Study On The Intervention Effect And Mechanism Of Adenophora Root And Radix Ophiopogonis Decoction On Experimental Pulmonary Fibrosi

Background:Idiopathic pulmonary fibrosis(IPF),a chronic and fatal pulmonary interstitial disease of unknown cause,is characterized by dry cough,progressive dyspnea,and eventual death due to respiratory failure.The pathogenesis of IPF is that sustained damage of alveolar epithelial cells causes dysregulation of repair response,leading to excessive deposition of extracellular matrix.The Epithelial-mesenchymal transition(EMT)and Fibroblast-myofibroblast transition(FMT)are key events in the mechanism.The Alternatively activated macrophage(M2)polarization is closely related to the IPF process.TCM has the advantages of multi-component,multi-target and multi-pathway in treating pulmonary fibrosis.Shashen Maidong decoction is a classic prescription for engendering liquid and moistening dryness,clearing heat and nourishing Yin.Modern pharmacological studies have shown that it has immune regulatory functions and plays an anti-fibrosis role by affecting a variety of biological pathways and signal transduction pathways.Therefore,by carrying out in vivo and in vitro experiments,this study explores the intervention effect and mechanism of Shashen Maidong decoction on pulmonary fibrosis.Methods:1.Study on efficacy of Shashen Maidong decoction on pulmonary fibrosis models in vitro and in vivo.In vivo,C57BL/6 mice were injected with bleomycin intratracheal to establish the animal model of pulmonary fibrosis.Administration began on the 14th day and was divided into control group,model group,Shashen Maidong decoction low-dose(1.5 g/kg),medium-dose(3 g/kg)and high-dose(6 g/kg)groups according to the intervention method.After 14 days of continuous administration,lung tissue and serum were collected to measure lung coefficient and hydroxyproline content in lung tissue.HE and Masson staining were performed to observe the pathological changes of lung tissue.RT-qPCR,Western blot,immunohistochemistry and ELISA were used to detect mRNA and protein of fibroblast-myofibroblast transformation(FMT)and epithelial interstitial transformation(EMT)markers and expression of proteins associated with TGF-β/Smads pathway.In vitro,TGF-β1-induced MRC-5 lung fibroblast-myofibroblast transformation(FMT)and TGF-β1-induced BEAS-2B human lung epithelial interstitial transformation(EMT)were constructed.The effect of Shashen Maidong decoction on cell proliferation activity was detected by CCK-8 method.The non-toxic dose range was obtained.RT-qPCR,Western blot,immunofluorescence and other methods were used to investigate the effect of Shashen Maidong decoction on FMT and EMT mRNA and protein in vitro.2.The active chemical constituents of Shashen Maidong decoction were screened from the database by the network pharmacological method,and the target of its active constituents was obtained based on the SwissTargetPrediction database.The target of pulmonary fibrosis disease was retrieved from GeneCards,CTD and other databases,and the potential target of treatment was obtained after intersection with the target of drug composition.The potential protein interaction information network was imported into STRING database to construct.According to Cytoscape software,the key targets were submitted to DAVID database for GO enrichment analysis and KEGG enrichment analysis.Finally,the pathway-target-component network map was constructed by software.3.Study on the mechanism of Shashen Maidong decoction in treating pulmonary fibrosis.Combined with the prediction results of network pharmacology,the specific mechanism of anti-fibrosis effect of Shashen Maidong decoction was verified through in vivo and in vitro experiments.In vivo,immunohistochemistry,Western blot,RT-qPCR and other methods were used to detect M1/M2 polarization markers of macrophages and TGF-β1 mRNA and protein expression levels in lung tissue of mice with pulmonary fibrosis.The M2-type polarization model of THP-1 macrophages induced by IL-4/IL-13 was established in vitro.The safe concentration range of Shashen Maidong decoction was detected by CCK-8 method,and the mRNA and protein expression levels of macrophage polarization markers were detected by RT-qPCR,Western blot and immunofluorescence.Finally,Western blot was used to detect the effect of Shashen Maidong decoction on the phosphorylation level of predicted pathway proteins in vivo and in vitro models.Results:1.Shashen Maidong decoction can reduce lung coefficient and hydroxyproline content in pulmonary fibrosis mice,and improve lung function indexes related to restrictive ventilation dysfunction,and reduce the damage of lung pathological structure and collagen deposition.mRNA and protein expression levels related to myofibroblast activation and proliferation were decreased.Epithelial marker genes and proteins were up-regulated,while interstitial marker genes and proteins were down-regulated.The safe dose range of Shashen Maidong decoction was obtained by CCK-8,which showed that it could significantly inhibit the mRNA and protein expression levels of type I collagen,type Ⅲ collagen and α-SMA in MRC-5 cell model induced by TGF-β1.The epithelial marker genes and proteins of BEAS-2B cell model induced by TGF-βl were inhibited,and the expression levels of interstitial marker genes and corresponding proteins were up-regulated.2.A total of 119 active chemical components of Shashen Maidong decoction were screened through database retrieval,872 active ingredient action targets were obtained through SwissTargetPrediction database,and 5469 pulmonary fibrosis disease targets were retrieved from GeneCards and CTD databases.After the intersection of drug targets and disease targets,577 potential targets of Shashen Maidong decoction for pulmonary fibrosis were obtained.Import STRING database to construct potential protein interaction information network.According to Cytoscape software,56 key targets were obtained for visualization analysis.The key targets were submitted to the DAVID database,and the GO enrichment analysis results showed that the potential core targets of Shashen Maidong decoction intervention in pulmonary fibrosis may be involved in the regulation of innate immune response,protein kinase B signal regulation and other biological activities.KEGG enrichment analysis showed that Shashen Maidong decoction interfered with pulmonary fibrosis mainly through PI3K/Akt signaling pathway and MAPK signaling pathway.The pathway-target-component network map was constructed,and A network consisting of 248 nodes and 1936 edges was obtained,in which Licochalcone B,Methyl Ophiopogonanone A,Phaseolinisoflavan,1-Methoxyphaseollidin,Medicarpin were the key active components.3.In vivo experiment results showed that the expression levels of M2-type macrophage marker gene and protein were significantly up-regulated in the pulmonary fibrosis mouse model group,and there was no significant difference in the expression of Ml-type macrophage polarization marker gene and protein.The levels of upregulated genes and proteins of M2 type could be decreased in Shashen Maidong decoction group.In vitro experiments showed that IL-4/IL-13 induced THP-1 cells could construct M2-type polarization model in vitro,and the administration of Shashen Maidong decoction could significantly reduce the level of M2-type marker genes and proteins in the model.In vitro and in vivo experiments,the protein phosphorylation levels of PI3K and Akt were significantly increased in the model group,and the phosphorylation levels of PI3K and Akt were down-regulated in the Shashen Maidong decoction group.Conclusions:1.Through in vivo and in vitro experiments,it was found that Shashen Maidong decoction could regulate TGF-β/Smads signaling pathway,inhibit lung FMT and EMT process in pulmonary fibrosis,reduce extracellular matrix deposition,and improve lung function.2.Network pharmacology results showed that Shashen Maidong decoction may interfere with pulmonary fibrosis by regulating PI3K/Akt signaling pathway,participating in biological activities such as innate immune response regulation,and playing an anti-pulmonary fibrosis role.3.In vitro and in vivo experiments were conducted to verify that Shashen Maidong decoction can improve the pro-fibrotic microenvironment of lung tissue and exert its anti-fibrosis effect by regulating M2-type macrophage polarization involved in PI3K/Akt signaling pathway.