The Effects Of Antler Stem Cells And Their Derived Exosomes On Pulmonary Fibrosis In Mice

Background and purpose:Pulmonary fibrosis(PF)is a severe pulmonary interstitial disease.Its pathological features include diffuse collagen deposition in lung tissue,poor elasticity,weakened regeneration capacity of lung epithelial cells,and alveolar structure destruction and fusion,which leads to airway remodeling and severely reduces lung ventilation.These processes are almost irreversible,which seriously affects the life quality and shortens the survival time of the patients.The pathogenesis of PF,including the activation of inflammatory cells and lung fibroblasts stimulated by harmful substances in the early stage,while in the middle and late stages,the infiltration of inflammatory cells,especially the secretion of various cytokines by macrophages(M2 type),such as transforming growth factor?(TGF-?),continuously activates lung fibroblasts and transforms them into myofibroblast phenotype,and obtains stronger extracellular matrix(ECM)secretion ability.Recently,due to air pollution,special pathogens,and other factors,the incidence of PF has gradually increased,and the age of onset has gradually decreased.Thus,how to treat and prevent PF is a problem that needs to be solved urgently.In recent years,stem cell therapies based on mesenchymal stem cells(MSCs)have achieved positive results in various animal models and clinical trials,because they have low immunogenicity and can create a role for tissue repair and regeneration friendly microenvironment.MSCs first reaches the lungs after vein transplantation which can be more effective in treating lung injury.Generally speaking,MSCs mainly treats PF in two ways:1)Replace damaged lung epithelial cells after differentiation,thereby promoting lung tissue regeneration.2)Regulate the phenotype of epithelial cells,immune cells,and fibroblasts through paracrine effects.According to data from the National Institutes of Health(NIH),there are currently 15(completed or ongoing)clinical trials of MSCs in PF treatment.MSCs is an undifferentiated stem cell population in almost all tissues,usually obtained from bone marrow,fat,umbilical cord blood,and placental tissues.However,with different donors,tissue types,uses,etc.,the applications and effects of MSCs are also different.Therefore,it is essential to select and expand the types of stem cell therapy.As the only accessory organ in mammals that can regenerate periodically,the antler exhibits two unique biological phenomena in its regeneration cycle:dead tissue(hard antlers)is attached for a long time but never inflamed,and giant wounds(after the hard antler falls off)heal quickly without leaving scars.The regulatory mechanism of inflammation and fibroblast phenotype has enlightening significance for the treatment of PF.Professor Li of our group has found that Antler stem cells(AnSC)is the cell source for antler regeneration,combines the properties of embryonic stem cells and mesenchymal stem cells,and can be induced to differentiate into osteogenic,adipogenic and chondrogenic.AnSC is also the leading cause of the biological phenomena mentioned above.Our team found that AnSC can significantly inhibit the proliferation of peripheral blood mononuclear cells(PBMC)activated by concanavalin A(Con A)and exert an immunosuppressive effect.It is also confirmed that AnSC can inhibit collagen deposits,reduce inflammation infiltration,and effectively promote the scarless healing of skin wounds,and alleviate liver fibrosis using rat skin injury and liver fibrosis model.Moreover,previous studies have found that compared with MSCs of human/mouse,AnSC has the advantages of low immunogenicity,stronger proliferation and division ability and stable source.Therefore,AnSC may be used as a new source of stem cells to treat inflammatory and fibrotic diseases.Exosomes(Exos)are tiny vesicles secreted by cells between 50-150nm and coated with a bilayer lipid membrane.Exos contains a variety of proteins,miRNA,lnc RNA,DNA,liposomes,and other components.Because of its special structure,it can enter cells through endocytosis,and it plays a crucial role in cell-to-cell communication.Recently,studies have found that stem cells mainly function in the form of paracrine,and Exos,as their main component of paracrine,has great therapeutic potential.In addition,Exos can avoid many risks related to cell transplantation(such as tumorigenesis and xenogeneic cell immunity,)and has the advantages of storage and transportation.Moreover,the functional components carried by Exos play a longer-lasting role in the body,which is more effective in the treatment of diseases.AnSC has ethical limitations for its direct clinical use as a heterogeneous animal cell,but its Exos can circumvent this limitation.Furthermore,compared to other MSC-Exos,AnSC-Exos may be more effective in treating PF due to the unique phenotype of AnSC.In This study,bleomycin(BLM)-induced PF mouse model was established and was intervened with AnSC or Antler stem cell derived exosomes(AnSC-Exos).The effects were evaluated,and the relative molecular mechanisms of fibroblast phenotypes and immune regulation were investigated.The main contents of this project are as followsThe first part is the study of the effect of AnSC on PF