ABSTRACT
OBJECTIVE@#To study the anti- fibrotic effect of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) and explore the mechanism.@*METHODS@#Twenty-four C57 BL/6 mice were divided into 4 groups (=6), including the control group treated with intratracheal injection of saline (3 mg/kg); lung fibrosis model group with intratracheal injection of 1.5 mg/mL bleomycin solution (prepared with saline, 3 mg/kg); EXOs1 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the next day after modeling); and EXOs2 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the 10th day after modeling). At 21 days after modeling, pulmonary index, lung tissue pathology and collagen deposition in the mice were assessed using HE staining and Masson staining. The expression level of TGF-β1 was detected using ELISA, and vimentin, E-cadherin and phosphorylated Smad2/3 (p-Smad2/3) were detected using immunohistochemical staining. CCK8 assay was used to evaluate the effect of hUCMSCEXOs on the viability of A549 cells, and Western blotting was used to detect the expression levels of p-Smad2/3, vimentin, and E-cadherin in the cells.@*RESULTS@#Compared with those in the model group, the mice treated with hUCMSC-EXOs showed significantly reduced the pulmonary index ( < 0.05), collagen deposition, lung tissue pathologies, lowered expressions of TGF-β1 ( < 0.05), vimentin, and p-Smad2/3 and increased expression of E-cadherin. hUCMSC-EXOs given on the second day produced more pronounced effect than that given on the 11th day ( < 0.05). CCK8 assay results showed that hUCMSC-EXOs had no toxic effects on A549 cells ( > 0.05). Western blotting results showed that hUCMSC-EXOs treatment significantly increased the expression of E-cadherin and decreased the expressions of p-Smad2/3 and vimentin in the cells.@*CONCLUSIONS@#hUCMSC-EXOs can alleviate pulmonary fibrosis in mice by inhibiting epithelialmesenchymal transition activated by the TGF-β1/Smad2/3 signaling pathway, and the inhibitory effect is more obvious when it is administered on the second day after modeling.
Subject(s)
Animals , Humans , Mice , Epithelial-Mesenchymal Transition , Exosomes , Gene Expression Profiling , Gene Expression Regulation , Mesenchymal Stem Cells , Cell Biology , Pulmonary Fibrosis , Therapeutics , Transforming Growth Factor beta1 , Genetics , Umbilical Cord , Cell BiologyABSTRACT
OBJECTIVE@#To study the anti- fibrotic effect of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) and explore the mechanism.@*METHODS@#Twenty-four C57 BL/6 mice were divided into 4 groups (=6), including the control group treated with intratracheal injection of saline (3 mg/kg); lung fibrosis model group with intratracheal injection of 1.5 mg/mL bleomycin solution (prepared with saline, 3 mg/kg); EXOs1 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the next day after modeling); and EXOs2 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the 10th day after modeling). At 21 days after modeling, pulmonary index, lung tissue pathology and collagen deposition in the mice were assessed using HE staining and Masson staining. The expression level of TGF-β1 was detected using ELISA, and vimentin, E-cadherin and phosphorylated Smad2/3 (p-Smad2/3) were detected using immunohistochemical staining. CCK8 assay was used to evaluate the effect of hUCMSCEXOs on the viability of A549 cells, and Western blotting was used to detect the expression levels of p-Smad2/3, vimentin, and E-cadherin in the cells.@*RESULTS@#Compared with those in the model group, the mice treated with hUCMSC-EXOs showed significantly reduced the pulmonary index ( < 0.05), collagen deposition, lung tissue pathologies, lowered expressions of TGF-β1 ( < 0.05), vimentin, and p-Smad2/3 and increased expression of E-cadherin. hUCMSC-EXOs given on the second day produced more pronounced effect than that given on the 11th day ( < 0.05). CCK8 assay results showed that hUCMSC-EXOs had no toxic effects on A549 cells ( > 0.05). Western blotting results showed that hUCMSC-EXOs treatment significantly increased the expression of E-cadherin and decreased the expressions of p-Smad2/3 and vimentin in the cells.@*CONCLUSIONS@#hUCMSC-EXOs can alleviate pulmonary fibrosis in mice by inhibiting epithelialmesenchymal transition activated by the TGF-β1/Smad2/3 signaling pathway, and the inhibitory effect is more obvious when it is administered on the second day after modeling.
Subject(s)
Animals , Humans , Mice , Epithelial-Mesenchymal Transition , Exosomes , Mesenchymal Stem Cells , Pulmonary Fibrosis , Transforming Growth Factor beta1 , Umbilical CordABSTRACT
OBJECTIVE:To establish the method for the content determination of rapamycin (RAPA) in human monocyte THP-1 derived foam cells,and to study the effects of RAPA targeting preparation(RAPA-NP-Apt)targeting at foam cells. METH-ODS:Foam cells model were established through THP-1 cells were induced by oxidized low density lipoprotein. Foam cells were incubated with 200 ng/mL RAPA or 200,400,800 ng/mL RAPA-NP-Apt for 60 min. The content of RAPA was determined by HPLC. The determination was performed on Diamonsil C18 column with mobile phase consisted of acetonitrile-water(90:10,V/V) at flow rate of 1.0 mL/min. The column temperature was set at 40 ℃,and the detection wavelength was 278 nm. The sample size was 20 μL. RESULTS:The concentration of RAPA ranged 50-6400 ng/mL (r=0.99996) with average recovery of 98.72%(RSD=0.62%,n=3). RSDs of inter-day and intra-day were not more than 6.15%(n=6),RSD of stability was lower than 2%(n=6),and RSD of repeatability was 1.64%(n=6). After foam cells were incubated with RAPA or low-concentration,medi-um-concentration and high-concentration of RAPA-NP-Apt,the contents of RAPA were 12,43,98,140 ng/106 cells. CONCLU-SIONS:The method is simple,stable and reproducible. It can be used for content determination of RAPA in foam cells. RA-PA-NP-Apt can improve the effects of RAPA targeting at foam cells.