Korean J Physiol Pharmacol.  2020 Nov;24(6):481-492. 10.4196/kjpp.2020.24.6.481.

AITC induces MRP1 expression by protecting against CS/CSE-mediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis

• Xu L
• Wu J
• Li N
• Jiang C
• Guo Y
• Cao P
• Wang D

• KMID: 2507731
• DOI: http://doi.org/10.4196/kjpp.2020.24.6.481

Abstract

The present study aimed to examine the effect of allyl isothiocyanate (AITC) on chronic obstructive pulmonary disease and to investigate whether upregulation of multidrug resistance-associated protein 1 (MRP1) associated with the activation of the PARK7 (DJ-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis. Lung function indexes and histopathological changes in mice were assessed by lung function detection and H&E staining. The expression levels of Nrf2, MRP1, heme oxygenase-1 (HO-1), and DJ-1 were determined by immunohistochemistry, Western blotting and reverse transcription-quantitative polymerase chain reaction. Next, the expression of DJ-1 in human bronchial epithelial (16HBE) cells was silenced by siRNA, and the effect of DJ-1 expression level on cigarette smoke extract (CSE)-stimulated protein degradation and AITC-induced protein expression was examined. The expression of DJ-1, Nrf2, HO-1, and MRP1 was significantly decreased in the wild type model group, while the expression of each protein was significantly increased after administration of AITC. Silencing the expression of DJ-1 in 16HBE cells accelerated CSE-induced protein degradation, and significantly attenuated the AITC-induced mRNA and protein expression of Nrf2 and MRP1. The present study describes a novel mechanism by which AITC induces MRP1 expression by protecting against CS/CSEmediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis.

Keyword

Allyl isothiocyanate; Chronic obstructive pulmonary disease; DJ-1/Nrf2; Multidrug resistance-associated protein 1

Figure

• 

  Fig. 1 Pathological evaluation of lung tissue. (A) Changes in lung histopathology observed following H&E staining. (B) Average score of airway inflammation. #p < 0.05 vs. control group, △p < 0.05 vs. model group, *p < 0.05 vs. WT model group (n = 3). (C) Destructive index of alveoli, #p < 0.05 vs. control group, *p < 0.05 vs. model group, △p < 0.05 vs. WT model group (n = 3). WT, wild type; AITC, allyl isothiocyanate; NAC, N-acetylcysteine; Nrf2, nuclear factor erythroid 2-related factor 2.

• 

  Fig. 2 Effect of Nrf2 gene knockout on MRP1 expression. (A) Immunohistochemistry results of Nrf2, DJ-1, and MRP1 expression in different groups. (B) Nrf2, DJ-1 and MRP1 relative optical density results in the WT groups. #p < 0.05 vs. control group, *p < 0.05 vs. model group (n = 5). (C) DJ-1 and MRP1 relative optical density results in the Nrf2-/- groups. #p < 0.05 vs. control group, *p < 0.05 vs. model group (n = 5). Nrf2, nuclear factor erythroid 2-related factor 2; DJ-1, PARK7; MRP1, multidrug resistance-associated protein 1; WT, wild type; AITC, allyl isothiocyanate; NAC, N-acetylcysteine.

• 

  Fig. 3 Protein expression in the different groups. (A) Western blot results of Nrf2, DJ-1, MRP1, and HO-1 expression in the WT and Nrf2-/- control groups. GAPDH was used as the internal reference. (B) Quantitative analysis of the results of protein expression for each group. Nrf2, nuclear factor erythroid 2-related factor 2; DJ-1, PARK7; MRP1, multidrug resistance-associated protein 1; HO-1, heme oxygenase-1; WT, wild type. *p < 0.05 vs. WT control group (n = 5).

