Tuberc Respir Dis.  2016 Oct;79(4):257-266. 10.4046/trd.2016.79.4.257.

Increased Cellular NAD⁺ Level through NQO1 Enzymatic Action Has Protective Effects on Bleomycin-Induced Lung Fibrosis in Mice

• Oh GS
• Lee SB
• Karna A
• Kim HJ
• Shen A
• Pandit A
• Lee S
• Yang SH
• So HS

• KMID: 2365317
• DOI: http://doi.org/10.4046/trd.2016.79.4.257

Abstract

BACKGROUND
Idiopathic pulmonary fibrosis is a common interstitial lung disease; it is a chronic, progressive, and fatal lung disease of unknown etiology. Over the last two decades, knowledge about the underlying mechanisms of pulmonary fibrosis has improved markedly and facilitated the identification of potential targets for novel therapies. However, despite the large number of antifibrotic drugs being described in experimental pre-clinical studies, the translation of these findings into clinical practices has not been accomplished yet. NADH:quinone oxidoreductase 1 (NQO1) is a homodimeric enzyme that catalyzes the oxidation of NADH to NAD+ by various quinones and thereby elevates the intracellular NAD⁺ levels. In this study, we examined the effect of increase in cellular NAD⁺ levels on bleomycin-induced lung fibrosis in mice.
METHODS
C57BL/6 mice were treated with intratracheal instillation of bleomycin. The mice were orally administered with β-lapachone from 3 days before exposure to bleomycin to 1-3 weeks after exposure to bleomycin. Bronchoalveolar lavage fluid (BALF) was collected for analyzing the infiltration of immune cells. In vitro, A549 cells were treated with transforming growth factor β1 (TGF-β1) and β-lapachone to analyze the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT).
RESULTS
β-Lapachone strongly attenuated bleomycin-induced lung inflammation and fibrosis, characterized by histological staining, infiltrated immune cells in BALF, inflammatory cytokines, fibrotic score, and TGF-β1, α-smooth muscle actin accumulation. In addition, β-lapachone showed a protective role in TGF-β1-induced ECM expression and EMT in A549 cells.
CONCLUSION
Our results suggest that β-lapachone can protect against bleomycin-induced lung inflammation and fibrosis in mice and TGF-β1-induced EMT in vitro, by elevating the NAD+/NADH ratio through NQO1 activation.

Keyword

Inflammation; Fibrosis; Transforming Growth Factor Beta1; Cell; NAD; Pulmonary Fibrosis; Mice; NQO1 Protein, Human

MeSH Terms

Actins
Animals
Bleomycin
Bronchoalveolar Lavage Fluid
Cytokines
Epithelial-Mesenchymal Transition
Extracellular Matrix
Fibrosis*
Idiopathic Pulmonary Fibrosis
In Vitro Techniques
Inflammation
Lung Diseases
Lung Diseases, Interstitial
Lung*
Mice*
NAD
Pneumonia
Pulmonary Fibrosis
Quinones
Transforming Growth Factor beta1
Transforming Growth Factors
Actins
Bleomycin
Cytokines
NAD
Quinones
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  Figure 1 Effect of β-Lap on BLM-induced lung inflammation in mice. Gross (A) and histological (B) analyses of lung tissues after bleomycin (5 mg/kg) and/or β-Lap (40 mg/kg) and BALF analysis for infiltration of immune cells (C, D). All procedures were followed, as described in the "Materials and Methods" section. *,**p<0.05 by one-way analysis of variance compared with control and bleomycin groups, respectively. β-Lap: β-lapachone; BALF: bronchoalveolar lavage fluid; BLM: bleomycin.

• 

  Figure 2 Effect of β-Lap on bleomycin-induced IL-6 expression in lung tissues. Lung tissues from bleomycin (5 mg/kg) and/or β-Lap (40 mg/kg)-treated mice were immunohistochemically stained with anti-mouse IL-6 antibody, as described in the "Materials and Methods" section. β-Lap: β-lapachone; BLM: bleomycin; IL-6: interleukin 6.

• 

  Figure 3 Effect of β-Lap on BLM-induced lung fibrosis in mice. Lung tissues from BLM (2 mg/kg) and/or β-Lap (5, 10, and 20 mg/kg)-treated mice were histologically stained with H&E (A) and picrosirius red (B). (C) Lung tissues from bleomycin and/or β-Lap–treated mice were further analyzed for hydroxyproline, as per the manufacture's instruction. *,**p<0.05 by one-way analysis of variance compared with control and BLM groups, respectively. β-Lap: β-lapachone; BLM: bleomycin.

• 

  Figure 4 Effect of β-Lap on BLM-induced lung fibrosis. Lung tissues from bleomycin (2 mg/kg) and/or β-Lap (20 mg/kg)–treated mice were immunohistochemically stained with anti-mouse TGF-β1 (A) and α-SMA (B) antibodies, as described in the Materials and Methods section. β-Lap: β-lapachone; BLM: bleomycin; TGF-β1: transforming growth factor β1; α-SMA: α-smooth muscle actin.

• 

  Figure 5 In vitro study for evaluating the inhibitory effect of β-Lap on TGF-β1–induced ECM expression, EMT, and morphological changes in A549 cells. (A) The effect of β-Lap on TGF-β1–induced collagen I expression and EMT. (B) The effect of β-Lap on TGF-β1–induced cellular morphological changes. β-Lap: β-lapachone; TGF-β1: transforming growth factor β1; ECM: extracellular matrix; EMT: epithelial-mesenchymal transition.

• 

  Figure 6 Effect of β-Lap on NQO1 activation and NAD+/NADH ratio, evaluated in vitro and in vivo. (A, B) Expression of NQO1 protein and assay for NQO1 enzymatic activation, and role of β-Lap and dicumarol from cytosolic fraction of A549 cell line. (C) Effect of β-Lap on intracellular NAD+/NADH ratio in A549 cells. (D) Effect of β-Lap on NAD+/NADH ratio in BLM-treated lung tissues of mice. NAD+/NADH ratio was analyzed following manufacture's instruction. *p<0.05 by one-way analysis of variance compared with the control, BLM, and β-Lap. β-Lap: β-lapachone; NQO1: NADH:quinone oxidoreductas; BLM: bleomycin.

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