The Influence Of Pulmonary Fibrosis Model’s TGF-β1/Smads Signaling Pathway By Kangfuxin Liquid Upon The MMPs/TIMPs

Part1: The effects of kangfuxin liquid on the pulmonary fibrosis cell modelObjective:To investigate effects of kangfuxin liquid on the pulmonary fibrosis cell model.Method: The pulmonary fibrosis cell model is established by treating human fetal lung fibroblast MRC-5 cell line with transforming growth factor-β1(TGF-β1).In addition,to detect effects of Kangfuxin liquid on MRC-5 cell by CCK8 assay,Kangfuxin liquid diluted by 80 times implements administration of drug on MRC-5 cell.Result:The pulmonary fibrosis cell model is established by treating human fetal lung fibroblast MRC-5 cell line with TGF-β1 at the dose of 10 ng.m L-1.Kangfuxin liquid could significantly alleviate pulmonary fibrosis degree and efficiently down-regulate expression levels of LN,FN,α-SMA,CTGF,Collagen I and Collagen III in the pulmonary fibrosis cell model,so that the degree of pulmonary fibrosis can be reduced.Conclusion:Kangfuxin liquid could significantly alleviate pulmonary fibrosis degree and might be a potential anti-fibrosis drug.Part2: The effects of kangfuxin liquid on the rat model of pulmonary fibrosisObjective:To investigate effects of kangfuxin liquid on the rat model of pulmonary fibrosis induced by infusing bleomycin.Method: 48 healthy Wistar rats were randomly divided into 4 groups: control(NS)group,model(BLM)group,kangfuxin liquid group,Dexamethasone(DXM)group.Pulmonary fibrosis model was established by injection of BLM into tracheal intubation at the dose of 5 mg.kg-1.control group was intratracheally instilled with equal normal saline.The rats of group kangfuxin liquid were given kangfuxin liquid 5 ml.kg-1.The rats of group DXM were given Dexamethasone 0.25 ml.kg-1.This treatment cycle is 21 days.To measure pulmonary fibrosis degrees in lung dyed with HE and Masson,and LN mRNA,FN mRNA,α-SMA mRNA in lung tissue was assessed by real-time quantitative PCR,and LN,FN,α-SMA in lung tissue was assessed by western blot(WB).Result:(1)pulmonary fibrosis degrees in group BLM were significantly higher than group NS;The groups of kangfuxin liquid and DXM could improve pulmonary fibrosis degree by comparison with group BLM;(2)LN,FN,α-SMA mRNA expression of the group BLM was higher than that in group NS;LN,FN,α-SMA mRNA expression of the kangfuxin liquid and DXM groups was lower than that in group BLM in PCR;(3)LN,FN,α-SMA expression of the group BLM was higher than that in group NS;LN,FN,α-SMA expression of thekangfuxin liquid and DXM groups was lower than that in group BLM in WB.Conclusion:Kangfuxin liquid could significantly alleviate pulmonary fibrosis degree,efficiently regulate expression levels of LN,FN,α-SMA mRNA in the lung tissues of pulmonary fibrosis rats induced by BLM,the degree of pulmonary fibrosis can be reduced.Part3:The Influence of pulmonary fibrosis model’s TGF-β1/Smads signaling pathway by Kangfuxin liquid upon the MMPs/TIMPsObjective: To investigate the underlying mechanism for effect of Kangfuxin liquid on the pulmonary fibrosis cell model is established by treating human fetal lung fibroblast MRC-5 cell line with transforming growth factor-β1(TGF-β1).Method:Human fetal lung fibroblast MRC-5 cell were cultured in vitro as research object.Human fetal lung fibroblast MRC-5 cell is treated by Kangfuxin liquid diluted by 80 times in three different time-points(1 hour before adding TGF-β1 at the dose of 10 ng.m L-1,1 hour after adding TGF-β1 at the dose of 10 ng.m L-1,3 hours after adding TGF-β1 at the dose of 10 ng.m L-1),and afterwards collecting these cells after 48 and 72 hours respectively.The expression of MMP1,MMP3,MMP9,TIMP1,SMAD2,SMAD7 and TGF-β1,as well as protein levels of MMP1,MMP3,MMP9,SMAD2 and SMAD7 in the cells treated above,were assessed by real-time fluorescent quantitative PCR technology and Western blot analysis,respectively.Result: Kangfuxin liquid could efficiently increase expression levels of MMP1,MMP9 and SMAD2 in the pulmonary fibrosis cell model is established by treating human fetal lung fibroblast MRC-5 cell line with transforming growth factor-β1.Kangfuxin liquid could down-regulate expression levels of SMAD7 in the pulmonary fibrosis cell model is established by treating human fetal lung fibroblast MRC-5 cell line with transforming growth factor-β1.Conclusion: Kangfuxin liquid has the prevention and treatment effect on the pulmonary fibrosis cell model is established by treating human fetal lung fibroblast MRC-5 cell line with transforming growth factor-β1,but it is not attributed to the regulation on the TGF-β1/Smads signaling pathway.