| Background and ObjectivePulmonary fibrosis(PF)is one of the clinically refractory lung diseases,and is a common pathological symptom in the late stages of a variety of lung diseases,immune diseases,connective tissue and other diseases.The clinical manifestations include cough,expectoration,and dyspnea.With yearly increasing incidence rate,high fatality rate and poor treatment prognosis,PF has become a medical problem of great concern.The pathogenesis of PF is complicated,but is closely associated with cells and cytokines related to inflammation and immune regulation.Among them,macrophages play an important role.Therefore,intervening in the polarization process of macrophages and regulating the immune microenvironment of PF has become a research focus in recent years.Yangfei Huoxue Decoction(YHD),which is a revised proved prescription by Professor Wang Canhui,a well-known doctor in Jiangsu Province,has a good clinical effect.In the early stage,the mechanism of its action was discussed in depth using evidence-based medicine,molecular biology,bioinformatics,and genomics methods in several national and bureau-level research projects.Studies have shown that the decoction can alleviate alveolitis and hyperplasia of fibrous connective tissue in PF animal models,and intervene in the phenotypic transformation of lung fibroblasts induced by TGF-β1.Through the intervention in the multiple links of PF immuneinflammatory reaction,the decoction can prevent and treat PF.On the basis of the previous studies,this paper firstly sorted out and summarized the basic understanding and treatment principles of traditional Chinese medicine on PF,reviewed the modern medical research literature on the intervening mechanism of immune inflammation in pulmonary fibrosis,and clarified the theoretical basis of intervening macrophage polarization to prevent and treat PF.Then,this paper focused on the main ingredients and used network phammacology to reveal the mechanism of action of the decoction from a deeper level.The results show that the YHD can play its role through multiple ingredients,multiple targets,multiple effects,and multiple pathways,and the regulation of immune inflammation and cytokines may be the key mechanism.Finally,a series of experimental studies were carried out on the polarization of macrophages.The influence of YHD on the polarization of macrophages in vivo and vitro was observed based on PF rat model,M1/M2 macrophage model,and M2 macrophages-primary lung fibroblasts co-culture model,hoping to provide more reliable and clear TCM methods for the clinical treatment of PF.Methodology1.Through literature study and theoretical discussion,the etiology,pathogenesis and treatment methods of pulmonary fibrosis were sorted out and summarized,and then the theoretical basis and experimental basis of using the clinically proved prescription Yangfei Huoxue Decoction to treat PF were explained in depth.Further,it summarized the understanding of modern medicine on the pathogenesis of PF from the immune inflammation perspective,and probed into the role and significance of intervention in macrophage polarization,regulation of immune inflammation,and regulation of cytokines in the outset and development of PF,so as to lay a foundation for subsequent network pharmacological research and related in-vivo and in-vitro experimental researches.2.Network pharmacology analysis:First,the active ingredients and target proteins were screened out in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and PF-related targets were searched in the OMIM database and GeneCards database.Second,the correlation degree of targets with pulmonary fibrosis was calculated using Gifts algorithm to screen out the targets with Gifts value≥30 degrees.Thirdly,the interactive targets were obtained from the drug active ingredient targets of YHD and PF disease targets.The screened interaction targets were imported into Cytoscape 3.6.1 software to construct a drug-target network diagram,and the screened interaction targets were input into the String database to draw a visualized PPI diagram and analyze their topological properties.Finally,R language was used to perforam GO enrichment and KEGG pathway enrichment analysis on the targets.3.In-vivo animal experiment:Bleomycin was injected in trachea of rats to establish PF animal models,which were divided into blank space group,dexamethasone positive control group,YHD high-dose group,middle-dose group,and low-dose group.Except for the blank space group,each group was given intragastric administration separately.Rats were sacrificed in batches for lung tissue sampling on the 7th,14th,and 28th days.Part of the lung tissue was fixed for HE and Masson staining,and then tested by Westernblot method for iNOS and ARG1 expression.4.In-vitro cell experiment:①The mouse macrophage cell line RAW264.7 was cultured in vitro,and were induced into M1 type with IFN-y+LPS and M2 type with IL-4.Serums with high,medium and low doses of YHD drug were used to intervene.Flow cytometry was used to detect the main surface markers CD 16/32 and CD206 of M1 and M2 cells,Westemblot method was used to detect the expression of marker proteins iNOS and ARG1,and ELISA method was used to detect the content of IL-1β and TNF-α in the M1 macrophages medium and the content of IL-10 and TGF-β in the M2 type macrophages medium.