Abstract
BackgroundOxidative 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.MethodsEffects 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.ResultsPFD 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.ConclusionsTheraputic 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.
Highlights
Oxidative stress is one of the important factors involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF)
Effects of PFD on the expressions of oxidants/antioxidants in mouse lung fibroblasts (MLF) stimulated by transforming growth factor-β1 (TGF-β1) To confirm the antioxidant capacity of PFD in MLF, we performed RT-PCR and Western blot to estimate the mRNA and protein levels, respectively, of antioxidant factors such as Nuclear factor-erythroid-related factor 2 (Nrf2), heme oxygenase-1 (Ho-1), glutathione peroxidase 1 (Gpx1), and Bach1 as a result of TGF-β1 stimulation with or without PFD in MLF (Fig. 1)
200 μg/mL PFD substantially improved the expression of an antioxidant gene, Ho-1, as compared to that with 100 μg/mL, but Effects of PFD on the expressions of oxidant/antioxidant in the lung tissues of mice with BLM- induced pulmonary fibrosis To extensively explore the antioxidant mechanisms of PFD, we investigated the expression of oxidants/antioxidants in lung tissues of mice after BLM administration
Summary
Oxidative stress is one of the important factors involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Idiopathic pulmonary fibrosis (IPF) is a disease characterized by diffused alveolar inflammation and extensive interstitial fibrosis, which is related with various cells, inflammatory factors, fibrosis related cytokines, and transforming growth factor-β1 (TGF-β1) signal transduction [1, 2]. It leads to high mortality, and the average survival time after diagnosis is about 2–5 years [3]. Pirfenidone (PFD) is approved drug for IPF treatment with anti-fibrotic, antioxidant, and anti-inflammatory effects [7] It inhibits the differentiation and proliferation of fibroblasts as well as the synthesis of collagen and generation of inflammatory cytokines [8, 9]. Despite that the exploration of the antioxidant mechanism of PFD is not intensive
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