| Objective:To investigate the therapeutic effect of umbilical cord mesenchymal stem cell-derived extracellular vesicle（MSC-EVs）on pulmonary fibrosis（PF）and the possible related mechanisms.Methods:（1）Isolation and identification of EVs: Umbilical cord tissues of newborns delivered normally in the Department of Obstetrics and Gynecology of Xi ’an Central Hospital were collected,cut into pieces on a sterile operating table and cultured with special culture medium for stem cells.A single passage was carried out when70%-80% of the cells had gathered.When passing to the fifth generation,cell supernatant was collected.Centrifuge was centrifuged at 300×g and 2000×g for 10 min,then centrifuge was centrifuged at 10000×g for 30 min,and centrifuge was set at 100,000×g for 70 min,and then re-suspended with inorganic phosphate buffer（PBS）to obtain exosome suspension.The collected particles were detected by transmission electron microscopy,and the size of the detected particles was tracked by nanoparticles.Finally,its surface antigen（Calnexin,CD9）was identified by Western Blot.（2）Establishment of PF model in vivo.Forty-five adult male C57BL/6 mice aged from 8 to 12 weeks and weighing about 20±2g were selected.Bleomycin（BLM）was selected for induction.Endotracheal intubation and endotracheal infusion were used for drug administration.Forty-five C57BL/6 mice were randomly divided into Sham group,BLM+PBS group and BLM+ MSC-EVs group.Mice were anesthetized by intraperitoneal injection of 4%chloral hydrate（10m L/kg）,and BLM（5U/kg）was dripped into the lungs of mice in BLM+PBS group and BLM+MSC-EVs group according to their body weight.3 h after administration,mice in BLM+ MSC-EVs group were intratracheal intubated again with50μg MSC-EVs（diluted with 50μl PBS）,and mice in BLM+PBS group were intratracheal injected with equal volume PBS.Mice were sacrificed 7,14 and 28 days after administration,and lung tissues were collected for subsequent experiments.（3）HE staining and Masson staining were performed on some lung tissues of mice in the above three groups to observe the pulmonary lesions of mice.According to the above staining results,the lung tissues of the three groups of mice were selected for m RNA sequencing on the 28 th day after drug administration.KEGG enrichment analysis and GO enrichment analysis were performed on the sequencing results to determine the biological process and related signal pathways that might be involved in BLM-induced PF in vivo.（4）The effect of MSC-EVs on PF and related mechanisms were verified in vitro.A549 cells were selected to establish in vitro PF model using TGF-β1.The cells were randomly divided into control group,Control + MSC-EVs group,control+TGF-β1 group and TGF-β1+ MSC-EVs group.The cells of each group were evenly spread into a six-well plate,and serum-free medium was added to starve for 24 hours to achieve synchronization,then the original medium was discarded.TGF-β1 was diluted in the medium without fetal bovine serum（FBS）to a final concentration of 10ng/ml,and cultured for 24 h to induce fibrosis in A549 cells in the control+TGF-β1 and TGF-β1+MSC-EVs groups,and the other groups were cultured with PBS at the same concentration for 24 h.After the induction of fibrosis,the original medium was discarded and cells from control group and Control+TGF-β1 group were collected.Control +MSC-EVs group and TGF-β1+ MSC-EVs group were added into serum-free medium diluted exosomes（10μg/ml）for induction for 12 h,and cells were collected.Western Blot was used to detect the expression of collagen deposition related molecules（Collagen I,α-SMA）,oxidative stress related molecules（Nrf2,HO-1）and inflammation related molecules（NF-κB P65,IL-2,IL-1β）.（5）According to m RNA sequencing and analysis results,Immunohistochemical staining（IHC）and Western Blot were performed on some lung tissues of mice at 14 and 28 days after administration.The therapeutic effects of MSC-EVs on PF and related mechanisms were verified from multiple perspectives.Results:（1）Under transmission electron microscope,the shape of foreign bodies isolated from MSC culture was elliptic vesicles with a diameter of 50-200 nm.Western Blot results showed that Calnexin was expressed in MSC group,while CD9 was only expressed in MSC-EVS group.（2）In vivo PF results showed that MSC-EVs could alleviate BLM-induced inflammatory cell infiltration,alveolar collapse or structural disappearance,and severe perialveolar hyperemia in mice,which was most obvious after28 days of administration.（3）Absolute value of difference multiple（FC）≥2,P<0.05 was the basic standard for screening differentially expressed genes.Between Sham group and BLM+PBS group,133 up-regulated genes and 124 down-regulated genes were screened.GO enrichment analysis showed that BLM-induced PF mainly involved molecular functions and cellular components.Molecular function mainly involves the structural components of extracellular matrix and immune response,while the cellular components are mainly concentrated in collagen aggregation.KEGG pathway enrichment analysis indicated that BLM may induce PF in vivo mainly by regulating the signal pathways of epithelial mesenchymal transformation,protein digestion and absorption,and immune inflammatory mechanism.（4）In vitro Western Blot results showed that MSC-EVs did not stimulate normal cells,but could regulate TGF-β1-induced collagen I,α-SMA,Nrf2,HO-1,NF-κB P65,IL-2 and IL-1β expression in A549 cells.（5）IHC staining and Western Blot showed that MSC-EVs could inhibit the expression of Collagen I,α-SMA,Nrf2,HO-1,NF-κB P65,IL-2 and IL-1β in BLM-induced lung tissues of mice.Conclusion:（1）The substances isolated from MSC culture are exosomes,which can be used for subsequent experimental studies.（2）MSC-EVs can alleviate pulmonary fibrosis in mice.（3）The occurrence of PF mainly involves molecular functions and cellular components,which may involve collagen deposition,nuclear receptor-related proteins,immune regulation and other related mechanisms.（4）MSC-EVs may treat PF by reducing extracellular matrix deposition,inhibiting oxidative stress and immune inflammatory response. |
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