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

BMC Pulm Med. 2017 Apr 18;17(1):63. doi: 10.1186/s12890-017-0405-7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / pharmacology*
  • Basic-Leucine Zipper Transcription Factors / metabolism*
  • Bleomycin / toxicity
  • Bronchoalveolar Lavage Fluid / chemistry
  • Bronchoalveolar Lavage Fluid / cytology
  • Collagen Type I / metabolism
  • Fibroblasts / metabolism
  • Glutathione Peroxidase / metabolism
  • Glutathione Peroxidase GPX1
  • Heme Oxygenase-1 / metabolism
  • Interleukin-6 / metabolism
  • Lung / pathology
  • Male
  • Malondialdehyde / metabolism
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Myofibroblasts / metabolism
  • NF-E2-Related Factor 2 / metabolism*
  • Oxidative Stress / drug effects*
  • Pulmonary Fibrosis / chemically induced
  • Pulmonary Fibrosis / drug therapy*
  • Pyridones / pharmacology*
  • RNA, Messenger / metabolism
  • Transforming Growth Factor beta / metabolism

Substances

  • Antioxidants
  • Bach1 protein, mouse
  • Basic-Leucine Zipper Transcription Factors
  • Collagen Type I
  • Interleukin-6
  • Membrane Proteins
  • NF-E2-Related Factor 2
  • Nfe2l2 protein, mouse
  • Pyridones
  • RNA, Messenger
  • Transforming Growth Factor beta
  • interleukin-6, mouse
  • Bleomycin
  • Malondialdehyde
  • pirfenidone
  • Glutathione Peroxidase
  • Heme Oxygenase-1
  • Hmox1 protein, mouse
  • Glutathione Peroxidase GPX1
  • Gpx1 protein, mouse