Oxidative Stress Contributes To The Persistence Of Pulmonary Fibrosis

[Rationale]Idiopathic pulmonary fibrosis (IPF) is a chronic, debilitating and lethal fibrotic disorder with progressive worsening of pulmonary function and characterized by persistent structural.alterations of lung parenchyma with an abnormal accumulation of fibroblasts/myofibroblasts and formation of fibroblastic foci in the lung, along with excessive deposition of extracellular matrix (ECM) components. The precise etiology of IPF remains elusive. Recent evidence suggests that, amongst other processes, oxidative stress and reactive oxygen species (ROS) may play a role in the pathobiology and progression of this fatal disease. Therefore,antioxidant enzymes, such as extracellular superoxide dismutase (ECSOD), may modulate the injury and repair components of the fibrogenic response. Accumulating evidence has demonstrated that lung specific transgenic overexpression of ECSOD could protect against bleomycin-or radiation-induced lung injury and thus interfered in the subsequent development of fibrosis. Furthermore, ECSOD knockout mice were more susceptible to pulmonary damage after bleomycin or asbestos exposure. However, whether ECSOD protects against ROS mediated damage and reduced requirement for repair and/or is involved in mediating the initiation and/or progression of fibrosis cannot be determined from these studies. Curcumin, a polyphenolic compound derived from a traditional herbal Chinese medicine Curcuma Longa, possesses antioxidant, anti-inflammatory and anti-carcinogenic properties. Curcumin has been found to protect lung structure alterations and suppress the increase in lung hydroxyproline content in bleomycin-induced pulmonary fibrosis. However, all the previous studies only provide evidence that curcumin possesses the ability to attenuate early inflammatory reactions associated with bleomycin administration, but do not test the hypothesis whether curcumin can exhibit an anti-TGFβ-1 activity or otherwise show a direct anti-fibrotic activity.[Objectives]To identify the presence of oxidative stress in TGF-β1 induced experimental lung fibrosis and the role of ECSOD in modulation of the oxidative stress and fibrotic changes induced by TGF-β1. To investigate curcumin's therapeutic effects in a rat model of established and persistent pulmonary fibrosis induced by TGF-β1.[Methods]Experiment 1:Rats were endotracheally intubated with an adenoviral vector encoding active TGF-β1 (AdTGF-β1) or a control vector (AdDL70). The amount and distribution of oxidative product in rat lung was determined by nitrotyrosine immunodot blot assay and immunohistochemistry respectively. Endogenous ECSOD expression in rat lung was evaluated by real-time PCR and immunohistochemistry. Furthermore, primary human lung fibroblasts, MRC-5 fibroblasts and A549 epithelial cells were exposed to recombinant active TGF-β1 and endogenous ECSOD gene expression was measured by real-time PCR.Experiment 2:ECSOD loss-of-function experiments were conducted in MRC-5 cells using small interfering RNA (siRNA) to target endogenous human ECSOD. An adenovirus vector that expresses.human ECSOD (AdECSOD) was constructed and rats were endotracheally intubated with an adenoviral vectors in combinations of AdTGF-β1/AdDL70 or AdTGF-β1/AdECSOD. The amount and of oxidative product in rat lung was determined by nitrotyrosine immunodot blot assay. The role of ECSOD in modulation of the fibrotic changes induced by TGF-β1 was evaluated by BAL cell count, ELISA, lung function, histologic analysis, hydroxyproline content and fibrosis-related gene expression.Experiment 3:Female Sprague Dawley rats were administrated with AdTGF-β1 via endotracheal intubation. Rats received daily intraperitoneal (IP) injection or oral administration of curcumin (200mg/kg) from day 1 to day 21. The effects of curcumin by gavaging and IP injection on TGFβ1-induced pulmonary fibrosis were determined by lung elastance measurements, histomorphology and hydroxyproline content 21 days after virus administration.[Results]Experiment 1:TGF-β1 alone induced fibrotic responses and significantly downregulated endogenous ECSOD gene expression both in vitro and in vivo and caused oxidative stress in rat lung. ECSOD protein was markedly reduced in the interstitium and fibrotic foci in TGF-β1 induced experimental lung fibrosis.Experiment 2:The fibrotic response caused by AdTGF-β1 was markedly attenuated by concomitant gene transfer using AdECSOD, detected by lung function measurements, histologic and morphometric analysis, hydroxyproline content and fibrosis-related gene expression. In addition, the oxidative stress and increased presence of activated TGF-β1 in rat lung induced by AdTGF-β1 was significantly reduced by ECSOD gene transfer.Experiment 3:As expected, transient overexpression of active TGF-β1 by adenoviral vector gene transfection in rat lung induced severe distortion of lung structure and formation of fibrous areas associated with increased lung elastance compared with the naive group. The pathologic changes in rat lung induced by TGF-β1 gene transfer were not significantly modified by either oral administration or IP injection of curcumin. In addition, lung compliance measurement and lung hydroxyproline content did not show a significant impact of either oral administration or IP injection of curcumin in TGF-β1 induced lung fibrosis.[Conclusions]These findings suggest a substantial role for oxidative stress in the pathogenesis of TGF-β1 driven persistent pulmonary fibrosis and enhanced presence of ECSOD can inhibit latent TGF-β1 activation by ROS and diminish subsequent fibrotic responses. In previous published studies, curcumin is administrated during an early phase of BLM-induced experimental fibrosis as a preventative treatment. In our current study, we show that neither IP injection nor oral administration of curcumin attenuates lung fibrosis induced by AdTGF-β1. This indicates that the therapeutic option for curcumin may only be confined to inflammation triggered fibrosis.