N-acetyl-l-cysteine inhibits TGF-β1-induced profibrotic responses in fibroblasts

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Abstract

Background

Excessive production of TGF-β1 plays a key role in the tissue remodeling or fibrotic process observed in bronchial asthma, chronic pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). TGF-β1 has been reported to decrease the intracellular glutathione level and stimulate the production of reactive oxygen species.

Objectives

The aim of this study was to evaluate whether the antioxidant N-acetyl-l-cysteine (NAC) can affect TGF-β1-mediated tissue remodeling in fibroblasts or modulate the production of fibronectin and vascular endothelial growth factor (VEGF) which are believed to be important mediators of tissue repair and remodeling.

Methods

To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess the effect of NAC on the TGF-β1-mediated contraction of floating gels and the TGF-β1-induced mediator production. In addition, the effect of NAC on the TGF-β1-induced differentiation to myofibroblasts was evaluated by assessing α-smooth muscle actin (α-SMA) expression.

Results

NAC significantly abolished the TGF-β1-augmented gel contraction (at 3 mM, gel size 63.4 ± 2.6% vs. 39.1 ± 4.1%; p < 0.01) compared with control in a concentration-dependent manner. NAC also significantly inhibited the TGF-β1-augmented fibronectin (p < 0.01) and VEGF (p < 0.01) production in the media of both the three-dimensional gel and monolayer culture. Furthermore, NAC reversed the TGF-β1-stimulated α-SMA expression (p < 0.01).

Conclusion

These results suggest that NAC can affect the TGF-β1-induced tissue remodeling or fibrotic process in vitro.

Introduction

Fibroblasts play a pivotal role in the normal wound healing or tissue repair process [1], whereas excessive stimuli for fibroblasts lead to the tissue remodeling or fibrous tissue that is observed in bronchial asthma [2], chronic obstructive pulmonary disease (COPD) [3] and idiopathic lung fibrosis (IPF) [4].

Transforming growth factor (TGF)-β1 is a multifunctional cytokine in a variety of physiological and pathological processes, including fibroblast repair responses [5], [6]. TGF-β1 regulates the fibroblast migration, proliferation, differentiation and production of matrix. In addition, TGF-β1 stimulates the fibroblast-mediated contraction of extracellular matrix [7].

N-acetylcysteine (NAC) is a non-toxic aminothiol that has a strong reductive capacity [8]. Because NAC is not only a precursor of GSH but also shows a direct ROS-scavenging capacity, NAC has been widely used as a potent antioxidant. In cultured fibroblasts, NAC inhibited the TGF-β1-stimulated collagen production [9]. Moreover, aerosolized administration of NAC attenuates the lung fibrosis induced by bleomycin in mice [10]. These results suggest that antioxidant may suppress TGF-β1 signaling in vitro and in vivo.

Recently, Meurer et al. clearly showed that NAC itself reduced the disulfide bonds of TGF-β1 and changed the bioactive form (dimmer) to the inactive form (monomer) [11]. Moreover, NAC changed the binding activity of TGF-β1 to its receptor in hepatic stellate cells [11], suggesting that the effect of antioxidant NAC is based on a direct blockade of TGF-β1 function and signaling. However, it has not been fully elucidated whether NAC can modulate the TGF-β1-induced tissue repair, mediator production and differentiation in human lung fibroblasts.

In the current study, we used a fibroblast-mediated collagen gel contraction assay as a tissue repair model in the current study and assessed the effect of NAC on the TGF-β1-stimulated collagen gel contraction. We investigated whether NAC affected the production of fibronectin and vascular endothelial growth factor (VEGF), which are believed to be important mediators of repair and remodeling. Furthermore, we evaluated the effect of NAC on the TGF-β-induced differentiation to myofibroblasts by assessing α-smooth muscle actin (α-SMA) expression.

Section snippets

Materials

Native type I collagen [rat tail tendon collagen (RTTC)] was extracted from rat tail tendons by a previously published method [12]. Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were removed carefully. Repeated washing with Tris-buffered saline (0.9% NaCl and 10 mM Tris, pH 7.5) was followed by dehydration and sterilization with 50%, 75%, 95% and pure ethanol. Type I collagen was then extracted in 6 mM hydrochloric acid at 4 °C. The collagen

Effect of NAC on the TGF-β1-augmented collagen gel contraction mediated by fibroblasts

To evaluate whether NAC can affect the TGF-β1-induced tissue repair process, we assessed the effect of NAC by using a collagen gel contraction assay. The cells were pretreated with various concentrations of NAC for 2 h prior to treatment with TGF-β1. TGF-β1 significantly augmented the three-dimensional (3D) collagen gel contraction compared with control on day 3 (at 100 pM, gel size was 39.1 ± 4.1% vs. 61.9 ± 1.1% of initial size; p < 0.01, Fig. 1A, B). NAC (0.3–3 mM) significantly blocked the TGF-β1

Discussion

The present study demonstrated that NAC inhibited the TGF-β1-augmented collagen gel contraction, mediator production and α-SMA expression. These results suggest that NAC can affect the tissue repair process in vitro.

NAC is a non-toxic aminothiol, a potent antioxidant, and a synthetic precursor of GSH. NAC is frequently used as an antioxidant in in vitro and in vivo studies. A previous report showed that NAC reduced the intracellular GSH level and production of reactive oxygen species (ROS) [15]

Acknowledgement

The authors thank Mr. Brent Bell for reading the manuscript.

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