Study On The Regulation Of Pulmonary Fibroblast Activation By M2 Macrophages And The Intervention Mechanism Of Maimendong Decoctio

BackgroundPulmonary fibrosis(PF)is a chronic,progressive and irreversible end-stage lung disease with an increasing prevalence and poor prognosis.At present,the main drugs for the treatment of PF are pirfenidone and nintedanib,but their clinical application is limited due to their many side effects.Therefore,it is urgent to find a drug with less side effects and can improve PF.The pathogenesis of PF is still unclear.In recent years,a large number of studies have shown that macrophages play an important role in the occurrence and development of PF.The previous study of our group also found that Maimendong Decoction(MMDD)can reduce the infiltration of macrophages in the consolidation area of fibrotic lung tissue,but the molecular mechanism of MMDD regulating macrophages to exert anti-pulmonary fibrosis is still unclear.ObjectiveIn this study,we first evaluated the anti-pulmonary fibrosis effect of MMDD,and predicted the potential targets and signaling pathways of MMDD against pulmonary fibrosis based on network pharmacology.Then,through pharmacological experiments in vitro and in vivo,we further explored whether MMDD could inhibit M2-type macrophages.Cellmediated pulmonary fibroblast activation exerts a mechanism of action against pulmonary fibrosis.Methods1.The anti-fibrotic effect of MMDD on pulmonary fibrosis mice:Healthy male C57BL/6N mice were randomly divided into normal group,model group,MMDD low-dose group(4g/kg/d),and MMDD medium-dose group(8g/kg/d),MMDD high-dose group(16g/kg/d),12 mices in each group.Except for the normal group,the other four groups used intratracheal instillation of BLM to construct a mouse pulmonary fibrosis model,and the normal group was given the same volume of sterile PBS as a control.On the 11th day after modeling,each administration group was given different doses of MMDD by gavage for 17days.The samples were collected on the 28th day after modeling,and the lung mass and lung coefficient of the mice in each group were calculated;by histopathological staining,hydroxyproline acid hydrolysis method,immunohistochemical staining(IHC)and Western blotting(WB)detection to assess the degree of improvement in pulmonary fibrosis in mice.2.Network pharmacology research of MMDD:All the active ingredients and targets of the six traditional Chinese medicines in MMDD were obtained by using the database of Traditional Chinese Medicine System Pharmacological Component Analysis Platform(BATMAN),and the active ingredient-target network was constructed by Cytoscape 3.2.1 software.PharmGkb,OMIM,TTD,Drugbank,NCBIGene,and GeneCards databases were used to search for PF disease-related targets,which were combined and deduplicated as potential targets of PF.The common targets of the two were constructed through the STRING platform to construct a target protein interaction network,and the DAVID database was used for GO analysis and KEGG pathway enrichment analysis.3.MMDD inhibits M2 macrophage-mediated fibroblast activation in vivo:The expression levels of M2 macrophage markers CD206 and arginase-1 Arg-1 in mouse lung tissue were detected by IHC,WB and IF double staining to observe the effect of MMDD on on M2 macrophages in mouse fibrotic lung tissue.The activation of fibroblasts was observed by staining α-SMA and col I in mouse lung tissue by IHC;The expression of α-SMA and COLI in mouse lung tissue was detected by WB to observe the effect of MMDD on fibroblast activation;The expression levels of key proteins such as Akt,p-Akt,FOXO3a,pFOXO3a(ser253)and p-FOXO3a(ser315)in the PI3K/Akt/FOXO3a signaling pathway in mouse lung tissue were detected by WB.4.The mechanism of MMDD inhibiting M2 macrophage-mediated fibroblast activation in vitro:① The effect of MMDD on M2 macrophages and their released profibrotic