Metformin Attenuates Experimental Autoimmune Arthritis through Reciprocal Regulation of Th17/Treg Balance and Osteoclastogenesis
Figure 3
Treatment with metformin Treg cells and decreases Th17 cells in CIA mice and in vitro Th17 polarizing condition. (a–c) Splenocytes were also obtained from metformin-treated CIA () and control CIA () on day 35 after first immunization. (a) Both isolated CD4+ T cells were stained with anti-CD25, anti-Foxp3, and anti-IL-17 antibody. The proportions of CD4+IL-17+ T cells and CD4+CD25+ Foxp3+ regulatory T cells were analyzed using flow cytometry. (b) The gene levels of IL-17, Ahr, RUNX1, RORT, and Foxp3 in splenocytes were determined by real-time PCR. (c) The expressions of phosphorylated AMPK were measured by western blot. The fold of control measured pAMPK/AMPK/-actin ratio (right). (d–f) Isolated CD4+ T cells of C57BL/6 mice were cultured with under Th17 polarizing conditions in the presence or absence of 1 mM metformin for 3 days. (d) The cells were stained with anti-CD4, anti-CD25, anti-IL-17, and anti-Foxp3. (e) The mRNA expression levels of IL-17, Ahr, RUNX1, RORγT, and Foxp3 were determined by real-time PCR. (f) CD4+ T cells were stimulated with IL-6 10 ng/mL in the presence or absence of metformin 1 mM for 1 hour. The activation of pAMPK was measured by western blot. The representative results are shown in the right panel. Data are presented as the mean ± SD of four independent experiments (; ; ).
