Fig. 5

EPC-EXOs-derived miR-222-3p promotes anti-inflammatory macrophages. A The relative expression of candidate genes was detected by qRT-PCR in EPCs and EPC-EXOs, n = 6, ^nsP > 0.05, *P < 0.05, ^&P < 0.001, ^#P < 0.0001. B The transfection efficiency of miR-222-3p mimic/mimic NC/inhibitor/inhibitor NC in BMMs was detected by qRT-PCR, n = 3, ^nsP > 0.05, *P < 0.05, **P < 0.01. C Representative immunofluorescence images of the groups: Vehicle + LPS, Mimic + LPS, Mimic NC + LPS, Inhibitor + LPS and Inhibitor NC + LPS interfering with CD86/ CD206 (red) and DAPI (blue) staining of F4/80⁺ macrophages (green). Scale bar: 50 μm. D, E Quantification of CD86⁺ or CD206⁺ cells in each group, n = 3, ^nsP > 0.05, **P < 0.01, ***P < 0,001. F Western blotting analysis of the expression levels of iNOS (pro-macrophage macrophages’ marker) and Arg-1 (anti-inflammatory macrophages’ marker) after LPS stimulation of macrophages with Mimic, Mimic NC, Inhibitor and Inhibitor NC. G qRT-PCR analysis of mRNA expression of pro-macrophage macrophages’ markers (iNOS, CD86 and TNF-α) and anti-inflammatory macrophages’ markers (CD206, Arginase-1 and IL-10) in macrophages treated with miR-222-3p-Mimic/Mimic NC/Inhibitor/Inhibitor NC, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. H The transfection efficiency of miR-222-3p mimic, inhibitor and NC in EPC-EXOs was detected by qRT-PCR, n = 3, ***P < 0.001, ****P < 0.0001. I, L Immunofluorescence images of F4/80⁺ macrophages (green), CD86⁺/CD206⁺ (red) and DAPI (blue) staining of spinal cord injury sections of mice representative of the groups: Vehicle, EPC-EXOs, EPC-EXOs Mimic, EPC-EXOs Mimic NC, EPC-EXOs Inhibitor, and EPC-EXOs Inhibitor NC at 7- and 14- days post-injury (dpi) in wild-type mice. Scale bar: 50 μm. J–N Immunofluorescence results were analyzed by ImageJ, GraphPad, and SPSS. n = 3, ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. O Western Blotting detected changes in the expression levels of iNOS and Arg-1 in macrophages in the region of injury at 7 and 14 days of spinal cord injury in the groups: EPC-EXOs-Mimic NC, Mimic, Inhibitor NC and Inhibitor-treated wild-type mice. P–S Statistical analysis of the expression levels of iNOS and Arg-1, n = 3, ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001