| ObjectiveIdiopathic pulmonary fibrosis(IPF)is a chronic progressive pulmonary interstitial disease.The onset of IPF is insidious,then gradually worsens,and eventually develops into respiratory failure,manifested by hypoxia and carbon dioxide retention.The prognosis of IPF is extremely poor.The average diagnostic survival rate if IPF is only 2-5 years,which is called ’tumor-like disease’.Traditional Chinese medicine classifies the disease as ’lung bi’ and ’lung wei’.The most common symptom is Qi deficiency and blood stasis,which is treated with Yiqi Huoxue prescription.The purpose of this study was to investigate the effect of Yiqi Huoxue prescription in mouse model of pulmonary fibrosis and vitro cell model.The network pharmacology method was used to predict the target of Yiqi Huoxue prescription and conduct experimental verification.The activation of CDK1/Src/Erk signaling pathway was used to provide experimental basis for the clinical application of Yiqi Huoxue prescription in the treatment of IPF.Methods1.Explore the effect of Yiqi Huoxue prescription on mice with pulmonary fibrosis.The model of pulmonary fibrosis was established by bleomycin injection into the trachea of mice.Two days after the model was established,the mice were treated daily with the Yiqi Huoxue prescription through gavage.The positive drug Pirfenidone(PF)was taken as the positive control.After 14 days of intervention,observed the general view of lung tissue,collected the required specimens,and verified the anti-fibrosis effect of Yiqi Huoxue prescription.Pulmonary pathological changes were observed by H&E/Masson staining of lung tissue.The expression of α-sma protein in lung tissues was detected by IHC method.Western-blot(WB)method was used to detect the effect of Yiqi Huoxue prescription on lung fibrosis-related proteins(Fibronectin and Collagen 1).2.Explore the effect of Yiqi Huoxue prescription on the transformation of fibroblasts into myofibroblasts.Transforming growth factor 1(TGF-β1)acted on mouse embryonic fibroblasts(NIH/3T3)to induce a cell model of fibroblasts transformed into myofibroblasts.MTS was used to detect the toxic effect of Yiqi Huoxue prescription on NIH/3T3 cells.The cells were divided into the control group,the model group,the Yiqi Huoxue prescription group and the positive drug group to observe whether Yiqi Huoxue prescription could inhibit the transformation of fibroblasts into myofibroblasts.WB and immunofluorescence(IF)are used to detect the expression of fibrosis markers such as a-sma and Collagen 1.3.Explore the effect of Yiqi Huoxue prescription on pulmonary fibrosis through CDKI/Src/Erk pathway.Through the network pharmacology method,the active ingredients of Ginseng,Chuanxiong and Sanqi were searched by TCMSP and SymMap website.According to’OB>30%’ and‘DL>0.18’,the effective active ingredients were selected,the targets of drug were searched,and the targets were interacted to obtain common targets.Cytoscape software was used to map the active ingredient-target and analyze the coacting targets.GPS software was used to analyze the sites of the proteins to predict the mechanism of Yiqi Huoxue prescription on pulmonary fibrosis.Then,experiments were conducted to verify the effect of targets after adding inhibitors of target protein.RT-PCR and WB were used to detect the activation of target proteins and pathways.Results1.Yiqi Huoxue prescription reduced the fibrosis in mice with pulmonary fibrosisBleomycin was used to construct pulmonary fibrosis model mice.The results of pulmonary surface view,body weight and H&E showed the presence of pulmonary lesions in model group mice.Furthermore,the model mice were treated with Yiqi Huoxue prescription and positive drugs.The H&E results showed that Yiqi Huoxue prescription could reduce the degree of pulmonary inflammation in mice with pulmonary fibrosis.Masson’s results showed that the degree of pulmonary fibrosis was significantly reduced after drug treatment.WB further showed that both Yiqi Huoxue prescription and positive drugs could reduce the levels of proteins(Fibronectin,Collagen 1)in lung tissue.It is suggested that the Yiqi Huoxue prescription can inhibit the degree of pulmonary fibrosis.2.Yiqi Huoxue prescription inhibits the transformation of fibroblasts into myofibroblasts.TGF-β1 at 5 ng/ml concentration for 24 h was selected as the induction condition of pulmonary fibrosis cell model.Ginseng,Chuanxiong and Sanqi were applied to the cell model alone.It was found that a certain concentration of a single Chinese medicine could inhibit the expression of fibrotic proteins in the cell model.And it was found that Ginseng,Chuanxiong and Sanqi had better anti-fibrosis effect when used in combination.Yiqi Huoxue prescription was used in the cell model of pulmonary fibrosis.Both IF and WB results showed that the expression levels of Collagen 1,Fibronectin and a-sma of fibrotic proteins could be reduced by Yiqi Huoxue prescription.3.Yiqi Huoxue prescription attenuates Pulmonary Fibrosis by Targetting CDK1/Src/Erk Pathway.Network pharmacology search database showed that there are 498 potential active ingredients in three traditional Chinese medicines.After screening according to ’OB>30%’ and ’DL>0.18’,there are 37 active ingredients.Combined with the drug active ingredients,32 active ingredients were obtained.Among them,Ginseng has 22 active ingredients,Chuanxiong has 8 active ingredients,and Sanqi has 7 active ingredients.Using the database to screen its target targets,we can find that Ginseng has 133 target targets,Chuanxiong has 88 target targets,and Sanqi has 150 target targets.After drawing the venn diagram,it was found that there are 17 common targets.The common targets and corresponding compounds were plotted on the network pharmacology map.Then selected the possible target CDK1,predicted its interaction site with phosphorylated Src,and calculated its interaction positions,amino acid names and sequences,scores and so on.The protein inhibitor(RO-3306/PP2)was added to further verify its action site and pathway,suggesting that Yiqi Huoxue prescription may act on the fibrosis process by inhibiting the CDK1/Src/Erk signaling pathway.ConclusionYiqi Huoxue prescription can reduce inflammation and fibrosis in a mouse model of pulmonary fibrosis and inhibit fibroblasts transforming into myofibroblasts.Yiqi Huoxue prescription can act on IPF through multiple targets,inhibit CDK1 protein activation,and inhibit the downstream Src/Erk signaling pathways against pulmonary fibrosis.In summary,Yiqi Huoxue prescription has a good anti-fibrotic effect on pulmonary fibrosis,which may be related to its regulation of CDK1/Src/Erk signaling pathway. |
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