Fig. 4

DMF applied in vitro induces M2 macrophage polarization. The murine macrophage cell line RAW 264.7 was used to verify DMF’s effect on phenotype shift. M1 macrophage phenotype was induced first by LPS (5 μg/ml) for 24 h, and then DMF (20, 50, or 100 μM) or vehicle was applied to the culture and allowed to incubate for 24 h. In vitro application of DMF reduced the immunocytochemical staining of iNOS⁺ cells (a) and increased the immunocytochemical staining of Arg1⁺ cells (b). The results were significant by semi-quantitative analysis of immunofluorescence for iNOS (c) and Arg1 (d). DMF application significantly attenuated mRNA expression of iNOS (e) and TNF-α (f). mRNA expression of Arg 1 (g) and IL-10 (h) increased significantly after DMF treatment. These results suggested a polarization shift from M1 to M2 phenotype. Scale bars in a and b are 50 μm. Comparisons between the DMF-treated groups and DMF-untreated control group (PBS) were performed using the Mann–Whitney U test. Results are expressed as means ± SEM (*p < 0.05 compared to the group stimulated with LPS only, n = 4)