Figure 7
Impacts of rapamycin and AGEs on macrophage polarization.
(a) RAW264.7 cells were treated with rapamycin (100 nM) or 3-MA for 24 h, LPS (100 ng/ml) was used simultaneously for 24 h to stimulate macrophage polarization to M1. CD11c (M1 marker) positive cells were analyzed by flow cytometry (n = 2). (b) RAW264.7 cells were treated with AGEs, AGEs plus 3-MA for 24 h, respectively. LPS was used for M1 polarization. CD11c (M1 marker) positive cells were analyzed by flow cytometry (n = 2). (c) After treatment with rapamycin or 3-MA for 24 h, IL-4 (10 ng/ml) was used simultaneously for 24 h to stimulate macrophage polarization to M2. CD206 (M2 marker) positive cells were analyzed by flow cytometry (n = 2). (d) RAW264.7 cells were incubated with rapamycin or 3-MA, and CQ was added simultaneously for inhibiting autophagic flux. After 24 h, western blotting was performed with LC3 and β-actin antibody. (e) RAW264.7 macrophage cells were treated with rapamycin or 3-MA for 24 h, respectively. LPS was used for M1 polarization. Relative mRNA levels of M1 related markers (tnf-a, inos, il-1b, il-6, mcp-1 and cox-2) were detected by real-time PCR (n = 3). Data are presented as the mean ± SD.*P < 0.05, **P < 0.01. (f) RAW264.7 cells were treated with AGEs, 3-MA, and AGEs plus 3-MA for 24 h, respectively. LPS was added simultaneously to stimulate M1 polarization. Relative mRNA levels of tnf-a, inos, il-1b, and il-6 were detected by real-time PCR (n = 3). Data are presented as the mean ± SD.*P < 0.05, **P < 0.01. (g) Relative mRNA levels of M2 related markers (arg-1, mrc-1, il-10 and mgl1) were detected in RAW264.7 cells after treatment with rapamycin or 3-MA plus IL-4 for 24 h. IL-4 was used for stimulating M2 polarization (n = 3). Data are presented as the mean ± SD.*P < 0.05, **P < 0.01.
