Experimental Study On The Effects Of Serum Containing Yiqi Huoxue Xiaozheng Prescription On Phenotypic Transformation Of Lung Fibroblasts And TGF-β1/Smad Signaling Pathwa

Background:Chronic obstructive pulmonary disease(COPD)is a common respiratory illness.The medicine workers pay more and more attention to COPD which owe to a great deal of COPD patients,increasing incidence,high case fatality rate and weighty burden to socialty.It is known that chronic inflammation,protease-antiprotease imbalance and oxidative stress are related to occurrence and development of COPD.An important feature of COPD is airway remodeling and the abnormal deposition of extracellular matrix is one of the main pathologic manifestations of it.Fibroblasts are differentiated into myofibroblasts under various stimuli which is related to TGF-β1/Smad signal pathway and they are the main source of ECM.Our team proposes that the theory of "micro Zhengjia obstructs lung collateral"is significant to explain occurance of airway remodeling.Both clinical research and animal experiments finished by us have provided powerful evidences for Yiqi Huoxue Xiaozheng formula to intervene development of airway remodeling.Yiqi Huoxue Xiaozheng formula can reduce the expression of MMP-9 and TIMP-1 in airway epithelial cells,depress the content of IL-7、TNF-α、IFN-y in serum,inhibit the proliferation of airway smooth muscle and activity of ERK1/2 signaling pathway of COPD rats.On the basis of previous research,this experiment is to observe the effect of Yiqi Huoxue Xiaozheng formula on the deposition of fibroblasts and ECM in COPD airway remodeling.What’s more,we try to find the possible targets of Yiqi Huoxue Xiaozheng formula based on the TGF-β1/Smad signaling pathway.Objective:Experiment one:To establish a model of LPS induced transformation of lung fibroblasts into myofibroblasts in vitro.Experiment two:To observe the effect of Yiqi Huoxue Xiaozheng formula on fibroblast phenotype transformation,explore possible targets from the protein expression level on the basis of TGF-β1/Smad signaling pathway and provide a basis for the theory of "micro Zhengjia obstructs lung collateral".Methodology:Experiment one:Stimulating MRC-5 cells for 24h with different concentrations of LPS,We hope to induce fibroblasts to transform into myofibroblasts.We detect the expression of α-SMA protein in cells by Western Blot.Experiment two:Yiqi Huoxue Xiaozheng formula will be broken up into Yiqi Xiaozheng Tongluo formula and Yiqi Huoxue Huatan formula.Three types drug containing serum will be maked according to these three formulas.We will use drug containing serum to intervene phenotype transdifferentiation of fibroblast to observe level of TGF-β1 using ELISA and α-SMA、Smad3、p-Smad3、Smad4、Smad7 using western blot.Results:Experiment one:50-1000ng/mL LPS can induce express α-SMA abundantly especially in the condition of 500ng/mL.Experiment two:1.Compared with the model group,Yiqi Huoxue Xiaozheng formula can suppress expression of α-SMA、TGF-β1、p-Smad3、Smad4 of MRC-5 cells.2.Compared with the model group,Yiqi Xiaozheng Tongluo formula can suppress expression of α-SMA、Smad3、p-Smad3、Smad4 and is similar to the way of Yiqi Huoxue Xiaozheng formula of MRC-5 cells.3.Compared with the model group,Yiqi Huoxue Huatan formula can suppress expression of p-Smad3 of MRC-5 cells.Conclusion:Experiment one:LPS can induce fibroblasts to transform into myofibroblasts in vitro.Experiment two:Yiqi Huoxue Xiaozheng formula can suppress transdifferentiation of fibroblast which may be related to down regulating the activity of TGF-β1/Smad signaling pathway and the target is to inhibit the expression ofα-SMA、TGF-β1、p-Smad3、Smad4 proteins in cells.The role of Yiqi Huoxue Xiaozheng formula in restraining airway remodeling of COPD prove the theory of "micro Zhengjia obstructs lung collateral".