| Pulmonary fibrosis is disease characterized by injury to alveolar epithelial cells,infiltration of inflammatory cells,enhanced proliferation of fibroblasts,aberrant accumulation of the extracellular matrix and abnormality in lung architectural remodeling.Recent evidence suggested that oxidative stress,due to the imbalance between the functions of oxidants and antioxidant proteins,has an important role in the progression of lung fibrosis,by activating redox-sensitive signaling pathways,modification of immune cell function,and activation of fibroblasts.Therefore,based on these studies,it was hypothesized that a strategy to upregulate the expression of endogenous multiple antioxidants maybe an efficient approach to prevent or treat lung fibrosis.The transcription factor nuclear factor erythroid2-related factor-2(Nrf2)has a primary role in regulating cellular antioxidant responsesand increasing the expression and activity of antioxidant enzymes.It has been reported that Nrf2 and its downstream antioxidants enzymes serve critical roles in a lung fibrosis model,where as deletion of the Nrf2 gene increases susceptibility to bleomycin due to reduced antioxidant activity.Previous studies have demonstratedthat the antitoxic and antioxidant properties of SFN in experimental models most likely involves activation of the Nrf2 gene.Therefore,the present study aimed to investigate the effect of SFN alleviate the development of BLM-induced lung fibrosis in a mouse model via the up-regulation of Nrf2.To explore the preventive effect of SFN on pulmonary fibrosis,lung fibrosis model was induced by BLM intratracheal instillation.Then the lung fibrosis model and the control mice were received a subcutaneous injection of SFN each day.Finally,mice from each group were sacrificed at days 7 or 28 after initial BLM or saline administration,and samples were collected.The lungs were investigated with histological examination including HE staining,Masson's trichrome staining,TUNEL and immumohistochemical staining to examine the effect of SFN on fibrosis model.The inflammatory,fibrotic and apoptosis processes were also confirmed by Western blotting assays for the IL-1?,TNF?,TGF-? and caspase-3 protein expressions.Pulmonary collagen content was analyzed with a Hydroxyproline assay kit.In addition,the protein levels of 3-NT,4-HNE,SOD1 and CAT were test by Western blot and the gene and protein levels of Nrf2 and its downstream antioxidant HO-1,NQO1,SOD1 and CAT targets were test by Western blot and Q-PCR.The vitro study aims to investigate the effect of SFN on Ang II induced oxidative stress in human lung fibroblast cells and TGF-? expression.Transfection with Nrf2 si RNA could downregulate the protection effect of SFN in human lung fibroblast cells.For SFN prevention of BLM induced pulmonary fibrosis experiment,SFN treatment significantly alleviated the degree of alveolitis at days 7 and 28,decreased in epithelial cell apoptosis compared at day 7 and declined the collagen accumulation in the interstitial areas and decreased the content of lung hydroxyproline at day 28.Blockade of pro-inflammatory proteins IL-1? and TNF-?,pro-apotosis protein caspase-3 and pro-fibrotic proteins TGF-? and prevention the expression levels of 3-NT and 4-HNE oxidative damage were documented concurrent with enhanced Nrf2 expression in SFN treated group.Mechanistic study using human lung fibroblast cells in vitro,SFN treatment significantly alleviated the levels of ROS and TGF-?induced by Ang II,accompany with the enhanced expression of Nrf2 and HO-1,which confirmed the role of Nrf2 activation since silencing the Nrf2 gene with its specific si RNA abolished SFN prevention of Ang II induced pro-fibrotic response.These results suggest that SFN's preventive effects on bleomycin-induced pulmonary fibrosis via Nrf2 activation and increased antioxidative enzyme activity,which can provides the basis for the therapeutic effect of SFN on the lung against oxidative damage,inflammation and fibrosis. |
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