Figure 6.

Magnolol attenuated the M1/M2 imbalance caused by cisplatin damage. Phenotypic changes in macrophages by magnolol treatment were analyzed in TA muscle tissue using immunostaining and flow cytometry. TA muscles were obtained from control (con), magnolol (mag) cisplatin (cis), or cis+mag mice at the end of the experiment (day 42). (A) Macrophage phenotyping using immunofluorescence staining with the pan-macrophage marker CD68 (green) and M2 regenerative macrophage specific marker CD163 (red). Magnification: ×40, Scale bar: 50 μm. Merged macrophages were enlarged in red box, Scale bar: 20 μm. (B) CD163⁺/CD163⁻ ratio calculated by dividing the numbers of CD68⁺CD163⁺ merged yellow pixel by those of CD68⁺CD163⁻ green pixel. (C) Flow cytometry analysis of CD86⁺CD206⁻ M1, CD86⁺CD206⁺ M2b, CD86-CD206⁺CD163⁻ M2a, and CD86⁻CD206⁺CD163⁺ M2c macrophages. Representative contour plots of macrophages characterized by CD86 and CD206 gated on CD45⁺CD11b⁺F4/80⁺ subsets (upper panel) and M2a or M2c macrophages characterized by CD163 and CD206 gated on CD86⁻CD206⁺ subsets (lower panel). (D) Bar graphs showing mean% of M1, M2b, and M2a/c macrophages in CD11b⁺F4/80⁺cells and (E) mean% of M2c macrophages in CD11b⁺F4/80⁺cells. (F) M1/M2c ratio calculated based on the number of M1 and M2c macrophages in CD45⁺CD11b⁺F4/80⁺ gated cells. Data represent mean ± SEM of 5 mice, and all data are representative from three individual experiments. *P < 0.05; **P < 0.01 compared to con, ^#P < 0.05; *P < 0.05 compared to mag, and ^$P < 0.05; ^$$P < 0.01; ^$$$P < 0.001 compared to cis using Turkey's test. The letters for no significance were not shown.