Mesenchymal stem cells-extracellular vesicles alleviate pulmonary fibrosis by regulating immunomodulators

Figure 5 Mesenchymal stem cells-extracellular vesicles may alleviate pulmonary fibrosis by inhibiting epithelial-mesenchymal transformation. A: The expressions of collagen I and α-smooth muscle actin (α-SMA) in A549 cells of each group were verified by western blot; B and C: The statistical analysis of the expression levels of each protein. Mesenchymal stem cells-extracellular vesicles (MSC-EVs) did not affect normal cell proliferation and differentiation (P > 0.05). Transforming growth factor (TGF)-β can induce epithelial-mesenchymal transformation of cells (collagen I: ^dP < 0.05 vs control group; α-SMA: ^fP < 0.001 vs control group). However, MSC-EVs can reverse the epithelial-mesenchymal transformation process caused by TGF-β (collagen I: ^aP < 0.05; α-SMA: ^cP < 0.001); D-I: The expression of TGF-β1, collagen I, and α-SMA in lung tissue was observed by immunohistochemical (IHC) staining (D, F, and H). The statistical analysis of the expression level of TGF-β, collagen I, and α-SMA in IHC staining (E, G, and I). On day 14 and day 28, TGF-β1 (14 d: ^fP < 0.001 vs sham group; 28 d: ^fP < 0.001 vs sham group), collagen I (14 d: ^fP < 0.001 vs sham group; 28 d: ^fP < 0.001 vs sham group) and α-SMA (14 d: ^dP < 0.05 vs sham group; 28 d: ^eP < 0.01 vs sham group) deposition were observed in the bleomycin + phosphate buffered saline group, and the above pathological changes could be reversed after MSC-EVs treatment [TGF-β1 (14 d: ^cP < 0.001; 28 d: ^cP < 0.001)], collagen I (14 d: ^cP < 0.001; 28 d: ^cP < 0.001) and α-SMA (14 d: ^bP < 0.01; 28 d: ^cP < 0.001); J: The western blot results of each protein’s expression in mice’s lung tissue in each group; K and L: The statistical analysis of western blot results of each protein in mouse lung tissue. No matter whether on day 7, 14, or 28, the expression of epithelial-mesenchymal transformation gene-related collagen I (7 d: ^eP < 0.01 vs sham group; 14 d: ^eP < 0.01 vs sham group; 28 d: ^eP < 0.01 vs sham group) and α-SMA (7 d: ^eP < 0.01 vs sham group; 14 d: ^eP < 0.01 vs sham group; 28 d: ^dP < 0.05 vs sham group) in lung tissue of mice was significantly higher than that in the sham group. After MSC-EVs treatment, the expression of collagen I and α-SMA decreased significantly at 7 d (collagen I: ^aP < 0.05; α-SMA: ^bP < 0.01) in the acute phase, 14 d in the subacute phase (collagen I: ^bP < 0.01; α-SMA: ^bP < 0.01) and 28 d in chronic phase (collagen I: ^aP < 0.05; α-SMA: ^bP < 0.01). These results indicate that the development of pulmonary fibrosis is long-term, and the anti-epithelial mesenchymal transformation of MSC-EVs is stable and long-lasting. MSC: Mesenchymal stem cell; EV: Extracellular vesicle; BLM: Bleomycin; PBS: Phosphate buffered saline; TGF: Transforming growth factor; α-SMA: Alpha smooth muscle actin.