Sulforaphane prevents bleomycin‑induced pulmonary fibrosis in mice by inhibiting oxidative stress via nuclear factor erythroid 2‑related factor‑2 activation

Yan,Bingdi; Ma,Zhongsen; Shi,Shaomin; Hu,Yuxin; Ma,Tiangang; Rong,Gao; Yang,Junling

doi:10.3892/mmr.2017.6546

Sulforaphane prevents bleomycin‑induced pulmonary fibrosis in mice by inhibiting oxidative stress via nuclear factor erythroid 2‑related factor‑2 activation

Authors:
- Bingdi Yan
- Zhongsen Ma
- Shaomin Shi
- Yuxin Hu
- Tiangang Ma
- Gao Rong
- Junling Yang
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Affiliations: Department of Respiratory Medicine, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China, Department of Respiratory Medicine, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130041, P.R. China
Published online on: May 3, 2017 https://doi.org/10.3892/mmr.2017.6546
Pages: 4005-4014
Copyright: © Yan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Lung fibrosis is associated with inflammation, apoptosis and oxidative damage. The transcription factor nuclear factor erythroid 2‑related factor‑2 (Nrf2) prevents damage to cells from oxidative stress by regulating the expression of antioxidant proteins. Sulforaphane (SFN), an Nrf2 activator, additionally regulates excessive oxidative stress by promoting the expression of endogenous antioxidants. The present study investigated if SFN protects against lung injury induced by bleomycin (BLM). The secondary aim of the present study was to assess if this protection mechanism involves upregulation of Nrf2 and its downstream antioxidants. Pulmonary fibrosis was induced in C57/BL6 mice by intratracheal instillation of BLM. BLM and age‑matched control mice were treated with or without a daily dose of 0.5 mg/kg SFN until sacrifice. On days 7 and 28, mice were assessed for induction of apoptosis, inflammation, fibrosis, oxidative damage and Nrf2 expression in the lungs. The lungs were investigated with histological techniques including haematoxylin and eosin staining, Masson's trichrome staining and terminal deoxynucleotidyl transferase UTP nick end labeling. Inflammatory, fibrotic and apoptotic processes were confirmed by western blot analysis for interleukin‑1β, tumor necrosis factor‑α, transforming growth factor‑β and caspase‑3 protein expressions. Furthermore, protein levels of 3‑nitro‑tyrosine, 4‑hydroxynonenal, superoxide dismutase 1 and catalase were investigated by western blot analysis. It was demonstrated that pulmonary fibrosis induced by BLM significantly increased apoptosis, inflammation, fibrosis and oxidative stress in the lungs at days 7 and 28. Notably, SFN treatment significantly attenuated the infiltration of the inflammatory cells, collagen accumulation, epithelial cell apoptosis and oxidative stress in the lungs. In addition, SFN treatment increased expression of the Nrf2 gene and its downstream targets. In conclusion, these results suggested that SFN treatment of pulmonary fibrosis mouse models may attenuate alveolitis, fibrosis, apoptosis and lung oxidative stress by increasing the expression of antioxidant enzymes, including NAPDH quinone oxidoreductase, heme oxygenase‑1, superoxide dismutase and catalase, via upregulation of Nrf2 gene expression. Thus, the results from the present study may facilitate the development of therapies for BLM‑toxicity and pulmonary fibrosis.