②The mouse primary lung fibroblasts and RAW264.7 M2 type macrophages were co-cultured in a transwell chamber,and the lung fibroblasts α-SMA,COL-1,COL-3,TGF-β1,Smad3,p38 were detected by Westernblot method for protein expression.The second-generation transcriptomics gene sequencing method was used to detect the RNA of M2 macrophage in the co-culture system.Bioinformatics methods was used to analyze its related biological functions and signal pathways,and fluorescent quantitative PCR was used to verify related differential genes.Westernblot method was used to detect the expression of Notch 1,NICD1,D114,and RBP-Jκ protein in the Notch signal pathway.Results1.There were 705 YHD compound targets screened out using network pharmacology method,and 126 compound ingredients were obtained after eliminating duplicates.217 human targets were matched.4623 PF-related targets were obtained in the OMIM database and GeneCards database.The protein-protein interaction network analysis using the String database shows that there are 183 nodes and 3450 interaction relationships in the network.The key targets obtained include TGFβ1,TNF,IL-6,IL-1β,CXCL8,MMP9,AKT1,MAPK8,MAPK1,VEGF-A,etc.Through GO enrichment analysis,178 biological processes were obtained.The main biological processes involved in the treatment of PF using YHD include nuclear receptor activity,cytokine receptor binding,cytokine activity,kinase regulation activity,and receptor and ligand activity.Through KEGG pathway analysis,a total of 184 targets were obtained.They are distributed in 164 signal pathways.The related signal pathways involved mainly include PI3K-AKT signal pathway,TNF signal pathway,Notch signal pathway,etc.2.YHD is capable of lowering the degree of alveolitis and fibrosis in PF model rats,reducing the expression of M1 macrophage marker iNOS and inflammation in the early stage of PF,reducing the expression of M2 macrophage marker ARG1 in the later stage,and intervening in the polarization of macrophages to M2 cells.3.The YHD drug-contained serum can inhibit the expression of lung fibroblast’s M1 phenotype markers iNOS,CD16/32 and M2 macrophage markers CD206 and ARG1.Moreover,the YHD drug-contained serum can also reduce the concentration of IL-1β and TNF-α in the culture medium of M1 macrophage model,reduce the concentration of IL-1β and TNF-α in the culture medium of M2 macrophage model,interfere in the polarization of macrophage,and promote the secretion of pro-inflammatory/anti-inflammatory cytokines.4.①YHD drug-contained serum can reduce the expression of α-SMA,COL-1,and COL-3 proteins of lung fibroblasts in the mouse lung fibroblast-M2 macrophage co-culture system,block the phenotypic transformation of fibroblasts to myofibroblasts,and reduce the expression of TGFβ1,Smad3,and p38 protein in the TGF-β/Smad signal pathway of lung fibroblasts.②The results of second-generation sequencing of the M2 macrophages transcriptome in the co-culture system show that there were 1433 differential expressed genes between the model group and the blank space group,and there were 481 differential expressed genes between the model group and the YHD group.The GO biological function enrichment and KEGG pathway analysis show that the effect of YHD on M2 macrophages may involve several biological processes include the immune system process,apoptosis process,negative regulation of cytokine production,cell proliferation,cell movement,and signal transduction,etc.The qRT-PCR experiment verified the expression of TNF,IRF4,CXCR4,CX3CR1,and Notchl genes,suggesting that the above sequencing results are reliable.③The detection of Notch signal pathway gene targets Notchl,NICD1,D114,and RBP-Jκ in M2 macrophages in the co-culture system indicates that YHD drug-contained serum can up-regulate the expression of these proteins,which is a possible mechanism for the decoction to regulate the polarization of macrophage.Conclusions1.YHD can intervene in the PF development through multiple ingredients,multiple targets,multiple effects,and multiple pathways.2.YHD can inhibit the polarization of macrophages in rats with PF and improve the degree of PF in rats.3.YHD drug-contained serum can inhibit the polarization of M1 and M2 macrophages and affect the immune environment of PF.4.YHD drug-contained serum can regulate the phenotypic transformation of lung fibroblastmyofibroblast induced by M2 macrophage co-culture,and its mechanism of action is possibly related to the intervention of TGF-β/Smad signal pathway and Notch signal pathway. |
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