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. 2016 Aug 30;17(1):107.
doi: 10.1186/s12931-016-0420-x.

Metformin Attenuates Lung Fibrosis Development via NOX4 Suppression

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Free PMC article

Metformin Attenuates Lung Fibrosis Development via NOX4 Suppression

Nahoko Sato et al. Respir Res. .
Free PMC article

Abstract

Background: Accumulation of profibrotic myofibroblasts in fibroblastic foci (FF) is a crucial process for development of fibrosis during idiopathic pulmonary fibrosis (IPF) pathogenesis, and transforming growth factor (TGF)-β plays a key regulatory role in myofibroblast differentiation. Reactive oxygen species (ROS) has been proposed to be involved in the mechanism for TGF-β-induced myofibroblast differentiation. Metformin is a biguanide antidiabetic medication and its pharmacological action is mediated through the activation of AMP-activated protein kinase (AMPK), which regulates not only energy homeostasis but also stress responses, including ROS. Therefore, we sought to investigate the inhibitory role of metformin in lung fibrosis development via modulating TGF-β signaling.

Methods: TGF-β-induced myofibroblast differentiation in lung fibroblasts (LF) was used for in vitro models. The anti-fibrotic role of metfromin was examined in a bleomycin (BLM)-induced lung fibrosis model.

Results: We found that TGF-β-induced myofibroblast differentiation was clearly inhibited by metformin treatment in LF. Metformin-mediated activation of AMPK was responsible for inhibiting TGF-β-induced NOX4 expression. NOX4 knockdown and N-acetylcysteine (NAC) treatment illustrated that NOX4-derived ROS generation was critical for TGF-β-induced SMAD phosphorylation and myofibroblast differentiation. BLM treatment induced development of lung fibrosis with concomitantly enhanced NOX4 expression and SMAD phosphorylation, which was efficiently inhibited by metformin. Increased NOX4 expression levels were also observed in FF of IPF lungs and LF isolated from IPF patients.

Conclusions: These findings suggest that metformin can be a promising anti-fibrotic modality of treatment for IPF affected by TGF-β.

Keywords: IPF; Metformin; NOX4; ROS; TGF-β.

