Molecular Mechanism Of Buyang Huanwu Decoction In Intervention Of EMT In Idiopathic Pulmonary Fibrosis Based On TGF-β₁ Induced Fibrosis Model Of A549

Objective:To study the expression changes of E-cadherin and N-cadherin in the EMT model of TGF-β1induced alveolar epithelial cells A549 cells,and the protein expression of a-SMA and Collagen-I in extracellular matrix deposits.To explore the preventive and intervention effects of Buyang Huanwu Decoction on early stage of idiopathic pulmonary fibrosis,and provide experimental evidence for prevention and treatment of idiopathic pulmonary fibrosis by traditional Chinese medicine.Methods:1.The effects of Buyang Huanwu Decoction and TGF-β1 on the cell viability of A549 were detected by MTT method.Screening analysis was performed on different concentrations of Buyang Huanwu Decoction and TGF-β1.2.Preparation of EMT in vitro model,divided into three groups,to observe cell morphology changes.Western blot was used to detect the EMT model of A549 cells induced by TGF-β1 for 72 hours.The expression of α-SMA,Collagen-I,E-cadherin and N-cadherin was observed.Results:1.MTT results showed that A549 cells were treated with Buyang Huanwu Decoction for 24h,and their concentrations of 62.5μg/ml,125 μg/ml,250 μg/ml and 500 μg/ml had no toxic effects on cell viability.Different concentrations of TGF-β1 induced cells for 24 h,and the cells showed cell proliferation at a dose concentration.2.When the model is 48h,the model cells begin to polarize,disperse each other,and grow into a spindle shape.At the time of modeling for 72 hours,the model showed cell proliferation,and the cell morphology became longer,and the transformation into interstitial cells was completed.3.Western blot analysis showed that in the control group,smooth muscle actin a-SMA was lowly expressed,and Collagen-I was not expressed.In the model group,a-SMA and Collagen-I were highly expressed,and the model group had higher expression than the control group,indicating that TGF-β1 can promote extracellular matrix deposition.Compared with the model group,the protein expression of the other 4 groups showed a downward trend,indicating that Buyang Huanwu Decoction plays a role in collagen formation and extracellular matrix deposition.4.Western blot analysis showed that in the control group,the cell’ s E-cadherin was highly expressed and N-cadherin was not expressed.In the model group,E-cadherin expression was down-regulated and N-cadherin expression was elevated.This is in line with the occurrence of polarization when epithelial cells are transformed into mesenchymal cells,and the tightness between cells is reduced.This indicates that TGF-β1 can promote the occurrence of EMT.Compared with the model group,Buyang Huanwu Decoction increased the E-cadherin protein compared with the model group at 125ug/ml.In terms of N-cadherin expression,the effect of Buyang Huanwu Decoction group was more obvious than that of the model group,indicating that Buyang Huanwu Decoction has the effect of preventing or reversing in the early stage of IPF.Conclusion:1.Buyang Huanwu Decoction effectively reduced the expression of Collagen-I and a-SMA in the EMT model of pulmonary fibrosis induced by TGF-β1.2.Buyang Huanwu Decoction effectively reduced the expression of N-cadherin in the EMT model of pulmonary fibrosis induced by TGF-β1,and increased the protein level of E-cadherin.3.The possibility of Buyang Huanwu Decoction in preventing and treating pulmonary fibrosis is related to the regulation of extracellular matrix formed by early EMT of IPF and degradation of fibrogenic factors.