• 

  Fig. 4 Effect of AITC on MRP1 expression. (A) Western blot results of Nrf2, DJ-1, MRP1, and HO-1 expression in different groups. GAPDH was used as the internal reference. (B) Quantitative analysis of the results of protein expression in the WT groups. #p < 0.05 vs. control group, *p < 0.05 vs. model group (n = 5). (C) Quantitative analysis of the results of protein expression in the Nrf2-/- groups. #p < 0.05 vs. control group, *p < 0.05 vs. model group (n = 5). Nrf2, nuclear factor erythroid 2-related factor 2; DJ-1, PARK7; MRP1, multidrug resistance-associated protein 1; HO-1, heme oxygenase-1; WT, wild type; AITC, allyl isothiocyanate; NAC, N-acetylcysteine.

• 

  Fig. 5 Protein expression in 16HBE cells following CSE treatment. (A) MTT assay was used to determine the cellular toxicity of CSE in 16HBE cells. (B) Protein expression of Nrf2, DJ-1, MRP1, and HO-1 in 16HBE cells following treatment for different times with CSE. GAPDH was used as the internal reference. (C) Quantitative analysis of the results of protein expression in each group. CSE, cigarette smoke extract; 16HBE, human bronchial epithelial; Nrf2, nuclear factor erythroid 2-related factor 2; DJ-1, PARK7; MRP1, multidrug resistance-associated protein 1; HO-1, heme oxygenase-1. *p < 0.05 vs. control group (n = 5).

• 

  Fig. 6 Protein expression in 16HBE cells following treatment with AITC and CSE. (A) Protein expression of Nrf2, DJ-1, MRP1, and HO-1 in 16HBE cells following treatment with different concentrations of AITC. (B) Quantitative analysis of the results of protein expression in each group. *p < 0.05 vs. control CSE group (n = 5). (C) Protein expression of Nrf2, DJ-1, MRP1, and HO-1 in 16HBE cells following treatment with AITC and CSE. GAPDH was used as the internal reference. (D) Quantitative analysis of the results of protein expression in each group. #p < 0.05 vs. control group, *p < 0.05 vs. CSE group (n = 5). 16HBE, human bronchial epithelial; Nrf2, nuclear factor erythroid 2-related factor 2; DJ-1, PARK7; MRP1, multidrug resistance-associated protein 1; HO-1, heme oxygenase-1; AITC, allyl isothiocyanate; CSE, cigarette smoke extract.

• 

  Fig. 7 Protein expression in 16HBE cells stimulated by CSE. (A) Protein expression of Nrf2, DJ-1, MRP1, and HO-1 in 16HBE cells following treatment with DJ-1siRNA/Lipofectamine 2000 complex and CSE. (B) Quantitative analysis of the results of protein expression in each group. 16HBE, human bronchial epithelial; Nrf2, nuclear factor erythroid 2-related factor 2; DJ-1, PARK7; MRP1, multidrug resistance-associated protein 1; HO-1, heme oxygenase-1; CSE, cigarette smoke extract. #p < 0.05 vs. control group, *p < 0.05 vs. si-Ctrl+CSE group (n = 5).

• 

  Fig. 8 Effect of DJ-1 on MRP1 and AITC-induced Nrf2 and MRP1 expression. (A) Protein expression of Nrf2, DJ-1, MRP1, and HO-1 in 16HBE cells following treatment with DJ-1siRNA/Lipofectamine 2000 complex and AITC. (B) Quantitative analysis of the results of protein expression in each group. #p < 0.05 vs. control group, *p < 0.05 vs. si-Ctrl+AITC group (n = 5). (C) mRNA expression of Nrf2, DJ-1, MRP1 and HO-1 in 16HBE cells following treatment with DJ-1siRNA/ Lipofectamine 2000 complex and AITC. GAPDH was used as the internal reference. #p < 0.05 vs. control group, *p < 0.05 vs. si-Ctrl+AITC group (n = 6). 16HBE, human bronchial epithelial; Nrf2, nuclear factor erythroid 2-related factor 2; DJ-1, PARK7; MRP1, multidrug resistance-associated protein 1; HO-1, heme oxygenase-1; AITC, allyl isothiocyanate.

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