Study On The Effect And Mechanism Of Bone Marrow Mesenchymal Stem Cell-derived Exosomes Ameliorate Silica-induced Pulmonary Fibrosis

?Objective?To observe the efficacy of intervention of bone marrow mesenchymal stem cell-derived exosomes on silica-induced pulmonary fibrosis by using a rat model that exposed silica dust,and explore initially the possible mechanisms involved.?Methods?1.Bone marrow mesenchymal stem cells(BMSCs)were isolated and cultured by whole bone marrow adherence method and identified by flow cytometry and three-lineage differentiation(osteogenic,adipogenic,and chondrogenic lineages).2.The conditioned medium of the third-generation BMSCs was collected,and the exosomes were separated and purified by differential ultracentrifugation,and identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western Blot.3.Recipient rats were randomly divided into control group,silica model group,BMSC transplantation group,and exosome transplantation group.The rat model of experimental silicosis pulmonary fibrosis was induced with 1.0mL of one-off infusing silica suspension using the non-exposed intratracheal instillation(50mg/mL/rat).On the second day after exposure to silica,rats in the control group and the silica model group were injected with1.0mL of sterile D-PBS through the tail vein.To the BMSC transplantation group,1.0mL BMSC suspension(2×10~6cells/mL/rat)was injected through a tail vein puncture.To the exosome transplantation group,1.0mL exosome suspension(200?g/mL/rat)was injected through a tail vein puncture.The rats were sacrificed 28 days after intervention,and the lung coefficient,hydroxyproline content and histopathological changes were detected to observe the effect of intervention.The mechanisms that may be involved in the intervention of exosomes on experimental silicosis pulmonary fibrosis model rats were then explored by detecting profibrotic factors TGF-?1,markers of EMT and expression of EMT-related signaling pathway proteins.?Results?1.Primary BMSCs of rats were initially non-homogeneous in morphology,most of them were round and a few were fusiform.As the number of passages increased,some non-adherent cells were gradually removed.When passed to the third generation,the cells were homogeneous,spindle shape,showing large flake radial or swirl-like colonies;Flow cytometric analysis demonstrated that third generation cells highly expressed of CD90,CD73,and CD44(99.58%,99.63%and 99.85%respectively),with little evidence of the expression of CD45 and CD11b(5.58%and 0.04%respectively);In addition,alizarin red S,oil red O and toluidine blue staining showed that BMSCs successfully differentiated into osteoblasts,adipocytes and chondrocyte lineages.The above results are consistent with the minimum criteria for the identification of mesenchymal stem cells(MSCs),confirming that the vast majority of cells in the third generation of this study were BMSCs.2.The results of TEM showed that the exosomes extracted by differential ultracentrifugation were circular or elliptical in shape,non-homogeneous in size,and had a complete membrane structure,which was typically cup-shaped and contained low electron density substances;The results of NTA showed that the main peak of the exosome particle size was 127.5nm,accounting for 97.5%of all particles,and the particle concentration was7.5E+6/mL;The results of Western Blot showed that the expression of the exosome marker protein CD63 could be detected.The above results are consistent with the identification criteria of exosomes,confirming that the success of extracting exosomes from CM of BMSCs using differential ultracentrifugation.3.Lung coefficient.After exposure to silica,the lung coefficients of the silica model group,BMSC transplantation group and exosome transplantation group was increased compared with the control group(P<0.05).Compared with the silica model group,the lung coefficient of the exosome transplantation group and the BMSC transplantation group decreased,but there was no significant difference between the three groups.4.Hydroxyproline content in lung tissue.After exposure to silica,the HYP content in the lung tissue of the silica model group was increased compared with the control group(P<0.05).However,after transplantation of exosomes and BMSCs,the content of HYP was decreased compared with the silica model group(P<0.05).5.Histopathological changes of lung.H&E staining and Masson trichrome staining showed that after exposure to silica,compared with the control group,the alveolar structure of the silica model group was severely damaged compared with the control group,accompanied by a large amount of inflammatory cell infiltration and the formation of silicotic nodules.In addition,blue-stained collagen fibers increased,indicating that the degree of pulmonary fibrosis was aggravated.However,after transplantation of exosomes and BMSCs,the number and area of inflammatory cells and silicotic nodules were reduced,the number of blue-stained collagen fibers was decreased,and the alveolar structure and fibrosis were improved.In addition,the modified Ashcroft semi-quantitative score was consistent with histopathological(P<0.05).6.The expression level of profibrotic factor TGF-?1 in serum.After exposure to silica,the expression level of TGF-?1 was increased in the silica model group compared with the control group(P<0.05).However,the expression level of TGF-?1 was decreased after transplantation of exosomes and BMSCs(P<0.05).7.Changes of EMT marker protein expression.After exposure to silica,the expression of epithelial markers CK19 and E-cadherin was decreased in the lung tissue of the silica model group compared with the control group,but the expression of the fibrosis marker?-SMA increased(P<0.05).However,after transplantation of exosomes and BMSCs,the expression of CK19 and E-cadherin increased,and the expression of?-SMA decreased(P<0.05).8.Expression of Wnt/?-catenin signaling pathway-related proteins.The results of IHC showed that the expression of?-catenin,P-GSK-3?and cyclin D1 was increased in the silica model group compared with the control group,while GSK-3?was decreased.On the other hand,after transplantation of exosomes and BMSCs,GSK-3?expression increased,while?-catenin,P-GSK-3?and cyclin D1 expression decreased.?Conclusions?1.Exogenous transplanted exosomes can exert similar effects as BMSCs in inhibiting silica-induced pulmonary fibrosis,indicting that exosomes can be used alone as a cell-free intervention for the treatment of silicosis.2.Transplantation of exosomes can alleviate silica-induced pulmonary fibrosis by reducing the level of profibrotic factor TGF-?1 and inhibiting the progression of EMT.3.After exposure to silica,the Wnt/?-catenin signaling pathway associated with the development of EMT is abnormally activated,while the transplantation of exosomes attenuates Wnt/?-catenin signaling,which may be one of the mechanisms involved in anti-fibrosis.