Schaeffeler E, Eichelbaum M, Schwab M and Zanger UM: Expression polymorphism of the blood-brain barrier component P-glycoprotein (MDR1) in relation to Parkinson's disease. Pharmacogenetics. 12:529–534. 2002. View Article : Google Scholar : PubMed/NCBI