Study On Epithelial-Mesenchymal Transition And Osteopontin In Mice Bleomycin-induced Pulmonary Fibrosis

Pulmonary fibrosis is a disease characterized by fibroblast accumulation,excessive collagen deposition,and matrix remodeling,which lead to distortion of the alveolar architecture,progressive decline in lung function,and ultimately death.This study confirms epithelial-mesenchymal transition(EMT) and the expression of osteopontin (OPN) in various periods of mice BLM-induced pulmonary fibrosis by semi-quantitative analysis and pathomorphological evaluation.78 healthy adult male C57BL/6 mice were randomly divided into the NS-control group(NS),the BLM-injured group(BLM) and BLM+TNL-treated group(BT),which was treated 50μl NS(NS-group) and 50μl BLM solution(BLM and BT group) by oropharyngeal aspiration respectively.Tranilast(250mg/kg,once daily) was administered orally to BLM+TNL-treated group,from modeling till the corresponding time point killing. The three groups mice were taken left lung tissue to perform paraffin imbedding and section,and taken right lung tissue to get western-blot analysis after liquid nitrogen frozen, separately on days 3,7,14,21,28 after treatment.The histopathological changes and total lung collagen content of mice were determined by hematoxylin-eosin staining and picrosirius staining.The expression of OPN,SP-C,E-Cadherin,FSP1 and vimentin were detected by immunohistochemical staining.The section-staining was measured integrated optical density(IOD) by Image-Pro plus 6.0,and then taken statistical analysis.The OPN content was taken SDS-PAGE and Western-blot analysis. The morphological changes of mice lung tissue in various periods were demonstrated by HE staining,and total lung collagen content of BLM-group increased during the pulmonary fibrosis progress by picrosirius-polarization staining(P＜0.05).In immunohistochemical staining,OPN was strongly expressed principally in alveolar macrophages in the lung of BLM-group mice,and the expression of OPN was associated with the development of the fibrotic process,peaking on day 21;and western-blot analysis indicated that the OPN content of BT-treated lung at day 21/28 was less than the content of BLM-injured lung at day21/28.E-Cad and SP-C were selected as the epithelial markers;respectively,FSP1 and vimentin were selected as the mesenchymal markers in the EMT analysis.Lots of SP-C positive typeⅡalveolar cell with normal size were observed in NS-group,and a progressive amount of positive cells in fibrous focal zone with bigger cell body was seen in BLM-group and BT-group;a few of E-Cadherin positive alveolar epithelial cell could be seen in the NS-treated lung and 14-day and 21-day BLM-injured lung,but little E-Cadherin positive cells were seen in fibrous focal zone of 28-day BLM-group and BT-group;FSP1 and vimentin were strongly expressed in fibroblasts accumulating in fibrotic areas in the lung of BLM-group and BT-group mice,but little FSP1 and vimentin positive cells were seen in the normal alveolar regions.These proteins expressions suggested the existence of EMT in mice BLM- induced pulmonary fibrosis,which was related to the increasing OPN potentially.It is hoped that this study will stimulate further consideration of the cellular mechanisms of pulmonary fibrosis,potentially leading to innovative avenues of investigation and treatment.