in miceMethods:AnSC was isolated and identified.PF mouse model was established by BLM tracheal instillation,and AnSC intervention(AnSC group)was given to the tail vein on the 7th day of modeling.Adipose MSC(ADSC)intervention(ADSC group)was used as a positive control and physiological saline as a negative control(BLM group).The body weight,survival rate,and other indicators of the mice were recorded,and histological samples were preserved to further judge the intervention effects.Results:The isolated AnSC expresses the MSC markers CD73,CD90 and CD105,and can differentiate into bone cells,chondrocytes,and adipocytes.Compared with the BLM group,the weight and survival rate of PF mice in the AnSC group were significantly increased.Lung tissue morphology showed that the alveolar structure of the intervention group was significantly improved.Sirius Red and Masson staining showed that collagen deposition of the intervention group was significantly improved,and the proliferation of lung epithelial cells(Ki67)was significantly increased.Fibronectin,?-SMA,Collagen-I,and Collagen-?were significantly reduced.A further comparison found that the therapeutic effect of AnSC on PF was significantly better than that of ADSC.It is suggested that AnSC may be used as a source of cells for the treatment of PF.The second part is the effect of AnSC-exos on PF in miceMethods:AnSC derived exosomes(AnSC-exos)was extracted and identified.PF mouse model was established by BLM tracheal instillation,and AnSC-Exos intervention(AnSC-Exos group)was given to the tail vein on the 7th day of modeling.AnSC intervention(AnSC group)was used as a positive control and physiological saline as a negative control(BLM group).The mouse body weight,survival rate,and other indicators were recorded,and histological samples were preserved to further judge the intervention effects.Results:AnSC-exos was successfully extracted.The AnSC-Exos obtained is a disc-shaped vesicle with a particle size of 40-100nm and expresses the Exos markers CD9,CD63,and TSG101.The evaluation of the therapeutic effects of PF mice showed that compared with the BLM model group,the weight and survival rate of PF mice in the AnSC-Exos group were significantly increased.Lung tissue morphology showed that the alveolar structure of the intervention group was significantly improved,and Sirius Red and Masson staining showed collagen deposition in the intervention group was significantly improved.The proliferation of lung epithelial cells(Ki67)was significantly increased,and Fibronectin,?-SMA,Collagen-I,and Collagen-?were significantly reduced.A further comparison found that the therapeutic effect of AnSC-Exos on PF is similar to that of AnSC,and there is no significant difference.It is suggested that AnSC-Exos may be used as a potential drug for the clinical intervention of PF.The third part discusses the mechanism of AnSC-exos intervention on PF in miceMethods:Immunofluorescence staining,Western Blot,and q RT-PCR methods were used to investigate the effect of AnSC-Exos on the heterogeneity of macrophages in the lung tissue of PF mice.Raw264.7 macrophages were used by in vitro Transwell co-culture experiments.Furthermore,the miRNAs in AnSC-Exos were subjected to high-throughput sequencing,and the Target Scan website was used for target prediction,and then miRNA mimics and inhibitors were used to verify in vitro Transwell co-cultivation experiments.Results:Immunofluorescence staining of lung tissue showed that compared with the BLM group,the macrophage infiltration in the AnSC-Exos group was significantly reduced,especially the number of interstitial macrophages(CD11b~+)derived from monocytes and macrophages in the blood;It was found that the infiltration of M2 type macrophages(CD163~+)in the lesion was also significantly reduced.In vitro co-cultivation experiments showed that AnSC-Exos could chemoattract the migration of Macrophages Raw264.7,but it cannot significantly inhibit its polarization toward M2.miRNA analysis showed that the content of let-7a and let-7b in AnSC-Exos are abundant(TOP2).Target prediction results showed that they have complementary base sites with CCL-7.In vitro co-culture experiments showed that fibroblast L929 could secrete CCL-7 to recruit Raw264.7 macrophages,and AnSC-Exos could inhibit this process.Furthermore,it was found that AnSC-Exos let-7a mimics and let-7b mimics can inhibit the CCL-7 expression of fibroblasts L929and recruit the migration of macrophages Raw264.7,while let-7a inhibitor and let-7b inhibitor are the opposite,which suggest AnSC inhibits the secretion of CCL-7 from lung fibroblasts to recruit mesenchymal macrophages through its miRNA let-7a and let-7b in Exos,however,it does not inhibit the polarization of macrophages to M2.Conclusion:Using the PF mouse model,we determined that AnSC has a significant therapeutic effect on PF,which may be due to its paracrine component miRNA let-7a and let-7b target binding to CCL-7,inhibiting the aggregation of interstitial macrophages,and reducing the overall number of macrophages,thereby reducing the infiltration of M2 macrophages(rather than inhibiting M2 polarization of macrophages).The discovery of the AnSC-Exos function and related mechanism of action provides a new strategy for the clinical treatment of PF.