| Objectives:Diabetes is a systemic metabolic syndrome that characterized by hyperglycemia, and its multiple chronic complications are the main reasons for the patients'low quality of life, death and disability. Researchers have focused the main strengths on complications of the heart, kidney, nerve and retina for a long time. However, there is relatively few study about how the diabetes impacts on lungs. Initial researches found there was abnormal extracellular matrix in lungs of diabetic rat, and cytokine that promoting fibrosis-- transforming growth factor (TGF)-?1 could be found significantly increased. Whether the increased extracellular matrix has anything to do with the over activation of the TGF-?1/Smad signal transduction pathway is not clear. It has been reported that, the basement membrane of capillary endothelial cells turn thinner, the expression of TGF-B1 and type III collagen decreased in the lungs of spontaneous type 2 diabetes rats that received the treatment of glucagons-like peptide (GLP-1). There is few study about whether GLP-1 can reduce the secretion of TGF-?1, then down-regulate the signal pathway of TGF-?1 Smad. Our study aims at the role of TGF-?1/Smad signal pathway in the changes of extracellular matrix in the lungs of diabetes, and explores whether GLP-1 can improve extracellular matrix in the lung by the signal pathway of TGF-?1/Smad, furthermore analyses the effect that GLP-1 has pulmonary function in diabetes.Methods:1.Twelve four-week-old OLETF rats and eight syngeneic non-diabetic LETO rats were housed in a sterile environment in individual cage and fed standard diet. Oral glucose tolerance tests were took regularly on the rats. Eight of the OLETF rats were qualified for the study at the age of thirty weeks. At forty-two weeks, executed them by cutting off femoral artery. Lung samples were collected.?Hematoxylin eosin stain and Masson stain were took to observe lung tissue structure and deposition of collagenous fiber of the rats;?Western blotting were took to detect the expression of Smad3,Smad7 and type III collagen in the lungs;?ELISA were took to detect the expression of TGF-?1 in the lungs; 2.Human embryonic lung fibroblast cell line (MRC-5) was cultivated. First,?the expressions of mRNA of GLP-1 receptor (GLP-1R) were detected by RT-PCR;?extracted membrane proteins of MRC-5 cell, and detected the expression of GLP-1R by western blotting to confirm that MRC-5 cell is the target cell of GLP-1. Then, cultivated the MRC-5 cell in different concentration of glucose, and extracted total mRNA and total protein after the treatment with GLP-1 (10nmol/L) at 24h,48h and 72h.?Detected the mRNA of fibronectin(FN), type?collagen of MRC-5 cell in different groups by RT-PCR;?Detected TGF-Bl,p-Smad3,Smad7,FN and type?collagen of MRC-5 cell in different groups on protein level by western blotting;?Detected TGF-?1 in the cell-culture medium by ELISA.Result:1.OLETF rats were much fatter than LETO rats, moving slowly, reluctantly and had dry and unmatted fur. The body weight, area under curve of blood glucose (OGTT-0-120min), area under curve of insulin (OGTT-0-120min) of LETO rats were less than those of OLETF rats at thirty weeks and forty-two weeks.?Hematoxylin eosin stain revealed that the alveolar in LETO rats was normal, while the tissue structure of lung in OLETF rats was in disorder, the wall of bronchial and alveolar turn thicker, alveolar epithelial cells were unclear, alveolar atrophy and collapsed, extracellular matrix in interstitial lung and around vessels increased, fibroblasts increased, and there were infiltrations of inflammatory cells.?Masson stain revealed there was few collagenous fiber in interstitial lung and around vessels in LETO rats, while there were plenty of collagenous fiber in interstitial lung and around vessels and structure disorder in OLETF rats.?The TGF-?1, Smad3, type?collagen were significantly increased in OLETF rats than LETO rats, while expression of Smad7 protein was significantly decreased in OLETF rats (P<0.05) There is a significantly positive relationship between TGF-B and Smad3 (?=0.853, P<0.05), and a significantly negative correlation between TGF-B and Smad7 (?=-0.720, P?0.05.2. There are GLP-1R mRNA expressing in MRC-5 cells, and we can detect GLP-1R protein in membrane proteins. In the high glucose environment, the expressions of TGF-?1,p-Smad3 proteins and FN, type?collagen mRNA of MRC-5 cells are significantly increased, while the expression of Smad7 protein is significantly decreased, and the expression of TGF-?1 in the cell-culture medium also significantly increased. All of the indexes above can be reversed after the treatment of GLP-1, and the differences are significant. Conclusions:?High glucose may over-activate the TGF-?1/Smad signal transduction pathway in OLETF rats'lung tissue, leading to the increase of collagenous fiber and the metabolic disorders of extracellular matrix.?GLP-1 may decrease the expression of FN and type III collagen in MRC-5 cell in high glucose condition by down-regulate TGF-?1/Smad signal transduction pathway. |
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