Figures

Fig. 1
Fig. 1
Metformin inhibits myofibroblast differentiation through AMPK activation in LF. a Western blotting (WB) using anti-type I collagen, anti-α-smooth muscle actin (SMA), and anti-β-actin of cell lysates from control (lane 1, 2), metformin (1 mM) (lane 3, 4), and metformin (10 mM) (lane 5, 6) treated LF. Metformin treatment was started 1 h before TGF-β (2 ng/ml) stimulation and protein samples were collected after 24 h treatment with TGF-β. In the right panels are the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05. b WB using anti-phospho-AMPK, anti-αSMA, and anti-β-actin of cell lysates from control (lane 1, 2) and metformin (10 mM) (lane 3, 4) treated LF. Metformin treatment was started 1 h before TGF-β (2 ng/ml) stimulation and protein samples were collected after 24 h treatment with TGF-β. In the right panels are the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05. c WB using anti-type I collagen, anti-αSMA, anti-phospho-AMPK, and anti-β-actin of cell lysates from control siRNA (lane 1, 2, 3, 4) and AMPK siRNA (lane 5, 6, 7, 8) transfected LF. Metformin treatment was started 48 h post transfection and 1 h before TGF-β (2 ng/ml) stimulation. Protein samples were collected after 24 h treatment with TGF-β. The right panels show the average (±SEM) of type I collagen and αSMA relative expression, which were taken from five to six independent experiments, respectively. Open bar is control and filled bar is TGF-β treated. *p < 0.05
Fig. 2
Fig. 2
Metformin-mediated AMPK activation is involved in suppression of TGF-β-induced NOX4 expression in LF. a WB using anti-NOX4, and anti-β-actin of cell lysates from control (lane 1, 2) and metformin (lane 3, 4) treated LF. Metformin treatment was started 1 h before TGF-β (2 ng/ml) stimulation and protein samples were collected after 24 h treatment with TGF-β. Lower panel is the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is metformin treated. *p < 0.05. b Left panel: LF were treated with TGF-β and mRNA samples were collected at indicated time points (n = 9). *p < 0.05. Right panel: LF were treated with TGF-β in the presence or absence of metformin (10 mM) and mRNA samples were collected after 12 h treatment with TGF-β (n = 6). Open bar is control and filled bar is metformin treated. Real time-PCR was performed using primers to NOX4 or β-actin, as a control. NOX4 expression was normalized to β-actin. Shown is the fold increase (±SEM) relative to control treated cells. *p < 0.05. c WB using anti-NOX4, anti-type I collagen, anti-α-smooth muscle actin (SMA) and anti-β-actin of cell lysates from control siRNA (lane 1, 2) and NOX4 siRNA (lane 3, 4) transfected LF. TGF-β (2 ng/ml) treatment was started 48 h post transfection. Protein samples were collected after 24 h treatment with TGF-β. In the right panels are the average (±SEM) taken from four independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05. d WB using anti-phospho-AMPK, anti-NOX4, anti-type I collagen, anti-αSMA, and anti-β-actin of cell lysates from control siRNA (lane 1, 2, 3, 4) and AMPK siRNA (lane 5, 6) transfected LF. Metformin treatment was started 48 h post transfection and 1 h before TGF-β (2 ng/ml) stimulation, and protein samples were collected after 24 h treatment with TGF-β. In the right panels are the average (±SEM) taken from five independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05
Fig. 3
Fig. 3
Metformin and NOX4 regulate SMAD phosphorylation in LF. a WB using anti-phospho-SMAD2, anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, and anti-β-actin of cell lysates from control siRNA (lane 1, 2) and NOX4 siRNA (lane 3, 4) transfected LF. TGF-β (2 ng/ml) treatment was started 48 h post transfection. Protein samples were collected after 30 min treatment with TGF-β. In the right panels are the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05. b WB using anti-phospho-SMAD2, anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, and anti-β-actin of cell lysates from control (lane 1, 2) and metformin (10 mM) (lane 3, 4) treated LF. Metformin treatment was started 1 h before TGF-β (2 ng/ml) stimulation and protein samples were collected after 30 min treatment with TGF-β. In the right panels are the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05
Fig. 4
Fig. 4
NOX4-mediated ROS is involved in the mechanisms for SMAD phospholylation and myofibroblast differentiation in LF. a Fluorescence intensity of CM-H2DCFDA staining for intracellular ROS production. After 24 h treatment with TGF-β, incubation with CM-H2DCFDA (10 μM) was performed for 30 min, fluorescence of DCF was measured by a fluorescence microplate reader. The fluorescence level in the control treated cells in the absence of metformin was designated as 1.0. Shown panels are the average (±SEM) taken from three independent experiments. *p < 0.05. b Fluorescence intensity of CM-H2DCFDA staining for intracellular ROS production. Metformin treatment was started 48 h post-siRNA transfection and 1 h before TGF-β (2 ng/ml) stimulation. After 30 min incubation with CM-H2DCFDA, fluorescence of DCF was measured by a fluorescence microplate reader. The fluorescence level in the control siRNA transfected cells without TGF-β and metformin treatment was designated as 1.0. Shown panels are the average (±SEM) taken from six independent experiments. *p < 0.05. c WB using anti-phospho-SMAD2, anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, anti-type I collagen, anti-αSMA, and anti-β-actin of cell lysates from control (lane 1, 2), NAC (1 mM) (lane 3, 4), and NAC (10 mM) (lane 5, 6) treated LF. NAC treatment was started 1 h before TGF-β (2 ng/ml) stimulation and protein samples were collected after 24 h treatment for type I collagen and anti-αSMA WB, but 30 min for SMAD WB. Shown panels are the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05 and **p < 0.001
Fig. 5
Fig. 5
Effect of metformin on bleomycin-induced lung fibrosis development in mice. a Body Wight (BW) changes after BLM treatment. BW at day 0 before treatment was designated as 1.0. *p < 0.05. b Photomicrographs of Masson trichrome and Hematoxylin-Eosin staining of mouse lungs at day 21. Upper panels are low magnification view of Masson trichrome staining. Original magnification × 40. Middle panels are High magnification view of Masson trichrome staining. Original magnification × 100. Lower panels are high magnification view of Hematoxylin-Eosin staining. Original magnification × 100. c Shown in the panel is the average (±SEM) soluble collagen measurement from Sircol assay using control (n = 13), BLM-treated (n = 18), and BLM-treated with subsequent metformin injection mouse lungs (n = 15) at day 21. Open bar is control, filled bar is BLM-treated, and horizontal crosshatched bar is BLM-treated with subsequent metformin injection. *p < 0.05. d Immunohistochemical staining of NOX4, p-SMAD3, αSMA in mouse lungs at day 21. Upper panels are high magnification view of NOX4 staining. Original magnification × 200. Middle panels are High magnification view of p-SMAD3 staining. Original magnification × 400. Lower panels are high magnification view of αSMA staining. Original magnification × 200. Bar = 100 μm e Immunohistochemical staining of NOX4 in human lungs. Upper panels are high magnification view of normal lungs. Original magnification × 200. Lower panels are High magnification view of IPF lungs. Original magnification × 400. Bar = 100 μm f WB using anti-NOX4, and anti-β-actin of cell lysates from normal LF (lane 1, 2, 3) and IPF LF (lane 4, 5, 6). Lower panel is the average (±SEM) taken from three patients shown as relative expression. Open bar is normal LF and filled bar is IPF LF. *p < 0.05
Fig. 6
Fig. 6
Hypothetical model of metformin-mediated inhibition of myofibroblast differentiation. Metformin-mediated AMPK activation is responsible for inhibiting NOX4 expression and ROS production, which is at least partly involved in the mechanisms for attenuation of TGF-β-induced SMAD phosphorylation and myofibroblast differentiation in relation to fibroblastic foci formation in IPF pathogenesis

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