Investigation On The Expression Changes And Intervention Of TGF-β1/Smad Signaling Pathway In OLETF Rats Lung

Objective:Diabetes mellitus (DM) is a chronic metabolic disorder, which character as persistent high blood glucose. Studies confirmed that lung damage is also a part of diabetes systemic damages and diabetes can significantly increase the risk of pulmonary fibrosis. TGF-β1 as a profibrotic cytokine plays an important role in the process in a variety of organ fibrosis. Smads proteins are the most important cytokins in the downstream of TGF-β1, which transfer the information of TGF-β1 from membrane to aim genes. Recent studies have found that, excessive activation of TGF-β1/Smad signaling transduction pathway is closely related to diabetes nephropathy and non-diabetes pulmonary fibrosis, while research on the relationship between TGF-β1/Smad signal transduction pathway and DM pulmonary fibrosis is little. Rosiglitazone is a synthetic ligand of peroxisome proliferator-activated receptor-y(PPAR-y), which can regulate metabolism, anti-inflammatory and anti-fibrosis.The aim of this study is to observe the changes of pulmonary structure by HE staining and the collagen aggradation by Masson staining, to observe the change of TGF-β1, Smad2, Smad3, Smad7,collagen I(COLⅠ) and collagenⅢ(COLⅢ) in OLETF rat pulmonary tissue by using immunohistochemistry.Establishing a rosiglitazone intervention group to observe the changes of these indicators, so that to explore whether rosiglitazone has a protective effect on diabetes pulmonary damage.Methods:30 OLETF rats, aged 4 weeks, are spontaneous type 2 diabetes mellitus model, and 8 LETO rats served as normal control group (N group). They were raised in single cage with standard feed in specific pathogen-free conditions. OGTT were performed every four weeks. There were 16 OLETF rats modelled successfully by the end of 30th week, which were randomly divided into treated group (RGT group) and untreated group (DM group) for 8 rats each group. Roziglitazone were given 3mg/kg daily to treated group for 12 weeks, while DM group and N group were administered with distilled water equal daily for 12 weeks. Then these three group rats were killed by the femoral artery bleeding and the lung samples were obtained, which were fixed in 10% neutral formalin for:①Observed the structural changes of lung tissue by HE staining;②Observed collagen deposition in lung tissue of each group by Masson staining;③Positioning TGF-β1,Smad2,Smad 3,Smad 7,COLⅠ,COLⅢby immunohistochemistry, and detected the expression of TGF-β1,Smad 2,Smad 3,Smad 7 under light microscope by observing the staining intensity and calculated the percentage of positive cells,and determined COLⅠ,COLⅢratios of the positive reactive area of view and mean optical density by the image analysis system.Results:(1) HE staining:N group had normal alveolar structure. The lung organizational structure of DM group disordered,bronchial wall,alveolar wall thickening,alveolar epithelial cells showed unclear alveolar atrophy,collapse,extracellular matrix and fibroblasts of pulmonary interstitial and perivascular increased, and there were inflammatory cell infiltration. Lung damage in RGT group were slighter than DM group, while didn't recover to normal either. (2) Masson staining:Pulmonary interstitial and perivascular of N group had little collagen fibers and in DM group they increased and disordered. Collagen deposition in RGT group was slighter than DM group, but still heavier than N group. (3) Immunohistochemistry:①TGF-β1 could be seen in the cytoplasm of bronchial epithelial cells, type II pneumocytes, vascular endothelium cells, alveolar macrophages and myofibroblasts.②Smad2 could be seen in the cytoplasm and nucleas of macrophages, myofibroblasts, vascular endothelium cells, bronchial epithelial cells and alveolar epithelial cells.③Smad3 could be seen in the cytoplasm and nucleas of macrophages, myofibroblasts, bronchial epithelial cells and alveolar epithelial cells.④Smad7 could be seen in the cytoplasm, sometimes nucleas aslo, of alveolar epithelial cells, bronchial epithelial cells and part of interstitial cells.⑤COLⅠ,Ⅲwere mainly scattered in the connective tissue column of small bronchial and vascular. The expression of TGF-β1,Smad2,Smad3,COLⅠ,COLⅢin DM group were heavier than N group and RGT group, while Smad7 was slighter(P<0.01).(5)Pearson correlation analysis:The expression of TGF-β1 had positive correlation with that of Smad2,Smad3,COLⅠand COLⅢratios of the positive reactive area of view and mean optical density in DM group (r=0.649, 0.714,0.974,0.948,0.932,0.879, P<0.01), while had negative correlation with Smad7 (r=-0.822, P<0.01)Conclusions:(1) Pathological changes and collagen fibers deposition occurred in lung tissue of DM group indicating that pulmonary was another target organ of diabetes. (2) The expression of TGF-β1,Smad2,Smad3,COLⅠ,COLⅢincreased in DM group, while Smad7 in DM group decreased.These all suggest that abnormal expression of TGF-β1/Smad signal transduction pathway may play a role in the pathogenesis of pulmonary damage. (3)The pathological changes of pulmonary in RGT group were slighter than DM group. The deposition of collagen and the expression of TGF-β1,Smad2,Smad3,COLⅠ,COLⅢin pulmonary were suppressed in RGT group, which suggest that roziglitazone can relieve the extent of pulmonary pathological changes in DM.