Fig. 5

hUC-EVs-ATO more effectively polarized M1 macrophages toward M2 macrophages in vitro. Bone marrow-derived macrophages (BMDMs) were collected from the femurs and tibias of 7-week-old C57BL/6 mice. On the sixth day, 100 ng/mL LPS (Sigma-Aldrich) and 20 ng/mL IFN-γ (PeproTech) were added to induce BMDM-M1. Twenty-four hours later, the stimulated macrophages were treated with hUC-EVs, ATO or hUC-EVs-ATO for 24 h. A Changes in the expression of M1- or M2-related markers in BMDM-M1 macrophages observed via flow cytometry after treatment with ATO, hUC-EVs or hUC-EVs-ATO. B Frequencies of M1 (CD86⁺) and M2 (CD206⁺) in BMDM-M1 macrophages analyzed by flow cytometry after treatment with ATO, hUC-EVs or hUC-EVs-ATO. C Protein expression of Arg1 and iNOS in the cell lysates of BMDM-M1 macrophages with different interferences was detected by immunoblotting analysis. D The fold changes at the mRNA level 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