factors:THP-1 monocytes were induced to THP-1 macrophages with 100ng/ml PMA,THP-1 macrophages were induced into M2 macrophages under the action of 20ng/ml IL-4 and 20ng/ml IL-13,and the macrophage markers CD68 and M2 macrophages were detected by cell IHC and WB,respectively The cell marker CD206 was used to judge whether THP-1 macrophages and M2 macrophages were successfully induced;the CCK-8 method was used to determine the dose of MMDD;The effect of different concentrations of MMDD on the expression levels of M2 macrophages and the pro-fibrotic factors released by MMDD was observed by WB method;②M2 macrophage culture medium supernatant containing MMDD had an effect on MRC-5 cell proliferation,Activated effects:The effect of M2 macrophage culture medium supernatant treated with different concentrations of MMDD on the proliferation of MRC-5 cells was detected by CCK-8 method;WB and IF methods were used to detect the effects of the supernatant of M2 type macrophages treated with different concentrations of MMDD on the activation of MRC-5 cells,the PDGFRB receptor of PDGFB on MRC-5 cells,and the expression of Akt,p-Akt,FOXO3a,p-FOXO3a(ser253)and pFOXO3a(ser315)in PI3K/Akt/FOXO3a signal pathway in MRC-5 cells.Results1.The anti-fibrotic effect of MMDD on mice with pulmonary fibrosis:MMDD can improve the body weight,reduce the degree of inflammatory infiltration and fibrosis in the lung tissue,reduce the expression of HYP in the pulmonary interstitium,and is accompanied by different degrees of recovery of alveolar cavity structure of BLM-induced fibrotic mice.The MMDD low-dose group had the most obvious effect.2.The results of network pharmacology of MMDD:89 active ingredients were screened in MMDD,involving 353 potential targets that MMDD and pulmonary fibrosis work together.Using the STRING platform,35 core targets were finally screened.KEGG enrichment analysis found that the PI3K/Akt signaling pathway is quite different,which may be one of the potential mechanisms.3.MMDD inhibits M2 macrophage-mediated fibroblast activation in vivo:MMDD can reduce the infiltration of M2 macrophages in the consolidation area of fibrotic lung tissue,reduce the pro-fibrotic factors TGF-β1 and PDGF-B receptor PDGF-RB expression in the lung tissue,reduce the expression of α-SMA and COL Ⅰ in the lung interstitium,and can significantly increase the expression of FOXO3a protein,significantly reduce the expression levels of Akt,p-Akt,p-FOXO3a(ser253)and p-FOXO3a(ser315)protein,and the effect was more obvious in the low-dose MMDD group.4.The mechanism of MMDD inhibiting M2 macrophage-mediated fibroblast activation in vitro:THP-1 monocytes can be induced to adherent THP-1 macrophages by 100ng/ml of PMA for 48h.20ng/ml IL-4 and 20ng/ml IL-13 acted on THP-1 macrophages for 24h and successfully induced M2 macrophages;After different concentrations of MMDD act on M2 macrophages,it can reduce M2 macrophages and the pro-fibrotic factors TGF-β1 released by them,and the effect of MMDD 25 group is more obvious;The supernatant of M2 macrophage culture medium containing MMDD at different concentrations had no effect on the proliferation of MRC-5 cells,but it could inhibit the expression of PDGF-B,α-SMA and COLI protein in MRC-5 cells,and significantly increase the expression of FOXO3a protein The expression levels of Akt,p-Akt,p-FOXO3a(ser253)and p-FOXO3a(ser315)were significantly reduced.Conclusions1.MMDD can significantly reduce the infiltration and inflammatory response of M2 macrophages in the fibrotic lung tissue of mice induced by BLM,reduce the deposition of collagen in the pulmonary interstitium and improve pulmonary fibrosis.2.MMDD exerts anti-pulmonary fibrosis effect by reducing the infiltration of M2 macrophages and their release of pro-fibrotic factors,thereby downregulating the PI3K/Akt/FOXO3a signaling pathway and inhibiting the activation of fibroblasts.