Mesenchymal Stem Cells Derived-exosomes Inhibit Macrophage Pyroptosis And Alleviate Acute Lung Injury,and Mechanism Of Shenfu Injection In The Treatment Of Pulmonary Fibrosis

Background:Acute respiratory distress syndrome(ARDS)is a serious diffuse lung disease.Although the current mechanical ventilation and medication(antibiotics,glucocorticoids,anticoagulants,etc.)have made significant progress,the average mortality rate of ARDS is still more than 40%.Therefore,in-depth study of the internal mechanism of ARDS and new clinical treatment targets may be the key to treatment.Acute lung injury(ALI)is an important pathological process of ARDS.During the development of ALI lung inflammation,alveolar macrophages(AMs)may affect other cells in the lung through different forms of death,such as necrosis,autophagy,and pyroptosis,causing cell death and tissue inflammation to form a positive feedback loop,but its specific mechanism is not completely clear.Therefore,it is of great significance to explore the role of different AMs death forms in ALI,and to develop new targets and new treatment modalities.Mesenchymal stem cells(MSCs)are multifunctional stromal cells that can differentiate into a variety of cell types and have multiple functions such as immune regulation and promotion of tissue repair.Advantages,studies have found that the function of MSCs mainly depends on their paracrine effects,of which exosomes are particularly important.Compared with MSCs,exosomes have the advantages of cell-free structure,low temperature resistance,easy penetration of the blood-brain barrier,and convenient storage and transportation.They are a potential new treatment method.The research content of this subject is divided into two parts:Study 1:The protective effect of Wharton’s Jelly-derived mesenchymal stem cells(WJ-MSCs)derived exosomes(MSCs-Exo)on lipopolysaccharide(LPS)-induced acute lung injury in mice;Study 2:The Role of AMs pyroptosis in the Pathogenesis of ALI and whether MSCs-Exo can reduce lung injury by inhibiting AMs pyroptosis.Method:Study 1:We firstly use tissue adhesion method to extract WJ-MSCs and verify that the extracted cells meet the criteria for mesenchymal stem cell identification.Secondly,the exosomes from WJ-MSCs and MRC-5 cell culture supernatants were extracted by ultracentrifugation and identified by electron microscopy,NTA,Western blot and other methods.Finally,intratracheal instillation of lipopolysaccharide to construct an ALI mouse model,two cell-derived exosomes were used to intervene on it,and inflammatory factors in serum and bronchoalveolar lavage fluid(BALF)were detected by Chest CT,lung wet/dry ratio,histopathology,ELISA,and other methods evaluated the effects of two different cell-derived exosomes on ALI.Study 2:First,the number of AMs and pyroptosis-related proteins in the BALF of ALI mice were analyzed to verify the role of AMs pyroptosis in ALI,and whether exosomes can inhibit AMs pyroptosis.Secondly,use LPS/Nigericin(Nig)to establish two macrophage pyroptosis models of the J774A.1 macrophage cell line and the primary AMs,and use MSCs-Exo to intervene.By cell morphology observed,PI staining,the release of inflammatory factors and LDH in the cell supernatant,and the detection of pyroptosis-related proteins,we can evaluate whether MSCs-Exo can play an anti-macrophage pyroptosis effect by inhibiting the activation of caspase-1.Result:Study 1:The cells derived from WJ are with the characteristics of mesenchymal stem cells,and the vesicles we extracted are exosomes.After constructing the ALI model,we used two kinds of cell-derived exosomes to intervene on it,and found that MSCs-Exo can reduce lung injury,reduce the release of inflammatory factors IL-1β and IL-18,and improve pulmonary vascular permeability.The above results suggest that exosomes derived from mesenchymal stem cells maintain the characteristics of their parental cells and have repair functions.Study 2:AMs deaths in BALF in the model group increased significantly,and MSCs-Exo intervention can reduce AMs deaths.Pyroptosis-related proteins in lung tissues suggest that the expression of NLRP3,caspase-1,Gasdermin D(GSDMD),IL-1β,IL-18,activated caspase-1,GSDMD and IL-1β protein in the model group increased significantly,while the expression and activation of pyroptosis-related proteins in the MSCs-Exo intervention group were inhibited.In vitro cell experiments found that MSCs-Exo can inhibit the activation of caspase-1,reduce the expression of NLRP3,caspase-1,GSDMD,IL-1β and Cleaved-GSDMD protein in the pyroptosis model,and caspase-1 p20 and IL-in the supernatant.1β p17 and IL-18 protein expression.Conclusion:1.We used two different cell-derived exosomes to intervene in the mouse ALI model.The results showed that the high-dose exosomes derived from MSCs can alleviate ALI in mice,and the effect of the high-dose group is better than that of the middle-dose group;exosomes are a paracrine part of the parental cells,which maintain the characteristics of the parental cells.Compared with other cells,exosomes derived from mesenchymal stem cells have therapeutic effects.2.In LPS-induced ALI,AMs pyroptosis can cause early tissue damage and inflammation,and play an important role in the occurrence of ALI.MSCs-Exo can inhibit the activation of caspase-1 and reduce the pyroptosis of AMs cells,thereby reducing lung injury.MSCs-Exo is a new treatment option in the early treatment of ALI.Background:Idiopathic pulmonary fibrosis(IPF)is disease with high mortality,and its effective treatment is limited.Shenfu injection is a traditional Chinese medicine which can improve circulation and protect cells.In this study,we aimed to investigate the feasibility and mechanism of Shenfu injection in the treatment of IPF.Method:The components and targets of Shenfu injection were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)database.The targets of Shenfu injection were standardized by UniProt database.The Genecards and OMIM databases was used to search for IPF-related genes.The Venn diagram of gene intersection was drawn using the OmicStudio tools,and the protein-protein interaction network was visualized using the Cytoscape 3.7.2 software.Moreover,the metascape online software was applied to explore the enriched Gene Ontology(GO)terms and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways,and the Cytoscape 3.7.2 software was used to construct the target-pathway network.Molecular docking was used to visualize the interactions between the main active compounds and targeted proteins.Animal experiments were performed to validate the effects and mechanisms of Shenfu injection.Result:We obtained 46 co-targets of Shenfu injection and IPF.Among the hub target genes,several genes with important functions were enriched,including TNF,IL-6,IL-1B,TP53,JUN,CASP3 and CASP8.The pathway enrichment analysis for the hub target genes identified pathways in cancer,tuberculosis,hepatitis B,apoptosis and IL-17 signaling.Molecular docking results showed that the main active compound Ginsenoside Re had high affinity to the core target proteins.These results suggested that Shenfu injection may have a positive effect in the treatment of IPF.Consistent with this finding,animal experiments showed that Shenfu injection significantly reduced pulmonary fibrosis in a bleomycin-induced mouse IPF model with inhibition of apoptosis and inflammation by downregulating IL-1β,caspase-3 and phosphorylated NF-κB.Conclusion:Our results demonstrated that Shenfu injection efficiently alleviate IPF through multi-targets in inflammation-,apoptosis-and cancer-related pathways,which provided first evidence and reference to the feasibility of Shenfu injection in the treatment of IPF.