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

Figure 6 Mesenchymal stem cells-extracellular vesicles may alleviate pulmonary fibrosis in mice by regulating oxidative stress response in lung tissue. A: The expressions of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) 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) can’t cause oxidative stress in cells (P > 0.05), and transforming growth factor (TGF)-β can cause obvious oxidative stress in A549 (Nrf2: ^eP < 0.01 vs control group; HO-1: ^fP < 0.001 vs control group), while MSC-EVs can reverse the above pathological process brought about by TGF-β (Nrf2: ^aP < 0.05; HO-1: ^cP < 0.001); D-G: The expression of Nrf2 and HO-1 in lung tissue was observed by immunohistochemical (IHC) staining (D and F). The statistical analysis of the expression level of each protein in IHC staining (E and G). On day 14 and day 28, Nrf2 (14 d: P > 0.05; 28 d: ^fP < 0.001 vs sham group), HO-1 (14 d: ^fP < 0.001 vs sham group; 28 d: ^fP < 0.001 vs sham group) deposition was observed in the bleomycin + phosphate buffered saline group, and the above pathological changes could be reversed after MSC-EVs treatment [Nrf2 (14 d: P > 0.05; 28 d: ^cP < 0.001), HO-1 (14 d: ^cP < 0.001; 28 d: ^cP < 0.001)]; H: The western blot results of the expression of Nrf2 and HO-1 in lung tissue of mice in each group; I and J: The statistical analysis of western blot results of each protein in mouse lung tissue. There were no significant changes in Nrf2 expression on day 7 (P > 0.05) and day 14 (P > 0.05) of pulmonary fibrosis development, but an increase in Nrf2 expression could be detected on day 28 (^fP < 0.001 vs sham group). No matter whether on day 7, 14, or 28, the expression of HO-1 (7 d: ^dP < 0.05 vs sham group; 14 d: ^fP < 0.001 vs sham group; 28 d: ^eP < 0.01 vs sham group) in lung tissue of mice was significantly higher than that in the sham group. After MSC-EVs treatment, the expression of HO-1 decreased significantly at 7 d (^aP < 0.05) in the acute phase, 14 d in the subacute phase (^cP < 0.001), and 28 d in the chronic phase (^bP < 0.01). However, the expression of Nrf2 decreased significantly only on day 28 (^cP < 0.001). MSC: Mesenchymal stem cell; EV: Extracellular vesicle; BLM: Bleomycin; PBS: Phosphate buffered saline; Nrf2: Nuclear factor E2-related factor 2; HO-1: Heme oxygenase-1.