Pirfenidone attenuates bleomycin-induced pulmonary fibrosis in mice by regulating Nrf2/Bach1 equilibrium

Abstract

Background: Oxidative stress is one of the important factors involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The equilibrium of Nuclear factor-erythroid-related factor 2 (Nrf2)/[BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1, Bach1] determines the expression level of antioxidant factors, further regulating the function of oxidation/antioxidation capacity. Pirfenidone (PFD) is one of two currently for IPF therapy approved drugs. PFD regulates intracellular antioxidants, inhibits secretion of inflammatory cytokines and collagen synthesis. However the mechanisms of its antioxidant effects remain elusive.

Methods: Effects of PFD treatment were studied in mouse lung fibroblasts (MLF) following induction by transforming-growth factor beta 1 (TGF-β1) and in mice following bleomycin-induced lung fibrosis. The mRNA and protein levels of oxidative stress-related factors Nrf2/Bach1 and their downstream antioxidant factors heme oxygenase-1 (Ho-1) and glutathione peroxidase 1 (Gpx1) were determined by RT-PCR and Western blot. Fibrosis-related cytokines interleukin-6 (IL-6) and myofibroblast markers type 1 collagen α1 (COL1A1) levels in supernate of MLF, serum, and bronchoalveolar lavage fluid (BALF) as well as malondialdehyde (MDA) in serum and BALF were detected by ELISA, reactive oxygen species (ROS) generation was measured by 2',7'- dichlorofluorescin diacetate (DCFH-DA) assay and lung pathological/morphological alterations in mice were observed by HE and Masson to assess the antioxidant mechanism and therapeutic effects on pulmonary fibrosis induced by bleomycin.

Results: PFD inhibited Bach1 mRNA and protein expressions in mouse lung fibroblasts induced by TGF-β1 and lung tissues with pulmonary fibrosis induced by bleomycin. Furthermore, it improved Nrf2, Ho-1 and Gpx1 mRNA and protein expressions. After PFD treatment, COL1A1and IL-6 levels in supernate of MLF, serum, and BALF as well as ROS in lung tissues and MDA in serum and BALF from a mouse with pulmonary fibrosis were significantly decreased, and the infiltration of lung inflammatory cells and fibrosis degree were alleviated.

Conclusions: Theraputic effects of PFD for IPF were involved in Nrf2/Bach1 equilibrium which regulated the capacity of oxidative stress. The study provided new insights into the antioxidant mechanism of PFD.

Keywords: Idiopathic pulmonary fibrosis; Nrf2/Bach1; Oxidative stress; Pirfenidone.

Figures

Fig. 1

Expression of oxidant/antioxidant factors in Mouse lung fibroblasts (MLF) incubated for 24 h with TGF-β1 and various concentrations of pirfenidone (PFD) for 48 h. MLF cells were stimulated with TGF-β1 (5 ng/mL) for 24 h before PFD (100 μg/mL, 200 μg/mL and 500 μg/mL) treatment for 48 h. The mRNA expression of nuclear factor-erythroid-related factor 2 (Nrf2), [BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap‘n’collar protein) homology 1, Bach1], heme oxygenase-1 (HO-1), and glutathione peroxidase 1 (GPx1) in MLF cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) (a, b, c, d). The protein levels of Nrf2, Bach1, Ho-1, and GPx1 in MLF cells were detected by Western blot (e). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, vs. TGF-β1 group

Fig. 2

Effects of PFD on oxidant/antioxidant factors in Bleomycin (BLM)-induced pulmonary fibrosis in mice (3 in each group). BLM (5 mg/kg, intratracheally instillation for 14d) administered mice were then treated with PFD (300 mg/kg/d orally administered) for 4 weeks. Control group was intratracheally administered 50 μL 0.9% saline), only PFD group was orally administrated with 300 mg/kg PFD daily for 4 weeks. The mRNA expressions of Nrf2, Bach1, HO-1, and GPx1 in the lung were analyzed by RT-PCR (a, b, c, d); protein levels of Nrf2, Bach1, HO-1, and GPx1 in the lung were detected by Western blot (e). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, vs. BLM group

Fig. 3

Analysis of type 1 collagen α1 (COL1A1) and interleukin-6 (IL-6) levels in the supernatant of cells, serum and bronchoalveolar lavage fluid (BALF). They were measured by enzyme-linked immunosorbent assay (ELISA). Data are expressed as the mean ± SD. a and b: COL1A1 and IL-6 levels in the supernatant of cells after TGF-β1 and various concentrations of PFD stimulation (*P < 0.05, **P < 0.01, vs. TGF-β1 group). c and d: Serum COL1A1 and IL-6 after BLM and PFD treatment (*P < 0.05, **P < 0.01, vs. BLM 14d and 42d group). e and f: COL1A1 and IL-6 levels in BALF after BLM and PFD treatment (*P < 0.05, **P < 0.01, vs. BLM 14d and 42d group)

Fig. 4

Effect of PFD on indicators of oxidative stress such as reactive oxygen species (ROS) generation and malondialdehyde (MDA) expression. a: ROS generation (%) in lung tissue was detected with 2,7-dichlorofluorescein diacetate (DCFDA) by flow cytometric. b and c: MDA expression in serum and BALF by ELISA. Data represent as the means ± SD. *P < 0.05, **P < 0.01, vs. BLM 14d and 42d group

Fig. 5

Histopathological changes of lung tissue in mice at the end point of the experiment (HE staining, ×200). BLM (5 mg/kg) intratracheally administered once for 2 weeks and then treated with pirfenidone (300 mg/kg/d orally administered) for 4 weeks. Lung histological data obtained on day 14 after BLM treatment and day 42 after PFD treatment (BLM 42 d). a: Lung inflammation, fibrosis, and integrity of the structure was evaluated by H&E (×200). Degree of fibrosis in lung tissues by Masson’s staining (×200). b and c: Degree of lung fibrosis was evaluated by inflammation and fibrosis scored. The data are expressed as mean ± SD,*P < 0.05, **P < 0.01, vs. BLM 14d and 42 d group