Fig. 6

hUC-EVs-ATO promoted M2 macrophage polarization by inducing autophagy in an mTOR-dependent manner. BMDM-M1 macrophages were obtained, and the corresponding intervention methods were identical to those above. A Electron micrography was performed on autophagic vesicles of unstimulated BMDM-M1 and BMDM-M1 macrophages stimulated with hUC-EVs, ATO or hUC-EVs-ATO for 24 h. Autophagosomes and autophagic lysosomes are indicated with red arrows. B The expression of p-mTOR, mTOR, P62, LC3 I and LC3 II was assayed in the cell lysates of BMDM-M1 macrophages with different interventions by Western blotting. C RAW264.7 cells were transfected with GFP-mCherry-LC3B fusion protein, and the number of autophagosomes in macrophages with different interventions was observed with confocal microscopy. Magnification: × 200. Scale bar, 50 μm. Autophagosomes presented with a yellow signal. D Number of autophagosomes in each macrophage with different interventions. The data shown are representative of three independent experiments and are presented as the mean ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001, n.s. = not significant. E MHY1485, an mTOR activator, or rapamycin, an mTOR inhibitor, was used to treat BMDM-M1 macrophages before hUC-EV-ATO stimulation. The expression of p-mTOR, mTOR, P62, LC3 I and LC3 II was assayed via Western blotting. F Changes in the expression of M1- or M2-related markers in BMDM-M1 macrophages and the frequencies of M1 (CD86⁺) and M2 (CD206⁺) in BMDM-M1 macrophages observed via flow cytometry after treatment with hUC-EVs-ATO. G The fold changes at the mRNA levels of TNF-α, IL-1β, iNOS, IL-10, TGF-β and Arg1 relative to BMDM-M1 macrophages without any intervention were calculated by the 2^−▲▲CT method. The data shown are representative of three independent experiments and are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, n.s. = not significant