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1	Todd NW, Luzina IG and Atamas SP: Molecular and cellular mechanisms of pulmonary fibrosis. Fibrogenesis Tissue Repair. 5:112012. View Article : Google Scholar :
2	Fernandez IE and Eickelberg O: New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis. Lancet. 380:680–688. 2012. View Article : Google Scholar
3	Shi K, Jiang J, Ma T, Xie J, Duan L, Chen R, Song P, Yu Z, Liu C, Zhu Q and Zheng J: Dexamethasone attenuates bleomycin-induced lung fibrosis in mice through TGF-β, Smad3 and JAK-STAT pathway. Int J Clin Exp Med. 7:2645–2650. 2014.
4	Barratt S and Millar A: Vascular remodelling in the pathogenesis of idiopathic pulmonary fibrosis. QJM. 107:515–519. 2014. View Article : Google Scholar :
5	Bocchino M, Agnese S, Fagone E, Svegliati S, Grieco D, Vancheri C, Gabrielli A, Sanduzzi A and Avvedimento EV: Reactive oxygen species are required for maintenance and differentiation of primary lung fibroblasts in idiopathic pulmonary fibrosis. PLoS One. 5:e140032010. View Article : Google Scholar :
6	Kinnula VL and Myllärniemi M: Oxidant-antioxidant imbalance as a potential contributor to the progression of human pulmonary fibrosis. Antioxid Redox Signal. 10:727–738. 2008. View Article : Google Scholar
7	Liu RM, Vayalil PK, Ballinger C, Dickinson DA, Huang WT, Wang S, Kavanagh TJ, Matthews QL and Postlethwait EM: Transforming growth factor β suppresses glutamate-cysteine ligase gene expression and induces oxidative stress in a lung fibrosis model. Free Radic Biol Med. 53:554–563. 2012. View Article : Google Scholar :
8	Felton VM, Borok Z and Willis BC: N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition. Am J Physiol Lung Cell Mol Physiol. 297:L805–L812. 2009. View Article : Google Scholar :
9	Odajima N, Betsuyaku T, Nagai K, Moriyama C, Wang DH, Takigawa T, Ogino K and Nishimura M: The role of catalase in pulmonary fibrosis. Respir Res. 11:1832010. View Article : Google Scholar :
10	Kinnula VL, Fattman CL, Tan RJ and Oury TD: Oxidative stress in pulmonary fibrosis: A possible role for redox modulatory therapy. Am J Respir Crit Care Med. 172:417–422. 2005. View Article : Google Scholar :
11	Sykiotis GP, Habeos IG, Samuelson AV and Bohmann D: The role of the antioxidant and longevity-promoting Nrf2 pathway in metabolic regulation. Curr Opin Clin Nutr Metab Care. 14:41–48. 2011. View Article : Google Scholar :
12	Nguyen T, Nioi P and Pickett CB: The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem. 284:13291–13295. 2009. View Article : Google Scholar :
13	Pekovic-Vaughan V, Gibbs J, Yoshitane H, Yang N, Pathiranage D, Guo B, Sagami A, Taguchi K, Bechtold D, Loudon A, et al: The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis. Genes Dev. 28:548–560. 2014. View Article : Google Scholar :
14	Kikuchi N, Ishii Y, Morishima Y, Yageta Y, Haraguchi N, Itoh K, Yamamoto M and Hizawa N: Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance. Respir Res. 11:312010. View Article : Google Scholar :
15	Kandhare AD, Bodhankar SL, Mohan V and Thakurdesai PA: Effect of glycosides based standardized fenugreek seed extract in bleomycin-induced pulmonary fibrosis in rats: Decisive role of Bax, Nrf2, NF-κB, Muc5ac, TNF-α and IL-1β. Chem Biol Interact. 237:151–165. 2015. View Article : Google Scholar
16	Mouratis MA and Aidinis V: Modeling pulmonary fibrosis with bleomycin. Curr Opin Pulm Med. 17:355–361. 2011. View Article : Google Scholar
17	Fahey JW and Talalay P: Antioxidant functions of sulforaphane: A potent inducer of Phase II detoxication enzymes. Food Chem Toxicol. 37:973–979. 1999. View Article : Google Scholar
18	Cui W, Bai Y, Miao X, Luo P, Chen Q, Tan Y, Rane MJ, Miao L and Cai L: Prevention of diabetic nephropathy by sulforaphane: Possible role of Nrf2 upregulation and activation. Oxid Med Cell Longev. 2012:8219362012. View Article : Google Scholar :
19	Noh JR, Kim YH, Hwang JH, Choi DH, Kim KS, Oh WK and Lee CH: Sulforaphane protects against acetaminophen-induced hepatotoxicity. Food Chem Toxicol. 80:193–200. 2015. View Article : Google Scholar
20	Szapiel SV, Elson NA, Fulmer JD, Hunninghake GW and Crystal RG: Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. Am Rev Respir Dis. 120:893–899. 1979.
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar
22	Cheresh P, Kim SJ, Tulasiram S and Kamp DW: Oxidative stress and pulmonary fibrosis. Biochim Biophys Acta. 1832:1028–1040. 2013. View Article : Google Scholar
23	Walters DM and Kleeberger SR: Mouse models of bleomycin-induced pulmonary fibrosis. Curr Protoc Pharmacol Chapter. 5:Unit 5 462008.
24	Cho HY, Reddy SP, Yamamoto M and Kleeberger SR: The transcription factor NRF2 protects against pulmonary fibrosis. FASEB J. 18:1258–1260. 2004.
25	Walters DM, Cho HY and Kleeberger SR: Oxidative stress and antioxidants in the pathogenesis of pulmonary fibrosis: A potential role for Nrf2. Antioxid Redox Signal. 10:321–332. 2008. View Article : Google Scholar
26	Jiang X, Bai Y, Zhang Z, Xin Y and Cai L: Protection by sulforaphane from type 1 diabetes-induced testicular apoptosis is associated with the up-regulation of Nrf2 expression and function. Toxicol Appl Pharmacol. 279:198–210. 2014. View Article : Google Scholar
27	Wang Y, Zhang Z, Sun W, Tan Y, Liu Y, Zheng Y, Liu Q, Cai L and Sun J: Sulforaphane attenuation of type 2 diabetes-induced aortic damage was associated with the upregulation of Nrf2 expression and function. Oxid Med Cell Longev. 2014:1239632014. View Article : Google Scholar :
28	Artaud-Macari E, Goven D, Brayer S, Hamimi A, Besnard V, Marchal-Somme J, Ali ZE, Crestani B, Kerdine-Römer S, Boutten A and Bonay M: Nuclear factor erythroid 2-related factor 2 nuclear translocation induces myofibroblastic dedifferentiation in idiopathic pulmonary fibrosis. Antioxid Redox Signal. 18:66–79. 2013. View Article : Google Scholar
29	Higgins LG, Kelleher MO, Eggleston IM, Itoh K, Yamamoto M and Hayes JD: Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents. Toxicol Appl Pharmacol. 237:267–280. 2009. View Article : Google Scholar
30	Miao X, Bai Y, Sun W, Cui W, Xin Y, Wang Y, Tan Y, Miao L, Fu Y, Su G and Cai L: Sulforaphane prevention of diabetes-induced aortic damage was associated with the up-regulation of Nrf2 and its down-stream antioxidants. Nutr Metab (Lond). 9:842012. View Article : Google Scholar :
31	Bai Y, Cui W, Xin Y, Miao X, Barati MT, Zhang C, Chen Q, Tan Y, Cui T, Zheng Y and Cai L: Prevention by sulforaphane of diabetic cardiomyopathy is associated with up-regulation of Nrf2 expression and transcription activation. J Mol Cell Cardiol. 57:82–95. 2013. View Article : Google Scholar
32	Oh CJ, Kim JY, Min AK, Park KG, Harris RA, Kim HJ and Lee IK: Sulforaphane attenuates hepatic fibrosis via NF-E2-related factor 2-mediated inhibition of transforming growth factor-β/Smad signaling. Free Radic Biol Med. 52:671–682. 2012. View Article : Google Scholar