Study On Regulation Of TGF-?1 / Smad / ERK Signaling Pathway In Lung Fibroblasts By Serum Containing Buyang Huanwu Decoction

Objective:Observed the effect of BYHWD containing serum acting on the expression of pulmonary fibrosis fibroblasts model TGF-β1, TGF-mRNA, Smad3, Smad4, Smad7, ERK1/2, p-ERK, preliminary study the possible molecular mechanisms of BYHWD with "Yiqihuoxue" efficacy antagonizing pulmonary fibrosis, broaden the clinical application space of BYHWD.Method:1. Using the classical modeling method of disposable drops bleomycin in tracheal for preparing a rat pulmonary fibrosis model, sacrificed the animals on the14th day, visual observation the appearance of morphological changes of lung tissues, prepared the pathological section, using hematoxylin-eosin staining (haematoxylin-eosine, HE), Masson trichrome staining, observed the lung histopathological changes by light microscope;2. Using tissue method to isolate and culture the primary fibroblasts of pulmonary fibrosis.Digested with0.25%trypsin, subcultured1:2. Used the4th generation in the experiment;3. Using ELISA method to detect the expression of TGF-β1in the supernatants;4. Using Real-Time PCR method to detect the expression of TGF-mRNA in cells;5.Using Western blot and immunohistochemical staining (SABC method) method to detect the expression of Smad3, Smad4, Smad7protein and ERK1/2, p-ERK in cells.Results:1.The lung appeared dark red, lacked glossiness, lung tissue surface was rough, scattered bleeding and bruising; HE staining results suggest that the alveolar walls thickened, the alveolar septa significantly widened, only a few inflammatory cells infiltration in the alveolar spaces, fibroblasts increased significantly; Masson staining showed that blue collagen deposition in levels of bronchial, vascular wall and thickening of the alveolar septa, suggesting ECM increased in this time to promote a large number of fiber proliferation.2. Explant attached adhered to the wall after72h, microscopy showed the fibroblasts climbed out, removed the tissue, around7d spindle cells locally integrated into the film, about20d cells nearly complete fusion. After passage, the second generation cells nearly complete fusion around7d, the fused time was5days of the third "generation after integration. Fibroblasts were long fusiform shape, often manifested swirling growth;3.Using ELISA method to detect the expression of TGF-β1in the supernatants, the results show that compared with the model group, the expression of TGF-β1in the pulmonary fibrosis fibroblasts were significantly reduced (P<0.01) in the positive group,20%serum containing group and10%serum containing group,5%serum containing cells expressing TGF-β1has a decreasing trend, three different concentrations BYHWD containing serum showed some effects of dose-effect relationship;4. Using REAL-TIME PCR method to detect the expression of TGF-β1mRNA in cells, the results show that compared with the model group, the expression of TGF-β1mRNA in the pulmonary fibrosis fibroblasts were significantly reduced (P<0.01) in the positive group and20%serum containing group, the expression of TGF-β1mRNA in the pulmonary fibrosis fibroblasts were reduced (P<0.05) in the10%serum containing group,5%serum containing cells expressing TGF-β1lmRNA has a decreasing trend, three different concentrations BYHWD containing serum showed some effects of dose-effect relationship;5. Using Western blot method to detect the expression of Smad3, Smad4, Smad7protein and ERK1/2, p-ERK in cells, the results show that compared with the model group, the expression of Smad3protein in the pulmonary fibrosis fibroblasts were significantly reduced (P<0.05) in the20%serum containing group, the positive group,10%serum containing group and5%serum containing cells expressing Smad3protein has a decreasing trend; The positive group,20%serum containing group,10%serum containing group and5%serum containing cells expressing Smad4protein has a decreasing trend; The expression of Smad7protein in the pulmonary fibrosis fibroblasts were significantly reduced (P<0.05) in the20%serum containing group, the positive group,10%serum containing group and5%serum-containing cells expressing Smad7protein has a decreasing trend;The expression of ERK1/2in the pulmonary fibrosis f ibroblasts were significantly reduced (P<0.05) in the20%serum containing group, the positive group,10%serum containing group and5%serum containing cells expressing ERK1/2has a decreasing trend; The positive group,20%serum containing group,10%serum containing group and5%serum containing cells expressing p-ERK has a decreasing trend.Conclusion:1. Using the method of disposable drops bleomycin in tracheal for preparing a rat pulmonary fibrosis model is successo2. Using tissue culture method for isolation and culture the fibroblasts is success.3. The administration and taking blood program of1time per day, continuous administration7d, fasting12h before taking blood,1h after the last administration is success。It was found that20%BYHWD serum containing inhibited pulmonary f ibrosis, regulated key cytokine in the TGF-β1/Smad/ERK signaling pathway, significantly better than the other concentrations, suggesting that the20%serum concentrations may be appropriate plasma concentration.4. BYHWD can effectively inhibit the overexpressing of the TGF-β1, TGF-β1mRNA, Smad3, ERK1/2and other cytokine in the TGF-β1/Smad/ERK signal transduction pathway. Prompting the role of BYHWD playing better anti pulmonary fibrosis and regulating the TGF-β1/Smad/ERK signaling pathway are related.